Transgenic Crop Plants: Volume 2: Utilization and Biosafety

Transgenic Crop Plants

Chittaranjan Kole Charles H. Michler Albert G. Abbott Timothy C. Hall l

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Editors

Transgenic Crop Plants Volume 2: Utilization and Biosafety

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Editors Prof. Chittaranjan Kole Department of Genetics & Biochemistry Clemson University Clemson, SC 29634, USA [email protected]

Prof. Albert G. Abbott Department of Genetics & Biochemistry Clemson University Clemson, SC 29634, USA [email protected]

Prof. Charles H. Michler Director Hardwood Tree Improvement and Regeneration Center at Purdue University NSF I/UCRC Center for Tree Genetics West Lafayette, IN, USA [email protected]

Prof. Timothy C. Hall Institute of Developmental & Molecular Biology Department of Biology Texas A&M University College Station, TX, USA [email protected]

ISBN: 978-3-642-04811-1 e-ISBN: 978-3-642-04812-8 DOI 10.1007/978-3-642-04812-8 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009939124 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Transgenic Plants – known also as Biotech Plants, Genetically Engineered Plants, or Genetically Modified Plants – have emerged amazingly fast as a boon for science and society. They have already played and will continue to play a significant role in agriculture, medicine, ecology, and environment. The increasing demands for food, feed, fuel, fiber, furniture, perfumes, minerals, vitamins, antibiotics, narcotics, and many health-related drugs and chemicals necessitate the development and cultivation of transgenic plants with augmented or suppressed trait(s). From a single transgenic plant (Flavr Savr tomato with a longer shelf-life) introduced for commercialization in 1994, we have now 13 transgenic crops covering 800 million ha in 25 countries of six continents. Interestingly, the 13.3 million farmers growing transgenic crops globally include 12.3 million (90%) small and resource poor farmers from 12 developing countries. Increasing popularity of transgenic plants is well evidenced from an annual increase of about 10% measured in hectares but actually of 15% in “trait hectares.” Considering the urgent requirement of transgenic plants and wide acceptance by the farmers, research works of transgenic plants are now being conducted on 57 crops in 63 countries. Transgenic plants have been developed in over 100 plant species and they are going to cover the fields, orchards, plantations, forests, and even the seas in the near future. These plants have been tailored with incorporation of useful alien genes for several desirable traits including many with “stacked traits” and also with silencing of genes controlling some undesirable traits. Development, applications and socio-political implications of transgenic plants are immensely important fields now in education, research, and industries. Plant transgenics has deservedly been included in the course curricula in most, if not all, leading universities and academic institutes all over the world, and therefore reference books on transgenic plants with a class-room approach are essential for teaching, research, and extension. There are some elegant reviews on the transgenic plants or plant groups (including a 10-volume series “Compendium of Transgenic Crop Plants” edited by two of the present team of editors C. Kole and T.C. Hall published by Wiley-Blackwell in 2008) and on many individual tools and

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techniques of genetic transformation in plants. All these reviews could surely serve well the purpose for individual crop plants or particular methodologies. Since transgenic plant development and utilization is studied, taught, and practiced by students, teachers, and scientists of over a dozen disciplines under basic science, agriculture, medicine, and humanities at public and private sectors, introductory reference books with lucid deliberations on the concepts, tools, and strategies to develop and utilize transgenic plants and their global impacts could be highly useful for a broad section of readers. Deployment of transgenic crop plants are discussed, debated, regulated, and sponsored by people of diverse layers of the society, including social activists, policy makers, and staff of regulatory and funding agencies. They also require lucid deliberations on the deployment, regulations, and legal implications of practicing plant transgenics. More importantly, depiction of the positive and realistic picture of the transgenic plants should and could facilitate mitigation of the negative propaganda against transgenic plants and thereby reinforce moral and financial support from all individuals and platforms of the society. Global population is increasing annually by 70 millions and is estimated to grow to eight billion by 2025. This huge populace, particularly its large section from the developing countries, will suffer due to hunger, malnutrition, and chemical pollution unless we produce more and more transgenic plants, particularly with stacked traits. Compulsion to meet the requirements of this growing population on earth and the proven innocuous nature of transgenic plants tested and testified for the last 13 years could substantiate the imperative necessity of embracing transgenics. Traditional and molecular breeding practiced over the last century has provided enormous number of improved varieties in economic crops and trees including wheat and rice varieties that fostered the “green revolution.” However, these crop improvement tools depend solely on the desirable genes available naturally, creatable by mutation in a particular economic species, or their shuffling for desired recombinations. Transgenic breeding has opened a novel avenue to incorporate useful alien genes from not only other cross-incompatible species and genera of the plant kingdom, but also from members of the prokaryotes including bacteria, fungi, and viruses, and even from higher animals including mice and humans. An array of plant genetic engineering achievements starting from the development of insect resistance cotton by transforming the cry genes from the bacteria Bacillus thuringiensis to the present-day molecular pharming that enables the expression of interferon- gene from human in tobacco evidence for this pan-specific gene transfer. Human and animal safety is another general concern related to transgenic food or feed. However, there is no reliable scientific documentation of these health hazards even after 13 years of cultivation of transgenic plants and consumption of about 1 trillion meals containing transgenic ingredients. Utilization of transgenic plants has reduced the pesticide applications by 359,000 tons that would otherwise affect human and animal health besides causing air, water, and soil pollution and also mitigated the chance of consumption of dead microbes and insects along with foods or feeds.

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Gene flow from transgenic crop species to their cross-compatible wild relatives is a genuine concern and therefore required testing of a transgenic crop plant before deployment followed by comprehensive survey of the area for presence of interfertile wild and weedy plants before introduction of a transgenic crop are being seriously conducted. Addition of novel genotypes with transsgenes in the germplasms is increasing the biological diversity rather than depleting it. Using the genetically engineered plants has also eliminated greenhouse gas emission of 10 million metric tons through fuel savings. In fact, 1.8 billion liters of diesel have been saved because of reduced tillage and plowing owing only to herbicide-resistant transgenic crops. Many transgenics are now being used for soil reclamation. Above all, cultivation of transgenic crops has returned $44 billion of net income to the farmers. Perhaps, these are the reasons that 25 Nobel Laureates and 3,000-plus eminent scientists appreciated the merits and safety and also endorsed transgenic crops as a powerful and safe way to improve agriculture and environment besides the safety of genetically modified foods. Many international and national organizations have also endorsed health and environmental safety of transgenic plants; these include Royal Society (UK), National Academy of Sciences (USA), World Health Organization, Food and Agriculture Organization (UN), European Commission, French Academy of Medicine and American medical Association, to name a few. Production, contributions, and socio-political implications of biotech plants are naturally important disciplines now in education, research, and industries and therefore introductory reference books are required for students, scientists, industries, and also for social activists and policy makers. The two book volumes on “Transgenic Crop Plants” will hopefully fill this gap. These two book volumes have several unique features that deserve mention. The outlines of the chapters for these two books are formulated to address the requirements of a broad section of readers. Students and scholars of all levels will obtain a lot of valuable reading material required for their courses and researches. Scientists will get information on concepts, strategies, and clues useful for their researches. Seed companies and industries will get information on potential resources of plant materials and expertise for their own R&D activities. In brief, the contents of this series have been designed to fulfill the demands of students, teachers, scientists, and industry people, for small to large libraries. Students, faculties, or scientists involved in various subjects will be benefited from this series; biotechnology, bioinformatics, molecular biology, molecular genetics, plant breeding, biochemistry, ecology, environmental science, bioengineering, chemical engineering, genetic engineering, biomedical engineering, pharmaceutical science, agronomy, horticulture, forestry, entomology, pathology, nematology, virology, just to name a few. It had been our proud privilege to edit the 23 chapters of these two books those were contributed by 71 scientists from 14 countries and the list of authors include one of the pioneers of plant transgenics, Prof. Timothy C. Hall (one of the editors also); some senior scientists who have themselves edited books on plant transgenics; and many scientists who have written elegant reviews on invitation for quality books and leading journals. We believe these two books will hopefully

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serve the purposes of the broad audience who are studying, teaching, practicing, supporting, funding, and also those who are debating for or against plant transgenics. The first volume dedicated to “Principles and Development” elucidates the basic concepts, tools, strategies, and methodologies of genetic engineering, while the second volume on “Applications and Safety” enumerates the utilization of transgenic crop plants for various purposes of agriculture, industry, ecology, and environment, and also genomics research. This volume also deliberates comprehensively on the legal and regulatory aspects; complies to the major concerns; and finally justifies the compulsion of practicing plant transgenics. Glimpses on the contents of this volume (Volume 2: Transgenic Crop Plants: Applications and Safety) will perhaps substantiate its usefulness. This volume enumerates the application of transgenic technologies in crop plants for particular objectives in the first ten chapters. Biotic stress resistant, specifically insect resistant, transgenics have been developed and commercialized in several crops. An example with Bt-expressing cotton and maize alone, with current market share of about $3.26 billion substantiates their success and popularity (Chap.1). Abiotic stresses, particularly drought, salinity, and temperature extremes, have always been difficult to manipulate. Still success stories are pouring in recently from works mainly in cereals and vegetables (Chap.2). Herbicide-resistant transgenic plants (in cotton and canola) were first deregulated in 1995 and in 2008 more than 80% of the transgenic plants grown globally possess a transgenic trait for herbicide resistance. Chapter 3 details the present and emerging herbicide-resistant transgenic plants. Although the first transgenic trait was developmental, shelf-life in tomato to be precise, transgenics research for these traits are yet to make significant commercial headway but started producing encouraging results (Chaps.4 and 5). Deployment of transgenic plants for biofuel, pharmaceuticals, and other bioproducts has been enunciated in three chapters (Chaps.6, 7, and 9). Transgenic plants have been labeled as a culprit for potential threats to ecology and environment by a few groups of social activists. Chapter 8 addresses these weird concerns with suitable examples of utilization of transgenic plants for phytoremediation, biomonitoring, and the production of bioplastics and biopolymers for amelioration of ecology and environment. Plant genomics has emerged fast within the last three decades and facilitated fine-scale view of the plant genes and genomes. Transgenic plants have provided enormous resources for functional genomics studies and expected to play their roles as more plants systems and genes are targeted (Chap. 10). Scientists practicing transgenics are no less aware of the potential risks of genetic engineering than the few people with antagonistic views. Neither are the regulatory agencies at institutional, state, national, and international level regulatory agencies unaware of the steps to be involved for inspection, monitoring, and approval of transgenic plants for commercial use. Chapter 11 delineates all these aspects with examples from US and other continents and countries. Any original innovation or effort deserves recognition and also an incentive. The scope of patenting and intellectual property rights for materials owned and generated and methodologies implemented have been appreciated and enforced legally. These aspects related to transgenic crop plants have been discussed in Chap.12.

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The concluding chapter (Chap.13) briefs the contributions and concerns with the compliances and compulsion of practicing plant transgenics for science and society. We thank all the 41 scientists from nine countries for their elegant and lucid contributions to this volume and also for their sustained support through revision, updating and fine-tuning their chapters. We also acknowledge for the recent statistics that have been accessed from the web sites of Monsanto Company on “Conversations about Plant Biotechnology” and “International Service for the Acquisition of Agri-Biotech Applications on ISAAA Brief 39-2008: Executive Summary” and used them in this preface and elsewhere in the volume. We enjoyed a lot of our Clemson–Purdue–Texas A&M triangular interaction, constant consultations, and dialogs while editing this book, and also our working with the editorial staff of Springer, particularly Dr. Sabine Schwarz who had been supportive since inception till publication of this book. We will look forward to suggestions from all corners for future improvement of the content and approach of this book volume. Chittaranjan Kole, Clemson, SC Charles H. Michler, West Lafayette, IN Albert G. Abbott, Clemson, SC Timothy C. Hall, College Station, TX

Contents

1

Transgenic Crop Plants for Resistance to Biotic Stress . . . . . . . . . . . . . . . 1 N. Ferry and A.M.R. Gatehouse

2

Transgenic Plants for Abiotic Stress Resistance . . . . . . . . . . . . . . . . . . . . . . 67 Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin

3

Transgenic Crops for Herbicide Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Stephen O. Duke and Antonio L. Cerdeira

4

Understanding and Manipulation of the Flowering Network and the Perfection of Seed Quality . . . . . . . . . . . . . . . . . . . . . . . . . 167 Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski, Paul Scott, Diaa Al-Abed, and Shobha D. Potlakayala

5

Biotechnological Interventions to Improve Plant Developmental Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe, Ajay Arora, Martı´n-Ernesto Tiznado-Herna´ndez, Ravinder K. Goyal, Anish Malladi, Pradeep S. Negi, and Autar K. Mattoo

6

Transgenics for Biofuel Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh

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Plant Produced Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown, Charles Spillane, and Lokesh Joshi

8

Biotech Crops for Ecology and Environment . . . . . . . . . . . . . . . . . . . . . . . . 301 Saikat Kumar Basu, Franc¸ois Eudes, and Igor Kovalchuk

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9

Algal Biotechnology: An Emerging Resource with Diverse Application and Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Cunningham Stephen and Joshi Lokesh

10

Transgenic Crops and Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . 359 Narayana M. Upadhyaya, Andy Pereira, and John M. Watson

11

Deployment: Regulations and Steps for Commercialization . . . . . . . . 391 Kelly D. Chenault Chamberlin

12

Patent and Intellectual Property Rights Issues . . . . . . . . . . . . . . . . . . . . . . . 411 Jim M. Dunwell

13

Transgenic Crop Plants: Contributions, Concerns, and Compulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Brian R. Shmaefsky

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

Contributors

Diaa Al-Abed Edenspace Systems Corporation, Manhattan, KS 66502, USA, [email protected] Ajay Arora Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi 110012, India Saikat Kumar Basu Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4; Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 Anjanabha Bhattacharya National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA, [email protected] Bradley C. Campbell School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia Antonio L. Cerdeira Brazilian Department of Agriculture, Agricultural Research Service, EMBRAPA/Environment, C.P. 69, Jaguariuna-SP-13820000, Brazil Kelly D. Chenault Chamberlin USDA-ARS, Wheat, Peanut, and Other Field Crops Unit, 1301 N. Western, Stillwater, OK 74075, USA, kelly.Chamberlint@ars. usda.gov Zhiping Deng Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA Stephen O. Duke Agricultural Research Service, United States Department of Agriculture, P. O. Box 8048, University, MS 38677, USA, [email protected]

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Contributors

Jim M Dunwell University of Reading, Whiteknights, Reading RG6 6AS, UK, [email protected] Franc¸ois Eudes Bioproducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1 N. Ferry School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK, [email protected] Joel Gaffe Laboratoire Adaptation et Pathoge´nie des Microorganismes, LAPM, UMR 5163 CNRS-UJF, Institut Jean Roget BP 170, 38042 Grenoble cedex 9, France A.M.R. Gatehouse School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK Jared Q. Gerlach Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Ian D. Godwin School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia, [email protected] Stephen L. Goldman Department of Environmental Sciences, The University of Toledo, Toledo, OH 43606, USA, [email protected] Ravinder K. Goyal Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 3P6 Avtar K. Handa Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA, [email protected] Margaret C. Jewell School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia Lokesh Joshi Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland, [email protected] Michelle Kilcoyne Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland

Contributors

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Igor Kovalchuk Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4, [email protected] Pawan Kumar National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA Joshi Lokesh Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland, [email protected] Anish Malladi 30602, USA

Department of Horticulture, University of Georgia, Athens, GA

Autar K. Mattoo Sustainable Agricultural Systems Laboratory, The Henry A. Wallace Beltsville Agric Research Center, Beltsville, MD 20705-2350, USA Peter McKeown Genetics and Biotechnology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland Michael G. Muszynski Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, [email protected] Pradeep S. Negi Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA Andy Pereira 24061, USA

Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA

Shobha D. Potlakayala Penn State Milton S. Hershey College of Medicine, Hershey, PA 17033, USA, [email protected] Sairam Rudrabhatla Environmental Engineering, College of Science, Engineering and Technology, Penn State University, Middletown, PA 17057, USA, [email protected] Paul Scott Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA, [email protected]; Department of Agronomy, USDA-ARS, Iowa State University, Ames, IA 50011, USA, paul.scott@ ars.usda.gov Brian R. Shmaefsky Lone Star College – Kingwood, HSB 202V, 20,000 Kingwood Drive, Kingwood, TX 77339-3801, USA, Brian.R.Shmaefsky@ lonestar.edu

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Contributors

Rippy Singh National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA Charles Spillane Genetics and Biotechnology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland Alka Srivastava Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA Cunningham Stephen Glycoscience and Glycotechnology Group and the Martin Ryan Institute, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Martı´n-Ernesto Tiznado-Herna´ndez Fisiologı´a y Biologı´a Molecular de Plantas, Coordinacio´n de Tecnologı´a de Alimentos de Origen Vegetal, Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., Hermosillo, Sonora, Me´xico Narayana M. Upadhyaya CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260, Australia, [email protected] John M. Watson Australia

CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260,

Abbreviations

1-FFT 1-MCP 1-SST 2,4-D 2D-PAGE 4C3H 4CL 6G-FFT 6-SFT AAFC ABA ABRE Ac ACC AChE ACP ADP ae1 AHK2/3 AL-PCD ALS AMGT AMPA ANVISA AOS AOX ap AP1 AP2

Fructan:fructan 1-fructosyltransferase 1-Methylcyclopropene Sucrose:sucrose 1-fructosyltransferase 2,4-Dichlorophenoxyacetic acid Two-dimensional polyacrylamide gel electrophoresis 4-Coumarate 3-hydroxylase 4-Hydroxycinnamoyl CoA ligase Fructan:fructan 6G-fructosyltransferase Sucrose:fructan 6-fructosyltransferase Agriculture and Agri-Food Canada Abscisic acid ABA responsive element Activator gene 1-Aminocyclopropane-1-carboxylate Acetylcholinesterase Acyl-carrier protein Adenosine di-phosphate amylose extender gene Arabidopsis histidine kinase Apoptotic-like programmed cell death Acetolactate synthase Agrobacterium-mediated gene transfer Aminomethylphosphonic acid National Agency for Health and Surveillance of the Ministry of Health Allene oxide synthase Altenative oxidase apetalla gene APETALA1 gene APETALA2/ Apetela2 gene

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APHIS APX ARF arsC Asc at AtCKX1 Avr AZ BA BADH BAP BAR BBC BC BGI-RIS bla BMR BRS Bt bZIP CAD CAL CAMBIA CaMV CAT CBER CBF cbLCV CBS CCR CDC CDF cDNA CEL CEPA CFIA CFSAN CGH-1 CGIAR cGMP CHO CHO

Abbreviations

Animal and Plant Health Inspection Service Ascorbate peroxidase Auxin response factor Arsenate reductase Ascorbate antherless gene Arabidopsis thaliana cytokinin oxidase gene Avirulence Abscission zone Benzyladenine Betaine aldehyde dehydrogenase 6-Benzylaminopurine Bialaphos resistance gene British Broadcasting Corporation Biotech crop Beijing Genomics Institute- Rice Information System Beta-lactamase Brown midrib Biotechnology Regulatory Service Bacillus thuriengensis Basic leucine zipper Cinnamoyl alcohol dehydrogenase CAULIFLOWER gene Center for the Application of Molecular Biology to International Agriculture Cauliflower mosaic virus Chloramphenicol acetyltransferase/catalase Centre for Biologics Evaluation and Research CRT binding factor Cabbage leaf curl virus Columbia Broadcasting System Cinnamoyl CoA reductase Centers for Disease Control Cycling DOF Factor Complementary-DNA Cellulase Canadian Environmental Protection Act Canadian Food Inspection Agency Centre for Food Safety and Applied Nutrition Cardenolide 16’-O-glucohydrolase Consultative Group on International Agricultural Research Current GMP Chinese hamster ovary Choline dehydrogenase

Abbreviations

chs CIGB CMO CMS CMS CNN CNR CO ConA CONABIA conz1 COR COR CP CpTI CRE CRIIGEN CRT Cry CTNBio CVM CV-N D2GT2A dab DDB DDT DEFRA DET DGDG DHAsc dlf1 DNMA DRE DREB driPTGS Ds DsE DsG DSL dzr1 EA EBV EC EDB

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chalcone synthase gene Cuban Centre for Biotechnology and Genetic Engineering Choline monooxygenase Cytoplasmic male sterility Cellular membrane stability Cable News Network Colorless non-ripening gene Constans gene Concanavalin A National Advisory Commission on Agricultural Biotechnology constans of Zea mays1 gene Cold responsive Cold responsive gene Chloroplast Cowpea trypsin inhibitor Cytokinin response Comite´ de Recherche et d’Information Inde´pendantes sur le Ge´nie C-Repeat Crystal National Technical Commission on Biosafety Centre for Vetinary Medicine Cyanovirin Diacylglycerol acyltransferase 2A delayed abscission gene Damaged DNA binding protein Dichlorodiphenyltrichloroethane Department of Environment, Food and Rural Affairs Detiolated gene Digalactosyldiacylglycerol Dehydroascorbate delayed flowering1 gene Directorate of Agricultural Markets Dehydration responsive element DRE binding protein Direct repeat-induced PTGS Dissociation gene Enhancer trap Ds Gene trap Ds Domestic Substance List delta zein regulator1 gene Environmental assessment Epstein-barr virus European Commission Ethylene dibromide

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EFSA EFSA EIN EIS EMEA EMS En EPA EPA EPSP EPSPS ER ERE ERS EST ETC ETH EU F F1 FAD1 FAD3 FAO FD FDA FFDCA FIFRA fl2 FONSI FPPA Fr FST FT FT-ICR-MS Fuc FucT FUL Fx G3P GA/ GA3 Gal GalNAc GalT GAT

Abbreviations

European Food Safety Authority European Food Standards Agency Ethylene-insensitive gene Environmental Impact Statement European Agency for Evaluation of Medicinal Products Ethylmethane sulfonate Enhancer transposon Eicosapentaenoic Environmental Protection Agency Enolpyruvyl-shikimate-3-phosphate 5-Enolpyruvyl-shikimate-3-phosphate synthase Endoplasmic reticulum Ethylene responsive element Economic Research Service Expressed sequence tag Electron transport chain Ethylene European Union Florigenic signal Fraction 1 anti-phagocytic capsular envelope protein Flavin adenine dinucleotide Omega-3 fatty acid desaturase Food and Agriculture Organization of the United Nations FLOWERING LOCUS D gene Food and Drug Administration Federal Food, Drug and Cosmetic Act Federal Insecticide, Fungicide and Rodenticide Act floury-2 gene Finding of no significant impact Federal Plant Pest Act Fertility restorer gene Flanking sequence tag Flowering Locus T/Flowering Transition gene Fourier-transform ion cyclotron mass spectrometry Fucose Fucosyltransferase FRUITFUL gene Fucoxantine Glycerol-3-phosphate Gibberellic acid Galactose N-Acetylgalactosamine Galactosyltransferase Glyphosate N-acetyltransferase

Abbreviations

GC GCase GCS GDP GE GFLV GFP GI gigz1 GlcNAc GlyBet GM GMHT GMO GMP GMP GMPO GMS GNA GOI GOX GPAT GPX GR GRC GS1 GSH GSSG GST GTN GUS HBcAg HBsAg HBV HCMV hEPO hGM-CSF HIV HMX HR HRC HS HSP HSV

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Glutathione synthetase Glucosyl-N-acylspingosineglycohydrolase g-Glutamylcysteine synthase Gross domestic product Genetic engineering/Genetically engineered Grapevine fanleaf virus Green fluorescent protein Gigantea gene gigantea of Zea mays1 gene N-Acetylglucosamine Glycine betaine Genetically modified Genetically modified herbicide tolerant Genetically modified organism Genetically modified plant Good manufacturing practise Genetically modified plant organism Genic male sterility Galanthus nivalis agglutinin Gene of interest Glyphosate oxidoreductase Glycerol-3-phosphate acyltransferase Glutathione peroxidase Glutathione reductase/ Glyphosate resistant Glyphosate resistant Crop Glutamine synthase gene 1 Glutathione/ Glutamate synthase Glutathione disulfide Glutathione S-transferase Glycerol trinitrate ß-Glucuronidase Hepatitis B core antigen Hepatitis B surface antigen Hepatitis B virus Human cytomegalovirus Human erythropoietin Human granulocyte-macrophage colony-stimulating factor Human immunodeficiency virus Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine Hypersensitive response/ Herbicide resistant Herbicide resistant crop Heat shock Heat shock protein Herpes simplex virus

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HT/Ht I i.p. iAc IBA ICTSD id1 ida IDD IFN IgA IL-12 IMI IPM IPP IPR IPT IR IRGSP ISAAA ISIS ISR JA LB LD LEA Leu LFY LOG LOX1/2/3 lpa1 LPS LRR LTB Lys MAb MAPK MGDG MHBsAg Mi MIP miRNA MPSS mRNA

Abbreviations

Herbicide tolerant/tolerance Inhibitor transposon Intraperitoneal Immobile Ac transposon Indole-3-butyric acid International Center for Trade and Sustainable Development indeterminate1 gene inflorescence deficient in abscission gene ID-domain Interferon Immunoglobulin A Interleukin-12 Imidazolinone Integrated pest management Isopentyl diphosphate Intellectual Property Rights Isopentenyl transferase/ Isopentyl transferase Insect resistant/resistance International Rice Genome Sequencing Project International Service of AgriBiotech Applications Institute for Science and International Security Induced systemic resistance Jasmonic acid Left border of T-DNA Long-day Late embryogenesis abundant Leucine Leafy gene Lonely guy gene Lipoxygenase gene Lysophosphatidic acid receptor Lipopolysaccharide Leucine rich repeats Heat-labile enterotoxin, subunit B Lysine Monoclonal antibody Mitogen-activated protein kinase Monogalactosyldiacylglycerol Middle HBsAg Meloidogyne incognita resistance gene Major intrinsic protein Micro-RNA Massively parallel signature sequencing Messenger-RNA

Abbreviations

MS MS MST MT MTP MTT Mu MuIL-12 MV MVL NAA NAM NAS NAS NBS NCBI NDV NEPA Neu5Ac NIH NIL NMR NO NOI NoV NR NST NUE o2 OECD OFB OMT ORF Ori OSTP PAHs PAL PAMPs PAT PC PCB PCD PCR PCS

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Mass spectrometry Murashige and Skoog (medium) Members of the Landless Rural Workers Movement Metallothionein Metal-tolerance protein Multi-tasking transgenics Mutator transposon Murine IL-12 Methyl viologen Microcystis viridis lectin a-Napthalene acetic acid Napthaleneacetamide National Academy of Sciences Nicotinamine synthase Nucleotide binding site National Center for Biotechnology Information Newcastle disease virus National Environmental Policy Act 5-N-Acetyl-D-neuraminic acid National Institute of Health Near-isogenic line Nuclear magnetic resonance Nitric oxide Notice of Intent Norovirus Nitrate reductase Nac secondary wall thickening promoter factor Nitrogen use efficiency opaque-2 gene Organization for Economic Cooperation and Development Office of Food Biotechnology O-Methyl transferase Open reading frame Origin of replication Office of Science and Technology Policy Polycyclic aromatic hydrocarbon Phenyalanine ammonia lyase Pathogen associated molecular patterns Phosphinothricin-acetyltransferance Phytochelatin Polychlorinated biphenyl Programmed cell death Polymerase chain reaction Phytochlelatin synthase

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PEG PETN PG PG PHB pi PiP PIP PL PLC PLD PLE PME PMP PNT PPO PR Pro PS PSI PSII PTGS Put PVP PVX PyMSP4/5 QPM QTL RAP-DB rasiRNA RB RB rDNA RDX Rf RFLP R-gene rGSII rhCVFVIII rhEPO rhIF RHS RID1 RIL

Abbreviations

Polyethylene glycol Pentaerythritol tetranitrate Polygalacturonase Phosphatidylglycerol Polyhydroxybutyrate pistillata gene Plant Incorporated Protectant Plasma membrane intrinsic protein Pectate lyase Phospholipase C Phospholipase D Phospholipid cleaving enzyme Pectin methylesterase Plant-made pharmaceutical Plant with novel trait Polyphenol oxidase Pathogenesis-related Proline Phytosiderophores Photosystem 1 Photosystem 2 Post-transcriptional gene silencing Putrescine Plant Variety Protection Potato virus X Murine P. yoelii merozoite surface protein 4/5 Quality protein maize Quantitative trait loci Rice Annotation Project-Database Repeat-associated siRNA Non-toxin B-chain from ricin Right border of T-DNA Recombinant-DNA Hexahydro-1,3,5-trinitro-1,3,5 triazine Restorer of fertility gene Restriction fragment length polymorphism Resistance-gene Recombinant Griffonia simplicifolia lectin II Recombinant human clotting factor VIII Recombinant human erythropoietin Recombinant human intrinsic factor Royal Horticultural Society Rice Indeterminate1 gene Recombinant inbred line

Abbreviations

rin Rip RNAi ROIs ROS RT-PCR RWC s.c. SA SA SAGE SAGPyA SAM SAM SAR scFv scN SD SE SENASA Ser SFI1 sh2 siRNA SIV SL SMT soc SOC1 SOD SOliD Spd Spm Spm SQDG ssRNA STP su1 SVN TA TAC TAGI tasiRNA tb1

xxv

ripening inhibitor gene Ribosome inactivating protein RNA-interference Reactive oxygen intermediates Reactive oxygen species Reverse transcriptase-PCR Relative water content Subcutaneously Salicylic acid Splice acceptor Serial analyses of gene expression Secretariat of Agriculture, Livestock, Fisheries and Food S-Adenosylmethionine Shoot apical meristem Systemic acquired resistance Single chain variable fragment Soyacystatin N Short-day Substantial equivalence/ equivalent National Agri-food Health and Quality Service Serine Segestria florentina venom peptide shrunken2 gene Short/Small interfering RNA Simian immunodeficiency virus Selenocysteine lyase Seleno-cysteine methyl transferase suppessor of overexpression of constans gene Suppressor of Overexpression of Constans1 gene Superoxide dismutase Supported Oligo Ligation Detection Spermidine Spermine Suppressor-Mutator transposon Sulfoquinovosyldiacylglycerol Single-stranded RNA Signal transduction pathway sugary1 gene Scytovirin Transcriptional activator Tiller angle control gene The Arabidopsis Genome Initiative Transacting siRNA teosinte branched1 gene

xxvi

TCE TCOH TCP T-DNA TDZ TET TETRYL TF TFL Thr ti TILLING TIP TMV TNT Trp TRV TSCA UAS uf UN UNCTAD US USAID USDA USPTO UV VB Vgt1 Vgt2 VIGS VIP VLP VRO WHO WT WUE XTH Xyl XylT YCF1 YFP ZCN zfl1

Abbreviations

2.4,6-Trichloroethylene Chloral and trichoethanol 2,4,6-Tricholorophenol Transferred-DNA Thidiazuron Transiently expressed transposase N-Methyl-N, 2, 4, 6-tetranitroaniline Transcription factor Terminal Flower gene Threonine Trypsin inhibitor allele Targeting induced local lesions in genomes Tonoplast intrinsic protein Tobacco mosaic virus Trinitrotoluene Tryptophan Tobacco rattle virus Toxic Substances Control Act Upstream activator sequence uniflora gene United Nations United Nations Conference on Trade and Development United States US Agency for International Development United States Department of Agriculture United States Patent and Trademark Office Ultraviolet Vector backbone Vegetative to generative transition1 gene Vegetative to generative transition2 gene Virus-induced gene silencing Vegetative Insecticidal Protein Virus-like particle Variety Registration Office World Health Organisation Wild type Water use efficiency Xyloglucan endotransglucosylase/hydrolase Xylose Xylosyltransferase Yeast vacuolar glutathione Cd transporter Yellow fluorescent protein Zea CENTRORADIALIS gene Zea FLO/LFY1 gene

Abbreviations

zfl2 ZFN ZMM4/5 ZmRap2 g-GCS o3

xxvii

Zea FLO/LFY2 gene Zinc-finger nuclease Zea mays FULL1-like gene Zea mays related to AP2 gene g-Glutamyl cysteine synthetase Omega 3

Chapter 1

Transgenic Crop Plants for Resistance to Biotic Stress N. Ferry and A.M.R. Gatehouse

1.1

Introduction

We couldn’t feed today’s world with yesterday’s agriculture and we won’t be able to feed tomorrow’s world with today’s. – Lord Robert May, President of the Royal Society, March 2002

The human population is everexpanding; conservative estimates predict that the population will reach ten billion by 2050 (United Nations Population Division), and the ability to provide enough food is becoming increasingly difficult (Chrispeels and Sadava 2003). The planet has a finite quantity of land available to agriculture and the need for increasing global food production has led to increasing exploitation of previously uncultivated land for agriculture; as a result wilderness, wetland, forest and other pristine environments have been, and are being, encroached upon (Ferry and Gatehouse 2009). The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. For nearly 50 years, mainstream science has told us that this would be impossible without chemical pesticides (Pimental 1997). The global pesticide market is in excess of $30 billion per year (Levine 2007); despite this, approximately 40% of all crops are lost directly to pest damage (Fig. 1.1). These figures are simplified rough estimates; in reality crop losses to biotic stress are extremely difficult to quantify and vary by crop, year, and region.

N. Ferry (*) School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_1, # Springer-Verlag Berlin Heidelberg 2010

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Fig. 1.1 The world agricultural cake

1.2 1.2.1

Biotic Stress due to Insect Pests Crop Losses due to Insect Pests

[A]nimals annually consume an amount of produce that sets calculation at defiance; and, indeed, if an approximation could be made to the quantity thus destroyed, the world would remain skeptical of the result obtained, considering it too marvelous to be received as truth. – John Curtis, 1860

Arthropods are the most widespread and diverse group of animals, with an estimated 4–6 million species worldwide (Novotny et al. 2002). While only a small percentage of arthropods are classified as pests, they cause major devastation of crops, destroying around 14% of the world annual crop production, contributing to 20% of losses of stored grains and causing around US$100 billion of damage each year (Nicholson 2007). Herbivorous insects and mites are a major threat to food production for human consumption. Larval forms of lepidopterans are considered the most destructive insects, with about 40% of all insecticides directed against heliothine species (Brooks and Hines 1999). However, many species within the orders Acrina, Coleoptera, Diptera, Hemiptera, Orthoptera and Thysanoptera are also considered agricultural pests with significant economic impact (Fig. 1.2). Insect pests may cause direct damage by feeding on crop plants in the field or by infesting stored products and so competing with humans for plants as a food resource. Some cause indirect damage, especially the sap-feeding (sucking) insects by transmitting viral diseases or secondary microbial infections of crop plants.

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Fig. 1.2 Phylloxera, a sap-sucking pest of grape, almost devastated the European wine industry in the nineteenth century

1.2.1.1

The Phylloxera Plague

In the late nineteenth century, a phylloxera epidemic destroyed most of the vineyards for wine grapes in Europe, most notably in France. Grape phylloxera (Daktulosphaira vitifoliae, family Phylloxeridae) is a pest of commercial grapevines worldwide, originally native to eastern North America. These minute, pale yellow sap-sucking insects feed on the roots of grapevines. In Vitis vinifera, the resulting deformations and secondary fungal infections can damage roots, gradually cutting off the flow of nutrients and water to the vine. Phylloxera was inadvertently introduced to Europe in the 1860s. The European wine grape V. vinifera was highly susceptible to the pest and the epidemic devastated most of the European winegrowing industry. Some estimates hold that between two-thirds and nine-tenths of all European vineyards were destroyed. Native American grapes Vitis labrusca are naturally Phylloxera-resistant. The grafting of European grape vines onto resistant grape rootstock is the preferred method to cope with the pest problem even today (http://www.calwineries.com). Thus, phylloxera provides a clear example of how a single insect pest can nearly devastate a whole industry. Innumerable examples exist of insect pests that are highly injurious to agricultural production. The most notable for their destructive capacity being the

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Fig. 1.3 Monument to the cotton boll weevil Source: Wikimedia Commons

migratory locust (Locusta migratoria), Colorado potato beetle (Leptinotarsa decemlineata), boll weevil (Anthonomus grandis), Japanese beetle (Popillia japonica), and aphids, which are among the most destructive pests on earth as vectors of plant viruses (many species in ten families of the Aphidoidea), and the western corn rootworm (Diabrotica virgifera virgifera), also called the billion dollar bug because of its economic impact in the US alone. Curiously, one of these pests, the cotton boll weevil, responsible for neardestruction of the cotton industry in North America, is also ultimately responsible for subsequent diversification of agriculture in many regions, thus warranting a monument in the town of Enterprise, Alabama, in profound appreciation of its role in bringing to an end the state’s dependence on a poverty crop (Fig. 1.3). The global challenge facing agriculture is to secure large and high-quality crop yields and to make agricultural production environmentally sustainable. Control of insect pests would go some way towards achieving this goal.

1.2.2

Insecticides

Insecticides have been, and still are, a highly effective method to control pests quickly when they threaten to destroy crops. The chemical nature of the

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insecticides used has evolved over time. In early farming practices, inorganic chemicals were used for insect control; however, with the advances in synthetic organic chemistry that followed the two world wars the synthetic insecticides were born. In the 1940s, the neurotoxic organochlorine, DDT, was the pesticide of choice, but following its indiscriminate use it was reported to bio-accumulate in the food chain where it affected the fertility of higher organisms – such as birds. Rachel Carson first highlighted this in the book Silent Spring published in 1962; while her presumptions have since been proven to be wrong, the book was nevertheless an important signature event in the birth of the environmental movement. This pesticide was subsequently replaced by the comparatively safer organophosphate and carbamate-based pesticides (both acetylcholinesterase inhibitors) and many of these were replaced in turn by the even safer pyrethroid-based pesticides (axonic poisons). Synthetic pyrethroids continue to be used today despite the fact that they are broad-spectrum pesticides. The major limiting factor on the insecticide strategy is the occurrence of resistance in insect populations. In fact, resistance to insecticides has now been reported in more than 500 species (Nicholson 2007). Furthermore, resistance has evolved to every major class of chemical. The underlying causes of insecticide resistance are manyfold. Owing to wide usage and narrow target range, arthropods have been put under a high degree of selection pressure (Feyereisen 1995). Insecticide resistance may be characterized by: (a) Metabolic detoxification (upregulation of esterases, glutathione-S-transferases, and monoxygenases) (b) Decreased target site sensitivity (via mutation of the target receptor) (c) Sequestration or lowered insecticide availability In addition, cross-resistance to different classes of chemicals has occurred because of the fact that many insecticides target a limited number of sites in the insect nervous system (Raymond-Delpech et al. 2005). The five target sites in insects comprise: nicotinic acetylcholine receptors (e.g., imidacloprid), voltagegated sodium channels (e.g., DDT, pyrethroids), g-aminobutyric acid receptors (e.g., fipronil), glutamate receptors (e.g., avermectins), and acetylcholinesterase (AChE) (e.g., organophosphates and carbamates). The world insecticide market is dominated by compounds that inhibit the enzyme AChE. Together, AChE inhibitors and insecticides acting on the voltage-gated sodium channel, in particular the pyrethroids, account for approximately 70% of the world market (Nauen et al. 2001). Unfortunately, as insecticide target sites are conserved between invertebrates and vertebrates, insecticides have undesirable nontarget effects and unacceptable ecological impacts. Insecticides are implicated in the poisoning of nontarget insects, other arthropods, marine life, birds, and humans (Fletcher et al. 2000). The poisoning of nontarget organisms has obvious implications for biodiversity.

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Integrated Pest Management and Organic Agriculture

In parallel to the development of modern insecticides, the specific microbial toxins produced by the soil-dwelling bacterium Bacillus thuringiensis (Bt) are increasingly being adopted as biopesticides. In fact, microbial sprays of Bt are used in organic agriculture. This shift is due in part to a demand for increased safety both for humans and for the environment. Organic agriculture is a form of agriculture that relies on crop rotation, green manure, compost, biological pest control, and mechanical cultivation to maintain soil productivity and control pests, excluding or strictly limiting the use of synthetic fertilizers and synthetic pesticides, plant growth regulators, and genetically modified organisms (Directorate General for Agriculture and Rural Development of the European Commission). Integrated pest management (IPM) has also been proposed as a sustainable control system for insects. Several control systems are combined, including the judicious application of chemicals and biopesticides, use of trap crops, biological control, rotation, good husbandry and cultural control to manage all the pests of a particular crop (Gatehouse and Gatehouse 1999). Increasing crop varietal resistance is critical to both IPM and organic agriculture. It is unfortunate that ultimately neither organic agriculture nor IPM will be able to feed the world. In order to feed an increasing world population, more food must be produced in the future and on either the same amount, or less land (Ferry and Gatehouse 2009). Neither of these farming methods will be as productive as will be necessary to meet increased demands (Amman 2009).

1.2.4

Transgenic (Genetically Modified) Crops

Genetically modified (GM) maize and cotton varieties that express insecticidal proteins derived from B. thuringiensis (Bt) have become an important component in agriculture worldwide. At present, 20.3 million hectares of land is planted with insect-protected transgenic Bt cotton and maize (James 2007), with economic benefits from Bt cotton estimated at US$9.6 billion and maize US$3.6 billion (James 2007). Significantly, Phipps and Park (2002) showed that on a global basis GM technology has reduced pesticide use. These authors estimated that pesticide use was reduced by a total of 22.3 million kg of formulated product in 2000 alone.

1.2.4.1

Bacillus thuringiensis Toxins

B. thuringiensis (Bt) is a soil-dwelling bacterium of major agronomic and scientific interest. Whilst the subspecies of this bacterium colonize and kill a large variety of

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Table 1.1 Insecticidal properties of Bt toxins Insect order Cry protein Lepidoptera Cry1A, Cry1B, Cry1C, Cry1E, Cry1F, Cry1I, Cry1J, Cry1K, Cry2A, Cry9A, Cry9I, Cry15A Coleoptera Cry1I, Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, Cry8C, Cry14A, Cry23A Diptera Cry2A, Cry4A, Cry10A, Cry11A, Cry11B, Cry16A, Cry19A, Cry20A, Cry21A Hymenoptera Cry22A Nematodes Cry5A, Cry6A, Cry6B, Cry12A, Cry13A, Cry14A Liver fluke Cry5A

host insects, each strain tends to be highly specific. Toxins for insects in the orders Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), Coleoptera (beetles and weevils), and Hymenoptera (wasps and bees) (Table 1.1) have been identified (de Maagd et al. 2001), but interestingly none with activity towards Homoptera (sap suckers) have, as yet, been identified, although a few with activity against nematodes have been isolated (Gatehouse et al. 2002). Further, there is little evidence of effective Bt toxins against many of the major storage insect pests. Bt toxins (also referred to as d-endotoxins; Cry proteins) exert their pathological effects by forming lytic pores in the cell membrane of the insect gut. On ingestion, they are solubilized and proteolytically cleaved in the midgut to remove the C-terminal region, thus generating an “activated” 65–70 kDa toxin. The active toxin molecule binds to a specific high-affinity receptor in the insect midgut epithelial cells. Following binding, the pore-forming domain, consisting of a-helices, inserts into the membrane; this results in cell death by colloid osmotic lysis, followed by death of the insect (de Maagd et al. 2001). A number of putative receptors in the insect gut have been identified and include aminopeptidase N proteins (Knight et al. 1994; Sangadala et al. 1994; Gill et al. 1995; Luo et al. 1997), cadherin-like proteins (Vadlamudi et al. 1995; Nagamatsu et al. 1998; Gahan et al. 2001) and glycolipids (Denolf 1996). Transgenic plants expressing Bt toxins were first reported in 1987 (Vaeck et al. 1987) and following this initial study numerous crop species have been transformed with genes encoding a range of different Cry proteins targeted towards different pest species. Since bacterial cry genes (genes encoding Bt toxins) are rich in A/T content compared to plant genes, both the full-length and truncated versions of these cry genes have had to undergo considerable modification of codon usage and removal of polyadenylation sites before successful expression in plants (de Maagd et al. 1999). Crops expressing Bt toxins were first commercialized in the mid-1990s, with the introduction of Bt potato and cotton. Currently, 20.3 million hectares of land is planted with Bt cotton and maize (James 2007). Bt Maize Lepidopteran pests such as European corn borer Ostrinia nubilalis, fall armyworm Spodoptera frugiperda, and corn earworm Helicoverpa zea perennially cause leaf

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and ear damage to corn. The Bt concept was particularly attractive for maize, since it made it possible to combat European corn borer larvae hidden inside the stem of the plant for the first time. Bt maize has now been grown on a large scale for over a decade, particularly in the US. In 2007, insect-resistant Bt maize was grown on 21% of the total maize cultivation area, and Bt maize with a combination of insect and herbicide resistance was grown on a further 28% (James 2007). Various Bt maize varieties are also authorized in the EU. In 2007, there was notable cultivation of Bt maize primarily in Spain, where it was grown on around 75,000 hectares (Ortego et al. 2009). Transgenic corn hybrids expressing the insecticidal protein Cry1Ab from B. thuringiensis (Bt) var. kurstaki were originally developed to control European corn borer, and offer the potential for reducing losses by fall armyworm and corn earworm. Several events of transgenic Bt corn have been developed with different modes of toxin expression (Ostlie et al. 1997). Amongst the most promising events were Bt11 expressing the cry1Ab gene from B. thuringiensis subsp. kurstaki (Novartis Seeds) and MON810 expressing a truncated form of the cry1Ab gene from B. thuringiensis subsp. kurstaki HD-1 (Monsanto Co.). In both events, the endotoxins are expressed in vegetative and reproductive structures throughout the season (Armstrong et al. 1995; Williams and Davis 1997). Crops containing either of these events are collectively referred to as having “YieldGard technology.” Furthermore, a modified cry9C gene from B. thuringiensis subsp. tolworthi strain BTS02618A is expressed in maize (tradename StarLink – marketed by Aventis CropScience). StarLink corn has only been approved in the US for livestock feed use. In recent years, there has been increasing focus on another maize pest, this time a Coleopteran (beetle); the western corn rootworm. Western corn rootworm is one of the most devastating corn rootworm species in North America. Its larvae are root pests and can destroy significant percentages of corn if left untreated. In the US, current estimates show that 30 million acres (120,000 km) of corn (out of 80 million grown) are infested with corn rootworms. The United States Department of Agriculture (USDA) estimates that corn rootworms cause US$1 billion in lost revenue each year. To make matters worse, this pest is extending its geographical range all of the time – including spreading throughout Europe. Bt maize which is resistant to the western corn rootworm has been authorized in the US since 2003 and has been grown on a large scale since. YieldGard Rootworm uses event MON 863 and expresses the Cry3Bb1 protein from B. thuringiensis (subsp. kumamotoensis) in the plant, protecting the plant against root feeding from the western and northern corn rootworm larvae. Products containing both YieldGard Corn Borer (MON810) and YieldGard Rootworm (MON 863) are marketed under the trade name YieldGard Plus (http://www.agbios.com). Corn rootworm-resistant maize is also produced by expression of the cry34Ab1 and cry35Ab1 genes from B. thuringiensis strain PS149B1 (DOW AgroSciences LLC and Pioneer Hi-Bred International, Inc.).

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Bt Cotton Cotton fibers used in textiles around the world come from the seed hairs of Gossypium hirsutum. Cotton develops in closed, green capsules known as bolls that burst open when ripe, revealing the white, fluffy fibers. But cotton is more than just a fiber for textiles. It is also an important source of raw materials used in animal feed and for various processed food ingredients, including cottonseed oil, proteinrich cottonseed meal (mostly used as animal feed) and even leftover fibers can be used as food additives. Lepidopteran, particularly heliothine, pests can have an enormously damaging effect on a cotton crop and controlling these insects in conventional farming involves treatment with a number of insecticide sprays. In 1996, Bollgard1 cotton (Monsanto) was the first Bt cotton to be marketed in the US. Bollgard cotton produces the Cry1Ac toxin from B. thuringiensis (subsp. kurstaki), which has excellent activity on tobacco budworm and pink bollworm. These two insects are extremely important as both are difficult and expensive to control with traditional insecticides and the damage caused by them directly impacts on the harvestable plant organ, the cotton bolls themselves. Bollgard II1 was introduced in 2003, representing the next generation of Bt cottons. Bollgard II contains Cry1Ac plus a second gene from the Bt bacteria which encodes the production of Cry 2Ab (also subsp. kurstaki). WideStrike (a Trademark of DowAgrosciences) was registered for use in 2004, and like Bollgard II, it expresses two Bt toxins but this time Cry1Ac and Cry1F were used in combination. Both Bollgard II and WideStrike have better activity on a wider range of caterpillar pests than the original Bollgard technology. GM Bt cotton has become widespread, covering a total of 15 million hectares in 2007, or 43% of the world’s cotton. Most GM cotton is grown in the US and China, but it can also be found in India, South Africa, Australia, Argentina, Mexico, and Columbia (http://www.agbios.com). Currently, 20% of the cotton grown commercially in China expresses Cry1Ac in combination with a plant protease inhibitor, cowpea trypsin inhibitor (CpTI) (He et al. 2009).

Bt Potato Potato (Solanum tuberosum L.) is a major world food crop. Potato is exceeded only by wheat, rice, and maize in terms of world production for human consumption (Ross 1986). Many commercial potato varieties are highly susceptible to damage by the Colorado potato beetle. In 1999, 93% of the 1.1 million potato acres grown in the US were treated with a total of 2.6 million pounds of insecticide (http://www. usda.gov/). To date, few traditionally bred varieties have been produced with resistance to this major pest. Unfortunately, many of the pesticides currently used are broad-spectrum pesticides, killing not only the target pest but most of its natural enemies as well. The Cry3A d-endotoxin from B. thuringiensis Berliner subsp. tenebrionis is toxic to coleopterans, particularly chrysomelids (Krieg et al. 1983;

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Bauer 1990; MacIntosh et al. 1990). It is insecticidal against the Colorado potato beetle, L. decemlineata (Ferro and Gerlernter 1989). In 1995, Bt.Cry3A (NewLeafTM) potato became the first Bt-crop to be commercialized, although they are currently withdrawn from the US market. 1.2.4.2

Evolution of Resistance in Pest Populations

Perhaps one of the most important issues surrounding the cultivation of Bt crops relates to the evolution of target pest resistance, which could limit the life span of the technology. In the case of Bt toxins, this is a major concern for the organic farming community, since the potential for insect populations to evolve resistance to Bt will not only limit the effectiveness of Bt-expressing crops but also Bt-based biopesticides. Bt resistance in insect pests has been reported to develop within 4–5 generations in the laboratory (Stone et al. 1989). To date, the mechanism of resistance to Cry toxins in insects has been most commonly ascribed to the loss or inactivation of specific toxin-binding sites on midgut cell membranes (Ferre´ and Van Rie 2002). Other resistance mechanisms that have been proposed include a defect in the toxin activation by midgut proteases (Oppert et al. 1994; Sayyed et al. 2001), or an increased repair and/or replacement rate of Cry-damaged midgut cells by stem cells (Forcada et al. 1999). Studies have also revealed evidence for novel resistance mechanisms based on active defensive responses (Rahman et al. 2004; Ma et al. 2005). When one considers the ability of insects to evolve resistance to chemical pesticides (French-Constant 2004), the development of field resistance is inevitable and in fact was recently reported to have already occurred (Tabashnik and Carrie`re 2009). Analysis of monitoring data shows that some field populations of H. zea have evolved resistance to Cry1Ac, the toxin produced by first-generation Bt cotton (Tabashnik et al. 2008). Nonetheless, resistance of H. zea to Cry1Ac has not caused widespread crop failures in the field for several reasons (Tabashnik et al. 2008). First, the documented resistance is spatially limited. Second, from the outset, insecticide sprays have been used to improve the control of H. zea on Bt cotton because Cry1Ac alone is not sufficiently effective to manage this pest. Finally, GM cotton producing two Bt toxins (Cry2Ab and Cry1Ac) was planted on more than one million ha in the US in 2006; control of H. zea by Cry2Ab would limit problems associated with resistance to Cry1Ac (Jackson et al. 2004). Considerable effort has been devoted to delaying the onset of evolution of resistance, e.g., the use of refugia has been required/recommended in most regions growing Bt-crops depending upon the country in question (Tabashnik and Carrie`re 2009). Gene-stacking and integrated pest management should be combined to control this problem. 1.2.4.3

Unexpected Benefits

Interestingly, the expression of Bt has resulted in improved crop quality as a consequence of decreased levels of Fusarium infestation and fumonisin mycotoxin

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production as a direct result of reduced levels of insect pest damage. This benefit is particularly important in food crops such as maize (Glaser and Matten 2003).

1.2.4.4

A Global Technology

Of the global total of 12 million biotech farmers in 2007, over 90% were small and resource-poor farmers from developing countries; the balance of one million were large farmers from both industrialized countries such as Canada and developing countries such as Argentina. Of the 11 million small farmers, most were Bt cotton farmers; 7.1 million in China (Bt cotton), 3.8 million in India (Bt cotton), and the balance of 100,000 in the Philippines (GM maize), South Africa (GM cotton, maize and soybeans often grown by subsistence women farmers) and the other eight developing countries which grew GM crops in 2007. This modest uptake by subsistence farmers contributes towards the Millennium Development Goals of reducing poverty by 50% by 2015 and is a very important development (James 2007).

1.2.5

Other Sources of Insecticidal Molecules

The concept of employing genes encoding Bt toxins to produce insect-resistant transgenic plants arises from the successful use of Bt-based biopesticides. A number of other strategies for protecting crops from insect pests actually exploit endogenous resistance mechanisms (Harborne 1998; Gatehouse 2002a, b). Genes encoding such defensive proteins are obvious candidates for enhancing crop resistance to insect pests.

1.2.5.1

Enzyme Inhibitors

Interfering with digestion, and thus affecting the nutritional status of the insect, is a strategy widely employed by plants for defense, and has been extensively investigated as a means of producing insect-resistant crops (Gatehouse 2002a, b). Insectdigestive proteases tend to fall into four mechanistic classes (serine, cysteine, aspartic or metallo proteases – depending on the enzyme-active site residue). Numerous studies since the 1970s have confirmed the insecticidal properties of a broad range of protease inhibitors from both plant and animal sources (Jouanin et al. 1998; Gatehouse 2002a, b). Proof of concept for exploiting such molecules for crop protection was first demonstrated with expression of a serine protease inhibitor from cowpea (CpTI), which was shown to significantly reduce insect growth and survival (Hilder et al. 1987). These studies were subsequently extended to include a greater range of target pests (Gatehouse et al. 1994; Graham et al. 1995; Xu et al. 1996), and a broader range of inhibitors and plant species, including economically

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important pest species, particularly lepidopterans (Broadway 1997; De Leo et al. 2001). Since many economically important coleopteran pests predominantly utilize cysteine proteases for protein digestion, inhibitors for this class of enzyme (cystatins) have also been investigated as a means for controlling pests from this order. Oryzacystatin, a cysteine protease inhibitor isolated from rice seeds, is effective towards both coleopteran insects and nematodes when expressed in transgenic plants (Leple et al. 1995; Urwin et al. 1995; Pannetier et al. 1997). Similarly, the cysteine/aspartic protease inhibitor equistatin, from sea anemone, is also toxic to several economically important coleopteran pests, including the Colorado potato beetle (Outchkourov et al. 2003). More recent studies have included the stacking of different families of inhibitors to increase the spectrum of activity (Abdeen et al. 2005). A major limitation, however, to this strategy for the control of insect pests arises from the ability of some lepidopteran and coleopteran species to respond and adapt to ingestion of protease inhibitors by either overexpressing native gut proteases, or producing novel proteases that are insensitive to inhibition (Bown et al. 1997; Jongsma and Bolter 1997). Thus, detailed knowledge about the enzyme–inhibitor interactions, both at the molecular and biochemical levels, together with detailed knowledge on the response of insects to exposure to such proteins is essential to effectively exploit this strategy. The concept of inhibiting protein digestion as a means of controlling insect pests has been extended to the inhibition of carbohydrate digestion. For example, inhibitors of a-amylase have been expressed in transgenic plants and shown to confer resistance to bruchid beetles (Shade et al. 1994; Schroeder et al. 1995).

1.2.5.2

Lectins

Lectins, found throughout the plant and animal kingdoms, form a large and diverse group of proteins identified by a common property of specific binding to carbohydrate residues, either as free sugars, or more commonly, as part of oligo- or polysaccharides. Many physiological roles have been attributed to plant lectins, including defense against pests and pathogens (Chrispeels and Raikhel 1991; Peumans and Vandamme 1995). Although some lectins are toxic to mammals, and are thus not suitable candidates for transfer to crops for enhanced levels of protection, this is by no means universal. Many lectins are not toxic to mammals, yet are effective against insects from several different orders (Gatehouse et al. 1995), including homopteran pests such as hoppers and aphids (Powell et al. 1995; Sauvion et al. 1996; Gatehouse et al. 1997). This finding has generated significant interest, not least since no Bts effective against this pest order have been identified to date. One such lectin is the snowdrop lectin (Galanthus nivalis agglutinin; GNA). Both constitutive and phloem-specific (Rss1 promoter) expression of GNA in rice is an effective means of significantly reducing survival of rice brown planthopper (Nilaparvata lugens), and green

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leafhopper (Nephotettix virescens) both serious economic pests of rice (Rao et al. 1998; Foissac et al. 2000; Tinjuangjun et al. 2000). GNA has been expressed in combination with other genes encoding insecticidal proteins, including the cry genes (Maqbool et al. 2001). Although lectins such as GNA, and ConA are not as effective against aphids as they are against hoppers, they nonetheless have significant effects on aphid fecundity when expressed in potato (Down et al. 1996; Gatehouse et al. 1997, 1999) and wheat (Stoger et al. 1999). The precise mode of action of lectins in insects is not fully understood although binding to gut epithelial cells appears to be a prerequisite for toxicity. In the case of rice brown planthopper, GNA not only binds to the luminal surface of the midgut epithelial cells, but also accumulates in the fat bodies, ovarioles and throughout the haemolymph, suggesting that the lectin is able to cross the midgut epithelial barrier and pass into the insect’s circulatory system, resulting in a systemic toxic effect (Powell et al. 1998; Du et al. 2000). As with protease inhibitors, the levels of protection conferred by the expression of lectins in transgenic plants are generally not high enough to be considered commercially viable. However, the absence of genes with proven high insecticidal activity against homopteran pests may well mean that transgenic crops with partial resistance may still find acceptance in agriculture, especially if expressed with other genes that confer partial resistance, or if introduced into partially resistant genetic backgrounds.

1.2.5.3

A Brief Aside; Plant Parasitic Nematodes

Plant parasitic nematodes include several groups causing severe crop losses. The most common genera are: Aphelenchoides (foliar nematodes), Meloidogyne (rootknot nematodes), Heterodera, Globodera (cyst nematodes) such as the potato cyst nematode, Nacobbus, Pratylenchus (lesion nematodes), Ditylenchus, Xiphinema, Longidorus, and Trichodorus. Several phytoparasitic nematode species cause histological damages to roots, including the formation of visible galls (Meloidogyne). Some nematode species transmit plant viruses through their feeding activity on roots. One of them is Xiphinema index, vector of GFLV (grapevine fanleaf virus), an important disease of grapes. Bt toxins, lectins (Burrows et al. 1999) and protease inhibitors have shown some promise for control, particularly the expression of cystatins (Cowgill et al. 2002). For a recent review of the topic, the reader is referred to Fuller et al. (2008).

1.2.5.4

Other Sources of Insecticidal Molecules

Generating insecticidal transgenic crops harboring genes from nonconventional sources is an extremely active area, with amongst others, foreign genes from plants (e.g., enzymes inhibitors and novel lectins) and animal sources including insects

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(e.g., biotin-binding proteins, neurohormones, venoms and enzyme inhibitors) being a major focus (Ferry et al. 2006). The development of second-generation transgenic plants with greater durable resistance might result from the expression of multiple insecticidal genes such as the Vip (vegetative insecticidal proteins) produced by B. thuringiensis during its vegetative growth. The benefit of such an approach is a broader insect target range than conventional Bt proteins and the proposed expectation to control current Bt resistant pests due to the low levels of homology between the domains of the two proteins classes (Christou et al. 2006). With Bt toxins as the classical reference, toxins from other insect pathogens provide a potential repository of novel insecticidal compounds. Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes, which are lethal to a wide range of insects (Chattopadhyay et al. 2004). Photorhabdus toxin expression in Arabidopsis caused significant insect mortality (see for review Ferry et al. 2006). Thus, toxins from other insect pathogens are also opening up new routes to pest control using transgenic-based strategies. Interesting recent developments include the use of novel proteins from insect biological control agents and insect hormones to generate transgenic crops. A teratocyte secretory protein from a hymenopteran endoparasitoid (a parasitic wasp often used in biocontrol programs) has been expressed in transgenic tobacco and shown to increase resistance to lepidopteran pests (Maiti et al. 2003). Similar protection has also been achieved with insect peptide hormones (Tortiglione et al. 2003). Interestingly, they replaced the tomato systemin peptide region of prosystemin (a plant signaling molecule) with the insect peptide and showed that this resulted in the production of biologically active insecticidal peptides. Reliance on the expression of a single gene product for pest control is a relatively short-term strategy that parallels the use of exogenously applied chemical pesticides. Thus, pyramiding (stacking) of genes encoding different Bt toxins has been developed as a method for preventing the onset of evolution of pest resistance, and for conferring greater levels of pest control (Boulter et al. 1990; Maqbool et al. 2001; Zhao et al. 2003). This strategy has now been adopted in commercially available crops (see above). For example, corn lines have recently been developed (Moellenbeck et al. 2001; Ostlie 2001) coexpressing two d-endotoxins from Bt for resistance to corn rootworm. Hybrid proteins have also been developed to enhance and extend the activity of Bt toxins. The use of a single Bt toxin in a crop is limited in that many insects attack a single crop and toxins generally show very high specificity towards a single pest species. Therefore, toxins have been engineered to modify their receptor recognition and pore formation. Each toxin consists of three domains. Domain I is involved in membrane insertion and pore formation. Domains II and III are both involved in receptor recognition and binding. Additionally, a role for domain III in pore function has been found. This approach has proved successful in both enhancing activity (Karlova et al. 2005) and extending host range (Singh et al. 2004). Such hybrid/fusion proteins offer an alternative/ complementary strategy to address potential limitations in conventional transgenic insect pest control.

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Transgenic Crop Plants for Resistance to Biotic Stress

1.2.5.5

15

Transgenic Plants Expressing Fusion Proteins

The concept of “gene stacking” has recently been extended to the development and use of fusion proteins. Such proteins not only provide a means of increasing durability, but also provide a “vehicle” for more effective targeting of insecticidal molecules, including peptides. It thus offers an alternative/complementary strategy to address potential limitations in conventional transgenic insect pest control. For example, recognition of binding sites in the insect gut is an important factor determining the toxicity of Bt. Enhancing toxin-binding capabilities should thus extend host range and delay resistance in pest populations. Bt is believed to bind primarily to aminopeptidase N or cadherin membrane proteins, while the generation of a fusion protein with the nontoxic B-chain from ricin (RB) was shown to extend the binding of Bt to include specific glycoproteins. Transgenic plants expressing the Bt fused RB demonstrated that the addition of the RBbinding domain provided a wider repertoire of receptor sites within target species and significantly enhanced the levels of toxicity of Bt. For example, survival of the armyworm Spodoptera littoralis, a species of insect not sensitive to Bt, was reduced by ~90% when feeding on transgenic maize expressing the fusion, compared to plants expressing either Bt Cry1Ac alone, or the RB-binding domain. Expression of the fusion protein resulted in the insect becoming susceptible to Bt (Mehlo et al. 2005). This strategy has shown great potential beyond just extending the toxicity of Bt. Zhu-Salzman et al. (2003) have generated fusion proteins with anchor regions to other insecticidal proteins to the insect gut epithelium. Using the legume lectin rGSII, they proposed a system to combat the ability of certain insect species to activate protease inhibitor-insensitive proteolytic enzymes. The soybean cysteine protease inhibitor soyacystatin N (scN) was covalently linked to the GlcNAc-specific legume lectin using a naturally occurring linker region from the potato multicystatin. In this instance, the fusion protein not only has a novel binding ability that is proposed to initiate a concentration effect by localizing the inhibitor at the anterior of the gut, but the fused lectin moiety additionally offers a degree of protection to the insecticidal moiety by blocking the access of scN-insensitive proteases, thereby preventing proteolytic destruction of the cystatin. Not only do fusion proteins have potential for use in transgenic crops, but also to improve the efficacy of biopesticide-based sprays. Neuropeptides potentially offer a high degree of biological activity, and thus provide an attractive alternative pest management strategy. There are major drawbacks to their use, particularly as topical sprays. They are unlikely to be rapidly absorbed through the insect cuticle to their site of action, and are prone to proteolysis and rapid degradation in the environment. Should they survive the application process and are then taken up by the insect, they are then unlikely to survive the conditions of the insect gut or be delivered to the correct targets within the insect. The discovery that snowdrop lectin (GNA) remains stable and active within the insect gut after ingestion, and that it is able to cross the gut epithelium, provided an opportunity for its use as a “carrier molecule” to deliver other peptides to the circulatory system of target insect

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species. This strategy effectively delivered the insect neuropeptide hormone, allatostatin, to the haemolymph of the tomato moth Lacanobia oleracea (Fitches et al. 2002). Subsequent expression of the fusion protein in potato further provided proof of concept for the efficacy of fusion proteins as a means of delivery. The results demonstrated significant reduction in mean larval weight when compared to the controls. GNA can be used to deliver insecticidal peptides isolated from the venom of the spider Segestria florentina (SFI1) to the haemolymph of L. oleracea (Fitches et al. 2004). Neither the GNA nor the SFI1 moieties alone were acutely toxic; however, the SFI1/GNA fusion was insecticidal to first stage larvae, causing 100% mortality after 6 days. This spider venom neurotoxin is believed to irreversibly block the presynaptic neuromuscular junctures. Such venom toxins show high degrees of specificity and thus lend themselves to environmentally benign pest management strategies.

1.2.6

Manipulation of Plant Endogenous Defenses

Alternative strategies for protecting crops from insect pests, that are not dependent on the expression of single or stacked genes, seek to exploit the induced endogenous resistance mechanisms exhibited by plants to most insect herbivores. For further information, the reader is referred to Chap. 10 of this volume. Such induced defenses are exemplified by the wounding response, first identified as the local and systemic synthesis of proteinase inhibitors (PIs), which block insect digestion in response to plant damage (Gatehouse 2002a, b). Many transgenic strategies have attempted to exploit the potential overexpression of plant PIs to protect crops from pest damage (Jouanin et al. 1998) but these have relied on the transfer of a single PI gene, and many insects have been able to adapt to this. More recent research has shown that induced defenses also involve the plant’s ability to produce toxic or repellent secondary metabolites as direct defenses, and volatile molecules, which play an important role in indirect defense (Kessler and Baldwin 2002). Insect herbivores activate induced defenses both locally and systemically via signaling pathways involving systemin, jasmonate, oligogalacturonic acid and hydrogen peroxide (Fig. 1.4). Ecologists have long understood that plants exhibit multi-mechanistic resistance towards herbivores, but the molecular mechanisms underpinning these complicated responses have remained elusive (Baldwin et al. 2001). However, recent studies investigating the plant’s herbivore-induced transcriptome, using microarrays and differential display technologies, have provided novel insights into plant–insect interactions. The jasmonic acid cascade plays a central role in transcript accumulation in plants exposed to herbivory (Hermsmeier et al. 2001). A single microarraybased study revealed that the model plant Arabidopsis undergoes changes in levels of over 700 mRNAs during the defense response (Schenk et al. 2000). In contrast, only 100 mRNAs were upregulated by spider mite (Tetranicus urticae) infestation in lima bean (Phaseolus lunatus), although a further 200 mRNAs were upregulated

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17

Herbivory physical forces local peptide hormone release eg. prosystemin

insect dervived elicitors

ethylene

systemin plant-plant interactions

pest deterrent/ attraction of natural enemies

receptor binding ABA linolenic acid SA

Voltiles e.g. -green leaf volatiles -C10, C15 terpenoids -indole

octadecanoid pathway methyl jasmonate jasmonic acid auxin, SA (-) ethylene, ABA (+) polygalactouronidase oligalacturonic acid vascular bundle

H202

Insecticidal compounds e.g. Protease Inhbitors

late genes (defence)

Cell wall

NAPDH oxidase

plant-plant interactions (systemic induction of Pls)

early genes signalling

mesophyll cells

Fig. 1.4 The generalized plant-wounding response. Generalized overview of the plant wounding response, and signalling molecules which can modulate it, showing the pathways necessary for both local and systemic induction of insecticidal proteins. (Adapted from: Ferry et al. 2004)

in an indirect response mediated by feeding-induced volatile signal molecules (Arimura et al. 2000). Deciphering of the signals regulating herbivore-responsive gene expression will afford many opportunities to manipulate the response. Signaling molecules such as salicylic acid, jasmonic acid and ethylene do not activate defenses independently by linear cascades, but rather establish complex interactions that determine specific responses. Knowledge of these interactions can be exploited in the rational design of transgenic plants with increased insect resistance (Rojo et al. 2003; De Vos et al. 2005; Giri et al. 2006).

1.2.6.1

A Special Case: Sap-Feeding Insects

While most herbivorous insects cause extensive damage to plant tissues when feeding, many insects of the order Homoptera feed from the contents of vascular tissues by inserting a stylet between the overlying cells, thus limiting cell damage and minimizing induction of a wounding response. In contrast to wounding, plant responses following attack by these insects have been shown to be typical of pathogen attack, with examples of gene-for-gene interactions being known (Walling 2000;

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Moran et al. 2002). However, these pathogen-induced pathways can induce expression of many of the genes upregulated by wounding because of pathway cross-talk. Moran and Thompson (2001) demonstrated that phloem feeding by the green peach aphid (Myzus persicae) on Arabidopsis induced expression of genes associated with salicylic acid (SA) responses to pathogens, as well as a gene involved in the jasmonic acid-mediated response pathway. These results suggest stimulation of response pathways involved in both pathogen and herbivore responses. Microarray data has identified genes involved in oxidative stress, calcium-dependent signaling, pathogenesis-related responses, and signaling as key components of the induced response (Moran et al. 2002). It may be that transgenic strategies that activate such signaling cascades could enhance plant resistance to these problematic pests. 1.2.6.2

Indirect Defense (Volatile Production)

The role of plant volatiles in indirect defense has been described as “top-down” defense (Baldwin et al. 2001). Some volatiles appear to be common to many different plant species, including C6 aldehydes, alcohols and esters (green leaf volatiles), C10 and C15 terpenoids, and indole, whereas others are specific to a particular plant species. Many volatiles are preformed and act in herbivore deterrence; furthermore, the wounding response also includes the formation of volatile compounds. Top-down control of herbivore populations is achieved by attracting predators and parasitoids to the feeding herbivore, mediated by these volatile organic compounds (VOCs). For example, genes involved in the biosythesis of the maize VOC bouquet are upregulated by insect feeding (Frey et al. 2000; Shen et al. 2000). In addition, herbivore oviposition has been shown to induce VOC emissions, which attract egg parasitoids (Hilker and Meiners 2002). Herbivoreinduced VOCs can also elicit production of defence-related transcripts in plants near the individual under attack (Arimura et al. 2000; Dicke et al. 2003). Exposure to herbivore-induced volatiles in lima bean results in transcription of genes involved in ethylene biosynthesis (Arimura et al. 2000). Manipulation of volatile biosynthesis can affect insect resistance. Transgenic potatoes in which production of hydroperoxide lyase (the enzyme involved in green leaf volatile biosynthesis) was reduced were found to support improved aphid performance and fecundity, suggesting toxicity of these volatiles to M. persicae (Vancanneyt et al. 2001). In a review of the topic Degenhardt et al. (2003) discuss the potential of modifying terpene emission with the aim of making crops more attractive to herbivore natural enemies. 1.2.6.3

Detoxification and Insect Modulation of the Wounding Response

However, insect pests are able to feed on plants despite their defenses, both constitutive and inducible. Many insects are able to detoxify potentially toxic secondary metabolites, using cytochrome P-450 monoxygenases and glutathioneS-transfereases. These enzymes are induced by exposure to toxic plant secondary

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19

compounds, for example, xanthotoxin (a furanocoumarin) induces P-450 expression in corn earworm (Li et al. 2000). More recently, Li et al. (2002) have shown that corn earworm uses signaling molecules from its plant host, jasmonate and salicylate, to activate four of its cytochrome P450 genes, thus making the induction of detoxifying enzymes rapid and specific. Recent strategies based on RNAi technology have shown that it is possible to overcome these insect responses (discussed later in this chapter).

1.2.7

RNAi

Disrupting gene function by the use of RNAi is a well-established technique in insect genetics based on delivery by injection into insect cells or tissues. The observation that RNAi could also be effective in reducing gene expression, measured by mRNA level, when fed to insects (Turner et al. 2006) has led to two recent articles in which transgenic plants producing double-stranded RNAs (dsRNAs) which are shown to exhibit partial resistance to insect pests. Transgenic maize producing dsRNA directed against V-type ATPase of corn rootworm showed suppression of mRNA in the insect and reduction in feeding damage compared to controls (Baum et al. 2007). Similarly, transgenic tobacco and Arabidopsis expressing dsRNA directed against a detoxification enzyme (Cytochrome P450 gene CYP6AE14) for the breakdown of gossypol (a defensive metabolite) in cotton bollworm caused the insect to become more sensitive to gossypol in the diet (Mao et al. 2007). This approach holds great promise for future development. It is also proving effective for nematode control (Bakhetia et al. 2005). For a recent review on RNAi-mediated crop protection against insect pests the reader is referred to Price and Gatehouse (2008). Advances in our understanding of induced responses in plants and their regulation, has refocused attention on potential exploitation of endogenous resistance mechanisms for crop protection. While plant resistance is an integral component of organic and IPM strategies, it does not afford similar levels of protection as those provided by the use of “direct” protective methods such as the expression of Bt toxins. The goal of the plant breeder, and now the biotechnologist, is to engineer durable multi-mechanistic resistance to insect pests in crops, and increased knowledge of induced defense mechanisms and their molecular control is likely to play an important role in realizing this aim.

1.2.8

Environmental Impact of IR Crops

Almost from the beginning of the production of transgenic crops there have been concerns over their use and introduction into the environment. There is international agreement that GM crops should be evaluated for their safety, including their environmental impact (Dale 2002). During the past 15–20 years, there have been

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extensive research programs of risk assessment, with several areas of major concern identified.

1.2.8.1

Impact on Nontarget Organisms

Assessing the consequences of pest control on nontarget organisms is an important precursor to their becoming adopted in agriculture. The expression of transgenes that confer enhanced levels of resistance to insect pests is of particular significance since it is aimed at manipulating the biology of organisms in a different trophic level to that of the plant. Potential risks to beneficial nontarget arthropods exist. Those groups most at risk include: nontarget Lepidoptera, beneficial insects (pollinators, natural enemies) and soil organisms. Exposure of nontarget Lepidoptera to insecticidal transgene products may occur through both direct consumption of transgenic plant tissues including via consumption of transgenic pollen; many nontarget Lepidoptera are rare butterflies having great conservation value. The case of the Monarch butterfly (Danaus plexippus), a conservation flagship species in the US, highlighted the need for ecological impact research. In a letter to Nature, Losey et al. (1999) claimed that both survival and consumption rates of Monarch larvae fed milkweed leaves (natural host) dusted with Bt pollen were significantly reduced, and that this would have profound implications for the conservation of this species. However, a series of ecologically based studies rigorously evaluated the impact of pollen from such crops on Monarchs and demonstrated that the commercial wide-scale growing of Bt-maize did not pose a significant risk to the Monarch population (Hellmich et al. 2001; Gatehouse et al. 2002). In fact, the initial experiments did not quantify the dose of pollen used, or indeed, if this was a realistic level likely to be encountered in the field; nevertheless, this work highlighted the importance of studying nontarget effects. In a separate field study Wraight et al. (2000) showed that Papilio polyxenes (black swallowtail) larvae were unaffected by pollen from Bt expressing maize event Mon810 at 0.5, 1, 2, 4 and 7 m from the transgenic field edge, highlighting the need for a case-by-case study of organisms considered to be at risk. In addition to the potential direct impacts of Bt toxins on susceptible target insects, as in the case of the Morarch butterfly, some Lepidoptera have been shown to have a reduced sensitivity to the lepidopteran-specific Bt toxins. For example, S. littoralis can survive on maize expressing Cry1Ab (Hilbeck et al. 1998) and thus present a route of exposure to the next trophic level. In the case of Bt Cry3Aa or Cry3Bb expressing potatoes or maize, some Lepidoptera may represent nontarget secondary pests, and whilst not directly affected by the transgene product themselves may again present a route of exposure to the next trophic level, as do other nontarget herbivores. Organisms such as those belonging to the orders Homoptera, Hemiptera, Thysanoptera, and Tetranychidae are not targeted by Bt toxins expressed in transgenic plants; however, they do utilize the Bt crop (Groot and Dicke 2002). The direct effect that this may have on these insects is dependent on the presence of Bt receptors in the first instance, and it is so far unclear whether such receptors are

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21

present in nontarget organisms (de Maagd et al. 2001). In addition, the fate of the toxin ingested by nontarget herbivores is unclear, since if it retains toxicity then this may have implications at the next trophic level. The impacts of insect-resistant transgenic crops at higher trophic levels have also been considered, where there are concerns over the risks to beneficial arthropod biodiversity (Schuler et al. 1999; Bell et al. 2001); in particular, predators and parasitoids, which play an important role in suppressing insect pest populations both in the field and under specialized cultivation systems (glasshouses). Natural enemies may ingest transgene products via feeding on herbivorous insects that have themselves ingested the toxin from the plant; such tritrophic interactions will be influenced by the susceptibility of the herbivore to the plant protection product. If, as in the case with Bt toxins, the prey item is susceptible to the toxin, then the predator will not come into contact with the toxin as the pest will effectively be controlled, and in target insects the toxin is bound to receptors in the midgut epithelium that are structurally rearranged and may lose their entomotoxicity (de Maagd et al. 2001). In nontarget insects (and resistant insects), the toxins do not bind and may thus retain biological activity. However, the overwhelming weight of evidence from independent laboratory and field studies show that Bt toxins have a limited ability to affect the next trophic level (reviewed in Sanvido et al. 2007; Ferry and Gatehouse 2009; Romeis et al. 2008). Pollinators represent another group of nontarget organisms highlighted as at risk from Bt toxins in GM crops. The current generation of transgenic crops produce Bt toxin in the pollen as well as in the vegetative tissues. Several studies have been conducted to determine toxicity of Bt toxins to pollinators (Vandenberg 1990; Sims 1995, 1997; Arpaia 1997; Malone and Pham-Delegue 2001); generally, they all conclude that neither the adults nor the larvae of bees were affected by Bt toxins. For a comprehensive review of the impact of transgenic crops on pollinators, the reader is referred to two recent reviews (Malone et al. 2008; Malone and Burgess 2009). Finally, nontarget species may come into contact with Bt toxins via the environment. Several studies have shown that Bt toxins released from transgenic plants bind to soil particles (Palm et al. 1996; Crecchio and Stotzky 1998; Saxena et al. 1999). Soil-dwelling and epigeic insects such as Collembola and Carabidae may thus be exposed to the toxins. Several studies (Saxena and Stotzky 2001; Ferry et al. 2007) show no differences in mortality or body mass of bacteria, fungi, protozoa, nematodes and earthworms or carabid beetles exposed to Bt. Exposure to the transgene products, however, does not necessarily imply a negative impact. Most studies to date have demonstrated that crops transformed for enhanced pest resistance have no deleterious effects on beneficial insects (reviewed in Ferry et al. 2003; Romeis et al. 2008).

1.2.9

Conclusions

Ultimately one must consider the impact of transgenic crops and specifically Bt toxins in comparison to other pest control strategies such as conventional crop

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protection using insecticides. While pesticides have no doubt brought vast yield improvements, they have well-documented undesirable nontarget effects (Devine and Furlong 2007). It is worth remembering that whilst potential risks do exist to the environment from the cultivation of GM crops, their potential to decrease reliance on external inputs (less insecticide sprays) and to increase the availability of genetic resources available to breeders is great (Ferry and Gatehouse 2009).

1.3 1.3.1

Biotic Stress due to Weeds Crop Losses due to Weeds

Weeds can compete with productive crops or pasture, or convert productive land into unusable scrub. Weeds are also often poisonous, distasteful, produce burrs, or thorns that interfere with the use and management of desirable plants by contaminating harvests or excluding livestock. Weeds tend to thrive at the expense of the more refined edible or ornamental crops. They provide competition for space, nutrients, water and light, although how seriously they will affect a crop depends on a number of factors. Some crops have greater resistance to competition than others, for example smaller, slower-growing seedlings are more likely to be overwhelmed than those that are larger and more vigorous. Weeds also differ in their competitive abilities, and this can vary according to specific conditions and the time of year. Tall-growing vigorous weeds such as fat hen (Chenopodium album) can have the most pronounced effects on adjacent crops. Chickweed (Stellaria media), a low-growing plant, can happily coexist with a tall crop during the summer, but plants that have overwintered will grow rapidly in early spring and may swamp crops. Simply put, weeds are any plant growing in an area where it is not wanted. Of over 250,000 plant species in the world, only a few hundred are troublesome weeds. Although no single characteristic clearly defines a weed, two attributes are common in the worst weeds: competitiveness and persistence. The world’s worst weeds are shown in Table 1.2 (Chrispeels and Sadava 2003).

1.3.2

Weed Management: Prevention, Control, and Eradication

As weeds are persistent, once they have established in a field they are extremely difficult to eradicate. One of the most successful attempts in the eradication of a weed is provided by witchweed (Striga sp.) in the US. Witchweed is a parasitic plant; its seeds germinate in response to a chemical, strigol, produced by the roots of the host plant. The germinated seedling attaches to the root through special haustoria, as this occurs underground infestation can go unnoticed, consequently serious

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Table 1.2 World’s worst weeds (from: Chrispeels and Sadava 2003) Plant family Weed examples Grass (Poaceae) Bermuda grass (Cynodon dactylon) Barnyard grass (Echinochloa crus-galli) Johnson grass (Sorghum halepense) Sedge (Cyperaceae) Purple nutsedge (Cyperus rotundus) Yellow nutsedge (Cyperus esculentus) Smallflower umberella sedge (Cyperus difformis) Sunflower (Asteraceae) Canada thistle (Cirsium arvense) Common cocklebur (Xanthium strumarium) Common dandelion (Taraxacum officinale) Buckwheat (Polygonaceae) Wild buckwheat (Polygonum convolvulus) Curly dock (Rumex crispus) Red sorrel (Rumex acetosella) Pigweed (Amaranthaceae) Smooth pigweed (Amaranthus hybridus)

Mustard (Brassicaceae)

Legume (Fabaceae)

Morning glory (Convolvulaceae)

Spurge (Euphorbiaceae)

Goosefoot (Chenopodiaceae)

Mallow (Malvaceae)

Nightshade (Solanaceae)

Spiny amaranth (Amaranthus sinosus) Redroot pigweed (Amaranthus retroflexus) Shepherd’s purse (Capsella bursa-pastoris) Hoary cress (Brassica draba) Wild mustard (Brassica kaber) Black medic (Medicago lupulina) Sensitive plant (Mimosa pudica) Kudzu (Pueraria lobata) Field bindweed (Convolvulvus arvensis) Field dodder (Cuscuta campestris) Swamp morning glory (Ipomoea aquatica) Leafy spurge (Euphorbia esula) Garden spurge (Euphorbia hirta) Spotted spurge (Euphorbia maculata) Common Lamb’s quarters (Chenopodium album) Russian thistle (Salsola iberica) Nettleleaf goosefoot (Chenopodium murale) Venice mallow (Hibiscus trionum) Velvetleaf (Abutilon theophrasti) Arrowleaf sida (Sida rhombifolia) Black nightshade (Solanum nigrum) Jimsonweed (Datura stramonium) Cutleaf groundcherry (Physalis angulata)

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Crop Wheat

None

Sunflower

Buckwheat

Grain amaranth

Canola

Soybean

Sweet potato

Cassava

Sugarbeet

Cotton

Potato

infestation leading to yield loss occurs before the parasitic plant even appears through the soil. Once the witchweed comes through the soil it becomes photosynthetically active, but still dependent on its host for water. Such parasitic weeds are extremely difficult to control and yield losses of approximately 50% can be expected under drought conditions. In the US, only quarantine of infected areas reduced witchweed infection from 150,000 hectares to a couple of 1,000 hectares, but this quarantine lasted for nearly 50 years (Chrispeels and Sadava 2003)!

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For this reason, preventative weed management is critical. This involves keeping weed seeds and vegetative propagules from getting to the field in the first place and is mainly achieved through seed cleaning and the purchase of certified clean seed. Good management practice and the cleaning of farm equipment moved between different areas are critical in preventative control. Ultimately weed control is achieved by mechanical, biological and predominately chemical means.

1.3.3

Herbicides

The economic importance of weeds is emphasized by the fact that the herbicides comprise as large a share of the world agrochemical market as all other pesticides combined, farmers spend an annual US$10 billion to control weeds in the US alone (Chrispeels and Sadava 2003). Herbicides have evolved over time, and herbicide use has seen a move towards more biodegradable chemicals that only need to be applied at a very low concentration of active ingredient. Thus, the new generation of herbicides are relatively environmentally benign. Many herbicides exploit the differences in plant physiology between the crop species and its weeds (usually the differences between monocots and dicots); they may be systemic or act on contact (Table 1.3). Major crops such as wheat, rice, and maize; and legumes such as soybean, common bean, and peanut come from the same plant families as their weeds, thus creating a control problem. The mode of action of common herbicides is varied (Naylor 2002). For example, inhibition of photosynthesis and light-dependent membrane destruction (acting on photosystems II and I, respectively) are the mode of action of the foliar-acting nonselective herbicides like atrazine, paraquat and diquat. Hormone herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D) induce abnormal plant growth by interring with auxin regulation. The sulfonyl ureas, imidazolines and the environmentally benign, but nonselective glyphosate (acting on 5-enolpyruvylshikimate3-phosphate synthase) inhibit amino acid synthesis. Others include inhibitors of lipid synthesis, inhibition of cell division and pigment synthesis. The advantages of herbicides are clear – they control multiple weed species, control perennial weeds, cause no injury to the crop plant, and can readily be applied to large areas. Table 1.3 Herbicide modes of action Herbicide type Contact herbicides destroy only that plant tissue in contact with the chemical spray. Generally, these are the fastest-acting herbicides. They are ineffective on perennial plants that are able to regrow from roots or tubers Systemic herbicides are foliar-applied and are translocated through the plant and destroy a greater amount of the plant tissue. Modern herbicides such as glyphosate are designed to leave no harmful residue in the soil Soil-borne herbicides are applied to the soil and are taken up by the roots of the target plant Pre-emergent herbicides are applied to the soil and prevent germination or early growth of weed seeds

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Nonchemical weed control includes: cultural weed control practices such as intercropping and rotation; mechanical removal of weeds; and biological control, all of which are labor intensive and unpredictable. Chemical treatment is by far the most effective method, but it is not without problems.

1.3.3.1

Resistance to Herbicides

A common consequence of repeated use of herbicide application is the evolution of resistance in weed populations because of strong selection pressure. The first example of a problematic herbicide resistant weed was reported in the 1970s (Chrispeels and Sadava 2003) when common groundsel resistant to triazine was identified in the US. The mechanism of herbicide resistance is usually a change in the target site with a reduction in herbicide affinity – this may be achieved through a single point mutation and alteration of a single amino acid. By 2001, over 150 weed species had resistance to at least one herbicide. Some biotypes of weeds have resistance to herbicides with different modes of action.

Rigid Ryegrass When a weed evolves resistance to a particular herbicide, the farmer is forced to use an alternative control strategy. This often involves the use of another herbicide with a different mode of action. Subsequent selection pressure may lead to a weed population acquiring multiple herbicide resistance. In nearly all the cases so far, resistance to only one or two chemical families has been reported. Rigid ryegrass is a notable exception. In the most severe cases, biotypes exist that are resistant to nine chemical families with five different modes of action! Resistance is conferred by multiple mechanisms; both altered sites of action and the ability to metabolize particular herbicides, via nonspecific monoxygenases.

1.3.4

Transgenic Crops for Weed Control

The adoption of transgenic herbicide-resistant (HR) crops has made remarkable changes to global agriculture within the last decade. Currently, an estimated 114.3 million hectares of transgenic crops are planted throughout a variety of agroecosystems in 23 developing and industrial countries. Approximately, 90% of the land area with transgenic crops includes a trait for glyphosate resistance (GR) (Owen 2008). While there are a number of herbicide-tolerant genetically modified crops that have been developed for several herbicides with different modes of phytotoxic action, the primary influence in world agriculture are glyphosate-resistant crops

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(GRCs) (Duke 2005; Duke and Powles 2008). More specifically, of the 23 countries that grow plant glyphosate-resistant crops, the US, Canada, Argentina and Brazil are the countries that account for most hectares (Owen 2008). In these countries, the principle GRCs planted include maize (Zea mays), soybean (Glycine max), cotton (G. hirsutum), and canola (Brassica napus). For further information, the reader is referred to Chap. 3 of this volume.

1.3.4.1

Round-up1 Ready Crops

Glyphosate (Round-up1) is a highly effective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a branch point enzyme in aromatic amino acid biosynthesis. A naturally occurring EPSP synthase gene (cp4) was identified from Agrobacterium sp. strain CP4, whose protein product provided glyphosate tolerance (Fig. 1.5) in plants (Padgette et al. 1995). COO– COO–

SHKP

+

P O

OH

CH2

P O

OH

phosphoenolpyruvate (PEP)

GLYPHOSATE

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase)

shkG H3PO4

COO–

COO–

H3PO4 CH2

P O

O

OH 5-enolpyruvylshikimate-3-phosphate (EPSP )

CH2

shkH COO–

chorismate synthase

O

COO–

OH chorismate (CHA)

Fig. 1.5 Glyphosate; mode of action. Glyphosate kills plants by interfering with the synthesis of the amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (SHKP) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (EPSP). EPSP is subsequently dephosphorylated to chorismate, an essential precursor in plants for the aromaticamino acids: phenylalanine, tyrosine and tryptophan. These amino acids are used as building blocks in peptides, and to produce secondary metabolites such as folates, ubiquinones and naphthoquinone

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Furthermore, glyphosate detoxification pathways are known in microbes involving glyphosate oxidoreductase (gox) genes (Jacob et al. 1988). These two genes confer glyphosate resistance in selected crops. Crops engineered for resistance to this broad-spectrum herbicide allow control of multiple weeds without injury to the main crop. The economic benefits to farmers of glyphosate-resistant crops are estimated at US$17.5 billion, mainly accrued from reduced sprays and lower inputs of labor. In addition, herbicide-tolerant crops reduce the amounts of active ingredient required for weed control; they also promote the usage of no/low till farming and thus lower fuel consumption with direct benefits to both soil structure and carbon emissions (James 2007).

1.3.4.2

Other Transgenic Herbicide-Tolerant Crops

Traits for resistance to three other classes of herbicides have been developed, but have not reached the same level of popularity as glyphosate resistance. Resistance to oxynil herbicides conferred by the BXN nitrilase from Klebsiella pneumoniae (subsp. ozaenae) (Stalker et al. 1988) was the first trait engineered in cotton (developed by Calgene, Davis, CA [now Monsanto]). Because glyphosate is less expensive and controls more weed species, interest in using the oxynil herbicides has waned and 2004 was the final year of BXN1 cotton sales. BXN canola was commercialized by Rhone-Poulenc Canada (now Bayer Crop Science, Monheim, Germany) and subsequently discontinued. Phosphinothricin acetyltransferase (PAT or BAR) detoxifies phosphinothricin- or bialaphos-based herbicides (glufosinate). The pat gene is native to Streptomyces viridichromogenes and bar is from S. hygroscopicus where they act in both the biosynthesis and detoxification of the tripeptide bialaphos (De Block et al. 1987). Like glyphosate, phosphinothricin herbicides control a broad spectrum of weed species and break down rapidly in the soil so that the problems with residual activity and environmental impact are greatly reduced. Bayer CropScience markets this trait as LibertyLink1 in several species. The pat and bar genes are also popular plant transformation markers in the research community. Finally, BASF (Ludwigshafen, Germany) markets nontransgenic CLEARFIELD1 imidazolinone-resistant canola, wheat, sunflower, corn, lentils, and rice, while DuPont (Wilmington, DE) markets nontransgenic STS1 soybeans with tolerance to sulfonylurea herbicides. A sulfonylurea-tolerant flax variety called CDC Triffid, developed by the University of Saskatchewan (Canada), was grown commercially in Canada in 2000 but is no longer offered. All these crops contain mutagenized versions of the acetohydroxyacid synthase, also called acetolactate synthase (ALS), which are not inhibited by imidazolinone and/or sulfonylurea herbicides (Devine and Preston 2000). Herbicides that inhibit ALS are considered low or very low use-rate herbicides with a good spectrum of weed control and are likely to remain an important part of weed resistance management programs (Castle et al. 2006).

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1.3.5

N. Ferry and A.M.R. Gatehouse

HT Canola

There are two GM traits for herbicide resistance in canola: glyphosate and glufosinate resistance. Resistance to glyphosate herbicides is conferred by the genes 5-enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 epsps) (event GT73, Monsanto) that is targeted to the chloroplast, and a glyphosate oxidoreductasegene from Achromobacter sp. strain LBAA (gox v247) (both Roundup Ready1). Resistance to phosphinothricin herbicides is conferred by phosphinothricin-acetyltransferance (pat gene), and marketed as LibertyLink1 by AgrEvo (now Bayer CropScience). Herbicide-resistant canola (oilseed rape) allows post-emergence application of a single herbicide with a wide spectrum of activity. For effective control, it has to be applied before the weeds reach 10 cm height. This extends the potential time period for spraying (Benbrook 2004). The timing is more flexible and the application of a single herbicide simplifies weed control (Firbank and Forcella 2000). Since many herbicides allow post-emergence applications, HT crops do not generally provide a new option. However, post-emergence spraying with non-GMHT oilseed rape is confined to a short 3–5 week period after crop emergence (Pallutt and Hommel 1998). Thus, GMHT canola offers greater flexibility (Owen 1999). With HT oilseed rape, the intention is to reduce the amount of active ingredient of herbicide used and to rely preferably on one broad-spectrum herbicide only (http://www.canola-councildemo.org). The intent is also to reduce the number of spraying rounds, which helps reduce soil compaction and erosion (Madsen et al. 1999). During the first years of cultivating GMHT oilseed rape, most farmers had reduced active ingredient rates and applications (Champion et al. 2003). GMHT oilseed rape was usually sprayed only once, with an average active ingredient amount that was either lower (Champion et al. 2003) or not significantly different from conventional oilseed rape (Schu¨tte et al. 2004). However, in Canada (where HT canola is widely cultivated), application of herbicide was seen to actually increase (Canola Council of Canada 2001). In fact, after years of continued GMHT oilseed rape cultivation, secondary adverse effects on application rates were reported. This was due to weeds becoming herbicide tolerant on- and offsite (Devos et al. 2004; Hayes et al. 2004). HT oilseed rape volunteers occurred in subsequent rotations (Devos et al. 2004), and multiple tolerances of volunteers to herbicides were recorded because of seed impurities and seed banks after outcrossing events between fields (Hall et al. 2000). Outcrossing to weedy relatives also occurred (Daniels et al. 2005); these weedy relatives, however, have not yet been proved to be fit enough to persist on cultivated fields (Hails and Morley 2005). Nevertheless, improved weed suppression with HT oilseed rape in many agronomic respects has been demonstrated (Westwood 1997; Bohan et al. 2005), and ultimately the technology remains popular with farmers because of its simplicity and reduced costs (Canola Council of Canada 2001; Graef et al. 2007). Genes have always moved between the natural and agroecosystem, and to date – despite the formation of hybrids between HT canola and wild relatives in Canada (Gressel

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2008) – the technology has proven safe and effective (Cerdeira and Duke 2006; Darmency et al. 2007; James 2007; Gressel 2008).

1.3.6

HT Maize

C. album, Amaranthus retroflexus, Abutilon theophrasti and several annual grass species cause economic losses in maize, if left uncontrolled. The option to use glyphosate in these systems is extremely valuable because it is a broad-spectrum herbicide that provides excellent control of a wide range of weed species. Resistance to glyphosate-herbicides in maize is conferred by the epsps gene in event GA21 (the GA21 trait for glyphosate-resistant maize relies on a modified maize epsps gene) (Roundup Ready), developed by DeKalb (now Monsanto) in 1998. This is now being largely replaced by event NK603, Roundup Ready corn 2, with two epsps expression cassettes under the transcriptional regulatory control of the rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced cauliflower mosaic virus 35S (P-e35S) promoters to impart fully constitutive expression. Maize has also been developed with resistance to phosphinothricin herbicides via transformation with the pat gene in events T14 and T25, developed by Aventis (now Bayer CropScience), and marketed as LibertyLink. Maize is an open-pollinated, wind-facilitated species and gene flow via pollen is well recognized (Haslberger 2001). Thus, the movement of GM traits is a significant consideration in maize production (Luan et al. 2001; Ma et al. 2004). Generally, the introgression of GR traits in seed maize can be managed successfully (
1% outcross) by establishing isolation distances of 200 m between fields (Ma et al. 2004). However, in typical maize production regions of the US, these isolation distances are not possible and GR trait introgression into non-GR fields is prevalent. The occurrence of the GR transgene in non-GM maize can have significant economic consequences, if the grower of the non-GM maize has a contract to provide a GM-free product. Furthermore, incidences of GM gene introgression in local landraces of maize in Oaxaca, Mexico have been reported (Quist 2001). The implications for transgene occurrence reflect concerns for the maintenance of the genetic resource of the landrace maize. However, the initial report of transgene introgression was followed by a second report that suggested that no transgenes existed in these landraces of maize (Ortiz-Garcia et al. 2005). Regardless, given the adoption of GR maize, the discovery of transgene introgression into landrace maize is likely in the longer term.

1.3.7

HT Cotton

Cotton is a slow-growing plant, and only a limited selection of herbicides can be used for weed control. These two factors sometimes make weed control difficult. Cotton is a semitropical, perennial plant, although it is grown as an annual crop.

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Its growth is especially slow in the northern cotton belt in the US, where it is often planted early into cool soils. Post-emergence broadleaf control is accomplished with herbicides that can injure cotton but that are applied in a directed spray that misses most of the cotton plant; thus, weed control in cotton is particularly difficult. Cotton with resistance to glyphosate herbicides has been developed based on the expression of the CP4 epsps in events MON1445/1698, Monsanto, Roundup Ready1. Resistance to phosphinothricin herbicides is based on the expression of the bar gene in LLCotton25, Bayer CropScience LibertyLink1. Herbicide-tolerant cotton expanded from around 2% of the cotton acreage in 1996 to 26% in 1998, and reached 46% in 2000 (James 2001). In 2008, adoption of GM cotton in the US with either or both herbicide tolerance and Bt reached 86% (James 2007). There are limited reports that cotton demonstrates introgression at low frequencies (Ellstand et al. 1999). Pollen movement in cotton is dependent on insects. Cotton is predominantly self-pollinated and natural outcrossing is typically quite low (Xanthopoulos and Kechagia 2000). Thus, there is a very low probability that the GR transgene would move into non-GR cotton cultivars via pollen flow. While gene flow via GR cotton seed can occur, the consequences of this are not thought to be important.

1.3.8

HT Soybean

Soybean engineered for resistance to glyphosate herbicides has been developed by Monsanto with the CP4 epsps gene, event GTS-40-3-2 and is marketed as Roundup Ready1. Glyphosate-resistant soybeans were one of the earliest transgenic crops brought to market and they have experienced rapid adoption to the point that over 85% of US soybeans and 56% of soybeans globally are now glyphosate-resistant (James 2007). Soybeans are an autogamous (self-fertilizing) species with limited opportunity for pollen-directed gene flow (Palmer et al. 2001). Spontaneous gene flow in cultivated soybeans ranges from 0.02 to 5% depending on distance and is facilitated by thrips (Thrips tabaci) and honeybees (Apis mellifera). While the movement of the GR transgene has been observed in soybeans, there are extremely limited opportunities for this occurrence and pollen-mediated gene flow in GR soybeans is essentially a nonissue (Abud et al. 2004; Owen 2005). However, gene flow by seed is highly probable and represents a significant economic problem (Swoboda 2002; Owen 2005).

1.3.9

Other GMHT Crops

In terms of commercial crops, glyphosate-resistant alfalfa had been developed and was launched in 2006 (James 2007).

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However, of special interest genetically engineered herbicide-resistant crops are being tested as a solution to the parasitic plants Orobanche spp. (broomrapes) and Striga spp. (witchweeds), which parasitize the roots of important crops, heavily reducing yields. Joel et al. (1997) showed that the use of three target-site resistances decimated Orobanche, demonstrating the potential of transgenic herbicide-resistant crops in the control of parasitic weeds. This is potentially of great importance as every year parasitic plant damage to crops accounts for an estimated US$7 billion in yield loss in sub-Saharan Africa, and affects the welfare and livelihood of over 100 million people. For this technology to have a great impact in Africa, crops adapted to African agroecologies need to be available to farmers.

1.3.10

Changes in Agronomic Practice

The adoption of GM Crops, and specifically glyphosate-resistant crops, has resulted in significant changes in agronomic practices. Most obviously, these changes include the increase glyphosate use at the cost of other herbicides and the manner and frequency in which glyphosate is used. In addition, the amount of tillage that is conducted for crop production has significantly changed (Young 2006; Service 2007a, b; Foresman and Glasgow 2008). This reduction in tillage has an important benefit of reducing the use of petroleum-based fuels as well as an implicit gain in time use efficiency by growers. A significant reduction in pesticide use has been attributed to the adoption of herbicide-tolerant (HT) crops (Sankula 2006). Furthermore, the benefits ascribed to herbicide-tolerant crops have dramatically changed the crop cultivars selected by growers and have hastened the development of new transgenic crops for commercial distribution worldwide (Duke 2005; Dill et al. 2008). Despite wide scale commercial plantings of GM soybean, canola, cotton and maize, several herbicide-resistant crops have been developed but have not been commercially introduced. Notably, sugarbeet (Beta vulgaris) cultivars that are resistant to glyphosate were deregulated in 1999, but not commercially offered until 2008 because of concerns about the acceptability of sugar refined from a GM crop (Duke 2005; Gianessi 2005). The development of GM rice (O. sativa) cultivars modified to be resistant to glufosinate herbicide proceeded from 1998 to 2001, but were withdrawn and commercial development terminated because of concerns about market acceptance of the GM rice (Gealy and Dilday 1997; Gealy et al. 2007). Similarly, wheat (Triticum aestivum) glyphosate-resistant cultivars were under development but the program was terminated in 2004 (Dill 2005). While the GR-based wheat production systems demonstrated excellent opportunities for improved weed management, concerns about the acceptance of the flour made from GR wheat cultivars as an export commodity in GM-adverse countries resulted in the decision to halt further development (Stokstad 2004; Howatt et al. 2006). In addition to concerns centered on consumer acceptability, several concerns over potential environmental impact have been raised. These are addressed below.

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1.3.11

N. Ferry and A.M.R. Gatehouse

Weeds, Gene Flow, Invasiveness, and Biodiversity Impacts

There have been significant concerns regarding the potential of GM crops, particularly glyphosate-resistant crops, to become major agricultural problems (by invasion, volunteerism) or to create “superweeds” by cross-pollination (Ellstrand 2003). 1.3.11.1

Gene Flow

While GM crops do have the potential to cross-pollinate other crops and wild relatives, there are four basic elements determining the likelihood and consequences of gene flow: first, the distance of pollen movement from the GM crop; second, the synchrony of flowering between crop and pollen recipient; third, sexual compatibility between crop and recipient; and fourth, ecology of the recipient species (Dale et al. 2002). Research has shown that pollination declines sharply with distance from the pollen source (Lutman 1999) and one could reduce the chances of GM pollen reaching other crops through the use of isolation or buffer zones, although pollen may travel further if plants are insect-pollinated. Ellstrand et al. (1999) reviewed the sexual compatibility of crops with weeds and feral species. For example, oilseed rape (canola), barley, wheat and beans can hybridize with weeds in some countries; however, in the UK, for example, the probability of hybridization with weeds is considered minimal for wheat, low for oilseed rape and barley, and high for sugarbeet. Although sugarbeet can readily hybridize, in the case of herbicide-tolerant varieties of sugarbeet the crop would be harvested before flowering and hence shed no pollen. Indeed, methods have been developed to block expression in the pollen of transgenic plants, including engineering of the chloroplast genome (Heifetz 2000) and transgene mitigation strategies (Gressel 2008). Transgene Mitigation Strategies Transgenic crops may interbreed with nearby weeds, increasing their competitiveness, and may themselves become a “volunteer” weed in the following crop. The desired transgene can be coupled in tandem with genes that would render hybrid offspring or volunteer weeds less able to compete with crops, weeds and wild species. Genes that prevent seed shatter or secondary dormancy, or that dwarf the recipient could all be useful for mitigation and may have value to the crop. Many such genes have been isolated in the past few years (Gressel 1999). Examples include: apomixis (asexual reproduction) as a fail-safe so that the seed is actually of vegetative origin and not from sexual pollination (Zemetra et al. 1998); chromosome-specific cytogenetic fail-safes (the risk of transgenic traits spreading into weeds can be reduced by orders of magnitude by using cytogenetic mapping to locate transgenes and releasing only those transgenic lines in which it is on a genome incompatible with local weeds (Gressel and Rotteveel 2000)); plastome-specific cytogenetic fail-safes (it is possible to introduce some traits into the chloroplast or

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mitochondrial genomes;thus, in species where these genomes are completely maternally inherited, the transgenes cannot move into other species (Daniell et al. 1998)); seed dormancy (the transgenic abolition of secondary dormancy is neutral to the crop but deleterious to weeds (Gressel 1999)); seed ripening and shattering (uniform ripening and anti-shattering genes are harmful to weeds but neutral for some crops (Schaller et al. 1998)), and dwarfing (dwarfing is disadvantageous for weeds, because they can no longer compete with the crop for light (Gressel 1999)). However, these strategies are not as of yet fully developed and in use, with the exception of plastid transformation. 1.3.11.2

Invasiveness

The potential exists for GM crops to become invasive. There has been a great deal of concern that such crops could persist in the wild and disperse from their cultivated habitat. However, recent studies (and experience from the field) have indicated that the ability of GMHT crops to invade and persist was actually no better than that of their conventional counterparts (Crawley et al. 2001). Finally, GM crops persisting in fields after harvest thus becoming a weed in a different crop may be dealt with in two ways; simple treatment with an appropriate herbicide or mitigation technologies that prevent the transgene being carried over to the next generation (Gressel 2008). 1.3.11.3

A Dose of Reality

In order to put these concerns into perspective, one must understand that flow from the agroecosystem to natural ecosystems has always occurred. Gene flow is a continuing process and is the source of biological diversity (Thies and Devare 2007). There has always been gene flow from commercial crops to relatives living in near proximity (Ferry and Gatehouse 2009). In reality, the vast majority of the major cultivated crops have no wild or weedy relatives outside of their centers of origin (Gressel 2008); however, some crops are grown in areas where gene flow may occur and appropriate measures must be taken in these areas.

1.3.12

Coexistence of GM and Non-GM Crops

A pervasive problem that exists with the production of GR crops is their coexistence with non-GM crops (Byrne and Fromherz 2003). The issue of coexistence includes three possibilities: (1) introgression of the trait via pollen (pollen drift), (2) containment of plant products during the production year (grain segregation), and (3) volunteer GRC plants in following years (Owen 2005). While GR crops and non-GM crops can coexist, growers must go to great lengths to accomplish segregation (Anonymous 2007). Grain segregation, while difficult to

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maintain, can be accomplished and will effectively minimize the impact of GR crops on non-GM crops. Given that the mixing of grain is equivalent to loss of intellectual property (IP), this can be costly to growers; thus, appropriate tactics to isolate GR crops from non-GM crops must be in place (Owen 2000). Controlling volunteer GR crops is also relatively easy, depending on the rotational crop, but does require diligence on the part of the grower (Owen 2005). The introgression of the HT trait via pollen movement is another possibility and management of this problem is considerably more difficult, particularly in open-pollinated crops such as maize (Luan et al. 2001; Palmer et al. 2001; Westgate et al. 2003; Abud et al. 2004). A number of factors affect the success of maize pollen movement and subsequent pollination, and generally, the greater the distance between the pollen source and the donor, the less likely is the introgression of the GR trait (Luan et al. 2001; Westgate et al. 2003). However, given the tolerance levels established for some GM traits in non-GM crops, the isolation distances required to mitigate the risks of gene flow are too large to be realistic (Matus-Cadiz et al. 2004). Other open-pollinated crops have also undergone a great deal of scrutiny (Charles 2007; Fisher 2007; Harriman 2007; Weise 2007). It is suggested that the issues of the coexistence of GRCs with non-GM crops will continue to be a concern as long as there are economic differences between the crop cultivar types (Hurburgh 2000; Ginder 2001; Hurburgh 2003). Nevertheless, coexistence guidelines and buffer zone (isolation zone) regulations are in existence (Boller et al. 2004).

1.3.13

Stacked Traits

Stacked products are a very important feature and future trend for GM crops, which meets the multiple needs of farmers and consumers; these are now increasingly deployed by ten countries – US, Canada, the Philippines, Australia, Mexico, South Africa, Honduras, Chile, Colombia, and Argentina (James 2007). In fact, 37% of all GM crops in the US in 2007 were stacked products containing two or three traits that delivered multiple benefits. Currently, there are a number of GM crops that have stacked herbicide resistance and Bt events (Owen 2006). In some events, the pat gene is used as a marker for the Bt – the pat gene also confers resistance for glufosinate herbicide. Interestingly, the Bt trait is combined with a GR hybrid, the resultant GM cultivar is resistant to both glyphosate and glufosinate. Monsanto has announced new transgenic maize cultivars that will combine several Bt events plus two herbicide resistance events. The Bt event functions ecologically differently compared to herbicide-resistant transgenes; the transgene for herbicide resistance can be considered benign to the weed population and has no impact until the herbicide is applied (Owen 2009). In contrast, Bt exerts continuous selection pressure on the target insect whether the insect population is at economic threshold or not. Furthermore, given the potential for the introgression of transgenes into near-relatives, Bt could potentially improve the fitness of compatible weed species (Snow and Pedro 1997). For example, the fitness of canola was increased by the inclusion of Bt (Steward et al. 1997); thus, gene flow

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was kept in check. However, there is no reason to believe that stacked traits will have any greater impact on nontargets than the crops with single traits, and for Bt non-target impacts have been shown to be minimal (Ferry and Gatehouse 2009).

1.3.14

Conclusions

Herbicide-resistant crops, specifically GR crops, have been globally adopted as the basis for the production of maize, soybean, cotton and canola (Dill et al. 2008). Their adoption provides economic benefits to agriculture and has major positive impacts on the environment; specifically, conservation tillage which can reduce soil erosion (Duke and Powles 2008; Shipitalo et al. 2008). However, growers must adopt measures to proactively sustain the technology (Mueller et al. 2005; Johnson and Gibson 2006; Sammons et al. 2007; Christoffoleti et al. 2008) and to steward GR crops to avoid the evolution of GR weeds (Duke and Powles 2008; Owen 2009). While there are tactics that are capable of mitigating some of the other concerns, including the risks of transgene introgression into near-relative plants (Snow and Pedro 1997; Gepts and Papa 2003), an improved process to assess the environmental risk of GR crop technologies and communicating those risks to the lay public should be in place (Owen 2009).

1.4 1.4.1

Biotic Stress due to Plant Pathogens Crop Losses due to Plant Pathogens

Plants are challenged constantly by many different potential pathogens (Table 1.4). There are hundreds of thousands of viral, bacterial, and fungal species in the world and thousands of these are pathogens that infect plants (Chrispeels and Sadava 2003). Any one pathogen can severely depress the yield of a given crop. Pathogens may reduce yield by causing tissue lesions; by reducing leaf, root, or seed growth; or by clogging up vascular tissue and causing wilt. Even in the absence of obvious symptoms, pathogens can still be a major metabolic drain that reduces productivity. They may also cause pre- or postharvest (stored products) damage to the harvested product. Table 1.4 Organisms that cause infectious disease in plants Types of plant pathogens Fungi Ascomycetes e.g., Fusarium, Verticillium Basidiomycetes e.g., Rhizoctonia, Puccinia (rust) Oomycetes e.g., Phytopthora (blight) Bacteria e.g., Xanthomonas, Pseudomonas Phytoplasmas and Spiroplasmas Viruses Viroids and virus-like organisms Nematodes, protozoa, and parasitic plants are also considered plant pathogens

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1.4.1.1

N. Ferry and A.M.R. Gatehouse

Current Disease Control Measures

Disease control in commercial crops is both economically important and environmentally controversial. Disease management includes: quarantine, cultural practices such as modification of the plant environment (e.g., soil management) to reduce pest numbers, host plant resistance, biological control and chemical control. Chemical control can be highly effective. The first fungicide was discovered in the 1880s when Bordeaux mixture (a mixture of copper sulfate and lime) was found to suppress grape downy mildew. Numerous compounds with antifungal or antibacterial activity have since been discovered, most often applied as sprays, dusts or seed coatings. Many older compounds are broad spectrum and toxic with the newer chemistries acting systemically with a narrower target range. Such chemicals are expensive and normally reserved for use on high-value fruit and vegetable crops (Ferry and Gatehouse 2009). They are expensive to manufacture and a great deal of investment must be put into human and ecological safety testing before a product can be released (Chrispeels and Sadava 2003). As with other chemical control strategies, pathogens can evolve resistance to these compounds. Thus, plant breeders have often relied on genetic disease resistance traits to manage pathogens of particular crops. In classical plant breeding, this has relied on crosses between elite crops and wild relatives (that are more genetically diverse) to introduce new disease resistance traits into the crops. Extensive backcrossing of the elite line is then required to eliminate the undesirable traits in the wild relative and thus makes traditional breeding a time-consuming process with a time of about 15 years required before a new resistant variety is available for release to growers. Nevertheless, such traditional plant breeding has had significant successes. For example, the development of F1 hybrid crops (derived from crossing two inbred lines) resulted in vastly improved yields (Chrispeels and Sadava 2003). Despite this enormous success, the F1 hybrids also serve as a warning as to the dangers of largescale monoculture and the need for effective and durable disease resistance. In 1970, the highly successful but genetically uniform F1 maize crops in the US were left devastated by a disease, southern corn leaf blight (Bipolaris maydis Nisik), which caused damage to 710 million bushels of maize (Ullstrup 1972). In extreme cases, crop disease can change political as well as agricultural landscapes!

1.4.1.2

The Irish Potato Famine

The Irish Potato Famine (the Great Hunger) started in 1845, lasted until 1849–1852 and led to the death of approximately one million people through starvation and disease; a further million are thought to have emigrated as a result of the famine. Some estimate that the population of Ireland was reduced by 25% (Kinealy 1995). The cause of the famine was a potato disease commonly known as late blight caused by the oomycete, Phytophthora infestans. Although blight ravaged potato crops throughout Europe, the impact and human cost in Ireland was high as a third of the

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population was entirely dependent on the potato for food; it was also exacerbated by a host of political, social and economic factors. The famine was a watershed in the history of Ireland. Its effects permanently changed the island’s demographic, political and cultural landscape. For both the native Irish and the resulting diaspora, the famine became a rallying point for nationalist movements. The fallout of the famine continued for decades afterwards and Ireland’s population still has not recovered to prefamine levels. 1.4.1.3

The Bengal Famine of 1943

The Bengal famine of 1943 occurred in British administered Bengal. It is estimated that around four million people died from starvation and malnutrition during the period. During the Second World War, the British had just suffered defeat in nearby Burma and British authorities feared a subsequent Japanese invasion of India by way of Bengal, so emergency measures were introduced to stockpile food for British soldiers and prevent access to supplies by the Japanese in case of an invasion. However, in the rice-growing season of 1942 weather conditions were exactly right to encourage an epidemic of the rice disease brown spot following a cyclone and flooding; brown spot in rice is caused by the fungus Helminthosporium oryzae. When food shortages became apparent, the Bengal government reacted to the crisis incompetently refusing to stop the export of food from Bengal to allied soldiers and failing to provide adequate famine relief in Bengal itself. Winston Churchill was Prime Minister at the time and while his involvement in the disaster, and indeed his knowledge of it remains unclear, it has been suggested that in response to an urgent request by the Secretary of State for India to release food stocks for India, Churchill responded with a telegram asking if food was so scarce, “why Gandhi hadn’t died yet” (Mishra 2007). Needless to say, whether this is completely true or is in fact an unfortunate misquote used out of context, the British Administration in India declined in popularity and there is little doubt that this incident fuelled feelings for Indian Independence. Given the vast array of diseases that threaten crops, and the scale of disaster that can ensue, it is a wonder that crops be produced at all?

1.4.2

Plant Defense Against Pathogens

Plants have evolved mechanisms to resist pathogen invasion that consist of different defense layers. Firstly, waxy coatings on epidermal cells provide a physical barrier; secondly, plants contain large amounts of preformed secondary metabolites that have antimicrobial activity (constitutive defenses including glucosides, saponins, alkaloids, antifungal proteins, antifeedants and enzyme inhibitors), these are effective in many cases – but pathogens have evolved enzymes capable of detoxifying these compounds. Often induced defenses are the plants’ last line of defense against pathogens and are sufficient to (partially) ward off invading microorganisms.

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Essentially, plants have two major types of induced disease resistance, basal defense and resistance (R) gene-mediated defense. All plants have basal defense, this is a general immune response to pathogens and other environmental stresses. R-gene-mediated defense is more specific and is only found in certain plant species. R-gene-mediated defense involves recognition of a specific pathogen effector by a plant ligand receptor. These pathogen effectors can suppress plant basal defense, making any plant without the R-gene defense susceptible. The ligand-effector recognition can result in a dramatic immune response such as cell death. In tomato, Solanum lycopersicum, the Mi gene, a member of a large family of R-genes, mediate resistance to potato aphids, whiteflies, and root-knot nematodes (Kaloshian and Walling 2005). Both types of plant defenses (R and basal) involve signaling via three major plant hormones: salicylic acid, jasmonic acid, and ethylene (ETH). In some instances, defense responses are induced distal to the site of infection and this is referred to as systemic acquired resistance (SAR). At least three nonspecific induced defense pathways are described which are triggered by these specific signaling molecules: (a) The salicylic acid (SA)-dependent pathway is induced by necrosis inducing pathogens and triggers systemic acquired resistance (SAR) (b) A second pathway is triggered by nonpathogenic rhizobacteria, it is dependent on jasmonic acid (JA) and ethylene (ETH) and constitutes induced systemic resistance (ISR) (c) JA and ETH regulate a third pathway that is effective against a different set of pathogens and not affected by ISR Most of the inducible defense-related genes are regulated by these signaling pathways (Delaney et al. 1994; Sticher et al. 1997; Van Loon 1997; Reymond and Farmer 1998; Knoester et al. 1998; Ananieva and Ananiev 1999). Defense gene regulation has been extensively studied and severa1 rapid processes characteristic of the hypersensitive response (HR) appear to involve primarily activation of preexisting components rather than changes in gene expression. One of these rapid processes is the striking release of reactive oxygen species.

1.4.2.1

Oxidative Burst

The oxidative burst, a rapid production of reactive oxygen species (ROS), is a welldocumented early plant response to biotic stress (e.g., Apel and Hirt 2004). ROS comprise radicals and other nonradical but reactive species derived from oxygen. Among them, the superoxide anion (O 2 ) and hydrogen peroxide (H2O2) exert various effects on cells, directly or in cooperation with other molecules. In excess, ROS pose a threat to important biomolecules and cell membranes. One of the consequences of ROS activity is oxidative damage of membrane integrity due to lipid peroxidation processes which may result in the generation of highly cytotoxic compounds. Glutathione-S-transferases (GSTs), induced upon pathogen attack, may detoxify lipid peroxides by conjugating them with glutathione (GSH). These

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enzymes can also catalyze the GSH-dependent reduction/inactivation of H2O2, forming glutathione disulfide (GSSG) and increasing GSH synthesis by feedback induction (Marrs 1996). On the other hand, numerous studies indicate an essential role of ROS in plant defense responses to biotic stress. In addition to direct antimicrobial activity and contribution to the strengthening of barriers against pathogens, recent reports point to H2O2 and O 2 as signal transduction agents activating defense pathways and as key mediators in cell death during hypersensitive response (HR) (Grant and Loake 2000). To maintain a balance between negative and beneficial functions of ROS, their levels are strictly controlled by a complex and flexible network of antioxidant systems (Mittler et al. 2004). The major enzymatic ROS-scavenging components of this network are superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Superoxide dismutases dismute O 2 to H2O2, an excess of which may be subsequently detoxified by CATs and/or APX. Ascorbate peroxidases, in contrast to CATs localized in peroxisomes, are present in almost all cellular compartments; they exhibit a high affinity to H2O2 and are considered to be responsible for the fine modulation of ROS level (Mittler 2002). Moreover, APX, in cooperation with two main low-molecular antioxidants, ascorbate (Asc) and glutathione (GSH), and enzymes for their regeneration, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase (GR), constitute the ascorbate–glutathione (Asc–GSH) cycle, believed to be the central part of the antioxidant network (Noctor and Foyer 1998). Ascorbate, present at high concentrations in all cellular compartments and capable of direct scavenging of O 2 and hydroxyl radicals, is considered to be the most powerful cell antioxidant (Noctor and Foyer 1998). Ascorbate and glutathione are the major cellular redox buffers, which together with their oxidized forms, dehydroascorbate (DHAsc) and glutathione disulfide (GSSG), enable cells to maintain a redox balance. Changes in the levels or redox state of ascorbate and glutathione pools as well as in H2O2 homeostasis, and thereby in cellular/compartment redox state, are considered to be pivotal signaling events influencing gene expression and modulating the plant defense response (Pastori and Foyer 2002; Foyer and Noctor 2005). Numerous genes are induced during the plant defense response and presumably these function in host plant pathogen defense. Induced defenses include the activation of phytoalexins (including terpenoids, glycosteroids and alkaloids) and PR proteins (including chitinase, b-1,3 glucanase and other antimicrobial proteins) and the production of reactive oxygen species (ROS) leading to a hypersensitive response (HR).

1.4.3

Utilizing Defense Mechanisms

Most conventional breeding strategies have relied on identifying major resistance genes (often in wild or ancestral plants) while fewer have concentrated on breeding for polygenic resistance. New opportunities and strategies to enhance disease

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resistance are afforded by genetic engineering of plants – these may follow the single gene model, or may involve manipulation of defense-activating mechanisms.

1.4.4

Transgenic Disease Resistance

Many transgenic-based approaches exist for conferring enhanced levels of disease resistance, as exemplified by Fig. 1.6, which provides a simplified model. 1.4.4.1

Expression of Single Genes

R-Genes A very effective defense mechanism in plants is gene-for-gene resistance, this induced resistance mechanism is based on the interaction between plant-derived Pathogen

ty

ici en g o th Pa tors c fa

(3) Pathogen mimicry

(1b) Induced defense

(2a) Detection of pathogen

(1a) Constitutive defense (2b) Defense regulation Host cell

Nucleus

Transgenic Strategies. 1a. Constitutive expression of antimicrobial factors, 1b. Pathogen induced expression of one or more genes, 2a. Altering recognition of the pathogen (e.g. R-genes) and 2b. altering downstream regulators, 3. Priming recognition of pathogens (genetic vaccination).

Fig. 1.6 Simplified model of transgenic strategies to enhance plant defense

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resistance (R) gene products and an avirulence (avr)-gene product (elicitor) produced by the pathogen. The interaction is generally very specific and results in the triggering of strong resistance responses including hypersensitive response (HR) and localized cell death at the point of infection. There is no common structure between avirulence gene products, except that most are secreted proteins. Because there would be no evolutionary advantage to a pathogen keeping a protein that only serves to be recognized by the plant, it is believed that the products of avr genes, sometimes known as effector proteins, play an important role in virulence in genetically susceptible hosts. The guard hypothesis model proposes that the R proteins interact, or guard, a protein known as the guardee which is the target of the Avr protein. When it detects interference with the guardee protein, it activates resistance. R-gene specificity (recognizing certain avr gene products) is believed to be conferred by the leucine-rich repeats (LLRs). LRRs are multiple, serial repeats of a motif of approximately 24 amino acids in length, with leucines or other hydrophobic residues at regular intervals. LRRs are involved in protein–protein interactions, and the greatest variation amongst resistance genes occurs in the LRR domain. LRR swapping experiments between resistance genes in flax rust resulted in the specificity of the resistance gene for the avirulence gene changing (Bergelson et al. 2001). Gene-for-gene resistance thus provides obvious targets to engineer disease resistance in transgenic plants. However, pathogens can often breakdown R-gene-mediated resistance if the corresponding avr gene mutates becoming inactivated. Pathogen adaptation is also seen in conventionally bred crops. The use of R-genes is limited by them conferring resistance to only a single pathogen or race of pathogen; however, they can provide effective (even broad spectrum) resistance if transformed by genetic engineering into new species or new genera of plant (Oldroyd and Staskawicz 1998). For example, Rxo1, an R-gene derived from maize, a nonhost of the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola, was successfully transformed into rice and shown to confer resistance against this pathogen (Zhao et al. 2005). Interspecies differences do radically influence R-gene function making it preferable to use R-genes from related species (Ayliffe and Lagudah 2004); however, the transgenic approach circumvents the tedious and time-consuming backcrossing required to introduce a single trait via traditional crop breeding.

Constitutive Expression of Inducible Antimicrobial Factors Several examples of transgenic strategies to enhance plant constitutive defenses are described below. This is an extension of the single gene paradigm that has worked so well for insect-resistant transgenic plants. Expression of Chitinases Chitinases are glycosyl hydrolases catalyzing the degradation of chitin, an insoluble linear b-1,4-linked polymer of N-acetylglucosamine. They are produced by a wide

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range of organisms from microbes to insects and mammals, including plants (Kasprzewska 2003). Plants produce chitinases as defense against chitin-containing fungal pathogens by inhibiting spore germination, germ tube elongation and degrading hyphal tips (Khan et al. 2008). Transgenic plants expressing chitinase ChiC genes have already been produced in several species. In carrot, the tobacco class I ChiC gene has shown resistance to Botrytis cinerea (Punja and Raharjo 1996). Transgenic tobacco transformed with bean class I ChiC exhibited enhanced resistance to Rhizoctonia solani (Broglie et al. 1991) and Alternaria alternata (Lan et al. 2000). The rice chitinase gene (RCC2) exhibited increased resistance to Sphaerotheca humuli in transgenic strawberry (Asao et al. 1997). Peanut (Arachis hypogae) plants transformed with tobacco ChiC gene were resistant to a fungal pathogen (Cercospora arachidicola), the causal organism of leafspot disease (Rohini and Rao 2001). Potato has been transformed with the Streptomyces griseus ChiC gene and resistance to Alternaria solani (causal agent of early blight) demonstrated (Kahn et al. 2008) and cotransfer and expression of ChiC, glucanase, and bialaphos resistance (bar) genes in creeping bent grass conferred resistance to the fungal pathogens Sclerotinia homoeocarpa and R. solani (Wang et al. 2003). Expression of Defensins Defensins are the best characterized cysteine-rich antimicrobial proteins in plants (Broekaert et al. 1995). All known members of this family have four disulphide bridges and are folded in a globular structure that includes three b-strands and an a-helix (Segura et al. 1998). Inhibition of fungal growth by defensins seems to occur by permeabilization of the plasma membrane through binding to a putative receptor (De Samblanx et al. 1997). Genes encoding plant defensins are developmentally regulated, with predominant expression in the outer cell layers, and are induced in response to pathogen infection and stress (Segura et al. 1998). Enhanced tolerance to the fungus Alternaria longipes is observed in transgenic tobacco overexpressing a radish defensin (Rs-AFP2) (Chiang and Hadwinger 1991). In support of this role, Manners et al. (1998) show that the promoter of the plant defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal pathogens and responds to methyl jasmonate, but not to salicylic acid. Expression of Oxalate Oxidase and H2O2 -Generating Enzymes Rapid generation of superoxide and accumulation of H2O2 is a characteristic early feature of the hypersensitive response following perception of pathogen avirulence signals. In germinating barley and wheat seeds as well as in barley leaves challenged with powdery mildew, oxalate oxidase activity has been identified as a generator of H2O2 (Lane et al. 1993; Zhou et al. 1998). Oxalate oxidase utilizes oxalic acid and oxygen as substrates producing H2O2 and CO2. For certain necrotrophic fungi, such as Sclerotinia sclerotiorum, oxalic acid is a major pathogenicity determinant, significantly altering environmental pH (Cessna et al. 2000).

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Pathogen-inducible oxalate oxidase both acts as a generator of H2O2, killing the invading pathogen, and simultaneously detoxifies the acid, which is phytotoxic at high concentrations. Sunflower plants expressing a wheat oxalate oxidase accumulate enhanced levels of salicylic acid and PR1 even in the absence of pathogen infection and display improved tolerance to Sclerotinia infection (Hu et al. 2003). This effect is most likely due to the production of H2O2 from endogenous oxalic acid. Amine oxidases are a class of enzymes mainly found in the plant apoplast that act on a variety of amine substrates, including mono-, di- and polyamines and release the corresponding aldehyde as well as NH3 and H2O2. Amines are present in the plant apoplast and accumulate in response to environmental stress (Bolwell et al. 1999). Thus, in plants several systems are available that can produce H2O2 following pathogen attack. The importance of H2O2 in plant defense has clearly been shown by several groups who have reported increased pathogen resistance in transgenic plants by introducing either H2O2-generating systems or by inhibiting H2O2-degrading systems. The expression of a fungal glucose oxidase resulted in enhanced resistance to P. infestans and Erwinia carotovora in potato (Wu et al. 1995). Similarly, the prevention of H2O2 breakdown resulted in higher levels of H2O2 and increased disease resistance (Chamnongpol et al. 1998). Whether this improved pathogen tolerance is due to the direct antimicrobial effect of H2O2, or due to the fact that the plant defense system is induced by the increased levels of H2O2, was, in either case, not investigated. Inducible Expression of Single Antimicrobial Factors Stilbene Synthase Production of stilbene, a phytoalexin, is a well-characterized defense reaction in grape vine. Stilbene plays an important role in resistance to fungal and bacterial infection in plants. It strongly inhibits the growth of fungi and sprout of spores. Stilbene synthase gene (Vst1) is responsible for the synthesis of resveratrol (trans3,40 5-trihydroxystilbene) when plants are challenged by fungal diseases. Thus, introduction of stilbene synthase genes into transgenic plants can be used to induce synthesis of phytoalexins. Vst1 has been transferred into many plants to enhance fungal resistance including common spring wheat using biolistic transformation, where plantlets with mild resistance to powdery mildew were identified (Leckband and Lo¨rz 1998). Other crops transformed to produce stilbene include: tomato (Thomzik et al. 1997); apple (Szankowski et al. 2003); and Vst1 overexpressed in its native grape (Fan et al. 2008). 1.4.4.2

Activation of Expression of Multiple Genes

Polygenic Resistance There are several types of disease resistance in terms of the effects of genes on the plant and pathogen. However, in terms of the number of genes involved, there are

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two general types of resistance. The first (as described above for R-genes) is called major-gene or single-gene resistance. This type of resistance is well defined and more easily measured; it is sometimes called qualitative resistance because plants are either resistant or susceptible, without intermediate levels. The second type, called polygenic resistance, involves several or many genes. This type of resistance is harder to define; exactly which genes are involved may be unknown. It usually is effective against all races of a pathogen. This type is often called quantitative, because there are intermediate levels ranging from resistant to susceptible. It is also harder to measure than major-gene or single-gene resistance. Often polygenic resistance does not give a plant as high a level of resistance as major gene resistance. Polygenic durable resistance to multiple diseases of a crop would be highly desirable. In nature, most host plant resistance is based on multiple genes and a diverse set of resistant factors. This diverse, polygenic resistance system helps to prevent plant-feeding microorganisms from overcoming the resistance in the host plant. At present, polygenic resistance may be bred into a crop via quantitative trait loci (QTL) analysis, and the use of molecular markers in molecular breeding approaches; promising transgenic strategies (as discussed below) activate polygenic resistance via elicitor-induced resistance and activation of multiple genes.

Detection of Pathogens; Expression of Elicitors and Defense Activators Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide binding site (NBS), and a putative amino-terminal signaling domain, termed NBS–LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the hypersensitive response (HR). It is thought that this halts pathogen growth. In the absence of specific recognition, a basal defense response also occurs, which is apparently driven by pathogen-associated molecular patterns (PAMPS), such as flagellin and lipopolysaccharides (LPS) elicitors of defense responses (Belkhadir et al. 2004). The basal defense response overlaps significantly with R-proteinmediated defense, but is temporally slower and of lower amplitude. However, Takakura et al. 2008 show that expression of a bacterial flagellin gene (N1141) in transgenic rice triggers disease resistance and enhances resistance against blast (Magnaporthe grisea). Furthermore, a number of transgenic plants expressing NBS–LRR proteins under the control of the CaMV 35S promoter have been described (Mindrinos et al. 1994; Ellis et al. 1999; Stokes et al. 2002). For example, Grant et al. (2003) show that an Arabidopsis mutant, adr1 (activated disease resistance), contains both elevated levels of SA and ROIs, accumulates a number of defense-related gene transcripts and exhibits resistance against a number of microbial pathogens. ADR1 encodes a distinct NBS–LRR protein which possesses N-terminal kinase subdomains. Furthermore, transient expression of ADR1 is sufficient to engage defense-related gene expression and establish disease resistance

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in the absence of significant seed yield penalty. ADR1 plants showed striking resistance against both P. parasitica (Noco2) and another biotrophic pathogen, E. cichoracearum (UED1).

Pathogen Phytosensing A related strategy for crop defense involves engineered phytosensors indicating the presence of key plant pathogens to provide an important first line of defense (Mazarei et al. 2008). Plant defense mechanisms are highly regulated on the transcriptional level, and can be induced by chemical elicitors produced by pathogens. These elicitors have been shown to cause changes in gene expression, which initiate a whole plant response from a localized encounter with a pathogenic organism (Metraux et al. 2002). This is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in HR, SAR, ISR, and pathogen specific responses, any of which could be useful in building phytosensors. Cis-acting elements are conserved among plant species, which enables them to be used efficiently as synthetic inducible promoters. Employing synthetic promoters with potential inducible elements to engineer plants that can sense the presence of plant pathogens at the molecular level provides novel technologies for monitoring and increasing resistance to diseases (Gurr and Rushton 2005). Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or “phytosensors,” by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Mazarei et al. (2008) have employed cis-acting elements from promoter regions of pathogen-inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the cauliflower mosaic virus CaMV 35S promoter to increase basal level of GUS expression. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies analyzed the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants. The end goal of such studies is to engineer transgenic plants for the purpose of early pathogen detection.

1.4.4.3

Regulation of Inducible Defenses

The overexpression of regulatory genes provides another tool to activate plant defenses in response to pathogen attack. For example, transduction of the SA signal requires the function of NPR1 (also known as NIM1), a regulatory protein that was

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identified in Arabidopsis through genetic screens for SAR-compromized mutants (Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Mutant npr1 plants accumulate normal levels of SA after pathogen infection but are impaired in their ability to express PR genes and to mount a SAR response. The NPR1 gene encodes a protein with a BTB/BOZ domain and an ankyrin-repeat domain (Cao et al. 1997). Upon induction of SAR, NPR1 is translocated to the nucleus (Kinkema et al. 2000) where it interacts with members of the TGA/OBF subclass of basic domain/leucine zipper (bZIP) transcription factors (Zhang et al. 1999; Despre´s et al. 2000; Zhou et al. 2000; Subramaniam et al. 2001; Fan and Dong 2002) that are involved in the SA-dependent activation of PR genes (Lebel et al. 1998; Niggeweg et al. 2000). Physical interaction between NPR1 and TGA transcription factors has been shown to be required for the binding activity of these factors to promoter elements that play a crucial role in the SA-mediated activation of PR genes (Despre´s et al. 2000; Fan and Dong 2002). In separate studies NPR1 overexpression and enhanced resistance are correlated with either elevated or earlier expression of PR transcripts (Cao et al. 1998). Genes with high sequence similarity to NPR1 are found in Arabidopsis, tobacco, tomato, rice and maize (Campbell et al. 2003). Overexpression of NPR1 in rice has been shown to enhance resistance to bacterial blight (X.o.o) (Chern et al. 2001).

1.4.5

Pathogen Mimicry and Virus Resistance

Numerous reports concern transgenic resistance to plant viruses (Fuchs and Gonsalves 2007) in which RNA-mediated gene silencing is the predominant strategy (viral RNA is degraded and viral DNA is deactivated by methylation). Most of these strategies can be categorized as pathogen mimicry. Transgenes constitutively expressed to provide RNA-mediated virus resistance fall into three main types: 1. Sense or antisense viral sequences 2. Inverted repeats/hairpin RNA of viral sequences, and 3. Sequences of engineered microRNAs targeted against the virus RNA-mediated resistance against viruses was first reported by Lindbo et al. (1993). Since the discovery of what is now generally called RNA silencing in plants, the same specific RNA degradation mechanism has been identified in nearly all eukaryotes (Baulcombe 2004). In plants, genetic and molecular analyses have revealed at least three natural pathways for RNA silencing: cytoplasmic short interfering RNA (siRNA) silencing; endogenous mRNA silencing by microRNA (miRNA); and the silencing associated with DNA methylation and suppression of transcription (Baulcombe 2004). All these pathways involve the cleavage of doublestranded RNA molecules into short 21–26 nuclotide RNAs, known as siRNAs and miRNAs (Baulcombe 2004). To date, it has primarily been the cytoplasmic siRNA silencing pathway that has been exploited by genetic engineering to confer resistance to plant viruses.

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The best documented examples of commercial transgenic resistance involve type (1) resistance, although the mechanism of resistance was not initially known. In the 1990s, the papaya industry in Hawaii suffered a 50% reduction in production because of papaya ringspot virus; the solution came through the expression of viral coat protein in transgenic plants (thought to be immune priming, analogous to vaccination).

1.4.5.1

Papaya Ringspot Virus

Papaya ringspot is a destructive disease (a potyvirus) characterized by yellowing and stunting of the crown of papaya trees, a mottling of the foliage, damage to younger leaves, water-soaked streaking of the petioles (stalks), and small darkened rings on the surface of fruit. Papaya ringspot virus is transmitted from infected papaya trees to healthy trees by the feeding action of various species of aphids, especially the green peach aphid and melon aphid. The virus is transmitted in a nonpersistent manner, meaning that the virus does not multiply within the aphid but is instead carried on its mouth parts and is transmitted from plant to plant while feeding. In 1995, American researchers developed a transgenic papaya resistant to the virus, by expressing a copy of a viral coat protein in the plant (Ling et al. 1991). It was field-tested in Hawaii where it was shown to be effective against the virus. The virus-resistant papaya is now widely used by commercial papaya producers in Hawaii. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral coat protein gene was later shown to be present in transgenic lines resistant to PRSV; thus it is posttranscriptional gene silencing involved in the establishment of resistance (Krubphachaya et al. 2007). Pathogen-derived resistance has also been shown to be effective against maize streak virus (Shepherd et al. 2007), potato leaf roll virus (Vazquez Rovere et al. 2001) and tomato yellow leaf curl (Yang et al. 2004). An important drawback of RNA-based approaches to enhanced resistance is the high level of sequence specificity required for RNA degradation. Viruses containing >10% nucleotide divergence are insensitive to RNA degradation. Another drawback is the size of the transgene, commonly >300 bp, required to trigger efficient RNA silencing. However, Parizotto et al. (2004) has provided experimental evidence using genetically engineered microRNA (miRNA) to show that a 21 nucleotide sequence complementary to GFP mRNA was sufficient to trigger complete GFP silencing in transgenic plants. In the future, similar miRNA-derived strategies could provide resistance to a large number of plant viruses. However, the major technical limitation for technologies based on RNA silencing is that many important plant crop species are difficult or impossible to transform, precluding the constitutive expression of constructs directing production of double-stranded RNA. Moreover, public concerns over the potential ecological impact of virus-resistant transgenic plants have so far significantly limited their use (Fermin et al. 2004). For DNA viruses (particularly, Gemini viruses), transgenic resistance involves both RNA-mediated resistance and mutated viral proteins exerting negative effects on viral replication (Vanderschuren et al. 2007).

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Conclusions

While insect-resistant and herbicide-tolerant transgenic plants are the most widely grown GM crops, very few strategies using genetically engineered plants for disease resistance have had a similar impact (Collinge et al. 2008). Given the effort put into biotechnological strategies for disease resistance over the last two decades, why have so few crops expressing these traits become commercialized crops? The development of molecular markers, which are particularly useful in screening for disease resistance and facilitate conventional breeding, provide a higher commercial incentive for seed companies because of strong public opposition to GM crops. Often, transgenic disease resistant plants have only partial resistance, or resistance to rapid breakdown (single genes), again giving a low economic incentive to developers. At present, clear field results show that transgenic virus resistance is effective; however, there are no signs that commercial bacterial- or fungal-resistant crops will be introduced onto the market at any point soon.

1.5

Transgenic Crops for Resistance to Biotic Stress: Conclusions

Approximately 10.3 million farmers in 22 countries grew transgenic (genetically modified) crops in 2006. Yet this technology remains one of the most controversial agricultural issues of current times. Many consumer and environmental lobby groups believe that GM crops will bring very little benefit to growers and to the general public, and that they will have a deleterious effect on the environment. The human population is currently 6.1 billion and it is predicted to increase to 9–10 billion in the next 50 years (Fig. 1.7). This is at a time when food and fuel are competing for land (Fig. 1.8) and climate change threatens to compromise current resources. Population growth, changing diets, higher transport costs and a drive towards biofuels are forcing food prices up (Fig. 1.9). The UN’s Food and Agriculture Organization (FAO) stated that the food crisis had thrown an additional 75 million people into hunger and poverty in 2007 alone. World Population Growth (billion people) 1950

1975

2000

2025

2050

2.5bn

4.1bn

6.1bn

8.0bn

9.2bn

Fig. 1.7 World population growth

1 Transgenic Crop Plants for Resistance to Biotic Stress Fig. 1.8 Demand for biofuels

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Demand for Biofuels Rise in use of course grains 2005-7 (FAO/OECD).

Total: 80m tonnes

Biofuel use: 47m tonnes

Price Rises in a Single Year (Mar 2007-Mar 2008) Source: Bloomberg, FAO/ Jackson Son & Co 130% 87% 74% 31%

Corn

Rice

Soya

Wheat

Fig. 1.9 Price rises of major food crops (2007–2008)

It is, and will continue to be, a priority for agriculture to produce more crops on less land. The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. Traditionally, agricultural production has kept pace, even outstripped human population growth; however, we currently face a set of unique challenges. One of the greatest dangers to agriculture is its vulnerability to global climate change. The expected impacts are for more frequent and severe drought and flooding, and shorter growing seasons. The performance of crops under stress will depend on their inherent genetic capacity and on the whole agroecosystem in which they are

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managed. It is for this reason that any efforts to increase the resilience of agriculture to climate change must involve the adoption of stress-resistant plants as well as more prudent management of crops, animals and the natural resources that sustain their production. Currently, we may be at the limit of the existing genetic resources available in our major crops (Gressel 2008). Thus, new genetic resources must be found and only new technologies will enable this.

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Chapter 2

Transgenic Plants for Abiotic Stress Resistance Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin

2.1

Introduction

Modern agricultural crop production relies on the growth of a few of the world’s plant species selected for their superior qualities and suitability as food, animal feed, fiber or industrial end uses. Centuries of selection and, more recently, scientific breeding for adaptation to biotic and abiotic stresses have been necessary to improve yield, yield stability, and product quality in agricultural species. Nevertheless, abiotic stresses remain the greatest constraint to crop production. Worldwide, it has been estimated that approximately 70% of yield reduction is the direct result of abiotic stresses (Acquaah 2007). The ever increasing pressure put on agricultural land by burgeoning human populations has resulted in land degradation, a cultivation shift to more marginal areas and soil types, and heavier requirements for agricultural productivity per unit area. Additionally, climate change has exacerbated the frequency and severity of many abiotic stresses, particularly drought and high temperatures, with significant yield reductions reported in major cereal species such as wheat, maize, and barley (Lobell and Field 2007). In many parts of the world, rainfall has become less predictable, more intense, and, due to increasing temperatures, subject to higher evapotranspiration. For example, in the major grain growing areas of eastern Africa, the predominant rainy season is starting later and ending earlier (Segele and Lamb 2005) with longer dry spells in between (Seleshi and Camberlin 2006). Agricultural practices to improve crop productivity per unit area have, in many cases, accelerated the rate of land degradation, with particularly marked effects in irrigated areas. Irrigation has led to salinity across large tracts of agricultural land,

I. D. Godwin (*) School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, Qld 4072, Australia e-mail: [email protected]

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with cases, such as in India, where it has reportedly led to the loss of seven million hectares from cultivation (Martinez-Beltran and Manzur 2005). In Australia, almost eight million hectares of land are under threat of dryland salinity (Munns et al. 2002). Higher yields are also only sustainable with higher nutrient use, and the heavy demand for fertilizers has caused rising costs for farmers worldwide. The environmental and economic consequences of increased nutrient use have been widely reported. For sustainability of crop production, there is a need to reduce the environmental footprint of food and fiber production, and nutrient use efficient crops are highly sought after. Transgenic approaches are one of the many tools available for modern plant improvement programs. Gene discovery and functional genomics projects have revealed multitudinous mechanisms and gene families, which confer improved productivity and adaptation to abiotic stresses. These gene families can be manipulated into novel combinations, expressed ectopically, or transferred to species in which they do not naturally occur or vary. Hence, the ability to transform the major crop species with genes from any biological source (plant, animal, microbial) is an extremely powerful tool for molecular plant breeding. Transgenic plants can be used as sources of new cultivars (or their germ plasm as new sources of variation in breeding programs) and they are also extremely useful as proof-of-concept tools to dissect and characterize the activity and interplay of gene networks for abiotic stress resistance. In this chapter, we will outline the major yield-limiting abiotic stresses faced by crop plants: drought, salinity, cold, nutrient deficiency, and metal toxicity. Within each section, we will then cite specific examples of transgenic crop approaches to overcome these stresses and also discuss a number of conserved plant stress response mechanisms, which have been demonstrated to confer better adaptation to a number of different abiotic stresses.

2.2

Water Scarcity and Agriculture

Drought is the most significant environmental stress on world agricultural production (Tuberosa and Salvi 2006; Cattivelli et al. 2008) and enormous effort is being made by plant scientists to improve crop yields in the face of decreasing water availability. During the twentieth century, the world’s population tripled from approximately 1.65 to 5.98 billion and population projections of 8.91 and 9.75 billion are expected to occur by 2050 and 2150, respectively. Developing countries in Africa and Asia account for approximately 80% of this growth and, with an estimated 800 million people in these countries already undernourished, the Food and Agriculture Organization (FAO) of the United Nations predicts that a 60% increase in world food production over the next two decades is required in order to sustain these populations. Agriculture accounts for approximately 70% of global water use and irrigation accounts for up to 90% of total water withdrawals in arid nations (World Water

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Council 2008; FAO 2009a). Approximately, 40% of all crops produced in developing countries are grown on irrigated arable land, which accounts for only 20% of the total arable land in these nations (FAO 2009c). The water withdrawal requirement for irrigation is expected to increase by 14% in developing countries by 2030 and strategies to decrease this demand by developing crops that require less irrigation will, therefore, play a vital role in maintaining world food supply. The area of plant drought tolerance research and improvement encompasses an enormous range of environmental, genetic, metabolic, and physiological considerations and an exhaustive discussion of all available avenues for developing droughtresistant crop varieties is beyond the scope of this chapter. Rather, this section attempts to provide an overview of some of the genetic mechanisms that have been manipulated in order to develop transgenic crops with improved drought tolerance and focuses on research that has involved long-term and field-based drought stress treatments performed on agronomic and horticultural crop species at yield determining plant life stages.

2.2.1

Improving Drought Tolerance in Agricultural Crops

All plants require water to complete their life cycle, with most plant cells consisting of at least 70% water on a fresh weight basis. When insufficient water is available, plant water status is disrupted, which causes imbalances in osmotic and ionic homeostasis, loss of cell turgidity, and damage to functional and structural cellular proteins and membranes. Consequently, water-stressed plants wilt, lose photosynthetic capacity, and are unable to sequester assimilates into the appropriate plant organs. Severe drought conditions result in reduced yield and plant death. The overall aim of genetically improving crops for drought resistance is to develop plants able to obtain water and use it to produce sufficient yields for human needs under drought conditions. While advances have been made in developing crops that are genetically improved with traits such as herbicide and pesticide resistance, attempts to improve plant drought resistance have been hindered by the complexity of plant drought resistance mechanisms at the whole plant, cellular, metabolic, and genetic levels. Interactions between these mechanisms and the complex nature of drought itself, adds another layer of intricacy to this problem.

2.2.2

Complexity of Drought and Plant Responses to Drought Stress

Drought (nonavailability of water for crop growth) and water deficit (insufficient plant water status) are variable, complex, and recurring features in most parts of the

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world. Even in areas with very high precipitation, many crops are likely to experience a certain level of water deficit at some stage during the growing cycle. Elucidation of plant drought resistance and response mechanisms has been compounded by the variable levels and forms of drought. Drought can be spatially and temporally variable; terminal, short-term, or sporadic; severe, moderate, or minor; and can occur at rates ranging from very sudden to gradual. In addition, the effects of drought and water deficit on crop productivity vary for different crops, macro- and microenvironments across a single field, plant life stages, and the plant material to be harvested. Furthermore, the effects of drought on crop productivity are often compounded by associated stresses such as heat, salt, and nutrient stress.

2.2.3

Plant Drought Resistance and Response

Plant drought resistance mechanisms can be broadly grouped into avoidance or tolerance mechanisms. Drought avoidance mechanisms are associated with physiological whole-plant mechanisms such as canopy resistance and leaf area reduction (which decrease radiation adsorption and transpiration), stomatal closure and cuticular wax formation (which reduce water loss), and adjustments to sink-source allocations through altering root depth and density, root hair development, and root hydraulic conductance (Beard and Sifers 1997; Rivero et al. 2007). Drought tolerance mechanisms are generally those that occur at the cellular and metabolic level. These mechanisms are primarily involved in turgor maintenance, protoplasmic resistance, and dormancy (Beard and Sifers 1997). Plants respond to water-limiting conditions by altering the expression of a complex array of genes (Fig. 2.1) and, although elucidation of biochemical pathways associated with many of these genes has been the focus of an enormous amount of research over the last two to three decades, the mechanisms by which these genes and their products interact remains relatively poorly understood. Abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth and productivity (Wang et al. 2001). Drought, salinity, extreme temperatures, and oxidative stress are often interconnected, and may induce similar cellular damage. For example, drought and/or salinization are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell (Serrano et al. 1999; Zhu 2001). Oxidative stress, which frequently accompanies high temperature, salinity, or drought stress, may cause denaturation of functional and structural proteins (Smirnoff 1998). As a consequence, these diverse environmental stresses often activate similar cell signaling pathways (Knight 2000; Shinozaki and Yamaguchi-Shinozaki 2000; Zhu 2001, 2002) and cellular responses, such as the production of stress proteins, upregulation of antioxidants and accumulation of compatible solutes (Vierling and Kimpel 1992; Zhu et al. 1997; Cushman and Bohnert 2000; Wang et al. 2003b).

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Signal sensing, perception, transduction

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Fig. 2.1 Plant responses to abiotic stress

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The Genetic Basis of Drought Tolerance

Expression studies have shown that drought-specific genes can be grouped into three major categories: (1) Genes involved in signal transduction pathways (STPs) and transcriptional control; (2) Genes with membrane and protein protection functions; and (3) Genes assisting with water and ion uptake and transport (Vierling 1991; Ingram and Bartels 1996; Smirnoff 1998; Shinozaki and Yamaguchi-Shinozaki 2000). Plants adapt to drought conditions by tightly regulating specific sets of these genes in response to drought stress signals, which vary depending on factors such as the severity of drought conditions, other environmental factors, and the plant species (Wang et al. 2003b). To date, successes in genetic improvement of drought resistance have involved manipulation of a single or a few genes involved in signaling/regulatory pathways or that encode enzymes involved in these pathways (such as osmolytes/compatible solutes, antioxidants, molecular chaperones/osmoprotectants, and water and ion transporters; Wang et al. 2003b). The disadvantage of this is that there are numerous interacting genes involved, and efforts to improve crop drought tolerance through manipulation of one or a few of them is often associated with other, often undesirable, pleiotropic and phenotypic alterations (Wang et al. 2003b). These complex considerations, when coupled with the complexity of drought and the plant–environment interactions occurring at all levels of plant response to water deficit, illustrate that the task plant researchers are faced with in engineering drought resistant crops is dauntingly multi-faceted and extremely difficult.

2.2.5

Engineering Improved Drought Avoidance in Crops

Most transformation studies to improve plant drought resistance have produced transformants that display a variety of both tolerance and avoidance traits. An exception was demonstrated by Rivero et al. (2007) who manipulated a leaf senescence gene. Leaf senescence is an avoidance strategy and is accelerated in drought-sensitive plants to decrease canopy size. In crop plants, accelerated senescence is often associated with reduced yield and is thought to be the result of an inappropriately activated cell death program. Therefore, suppression of droughtinduced leaf senescence in tobacco plants was investigated as a tool to enhance drought resistance. Transgenic plants were developed by expressing isopentyl transferase (IPT), a key enzyme in the biosynthesis of cytokinin (a leaf senescence inhibitor) under the control of the senescence-associated receptor protein kinase promoter (PSARK). The SARK gene, which is induced during late maturation and drought and decreased during senescence development, encodes a maturation/sensescence-dependent protein kinase. Although transgenic plants wilted under a 15-day glasshouse drought stress treatment, senescence did not occur and a reduced

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photosynthetic capacity was maintained. Following rewatering, transgenic plants recovered full leaf turgor and resumed growth and maximum photosynthetic capacity, while the control plants were unable to recover from the drought stress. Water use efficiency (WUE) of the transgenic plants was also markedly higher than wild-type (WT) plants, and was two to three times higher after rewatering than before the drought treatment. An experiment to assess whether the transgenic plants could produce significant yields under water-limiting conditions determined that biomass and seed yield were not as affected in transgenic plants than in WT controls, although this result was not significant (Rivero et al. 2007). Other drought avoidance/whole-plant traits that have been investigated include stay-green and cuticular biosynthesis. Stay-green is a variable and quantitative trait, which generally refers to delayed senescence. It has not yet been used to successfully produce transgenic plants with increased drought resistance in the field. Cuticular biosynthesis was investigated by transgenic expression of AtMYB41, which encodes an R2R3-MYB transcription factor (TF) in Arabidopsis. AtMYB41 is expressed at high levels in response to drought, abscisic acid (ABA; Sect. 2.2.6.1), and salt treatments, and was demonstrated to have a role in cell expansion and cuticle deposition. The transformation of Arabidopsis with AtMYB41 was associated with undesirable pleiotropic phenotypes including dwarfism, enhanced sensitivity to desiccation, and enhanced permeability of leaf surfaces (Cominelli et al. 2008).

2.2.6

Improving Plant Drought Tolerance

2.2.6.1

Absicisic Acid and Transcriptional Regulation

The plant hormone ABA regulates the plant’s adaptive response to environmental stresses such as drought, salinity, and chilling via diverse physiological and developmental processes. ABA has functional roles ranging from seed maturation processes to lateral root development (McCourt and Creelman 2008; Wasilewska et al. 2008). Under abiotic stress, ABA induces stomatal closure, reduces water loss via transpiration, and induces gene expression (Chandler and Robertson 1994). Gene expression and biochemical studies into ABA synthesis in Arabidopsis and some other model plants have largely elucidated the basic ABA biosynthetic pathway (Schwartz et al. 2003) and many of the key enzymes involved in ABA synthesis have been investigated transgenically in relation to improving plant drought tolerance. For example, transgenic Arabidopsis plants constitutively overexpressing the zeaxanthin epoxidase gene, AtZEP, which encodes an enzyme required for an initial step in ABA synthesis from isopentyl diphosphate (IPP) and b-carotene (Schwartz et al. 2003) showed increased tolerance to drought and salinity stress. The increased drought stress tolerance was attributed to increased leaf and lateral root development, longer primary roots, higher fresh weight, and increased survival compared with control plants following drought treatment.

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Furthermore, compared with WT plants, the rate of water loss was lower, the levels of ABA were higher, the expression of stress responsive genes such as Rd29a was much higher, and stomatal aperture was smaller under salt and/or drought stress. Overall, the increased stress resistance was attributed to increased ABA levels in response to osmotic stress, which resulted in enhanced expression of ABAresponsive stress-related genes (Park et al. 2008). Many of the drought stress response pathways that have been identified to date appear to be under transcriptional regulation and ABA plays a key role in this process (Fig. 2.2). Transcriptional regulation involves interaction between TFs and specific cis-acting elements located within or near the promoter region upstream of expressed genes. Figure 2.2 shows links between responses to low temperature and dehydration stress at the transcriptional level. It can be seen that ABA is involved in both types of abiotic stress. Many transcriptional responses to drought stress have been well characterized and are classified as being ABA-dependent, ABA-independent, or both. ABA is Low Temperature

Dehydration

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CBF1, 2, 3 / DREB1a, b, c DREB2

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bZIP

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DREB2 AREB/ABF

CRT/DRE (Rd29a, Cor15a)

ABRE (Rd29a, Rd29b)

MYCR/MYBR (Rd22, AtADH1)

Fig. 2.2 Plant transcriptional processes induced by dehydration and low temperature stress. Displayed are transcription factors (rounded rectangles) both ABA-dependent (shaded) and ABA-independent (unshaded), posttranscriptional modification such as phosphorylation (elipses), transcription factor binding sites and representative promotors (rectangles), possible regulation points (dotted arrows), and possible cross-talk (bidirectional arrows)

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often used in stress and stress acclimation studies because it is produced in response to stress. ABA induces expression of many genes that are also induced by drought, cold, and salinity when applied exogenously (Shinozaki and Yamaguchi-Shinozaki 1996). There are two types of ABA-dependent transcription. The “direct” pathway involves cis-acting ABA-responsive elements (ABREs), which are directly activated by binding with TFs such as basic-domain leucine zipper (bZIP)-type DNAbinding proteins (Shinozaki and Yamaguchi-Shinozaki 1996; Kobayashi et al. 2008). Alternatively, the “indirect” ABA-dependent transcription pathway involves other cis-acting elements, such as MYC and MYB. These elements are activated through binding with ABA- or drought-inducible TFs, such as basic helix–loop– helix (bHLH)-related protein AtMYC2 and an MYB-related protein, AtMYB2 (Abe et al. 2003). An example of the indirect pathway can be seen in the expression of rd22 from Arabidopsis (Shinozaki and Yamaguchi-Shinozaki 1996). Some genes are induced by drought stress but are not expressed in response to exogenous ABA applications and these genes are the product of ABA-independent STPs. One such gene is rd29a (also known as lti78 and cor78). YamaguchiShinozaki and Shinozaki (Yamaguchi-Shinozaki and Shinozaki 1994) identified a dehydration-responsive element (DRE) in the promoter region of rd29a and the DRE-binding (DREB) protein transcription pathway has since been explored for its important roles in drought, cold, and salinity stress (Shinozaki and YamaguchiShinozaki 1996; Qin et al. 2007). Several C-repeat (CRT) binding factor (CBF)/ DREB proteins have now been identified from the promoter regions of other stressinducible Arabidopsis genes, such as cor15a, kin1, cor6.6 and cor47/rd17, and the CBF/DREB pathway has been shown to be conserved across species (Benedict et al. 2006; Pasquali et al. 2008). CBF/DREB1 and DREB2, belong to the ethyleneresponsive element/apetela 2 (ERE/AP2) TF family; their expression is induced by cold or drought stress and both activate expression of genes possessing a CRT/DRE cis-element (Stockinger et al. 1997; Liu et al. 1998). Likewise, DREB2A positively regulates expression of many abiotic stress-related genes possessing DRE sequences in their 5’-upstream regions. DREB2A overexpression in Arabidopsis confers significant drought tolerance in transgenic plants (Sakuma et al. 2006a, b). DREB genes have been used in transformation of several crops, including wheat and rice, in attempts to increase drought tolerance (Chen et al. 2008; Kobayashi et al. 2008). Although DREs are cis-acting elements that were first thought to activate ABA-independent stress-responsive gene expression, some are also implicated in ABA-dependent expression (Shinozaki and Yamaguchi-Shinozaki 2000). CBF4 is an apparent homolog of the CBF/DREB1 proteins that is thought to be a critical regulator of gene expression in drought stress signal transduction. The action of CBF4 is thought to be through its binding with CRT/DRE elements in promoter regions of drought- and cold-inducible genes (Haake et al. 2002). CBF4 gene expression has been shown to be upregulated in response to drought and ABA; however, constitutive expression of CBF4 was found to result in expression of cold- and drought-induced genes under nonstress conditions and this was associated with retarded growth, shorter petioles, darker green leaves, and delayed time to

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flowering in Arabidopsis seedlings (Haake et al. 2002). Another study showed that CBF4 expression was induced by salt, but not by drought, cold, or ABA (Sakuma et al. 2002). Similar observations, and observations of higher levels of soluble sugars and proline, have been recorded during many CBF overexpression studies, which suggest that the use of constitutively expressed CBF/DREB genes may not be applicable to the development of crops with improved drought tolerance. It is thought that the use of stress-inducible promoters that have low expression levels under non-stress conditions could be used in conjunction with CBF genes to alleviate the retarded growth observed in CBF overexpression studies (Zhang et al. 2004). Many studies have illustrated the potential of manipulating CBF/DREB genes to confer improved drought tolerance. For example, overexpression of CBF1/ DREB1B from Arabidopsis was able to improve tolerance to water-deficit stress in tomato. Furthermore, when driven by three copies of an ABA-responsive complex (ABRC1) from the barley HAV22 gene, transgenic tomato plants expressing CBF1 exhibited enhanced tolerance to chilling, water deficit, and salt stress, and maintained normal growth and yield under normal growing conditions when compared with control plants (Lee et al. 2003a). Other studies have also found that expression of CBF/DREB genes under stress-inducible promoters result in transgenic plants that do not express detectable levels of these genes under non-stress conditions, minimizing growth retardation and other adverse effects (Al-Abed et al. 2007). The CRT/DRE motif also acts as one of the binding sites for the ERF family of TFs (Trujillo et al. 2008). A novel ERF from sugarcane, SodERF3, was found to enhance salt and drought tolerance when overexpressed in tobacco plants. Under drought treatment, transgenic plants were significantly taller than controls and were able to flower under an extended growth period. Furthermore, the absence of observable differences in height, number of leaves, leaf area, leaf weight, and stalk weight between transgenic and control plants illustrates that this gene has potential for engineering drought stress tolerance in plants (Trujillo et al. 2008). Other TFs involved in mediation of ABA-dependent and ABA-independent signal transduction and gene expression include NAC, WRKY, RING finger, and zinc-finger TFs (Seki et al. 2003; Zhang et al. 2004; Chen et al. 2006). Nelson et al. (2007) showed that constitutive expression of a TF from the nuclear factor (NF-Y) family, AtNF-YB1, which belongs to the CCAAT-binding TF family, improved performance of Arabidopsis under drought conditions. Consequently, an orthologous maize TF gene, ZmNF-YB2, was constitutively expressed in maize. Transgenic lines were exposed to both glasshouse-based and field-based drought stress treatments. Transgenic lines exhibited less wilting and faster recovery and re-established growth more rapidly than WT (on average) under glasshouse-based drought treatment. Transgenic lines subjected to field-based drought stress at the late vegetative stage exhibited superior health, higher chlorophyll indices and photosynthetic rates, lower leaf temperatures, higher stomatal conductance, and less yield reduction than WT plants. Furthermore, under favorable conditions, transgenic plants were greener, flowered 1–3 days earlier, and had slightly compressed internodes. Most importantly, the stress adaptation response contributed to a yield advantage in

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transgenic maize grown within drought environments, suggesting that ZmNF-YB2 has a realistic application for use in commercial agriculture under severe waterlimiting conditions (Nelson et al. 2007). Another TF that has been manipulated in order to increase plant drought tolerance is the HARDY (HRD) gene, which has been linked to increased transpiration efficiency related to stomatal adjustment. HRD is an AP2/ERF-like TF isolated from hrd-dominant (hrd-D) Arabidopsis mutants, which displayed vigorous rooting and dark green leaves that were smaller and thicker than WT plants. Karaba et al. (2007) isolated the HRD gene and constitutively expressed it in Arabidopsis under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The thicker leaves, higher root density, and increased root strength were associated with abundant chloroplasts, increased secondary and tertiary root initiation and proliferation, and extra corticle cell layers and more compact, stele bearing vascular tissue, respectively. Furthermore, the mutants survived longer periods of drought stress and could reach full maturity under high levels of salt stress. HRD was also constitutively expressed in rice and transgenic plants displayed no reduction in growth, seed yield, or germination, but had significantly increased leaf canopy with more tillers under normal greenhouse conditions compared with WT controls. Under drought stress, the transgenic plants were of deeper green color (attributable to increased number of bundle sheath cells), displayed distinctive drought tolerance and lower stomatal conductance, had higher net carbon assimilation and photosynthetic rates, and possessed higher root biomass (Karaba et al. 2007). Recently, a novel drought-tolerant gene, HDG11, which encodes a protein from the homeodomain (HD)-START TF family (also known as the Class IV HD-leucine zipper TF family) was identified in Arabidopsis and was found to confer drought resistance via enhanced root growth and decreased stomatal density when constitutively overexpressed in transgenic tobacco (Yu et al. 2008). The constitutive expression of the gene was not associated with retarded growth or any other observable deleterious phenotypic effects and, the gene was also shown to transactivate a number of other genes involved in the drought stress response including ERECTA (Sect. 2.2.6.2.1; Yu et al. 2008). SNAC1 from rice has also been shown to have trans-activation activity. NAC TFs comprise a large gene family with proteins exhibiting a highly conserved N-terminal DNA-binding domain and a diversified C-terminal domain. NAC was derived from the names of the first three described proteins containing the DNA-binding domain, namely, NAM (no apical meristem), ATAF1-2, and CUC2 (cup-shaped cotyledon; Souer et al. 1996; Aida et al. 1997). NAC is a plant-specific TF family with diverse roles in development and stress regulation. When constitutively overexpressed in rice, SNAC1 was found to significantly improve plant resistance to severe drought stress during reproductive and vegetative growth and was not associated with any negative phenotypic effects or yield penalty (Hu et al. 2006). Transgenic plants were more sensitive to ABA and closed more stomata than WT plants but maintained continual photosynthetic activity. There was no difference between root morphologies of transgenic and WT plants indicating that the improved drought resistance was not related to increased root-water uptake.

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The WRKY superfamily of plant TFs has a conserved sequence (WRKYGQK) at their N-terminal ends (Wu et al. 2008b). Transgenic rice seedlings, expressing OsWRKY11 under the control of a rice heat shock protein (HSP) promoter, HSP101, were shown to survive longer and lose less water under a short, severe drought treatment, than WT plants (Wu et al. 2008b). A TFIIIA-type zinc-finger protein gene, ZFP252, was also found to confer improved drought stress resistance in rice. Young transgenic rice plants overexpressing ZFP252 survived longer, displayed less relative electrolyte leakage, and accumulated more compatible osmolytes than WT plants or plants with ZFP252 knocked out during a 14-day period of drought stress (Xu et al. 2008a). A salt- and drought-induced RING-finger protein, SDIR1, was found to confer enhanced drought tolerance to tobacco and rice (Zhang et al. 2008b). Arabidopsis E3 ligase SDIR1 is a positive regulator in ABA signal transduction. Tobacco and rice plants constitutively overexpressing the SDIR1 gene displayed less leaf wilting and rolling, longer survival, and improved recovery under drought conditions than control plants. The mechanism of drought tolerance was thought to be due to decreased stomatal aperture, which increased transpiration efficiency of transgenic plants. Some genes have been shown to suppress expression of drought-response transcription pathways. For example, Jiang et al. (2008) recently characterized SAZ, an Arabidopsis gene from the SUPERMAN (SUP) family of plant-specific zinc-finger genes, which encode proteins containing single C2H2-type zinc-finger motif with a conserved short amino acid sequence and a class II ERF-associated amphiphilic repression (EAR) motif-like TF domain at the carboxy-terminal region. SAZ was found to be rapidly downregulated in response to drought and other abiotic stresses and SAZ gene knockouts resulted in elevated expression of the ABA-responsive genes rd29B and rab18 under stressed and unstressed conditions. This shows that gene knockouts and gene silencing may also be applicable to the development of crops with improved drought resistance.

2.2.6.2

Signal Sensing, Perception, and Transduction

Prior to transcriptional activation of genes, drought stress signals are received and messages conveyed to the appropriate components of the downstream pathway (Xiong and Ishitani 2006). In general, STPs involve perception of stress by specific receptor molecules, which vary in identity, structure, perception, signal relay mechanism, and location within the cell (Xiong and Ishitani 2006). Plant stress STPs often involve secondary messengers, which may modify signals (often via reversible protein phosphorylation) prior to conveying them from receptor molecules to the activators of the appropriate gene expression pathway (Xiong and Ishitani 2006). Other molecules may also be involved in stress STPs and the functions of these include recruitment and assembly of signaling complexes, targeting of signaling molecules, and regulation of signaling molecule lifespan (Xiong and Ishitani 2006).

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The major molecules involved in drought stress signal sensing, perception, and transduction include receptor molecules/osmosensors, phospholipid-cleaving enzymes (PLEs), reactive oxygen species (ROS), mitogen-activated protein kinases (MAPK), and Ca2+ sensors.

Receptor Molecules/Osmosensors Receptor molecules/osmosensors are the initial stress signal perceivers and they convey the signal to the appropriate molecule to initiate STPs. On the basis of analyses of plants and other species, receptor molecules are thought to include receptor-like kinases, two-component receptors, receptor tyrosine kinases, G-protein-coupled receptors, iontropic channel-related receptors, histidine kinases, and nuclear hormone receptors. Receptor molecules that have been identified to date in plants include: ROP10, a small G protein from the ROP family of Rho GTPases, that negatively regulates ABA response in Arabidopsis (Zheng et al. 2002); ATHK1, a putative homolog of the yeast SLN1, which is a functional histidine kinase feeding into the HOG MAPK pathway (Urao et al. 1999; Reiser et al. 2003); NtC7, a receptor-like membrane protein from tobacco (Tamura et al. 2003); and Cre1, a putative cytokinin sensor and histidine kinase from Arabidopsis (Reiser et al. 2003). The ERECTA gene from Arabidopsis is a putative leucine-rich repeat receptorlike kinase (LRR/RLK). It was the first gene to be shown to act on the coordination between transpiration and photosynthesis (Masle et al. 2005). ERECTA was analyzed by its transgenic expression in null-mutants and was shown to have roles in lowering stomatal conductance, controlling leaf photosynthesis and organogenesis, modulation of cell expansion, cell division, cell–cell contact, cell–cell and tissue–tissue signaling, cell proliferation, and inflorescence differentiation. Owing to the range of traits attributed to ERECTA expression, ERECTA is thought to act as a master gene in transpiration regulation (Masle et al. 2005). No known studies have yet involved transgenic expression of ERECTA in economic crops; however, initial studies suggest that this gene may be useful in the design of crops with improved transpiration efficiencies, reduced stomatal limitations, and increased yield potentials.

Phospholipid-Cleaving Enzymes PLEs degrade phospholipid membranes, catalyzing the release of lipid and lipidderived secondary messengers (Chapman 1998; Sang et al. 2001). Phospholipases C (PLC) and D (PLD) are both involved in ABA-mediated signal transduction and drought stress tolerance perception in plants. Phosphatidic acid (PtdOH), a product of the PLC and PLD pathways, is also important in the signaling process (Bartels et al. 2007; Wang et al. 2008a).

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Wang et al. (2008a) successfully produced maize plants constitutively overexpressing ZmPLC1, a phospholipase catalyzing the hydrolysis of 4,5-bisphosphate to form diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the products of which are the second messengers Ca2+ and PtdOH, respectively. This pathway is important in a wide variety of abiotic stress-responsive processes. Transgenic maize plants carrying the ZmPLC1 gene were shown to have increased photosynthetic activity, reduced anthesis to silking interval (ASI; an indicator of maize yield potential), better recovery, and higher grain yield than WT plants when subjected to 21 days of drought stress at the ten-leaf stage. Because there was no significant difference between stomatal conductances of WT and transgenic plants, the higher photosynthetic rate was attributed to better photochemical activity rather than the improved guard cell signaling. This has also been demonstrated in other studies (Staxen et al. 1999; Hunt et al. 2003; Mills et al. 2004). Sang et al. (2001) showed that overexpression of PLD results in enhanced sensitivity of transgenic tobacco and plays a key role in controlling stomatal movements and plant response to water stress.

Reactive Oxygen Species ROS are generated in plants as photoreaction and cellular oxidation byproducts under normal conditions and can cause cellular damage under water deficit when they accumulate to toxic levels. Some of these species also have important roles in early stress response through activation of cellular defense mechanisms and mitigation of cellular damage. While plant mechanisms must be in place to detoxify high levels of ROS that occur under drought, low levels of these beneficial ROS must also be maintained. Those ROS known to have important signaling roles in plant stress STPs include nitric oxide (NO) and hydrogen peroxide (H2O2).

Mitogen-Activated Protein Kinases MAPKs are enzymes that catalyze reversible phosphorylations, important for relaying signals. They function via cascades, which involve sequential phosphorylation of a kinase by its upstream kinase (Xiong and Ishitani 2006). Recently, the MKK2 pathway was identified in Arabidopsis as having involvement in cold and osmotic stress signal transduction. An example of a MAPK having specific involvement in drought and salt stress is the p44MMK4 kinase from alfalfa (Medicago sativa; Jonak et al. 1996). Phosphatases involved in the sequential phosphorylation of MAPKs and other protein kinases are also important for stress signaling. For example, the ABI1 and AB12 proteins from Arabidopsis have been shown to act in a negative regulatory feedback loop of the ABA signaling pathway (Merlot et al. 2001). Some MAPK and MAPKK proteins have also been shown to activate the Rd29a stress pathway in Arabidopsis (Hua et al. 2006). Other protein kinases involved in stress signaling include calcium-dependent protein kinases (CDPKs),

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kinases from the SNF1 family of protein kinases, and serine-threonine-type protein kinases (Xiong and Ishitani 2006; Bartels et al. 2007). Ca2+ Sensors Ca2+ sensors are important for coupling extracellular signaling to intercellular responses and comprise calmodulin (CaM) and CaM-related proteins (Sneddon and Fromm 1998; Sneddon and Fromm 2001), calcineurin B-like proteins (CBL; also known as SCaBP/SOS3-like calcium-binding proteins; Kudla et al. 1999), and CDPKs (Harmon et al. 2000). Ca2+ sensors that have been attributed with roles in drought tolerance in plants include the CBL1 gene (Kudla et al. 1999) and the AtCAMBP25 protein (Perruc et al. 2004) from Arabidopsis. 2.2.6.3

Stress-Responsive Mechanisms

The outcome of stress signal perception, transduction, and transcriptional up- or downregulation of genes is the production of molecules with various plant protection, repair, and stabilization functions. These molecules can be broadly grouped into five functional groups: (1) detoxification; (2) chaperoning; (3) late embryogenesis abundant (LEA) protein functions; (4) osmoprotection; and (5) water and ion movement. Detoxification To prevent stress injury, cellular ROS need to remain at nontoxic levels under drought stress. Antioxidants involved in plant strategies to degrade ROS include: (1) enzymes such as catalase, superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase; and (2) nonenzymes such as ascorbate, glutathione, carotenoids, and anthocyanins (Wang et al. 2003b). Some proteins, osmolytes, and amphiphilic molecules also have antioxidative functionality (Bowler et al. 1992; Noctor and Foyer 1998). Chaperoning Chaperone functions involve specific stress-associated proteins, which are responsible for protein synthesis, targeting, maturation and degradation, and function in protein and membrane stabilization, and protein renaturation. HSPs, which can be divided into five conserved families, have been shown to have particularly important stress-related chaperone functions in plants (Hendrick and Hartl 1993; Boston et al. 1996; Hartl 1996; Waters et al. 1996; Torok et al. 2001). HSPs, which are induced by heat, have been implicated in plant cell protection mechanisms under drought stress. Protein denaturation occurs under drought stress because decreased

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cellular volume increases the likelihood of degradative molecular interactions (Cho and Hong 2006). HSPs maintain or repair companion protein structure and target incorrectly aggregated and non-native proteins for degradation and removal from cells (Cho and Hong 2006). One such protein, NtHSP70-1, was constitutively overexpressed in tobacco to ascertain its role in plant drought response and tolerance (Cho and Hong 2006). The drought tolerance of transgenic seedlings was increased and their optimum water content was maintained after progressive drought stress (Cho and Hong 2006). Few other studies have involved transforming plants with HSPs; however, HSP24 from Trichederma harzianum was found to confer significantly higher resistance to salt, drought, and heat stress when constitutively expressed in Saccharomyces cerevisiae (Liming et al. 2008).

Late Embryogenesis Abundant Protein Functions LEA proteins are produced in response to dehydration stress and function in water status stabilization, protection of cytosolic structures, ion sequestration, protein renaturation, transport of nuclear targeted proteins, prevention of membrane leakage, and membrane and protein stabilization. LEA and LEA-type genes are found universally in plants. They accumulate in seeds during the late stages of embryogenesis and are associated with the acquisition of desiccation tolerance under drought, heat, cold, salt, and ABA stress (Sivamani et al. 2000; Bartels et al. 2007). They are also present in the biomass tissue of resurrection plants and are upregulated in many desiccation-sensitive plants in response to drought stress (Bartels et al. 2007). LEA proteins are divided into groups based on conserved sequence motifs (Zhang et al. 2000; Wise 2003). Five of these groups have been characterized at the molecular and structural level (Table 2.1); however, recent research indicates that additional groups of LEA and LEA-like proteins are still being identified (Park et al. 2003; Wang et al. 2006; March et al. 2007). Common Table 2.1 The five groups of LEA proteins LEA group Description Group 1 Contain a 20-amino acid motif and are represented by the wheat Em protein, for which gene homologs have been identified in a wide range of plant species Group 2 The most extensively studied group. They contain a lysine-rich 15-amino acid (dehydrins) motif (K-segment; EKKGIMDKIKEKLPG), which is predicted to form an amphipathic a-helix, a tract of contiguous serine residues and a conserved motif containing the consensus sequence DEYGNP in the N-terminal section of the protein Group 3 Contain a characteristic repeat motif of 11 amino acids, which have been predicted to form an amphipathic a-helix with possibilities for intra- and inter-molecular interactions Group 4 Have a conserved N-terminus, which is proposed to form a-helices and a diverse C-terminal region with a random coil structure Group 5 Contain more hydrophobic residues than the other groups, are insoluble after boiling, and are likely to adopt a globular structure Source: Bartels and Salamini (2001), Ramanjulu and Bartels (2002)

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features of LEA proteins generally include hydrophilicity (Garay-Arroyo et al. 2000; Park et al. 2003), heat stability (Close and Gallagher-Ludeman 1989; Ceccardi et al. 1994; Houde et al. 1995; Thomashow 1998, 1999), and transcriptionally regulated and ABA-responsive gene expression (Close and Gallagher-Ludeman 1989). It is generally assumed that they play a role in water-deficit tolerance and the possible functions of LEA proteins include binding and replacement of water (Dure 1993), ion sequestration (Bray 1993), maintenance of protein and membrane structure (Baker et al. 1988), molecular chaperones (Close 1996), membrane stabilization (Koag et al. 2003), and nuclear transport of specific molecules (Goday et al. 1994). One class of LEAs, the dehydrins, which have detergent and chaperone-like properties, stabilize membranes, proteins, and cellular compartments (Close 1996). LEA genes have been manipulated in many plants in order to increase drought resistance. For example, a wheat dehydrin, DHN-5, was ectopically overexpressed in Arabidopsis and transgenic plants displayed superior growth, seed germination rate, water retention, ion accumulation, more negative water potential, and higher proline contents than WT plants under salt and/or drought stress (Brini et al. 2007a). The barley (Hordeum vulgare L.) group 3 LEA gene, HVA1 was constitutively overexpressed in rice plants to increase drought tolerance. Transgenic plants displayed significantly increased tolerance to water deficit and salinity, which was associated with higher growth rates, delayed onset of stress damage symptoms, and improved recovery following stress removal (Xu et al. 1996). A more recent study involving the overexpression of this gene in rice showed that transgenic plants had significantly higher relative water content (RWC), improved turgor, less reduction in shoot and root growth, and improved cell membrane stability under prolonged drought conditions. It was found that HVA1 did not function as an osmolyte and that membrane protection was the mechanism, which inferred drought resistance in rice plants (Chandra Babu et al. 2004). HVA1 was also expressed in Basmati rice under control of either a constitutive rice promoter or a stress-inducible promoter. Transgenic plants exhibited increased stress tolerance in terms of cell integrity and growth, and it was found that inducible expression of HVA1 resulted in transgenic plants that were able to grow normally under nonstress conditions (Rohila et al. 2002). Transgenic wheat plants expressing HVA1 displayed more root fresh and dry weights, and shoot dry weight than WT plants under water-deficit conditions (Sivamani et al. 2000). Similarly, HVA1 overexpressing transgenic mulberry, Morus indica, exhibited improved cellular membrane stability, photosynthetic yield, less photo-oxidative damage, and superior WUE than WT plants under salt and drought stress (Lal et al. 2008). The discussion of expression of HVA1 in mulberry will be continued in Sect. 2.3.5. Other group 3 LEA genes that have been manipulated in order to improve plant drought tolerance include a Brassica napus group 3 LEA gene, which conferred improved salt and drought tolerance when constitutively expressed in Chinese cabbage (Park et al. 2005a), and TaLEA3 from wheat, which increased RWC, leaf water potential, and relative average growth rate of transgenic plants compared to WT plants under drought stress when constitutively overexpressed in the perennial grass Leymus chinensis (Wang et al. 2008b). Two group 4 LEA proteins,

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BhLEA1 and BhLEA2 from the resurrection plant Boea hygrometrica, conferred improved drought tolerance in transgenic tobacco. This was associated with plant cell protection and increased membrane and protein stability during dehydration (Liu et al.). A novel LEA gene from Tamarix androssowii also conferred increased drought tolerance when expressed in transgenic tobacco (Wang et al. 2006).

Osmoprotection Osmoprotection involves the upregulation of compatible solutes (osmolytes) that function primarily to maintain cell turgor, but are also involved in antioxidation and chaperoning through direct stabilization of membranes and/or proteins (Yancey et al. 1982; Bohnert and Jensen 1996; Lee et al. 1997; Hare et al. 1998; McNeil et al. 1999; Diamant et al. 2001). Compatible solutes are low molecular weight, highly soluble compounds that are usually nontoxic at high cellular concentrations. The three major groups of compatible solutes are amino acids (such as proline), quaternary amines (glycine betaine (GlyBet), polyamines, and dimethylsulfonioproprionate), and polyol/sugars (such as mannitol, galactinol, and trehalose; Wang et al. 2003b). Many genes involved in the synthesis of these osmoprotectants have been explored for their potential in engineering plant abiotic stress tolerance (Vinocur and Altman 2005). GlyBet and trehalose act as osmoprotectants by stabilizing quaternary structures of proteins and highly ordered states of membranes. Mannitol serves as a freeradical scavenger. Proline serves as a storage sink for carbon and nitrogen and a free-radical scavenger. It also stabilizes subcellular structures (membranes and proteins), and buffers cellular redox potential under stress. Many crops lack the ability to synthesize the special osmoprotectants that are naturally accumulated by stress tolerant organisms. It is believed that osmoregulation would be the best strategy for abiotic stress tolerance, especially if osmoregulatory genes could be triggered in response to drought, salinity, and high temperature. Therefore, a widely adopted strategy to develop stress-tolerant crops has been to engineer or overexpress certain osmolytes in plants (Bhatnagar-Mathur et al. 2008). GlyBet is a compatible solute that has been extensively studied for its role in drought stress response and increasing the levels of GlyBet in plants via genetic engineering has enhanced the drought tolerance of many model plants (Sakamoto and Alia 1998; Sakamoto and Murata 2000; Mohanty et al. 2002). A two-step enzymatic process accomplishes production of GlyBet in plants. The first step involves conversion of choline to betaine aldehyde by choline monoxygenase (CMO), a stromal enzyme with a Rieske-type (2Fe-2S) center (Brouquisse et al. 1989), and the second step involves betaine aldehyde dehydrogenase (BADH), a nuclear-encoded chloroplast stromal enzyme, which converts betaine aldehyde to GlyBet (Weigel et al. 1986). Quan et al. (2004) reported one of the first attempts to increase the GlyBet expression levels of maize by overexpressing the betA gene, which encodes choline dehydrogenase (CHO), another key enzyme in the choline–betaine aldehyde reaction (Zhang et al. 2008a). The study showed that

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transgenic maize plants were more drought tolerant than WT plants at three different life stages, including the ten-leaf-flowering stage, and also that yields of transgenic plants were less affected by drought stress than WT. Tobacco lacks GlyBet; however, it possess some BADH activity and the transfer of CMO is, therefore, a means of installing the GlyBet pathway in tobacco. Furthermore, because conversion of choline to GlyBet occurs in the chloroplast, it is also possible to use chloroplast genetic engineering to transfer CMO into GlyBet non-accumulators (Zhang et al. 2008a). Zhang et al. (2008a) transformed tobacco with a gene for CMO from beetroot via chloroplast genetic engineering and found that the transgenic plants accumulated GlyBet in leaves, roots, and seeds, and exhibited improved tolerance to toxic choline levels and salt and drought stress. GlyBet accumulation in the chloroplasts may be more effective than in other organelles, such as the nucleus, for abiotic stress protection because of protection and stabilization of chloroplast proteins, membrane, and photosynthesis systems under stress conditions (Zhang et al. 2008a). Lv et al. (2007) found that transgenic cotton plants constitutively overexpressing betA had increased RWCs, increased photosynthesis, better osmotic adjustment, decreased percentage of ion leakage, decreased lipid membrane peroxidation, and increased yield in response to drought stress at the seedling, squaring, and anthesis stages.

Water and Ion Movement Water and ions move through plants via transcellular and intracellular pathways. Aquaporins (major intrinsic proteins; MIPs), which are either tonoplast- (TIP) or plasma membrane- (PIP) localized, facilitate water, glycerol, small molecule, and gas transfer through membranes and, therefore, have a role in water homeostasis (Bartels et al. 2007). Active transport of solutes into the cell and cellular organelles, particularly the vacuole, is another means of cell turgor maintenance as increased solute potential facilitates the passive movement of water into cells and cellular compartments (Li et al. 2008). Successful attempts made in engineering plants expressing genes for enzymes involved in proton pumps that generate energy for tonoplast transport of solutes into vacuoles include the overexpression of the Arabidopsis H+-pyrophosphatase (H+-PPase; AVP1) in Arabidopsis (Gaxiola et al. 2001), upregulation of AVP1 in tomato (Park et al. 2005c), heterologous expression of the Thellungiella halophila vacuolar-H+-PPase (V-H+-PPase; TsVP) in tobacco (Gao et al. 2006), and overexpression of the wheat Na–H+ antiporter, TNHX1, and H+-PPase, TVP1, in Arabidopsis (Brini et al. 2007b; Li et al. 2008). In all the cases, the transgenic plants displayed superior drought and/or salinity resistance compared with WT plants with resistance being attributed to mechanisms such as increased vacuolar H+ to drive secondary uptake of ions into the vacuole and more enhanced development and robustness of root systems (Gaxiola et al. 2001; Li et al. 2005a; Park et al. 2005c; Gao et al. 2006; Brini et al. 2007a, b). Recently, Li et al. (2008) reported that

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heterologous expression of the potassium-dependent TsVP gene from the halophyte T. halophyta in maize under the control of the maize ubiquitin promoter could infer drought tolerance. Under drought stress, transgenic plants had a higher percentage of seed germination, better-developed root systems, more biomass, increased solute accumulation, less cell membrane damage, less growth retardation, shorter ASI, and much higher grain yields than WT plants. Attempts have also been made to improve drought tolerance of plants by altering the expression of aquaporins (Aharon et al. 2003; Porcel et al. 2005; Yu et al. 2005; Peng et al. 2006; Jang et al. 2007; Cui et al. 2008; Miyazawa et al. 2008; Zhang et al. 2008c). Aquaporins facilitate transport of water and other small solutes and ions across membranes via the apoplastic route (Aharon et al. 2003; Cui et al. 2008; Jang et al. 2007; Peng et al. 2006; Zhang et al. 2008c). Research into the role of aquaporins in plant drought tolerance has shown that various aquaporins function differently depending on the severity and type of stress. For example, some aquaporins, such as the Arabidopsis Rd28, and rice RWC3, are upregulated under drought stress and others, such as NtQP1 and AtPIP1, remain unchanged under drought stress (Cui et al. 2008). Additionally, some aquaporins genes, such as AtPIP1b have been shown to diminish the drought tolerance capability of some plants, while others, such as the Vicia faba PIP1, Panax ginseng PgTIP1, Brassica napus BnPIP1, and Brassica juncea BjPIP1, have been shown to improve drought tolerance (Aharon et al. 2003; Yu et al. 2005; Peng et al. 2006; Cui et al. 2008; Zhang et al. 2008c). There is also evidence that overexpression of aquaporins in some plants causes them to respond differently to different stresses. For example, Jang et al. (2007) found that Arabidopsis and tobacco plants overexpressing Arabiopsis PIP’s displayed enhanced water flow and improved germination under cold stress, but exhibited rapid water loss, retarded seedling growth, and inferior germination under drought conditions. It is therefore thought that different aquaporin isoforms are associated with different physiological processes and that plants respond to drought conditions either by increasing aquaporin expression, which facilitates water movement (especially into the tonoplast in order to maintain cell-turgor) or downregulating aquaporin expression to avoid excessive water loss (Aharon et al. 2003; Peng et al. 2006). Overexpression of aquaporins has also been implicated in conferring heavy metal tolerance to transgenic plants by alleviation of metal ion-induced water deficit and oxidative damage caused by metal ions (Zhang et al. 2008c).

2.3 2.3.1

Engineering Salt Tolerance in Plants Impacts of Salinity on Agricultural Production

The damaging effects of salt accumulation in agricultural soils have severely affected agricultural productivity in large swathes of arable land throughout the world. Salt-affected land accounts for more than 6% of the world’s total land area

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(FAO 2009c) and is distributed largely amongst coastal salt marshes or inland desert sands. These have primarily arisen naturally through mineral weathering (which leads to the release of soluble salts such as chlorides of calcium, magnesium and sodium, and, to a lesser extent, sulfates and carbonates) or wind and rain deposition of oceanic water (Szabolcs 1989; Munns and Tester 2008; FAO 2009b). Secondary salinization occurs when irrigation and tree clearing of agricultural land cause water tables to rise and concentrate salts in the root zone (Rengasamy 2006). Approximately 20% of the world’s irrigated land, from which one-third of the world’s food supply is produced, is presently affected by salinity (Ghassemi et al. 1995). With the expected increase in world population, the loss of arable land due to salinity presents a serious challenge to food sustainability and productivity. Removal of salts from the root zone (reclamation) is perhaps the most effective way to ameliorate the detrimental effects of salinity; however, this is a slow and expensive process. The use of plant breeding and genetic engineering technologies to alter the salt tolerance of crops will, therefore, play an important role in maintaining global food production in the future.

2.3.2

Improving Salinity Tolerance of Agricultural Crops

Plants have evolved a complex adaptive capacity to perceive and respond to salt stress. The existence of salt-tolerant flora (halophytes) and differences in salt tolerance between genotypes within the salt-sensitive plant species (glycophytes) give rise to the belief that salt tolerance has a genetic basis (Yamaguchi and Blumwald 2005). As for drought, efforts to improve the salt tolerance of crops have met with limited success because of the physiological and genetic complexity of the trait. Salinity tolerance is a multi-genic trait, with quantitative trait loci (QTL) identified in barley, wheat, soybean, citrus, rice, and tomato (Flowers and Flowers 2005; Jenks et al. 2007). Genetic approaches currently being used to improve salinity tolerance include the exploitation of functional genomics, bioinformatics, and natural genetic variations, either through direct selection in stressful environments or through the mapping of QTLs and subsequent marker-assisted selection (Yamaguchi and Blumwald 2005), or the generation of transgenic plants (Vij and Tyagi 2007).

2.3.3

Physiological Effects of Salinity on Plants and Salinity Tolerance Mechanisms

Salinity imposes a variety of stresses on plant tissues. Two of these are osmotic stress, which results from the relatively high soil solute concentrations, and ion

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cytotoxicity. The decreased rate of leaf growth that occurs after an increase in soil salinity is primarily due to the osmotic effect of the salt around the roots, which inhibits plant water uptake and causes leaf cells to lose water. However, this loss of cell volume and turgor is transient and reductions in cell elongation and also cell division lead to slower leaf appearance and smaller final size over the longer term (Bartels and Sunkar 2005; Munns and Tester 2008). Under prolonged salinity stress, inhibition of lateral shoot development becomes apparent within weeks and, within months, there are effects on reproductive development, such as early flowering and reduced floret number. Concomitantly, older leaves may die while the production of younger leaves continues. The cellular and metabolic processes involved are similar to those occurring in drought-affected plants and are responses to the osmotic effect of salt (Yeo et al. 1991; Munns and Tester 2008). Ion cytotoxicity occurs when salt accumulates to toxic concentrations in fully expanded leaves (which, unlike younger leaves, are unable to dilute high salt concentrations), causing leaf death. Replacement of K+ by Na+ in biochemical reactions leads to conformational changes and loss of protein function, as Na+ and Cl– ions penetrate hydration shells and interfere with noncovalent interactions between amino acids. If the rate of leaf death generated by ion cytotoxicity is greater than the rate at which new leaves are produced, the photosynthetic capacity of the plant will no longer be able to supply the carbohydrate requirement of young leaves, which further reduces their growth rate (Munns and Tester 2008). Halophytes, though taxonomically widespread, are relatively rare amongst the flowering plants and virtually all crop plants are glycophytes (Flowers and Flowers 2005). However, there is considerable variability in the tolerance of glycophytes to salt. Munns and Tester (2008), categorize salinity tolerance under three broad categories: (1) Tolerance to osmotic stress, which immediately reduces cell expansion in root tips and young leaves, and causes stomatal closure; (2) Na+ exclusion from leaf blades, which ensures that Na+ remains at nontoxic concentrations within leaves; and (3) Tissue tolerance to Na+ or Cl–, which requires compartmentalization of Na+ and Cl– at the cellular and intracellular level to avoid accumulation of toxic concentrations within the cytoplasm.

2.3.4

Salt Tolerance Using Transgenic Approaches

2.3.4.1

Osmoprotectants

Osmoprotectants were discussed previously (Sect. 2.2.6.3.4) in relation to their use in developing drought-tolerant crops and the transfer of GlyBet intermediates have improved the drought and salt tolerance of transgenic plants in many cases. Mohanty et al. (2002) demonstrated that Agrobacterium-mediated transformation of an elite indica rice cultivar to increase GlyBet synthesis through the incorporation of the codA gene, which encodes choline oxidase, was an effective way to

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improve salinity tolerance. Challenge studies performed with R1 plants by exposure to salt stress for one week, followed by a recovery period, revealed that in some cases more than 50% of the transgenic plants could survive salt stress and set seed whereas WT plants failed to recover. A more recent example of enhanced GlyBet synthesis experiments involved transformation of maize with the BADH gene, introduced by the pollen-tube pathway (Wu et al. 2008a). Transgenic lines were examined for tolerance to NaCl by induced salt stress and, after 15 days of treatment, most transgenic seedlings survived and grew well, whereas WT seedlings wilted and showed loss of chlorophyll. The amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental stresses (Kavi Kishore et al. 2005; Ashraf and Foolad 2007). In plants, the precursor for proline biosynthesis is l-glutamic acid. Two enzymes, pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), play major roles in the proline biosynthetic pathway (Delauney and Verma 1993; Ashraf and Foolad 2007). Su and Wu (2004) established that the rate of growth of transgenic rice plants expressing mothbean D1-pyrroline-5-carboxylate synthetase (p5cs) cDNA under either a constitutive or stress-inducible promoter led to the accumulation of p5cs mRNA and proline in third-generation (R2) transgenic rice seedlings. Significantly higher salinity and water-deficit stress tolerance of R2 seedlings were attributed to faster growth of shoots and roots in comparison with non-transformed plants. Stressinducible expression of the p5cs transgene showed significant advantages over constitutive expression in increasing the biomass production of transgenic rice grown in soil under stress conditions. The osmoprotectant role of proline has been verified in other plants such as potato, where salt tolerance, measured by comparing tuber yield of transgenic lines cultivated in a greenhouse and watered with saline water to that of plants watered with normal tap water, had a less significant effect on tuber yield of transgenic plants than WT (Hmida-Sayari et al. 2005). Polyamines, including spermidine (Spd, a triamine), spermine (Spm, a tetramine), and their obligate precursor putrescine (Put, a diamine), are aliphatic amines widely present in living organisms. The polyamine biosynthetic pathway is depicted in Fig. 2.3. Recently, it has been demonstrated that plant polyamines are involved in the acquisition of tolerance to such stresses as high and low temperatures, salinity, hyperosmosis, hypoxia, and atmospheric pollutants (Liu et al. 2007). Furthermore, genetic transformation of several plant species with polyamine biosynthetic genes encoding arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), or Spd synthase (SPDS) led to improved environmental stress tolerance (Liu et al. 2007). He et al. (2008) tested transgenic apple engineered with (SPDS)-overexpressing transgenic European pear (Pyrus communis L. “Ballad”) for changes in enzymatic and nonenzymatic antioxidant capacity in response to NaCl or mannitol stress. Their research revealed that transgenic plants accumulated more Spd than WT. The transgenic line contained higher antioxidant enzyme activities (less malondialdehyde and H2O2) than the WT, implying that it suffered from less injury and enhanced enzymatic and nonenzymatic antioxidant capacity (He et al. 2008).

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N

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Fig. 2.3 Polyamine biosynthesis

Mannitol is a primary photosynthetic product that is associated with exceptional salt tolerance. In celery, mannitol metabolism is clearly altered by salt stress, with several lines of evidence indicating a connection between mannitol and salt tolerance, including increases in the capacity for mannitol biosynthesis and accumulation and decreases in catabolism (Williamson et al. 2002). Sickler et al. (2007) showed that Arabidopsis plants transformed with celery’s mannose6-phosphate reductase (M6PR) gene produced mannitol and grew normally in the absence of stress. However, in the presence of salt stress, daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant, and specific leaf weight) over a 12-day period showed less effect of salt on transformants than WT plants. The daily energy use efficiency for photochemistry by photosystem 2 (PSII) was also measured and demonstrated that, unlike transformed plants, which were not affected, WT plants treated with 100 mM NaCl displayed a reduction in PSII yield after 6 days with a 50% loss in yield after 12 days. Similarly, under atmospheric levels of CO2, growth with 200 mM NaCl caused an increase in sub-stomatal levels of CO2 in WT plants but not in transformants. Trehalose is a disaccharide sugar widely distributed in bacteria, fungi, insects, plants and invertebrate animals. In microbes and yeast, trehalose is produced from glucose by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), and functions in sugar storage, metabolic regulation, and protection against abiotic stress (Strom and Kaasen 1993; Wiemken 1990). Trehalose acts as a compatible solute, protecting membranes and proteins and conferring desiccation

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tolerance on cells in the absence of water (Crowe et al. 1984). Ge et al. (2008) illustrated the protective role of trehalose in higher plants. Expression analysis demonstrated that OsTPP1 isolated and cloned from rice, was initiated and transiently upregulated after salt, osmotic, and ABA treatments but slowly upregulated under cold stress. OsTPP1 overexpression in rice enhanced salt and cold stress tolerance. Tolerance of transgenic plants to abiotic stress was examined by observing 2-week-old seedlings exposed to salt. Following one week of exposure, seedlings exhibited salt-induced damage symptoms such as wilted leaves. However, after prolonged salt treatment, transgenic lines were more vigorous and displayed increased leaf greenness and viability over control plants. Generally, two broad themes have emerged from the results of attempts to engineer overexpression of osmoprotectants. The first is that metabolic limitations have been encountered in generating absolute levels of target osmolytes, especially when compared with salt-tolerant halophytes and the second is that the degree to which transformed plants are able to tolerate salinity stress is not necessarily correlative with the levels of osmoprotectants attained.

2.3.4.2

Transporter Genes

Mechanisms that confer salt tolerance vary with the plant species; however, the ability to maintain low cytosolic Na+ is thought to be one of the key determinants of plant salt tolerance (Tester and Davenport 2003). Salt “inclusion” and “exclusion” are recognized as different mechanisms by which higher plants tolerate salinity. The functional removal of Na+ from the cytoplasm of plant cells and the maintenance of low cytosolic Na+ concentrations under salinity conditions (Blumwald et al. 2000) is accomplished by either pumping Na+ out of cells (plasma membrane antiporter) or into vacuoles (vacuolar antiporter) in exchange for H+. Na+/H+ antiporter activity is driven by the electrochemical gradient of protons (H+) generated by the H+ pumps (H+-ATPase) in the plasma membrane or the tonoplast (Chinnusamy and Zhu 2003; Tester and Davenport 2003). In Arabidopsis, active exclusion of Na+ is mediated by the plasma membrane-localized Na+/H+ antiporter, AtSOS1 (Shi et al. 2003). In contrast, the sequestration of excess Na+ into the vacuole is mediated by the vacuolar membrane-localized Na+/H+ antiporter, AtNHX1 (Gaxiola et al. 1999; Shi et al. 2008). In a similar way, overexpression of the S. cerevisiae HAL1 gene (Gaxiola et al. 1992) conferred salt tolerance in yeast by increasing intracellular K+ and decreasing Na+ levels. The successful use of transporter genes has been demonstrated in several plants. He et al. (2005) created transgenic cotton plants expressing AtNHX1 and found that transgenic plants generated more biomass and produced more fibers under salt stress in a greenhouse. It was suggested that the increased fiber yield was due to superior photosynthetic performance and higher nitrogen assimilation rates observed in the transgenic plants compared with WT. Interestingly, the researchers demonstrated that field-grown irrigated AtNHX1-expressing cotton plants produced higher fiber yields (fiber plus seeds) than WT, with an average increase of more

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than 25% per line. Furthermore, the fibers produced by transgenic plants were generally more uniform, stronger, and longer than those of WT. Similarly, Chen et al. (2007) engineered maize plants overexpressing the rice OsNHX1 gene. Transformants accumulated more biomass under greenhouse-based salt stress. Higher Na+ and K+ content was observed in transgenic leaves than in WT when treated with 100–200 mM NaCl, while the osmotic potential and the proline content in transgenic leaves was lower than in WT. Salt stress field trials revealed that the transgenic maize plants produced higher grain yields than WT plants at the vegetative growth stage. Biochemical studies suggest that Na+/H+ exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis by transporting Na+ ions out of the cell (Qiu et al. 2002). SOS1 encodes a plasma membrane Na+/H+ exchanger in Arabidopsis (Qiu et al. 2002) and the important role of the plasma membrane Na+/H+ exchangers for plant salt tolerance was supported by the finding that overexpression of SOS1 improved plant salt tolerance (Shi et al. 2003). Zhao et al. (2006) demonstrated that expressing the plasma membrane Na+/H+ antiporter SOD2 from yeast (Schizosaccharomyces pombe) in transgenic rice also increased salt tolerance. Transgenic plants accumulated more K+, Ca2+, and Mg2+ and less Na+ in their shoots compared with non-transformed controls. Moreover, measurements on isolated plasma membrane vesicles derived from the SOD2 transgenic rice plant roots showed that the vesicles had enhanced P-ATPase hydrolytic activity as well as being able to maintain higher levels of photosynthesis and root proton exportation capacity. Martinez-Atienza et al. (2007) identified an AtSOS1 homolog, OsSOS1, in rice, which demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (S. cerevisiae) cells and reduced their net cellular Na+ content. OsSOS1 was also shown to suppress the salt sensitivity of an sos1-1 mutant of Arabidopsis. In relation to the introduction of genes that modulate cation transport systems, many researchers have sought to employ the overexpression of the S. cerevisiae HAL1 gene, which has conferred salt tolerance in yeast by facilitating intracellular K+ accumulation and decreasing intracellular Na+ (Gaxiola et al. 1992; Rios et al. 1997). Rus et al. (2001) established ectopic expression of HAL1 in transgenic tomato plants, and showed that transformants were able to minimize the reduction in fruit production caused by salt stress. Maintenance of fruit production by transgenic plants was correlated with enhanced growth under salt stress of calli derived from the plants. The HAL1 transgene enhanced water and K+ contents in leaf calli and leaves in the presence of salt, which indicates that, similar to the yeast gene, plant HAL1 functions by facilitating K+/Na+ selectivity under salt stress. Ellul et al. (2003), utilizing an optimized Agrobacterium-mediated gene transfer protocol, developed HAL1-expressing watermelon (Citrullus lanatus). Salt tolerance of transgenic plants was confirmed in a semi-hydroponic system on the basis of the higher growth performance of transgenic lines compared to control plants. The halotolerance observed supports the potential usefulness of the HAL1 gene as a molecular tool for genetic engineering salt-stress protection in other crop species.

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Detoxifying Genes

The mechanisms of plant detoxification of ROS under drought stress were introduced in Sects. 2.2.6.2 and 2.2.6.2.3. As an antioxidant enzyme, glutathione peroxidase (GPX) reduces hydroperoxides in the presence of glutathione to protect cells from oxidative damage, including lipid peroxidation (Maiorino et al. 1995). Gaber et al. (2006) generated transgenic Arabidopsis plants overexpressing GPX2 genes in cytosol (AcGPX2) and chloroplasts (ApGPX2). The activities toward a-linolenic acid hydroperoxide in ApGPX2- and AcGPX2-expressing plants were 6.5–11.5 and 8.2–16.3 nmol min–1 mg protein–1, respectively, while no activity was detected in the WT plants. Both transgenic lines showed enhanced tolerance to oxidative damage caused by the treatment with H2O2, Fe ions, or methylviologen (MV) and environmental stress conditions, such as chilling with high light intensity, high salinity or drought. The degree of tolerance of the transgenic plants to all types of stress was correlated with the levels of lipid peroxide suppressed by the overexpression of the GPX-2 genes. SOD is the first enzyme in the enzymatic antioxidative pathway and halophytic plants, such as mangroves, reported to have a high level of SOD activity. SOD plays a major role in defending mangrove species against severe abiotic stresses. Prashanth et al. (2008) further characterized the Sod1 cDNA (a cDNA encoding a cytosolic copper/zinc SOD from the mangrove plant Avicennia marina) by transforming it into rice. Transgenic plants were more tolerant to MV-mediated oxidative stress in comparison to WT and withstood salinity stress of 150 mM of NaCl for a period of 8 days while WT plants wilted at the end of the hydroponic stress treatment. Pot-grown transgenic plants tolerated salinity stress better than the WT when irrigated with saline water. In plant cells, APXs are directly involved in catalyzing the reduction of H2O2 to water, which is facilitated by specific electron donation by ascorbic acid. APXs are ubiquitous in plant cells and are localized in chloroplasts (Takahiro et al. 1995), peroxisomes (Shi et al. 2001), and cytosol (Caldwell et al. 1998). Xu et al. (2008b) transformed Arabidopsis plants with a pAPX gene from barley (HvAPX1). The transgenic line was found to be more tolerant to salt stress than the WT. There were no significant differences in Na+, K+, Ca2+, and Mg2+ contents and the ratio of K+ to Na+ between pAPX3 and WT plants, which indicated that salt tolerance in transgenic plants was not due to the maintenance and re-establishment of cellular ion homeostasis. However, the degree of H2O2 and lipid peroxidation (measured as the levels of malondialdehyde) accumulation under salt stress was higher in the WT than in transgenic plants. The mechanism of salt tolerance in transgenic plants was explained by a reduction of oxidative stress injury. Apart from catalase and various peroxidases and peroxiredoxins (Dietz 2003), four enzymes, APX, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase (GR), are involved in the ascorbate-glutathione cycle, a pathway that allows the scavenging of superoxide radicals and H2O2 (Asada 1999). Most of the ascorbate-glutathione cycle enzymes are located in the stroma, cytosol, mitochondria, and peroxisomes (Jimenez et al. 1998). APX and GR, the first and

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last enzymes in this cycle, respectively, are responsible for H2O2 detoxification in green leaves (Foyer et al. 1994). GR has a central role in maintaining the reduced glutathione (GSH) pool during stress (Pastori et al. 2000). Lee and Jo (2004) introduced BcGR1, a Chinese cabbage gene that encodes cytosolic GR into tobacco plants via Agrobacterium-mediated transformation. Homozygous lines containing BcGR1 were generated and tested for their acquisition of increased tolerance to oxidative stress. When ten-day old transgenic tobacco seedlings were treated with 5 to 20 mM MV, they showed significantly increased tolerance compared to WT seedlings. The most drastic difference was observed at a concentration of 10 mM MV. In addition, when leaf discs were subjected to MV, the transgenic plants were less damaged than the WT with regard to their electrical conductivity and chlorophyll content.

2.3.5

Late Embryogenesis Abundant (LEA) Proteins

The LEA proteins were introduced in Sect. 2.2.6.3.3 in relation to their use in improving plant drought tolerance. These proteins have also been used in engineering salt-tolerant crops. Park et al. (2005b) introduced a B. napus LEA protein gene, ME-leaN4 (Wakui and Takahata 2002) into lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation. Transgenic lettuce demonstrated enhanced growth ability compared with WT plants under salt- and water-deficit stress. After 10-day growth under hydroponic 100 mM NaCl conditions, average plant length and fresh weight of transgenic lettuce were higher than those of WT and the increased tolerance was also reflected by delayed leaf wilting caused by water-deficit stress. Brini et al. (2007a) analyzed the effect of ectopic expression of dehydrin (Dhn-5; Table 2.1) cDNA in Arabidopsis under salt and osmotic stress. When compared to WT plants, the Dhn-5-expressing transgenic plants exhibited stronger growth under high concentrations of NaCl or water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in WT than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in WT plants and the transgenic lines had higher proline contents and lower water loss rates under water stress. Na+ and K+ also accumulated to a greater extent in the leaves of the transgenic plants. Lal et al. (2008) reported the effects of overexpression of the HVA1 gene in mulberry under a constitutive promoter. HVA1 is a group 3 LEA (Table 2.1) isolated from barley aleurone layers and has been found to be inducible by ABA. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of HVA1 in conferring tolerance. Using leaf discs as explants, growth performance under salt-stress and water-deficit conditions were carried out from 8- to 10-month-old transgenic plants. Leaf discs of uniform size were cut and used for simulated salt and water-deficit stress treatments for different durations. After the stress treatments, leaf discs were analyzed for proline content,

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photosynthetic yield, RWC, and cellular membrane stability (CMS). Transgenic plants showed better CMS, photosynthetic yield, less photooxidative damage, and better WUE as compared with the nontransgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants showed a manyfold increase in proline concentration over WT plants and, under water-deficit conditions, proline accumulated only in the nontransgenic plants. Results also indicated that the production of HVA1 proteins enhanced the performance of transgenic mulberry by protecting plasma and chloroplast membrane stability under abiotic stress.

2.3.6

Transcription Factors

The importance of TFs in plant stress response was discussed in Sect. 2.2.5. In addition to binding to cis-acting elements in the promoters of environmental stressresponsive genes, TFs can activate and repress gene expression through interactions with other TFs, thus playing a central role in plant response to environmental stresses (Chen and Zhu 2004). It is generally accepted that activation or ectopic expression of a specific TF can result in expression of many functional genes related to stresses. CBF/DREBs are key regulatory factors that function primarily in freezing tolerance by activating a network of target genes (Fowler and Thomashow 2002; Maruyama et al. 2004). Oh et al. (2007) isolated a barley gene, HvCBF4, whose expression is induced by low temperature stress. Transgenic overexpression of HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity, and low temperature stresses without stunting growth. Interestingly, under low temperature conditions, the maximum photochemical efficiency of PSII in the dark-adapted state in HvCBF4 plants was higher by 20 and 10% than that in nontransgenic and CBF3/DREB1A-expressing plants, respectively. Using the 60K Rice Whole Genome microarray, 15 rice genes were identified that were activated by HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, 5 genes were common to both HvCBF4 and CBF3/DREB1A, and 10 and 7 genes were specific to HvCBF4 and CBF3/DREB1A, respectively. Results suggested that CBF/ DREBs of barley acted differently from those of Arabidopsis in transgenic rice. The NAC family of TFs (Sect. 2.2.6.1) has applicability for generating salttolerant crops. Hu et al. (2008) characterized a stress-responsive NAC gene (SNAC2) isolated from upland rice for its role in stress tolerance. Northern blot and SNAC2 promoter activity analyses demonstrated that SNAC2 expression was induced by drought, salinity, cold, wounding and ABA treatment. The SNAC2 gene was overexpressed in japonica rice to test the effect on improving stress tolerance. To test salinity tolerance, germinated positive transgenic and WT seeds were transplanted on Murashige and Skoog (MS) medium containing 150 mM NaCl and the normal MS medium without NaCl as a control. Under saline conditions, transgenic seedlings grew faster and their shoots were significantly longer than WT after 20 days. However, there was no difference in root length or root numbers

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between transgenic and WT seedlings grown under saline conditions and no difference in growth performance was observed between transgenic and WT seedlings in the normal MS medium. Hu et al. (2008) also evaluated germination ability of transgenic lines harboring SNAC2 under salt-stress conditions. After 4 days of germination on the medium containing 150 mM NaCl, only 40% of WT seeds were poorly germinated, whereas more than 70% of transgenic seeds germinated efficiently. In the MS medium, more than 90% of both transgenic and WT seeds germinated well and there was no significant difference in germination rates, suggesting that overexpression of SNAC2 does not affect seed germination under normal conditions. The significantly higher germination rate of transgenic seeds than that of WT under saline conditions further supported the improved salt tolerance of SNAC2-overexpressing plants. DNA chip profiling analysis of the transgenic plants revealed many upregulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, HSP, GDSL-like lipase, and phenylalanine ammonia lyase. This data suggests that SNAC2 is a novel stress-responsive NAC TF that possesses potential utility in improving stress tolerance of rice. The TFIIIA-type zinc-finger proteins, first discovered in Xenopus, represent an important class of eukaryotic TFs (Miller et al. 1985). More than 40 TFIIIA-type zinc-finger protein genes have been identified from various plants, including petunia, soybean, Arabidopsis, and rice (Kim et al. 2001; Sugano et al. 2003; Mittler et al. 2006; Huang and Zhang 2007) and these genes have been shown to be induced by various abiotic stresses. Xu et al. (2008a) have recently reported the functional analysis of ZFP252 (a salt and drought stress responsive TFIIIA-type zinc-finger protein gene from rice), using gain- and loss-of-function strategies. They discovered that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes, and enhanced rice tolerance to salt and drought stresses compared with ZFP252 antisense and nontransgenic plants. Their findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced drought and salt tolerance (Xu et al. 2008a).

2.3.6.1

Signal Transduction Genes

Plant salt-stress-response genes are involved in many plant cellular processes, including physiological metabolism, cell defense, energy production and transportation, ion transfer and balance, and cell growth and division. These genes function through certain coordination mechanisms to maintain the normal growth of plants under salt stress. As discussed previously, components of the STP may also be shared by various stress factors such as drought, salt, and cold (Shinozaki and Yamaguchi-Shinozaki 1999). Signal molecules, H2O2 and NO, are involved in the ABA-induced stomatal closure and gene expression and activities of antioxidant enzymes (Zhang et al. 2006, 2007a). ABA-induced H2O2 production mediates NO generation, which in

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turn activates MAPK and results in upregulation of the expression and activities of antioxidant enzymes (Zhang et al. 2007a). The importance of ABA in plant environmental stress responses was discussed in Sect. 2.2.6.1. The oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycartenoid dioxygenase (NCED) is the key regulatory step of ABA biosynthesis in higher plants. Overexpression of SgNCED1 in transgenic tobacco plants resulted in 51–77% more accumulation of ABA in leaves (Zhang et al. 2008d). Transgenic tobacco plants were shown to have decreased stomatal conductance and transpiration and photosynthetic rates and increased activities of SOD, catalase, and APX activities. H2O2 and NO in leaves were also induced in the transgenic plants. Compared with WT, the transgenic plants displayed improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl induced salinity stress. It was suggested that the ABA-induced H2O2 and NO generation upregulates stomatal closure and antioxidant enzymes and, therefore, increases drought and salinity tolerance in transgenic plants. Salt stress is known to trigger a rapid and transient increase of free calcium concentration in plant cells (Knight 2000; Pauly et al. 2000). As such, Ca2+ signaling processes are one of the earliest events in salt signaling and may play an essential role in the ion homeostasis and salt tolerance in plants ( Zhu 2003; Reddy and Reddy 2004). CBLs represent a unique family of calcium sensors in plants and function as a positive regulator in the salt-stress-response pathway. Extensive studies have progressed toward understanding of Arabidopsis CBLs, yet knowledge of their functions in other plant species is still quite limited. Wang et al. (2007a) have reported the cloning and functional characterization of ZmCBL4, a novel CBL gene from maize. ZmCBL4 encodes a putative homolog of the Arabidopsis CBL4/SOS3 protein. Under normal conditions, ZmCBL4 was shown to be expressed differentially at a low level in various organs of maize plants and its expression was regulated by NaCl, LiCl, ABA and PEG treatments. Expression of 35S::ZmCBL4 not only complemented the salt hypersensitivity in an Arabidopsis sos3 mutant, but also enhanced the salt tolerance in Arabidopsis WT plants at the germination and seedling stages. ZmCBL4-expressing Arabidopsis lines accumulated less Na+ and Li+ as compared with WT plants. Wang et al. (2007a) concluded that the maize CBL gene functions in salt-stress-elicited calcium signaling and thus in maize salinity tolerance.

2.4 2.4.1

Engineering Cold Tolerance in Plants Impacts of Cold Stress on Agricultural Production

Agricultural borders for crop species are defined geographically by occurrences of low temperatures and frost, which cause severe yield losses in marginal areas. Approximately two-thirds of the world’s landmass is annually subjected to temperatures below freezing and half to temperatures below –20 C.

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Most crops of tropical origin, as well as many of subtropical origin, are sensitive to chilling temperatures. Amongst the major world food crops, maize and rice are sensitive to chilling temperatures and yield loss or crop failure of these species can occur at temperatures below 10 C. Many other crops, such as soybean, cotton, tomato, and banana, are injured at temperatures below 10–15 C (Lynch 1990). The temperature below which chill injury can occur varies with species and regions of origin and ranges from 0 to 4 C for temperate fruits, 8 C for subtropical fruits, and about 12 C for tropical fruits (Lyons 1973). Cold acclimation, also known as cold hardening, describes an increase in tolerance over time to cold temperatures and cellular desiccation in response to conditions such as cold temperature, short photoperiods, and mild drought and results from changes in gene expression and physiology (Xin and Browse 2000; Kalberer et al. 2006). Most temperate plants can cold-acclimate and acquire tolerance to extracellular ice formation in their vegetative tissues. Winter-habit plants such as winter wheat, barley, oat, rye, and oilseed rape have a vernalization requirement, which allows them to survive freezing stress as seedlings during winter. However, after vernalization and at the end of the vegetative phase, the cold acclimation ability of winter cereals gradually decreases, making them sensitive to freezing injuries (Fowler et al. 1996; Chinnusamy et al. 2007). Therefore, it is not surprising that the impacts of cold stress on plant life have been comprehensively studied. Many attempts have been made to improve cold resistance of important crop plants; however, progress in achieving frost hardiness of plants either by classical breeding or by gene transfer is difficult because of the fact that cold resistance is not a quality conferred by the product of one gene, but, as for most abiotic stress tolerance mechanisms, is quantitative in nature (Mahajan and Tuteja 2005).

2.4.2

Physiological Effects of Cold Stress on Plants and Cold Tolerance Mechanisms

The symptoms of chilling-induced stress injury in cold-sensitive plants are variable and generally manifest within 48 to 72 h of stress exposure. Observed phenotypic symptoms in response to chilling stress include reduced leaf expansion, wilting, chlorosis, and necrosis (Mahajan and Tuteja 2005). Chilling also severely inhibits plant reproductive development, with species such as rice displaying sterility when exposed to chilling temperatures during anthesis (Jiang et al. 2002). The extent of plant damage caused by exposure to low temperature depends on factors such as the developmental stage, the duration and severity of the frost, the rates of cooling (and rewarming), and whether ice formation takes place intra- or extra-cellularly (Beck et al. 2004). Chilling stress (<20 C) is a direct result of low temperature effects on cellular macromolecules, which leads to slowing of metabolism, solidification of cell

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membranes, and loss of membrane functions. This kind of damage results primarily from loss of function of biomembranes associated with decreased fluidity and inactivation or deceleration of membrane-bound ion pumps. Absorbance of light energy, which occurs independently of temperature, results in oxidative stress if metabolism cannot keep pace with the excitation of the photosynthetic components. Freezing stress (<0 C), which causes extracellular ice-crystal formation, freeze-induced dehydration, and concentration of cell sap, has major mechanical impacts on cell walls and plasma membranes and leads to cell rupture (Margesin et al. 2007). Generally, freezing results in loss of membrane integrity and solute leakage. The integrity of intracellular organelles is also disrupted under freezing stress and leads to loss of compartmentalization and reduction and impaired photosynthesis, protein assembly, and general metabolic processes. The primary environmental factors responsible for triggering increased tolerance against freezing are collectively known as “cold acclimation” (Mahajan and Tuteja 2005). Frost resistance can be achieved by two main mechanisms: (1) avoidance of ice formation in tissues; or (2) tolerance of apoplastic extracellular ice. An individual plant may employ both mechanisms of frost resistance in different tissues (Sakai and Larcher 1987; Margesin et al. 2007). A key function of cold acclimation is to stabilize membranes against freezing injury through mechanisms such as adjustment of lipid composition and accumulation of protective sugars, hydrophilic and LEA proteins, and antioxidants (Thomashow 1999).

2.4.3

Cold Tolerance Using Transgenic Approaches

2.4.3.1

ROS Detoxifying Substances

The mechanisms of ROS accumulation and detoxification were discussed in Sects. 2.2.6.2 and 2.3.4.3. The decrease in the amount of unsaturated fatty acids during lipid peroxidation elevates membrane viscosity, promotes lipid transition from a liquid crystalline phase to a gel phase, raises proton permeability of membranes, diminishes membrane electric conductance, and causes inactivation of membrane-localized enzymes. The adaptation of plant cells to low temperature is based on their ability to maintain saturation of fatty acids in membrane lipids, thus modifying membrane fluidity (Szalontai et al. 2003). Demin et al. (2008) examined the role of D12-acyl-lipid desaturase in plant resistance to hypothermia-induced oxidative stress. The study focused on modulation of free-radical processes occurring at low temperature in leaf cells of potato plants transformed with the gene for D12-acyl-lipid desaturase from cyanobacterium. Plants were grown in vitro on MS agar medium containing 2% sucrose. During hypothermia (–9 C), the rate of O2– generation and H2O2 concentration decreased significantly. In addition, the contents of both primary products (conjugated dienes and trienes) and secondary products (malonic dialdehyde) of lipid

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peroxidation were lower in transformed plant leaves than in leaves of WT plants. It was hypothesized that insertion of D12-acyl-lipid desaturase into the plant genome stabilizes the composition and physical properties of biomembranes by promoting polyunsaturation of fatty acids, which averts the accelerated generation of O2– and suppresses lipid peroxidation during hypothermia. Evidence suggests that ROS are the cause of photosystem 1 (PSI) inactivation in chilling-sensitive plants during cold stress in the light, and that H2O2 accumulation is a major factor leading to the decline in PSI activity (Sonoike 1996). Cotton is considered to be a chilling-sensitive species; its photosynthetic performance and ability to rapidly recover photosynthetic activity following chilling in the light diminish considerably with time of exposure (Koniger and Winter 1993). Kornyeyev et al. (2003) compared the photosynthetic performance between leaf discs of WT cotton and transgenic cotton overexpressing chloroplast stroma-based APX+ plants during exposure to 10 C and 500 mmol photons m–2 s–1. They showed that APX+ leaves did not exhibit as large an increase in cellular H2O2 as WT leaves shortly after the imposition of the chilling treatment. In addition, APX+ leaves exhibited slightly but significantly less PSI and PSII photoinhibition. The electron transport chain (ETC) of plant mitochondria has also been researched in relation to production of freeze-tolerant transgenic plants. Plant ETCs have unique features compared with other eukaryotes, including the ubiquitous presence of a terminal alternative oxidase (AOX) that competes for electrons with the standard cytochrome (Cyt) pathway (Finnegan et al. 2004). The maintenance of ETC redox balance is critical because electron input in excess of ETC capacity can be responsible for the production of ROS, particularly O2– and H2O2. It has been proposed that AOX may be an important factor in allowing plants to tolerate chilling-induced ROS damage to membrane phospholipids (Purvis and Shewfelt 1993). Evidence suggests that the AOX pathway of plant mitochondria uncouples respiration from mitochondrial ATP production and may ameliorate plant performance under stressful environmental conditions, such as cold temperatures, by preventing excess accumulation of ROS (Wagner et al. 1998). Fiorani et al. (2005) tested this model in whole tissues by growing AtAOX1a-transformed Arabidopsis plants at 12 C. Twenty-one days after sowing, antisense mutants showed (on average) a 27% reduction in leaf area and 25% smaller rosettes versus 30% increased leaf area and 33% larger rosette size for overexpressing lines compared with WT plants. These results demonstrate that AOX activity plays a role in shoot acclimation to low temperature in Arabidopsis.

2.4.3.2

Membrane Modifications

Chilling-resistant plants have a greater abundance of unsaturated fatty acids than chilling-sensitive plants and it has been shown that the proportion of unsaturated fatty acids increases during acclimation to cold temperature (Palta et al. 1993). This modification allows membranes to remain fluid by lowering the temperature at which the membrane lipids experience a gradual phase change from fluid to

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semi-crystalline. Thus, desaturation of fatty acids provides protection against damage from chilling temperatures (Khodakovskaya et al. 2006). Khodakovskaya et al. (2006) developed a cold-inducible genetic construct cloned using a chloroplastspecific omega-3-fatty acid desaturase gene (FAD7) under the control of a coldinducible promoter (cor15a) from Arabidopsis and expressed it in young tobacco plants. When seedlings were exposed to low-temperature (0.5, 2, or 3.5 C) for up to 44 days, survival within independent cor15a-FAD7 transgenic lines (40.2%–96%) was far superior to the WT (6.7%–10.2%). In addition, the major trienoic fatty acid species remained stable in cold-induced cor15a-FAD7 plants under prolonged cold storage while the levels of hexadecatrienoic acid (16:3) and octadecatrienoic acid (18:3) declined in WT plants under the same conditions (79 and 20.7%, respectively). Electron microscopy showed that chloroplast membrane ultrastructure in cor15a-FAD7 transgenic plants was unaffected by prolonged exposure to cold temperatures. In contrast, WT plants experienced a loss of granal stacking and disorganization of the thylakoid membrane under the same conditions. In higher plants, the most abundant lipids of thylakoid membranes are glycolipids (monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)). PG is the only phospholipid in thylakoid membranes. The chilling resistance of higher plants is apparently closely correlated with the level of cis-unsaturated fatty acids in PG from chloroplast membranes (Nishida and Murata 1996). Chilling-resistant plants contain a large proportion of cis-unsaturated fatty acids at the sn-1 position of PG and there are few cis-unsaturated fatty acids in chilling-sensitive plants. The sn-2 position is occupied mainly by saturated and trans-unsaturated fatty acids (Bertrams and Heinz 1981) and hence the content of cis-unsaturated fatty acids at the sn-1 position of PG determines plant chilling resistance. The dominant factor that determines the level of cis-unsaturated fatty acids in PG is the substrate selectivity of glycerol-3-phosphate (G3P) acyltransferase (GPAT: EC2.3.1.15) in chloroplasts, which catalyzes the first step of glycerolipid biosynthesis by transferring the acyl group of acyl-(acyl carrier protein; ACP) to the sn-1 position of G3P to yield 1-acylglycerol-3-phosphate (lysophosphatidate; LPA; (Roughan and Slack 1982)). GPAT from chilling-resistant plants prefers oleoyl-ACP (18:1-ACP) to palmitoyl-ACP (16:0-ACP) as a substrate resulting in a high level of cis-unsaturated fatty acids in PG. The enzymes from chilling-sensitive plants hardly distinguish 18:1-ACP from 16:0-ACP resulting in a low level of cis-unsaturated fatty acids at the sn-1 position of PG (Weber et al. 1991). In this way, fatty acids remain saturated, which renders the plants sensitive to chilling stress. Sui et al. (2007) isolated a tomato GPAT gene (LeGPAT), which despite the chilling sensitivity of tomato exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in the thylakoid membrane. Chilling treatment (4 C for 4 h) induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than WT plants under optimal

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conditions. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membranes, which was beneficial for the recovery of chilling-induced PSI photoinhibition in tomato.

2.4.3.3

LEA and Chaperoning Modifications

Several proteins are expressed in plants upon exposure to low temperature. These are either located in the cytosol or secreted to the apoplast. They have various putative functions, including cryoprotection, altered lipid metabolism, protein protection, desiccation tolerance, and sugar metabolism (Hiilovaara-Teijo and Palva 1999; Margesin et al. 2007). During cold acclimation, several stress proteins that may function as chaperones and membrane stabilizers during freeze dehydration are expressed in the cytosol (Puhakainen et al. 2004). Of the many low temperature-responsive genes characterized to date, several have been predicted to encode proteins with the characteristics of the dehydrin class of LEA proteins (Table 2.1). Houde et al. (2004) reported an improvement of the selection procedure and the transfer of the wheat Wcor410a acidic dehydrin gene to strawberry. The WCOR410 protein was expressed in transgenic strawberry at a level comparable with that in cold-acclimated wheat. Freezing tests showed that cold-acclimated transgenic strawberry leaves had a 5 C improvement in freezing tolerance over WT leaves or transformed leaves not expressing the WCOR410 protein. However, no difference in freezing tolerance was found between the different plants under non-acclimated conditions, suggesting that the WCOR410 protein needs to be activated by another factor induced during cold acclimation. The data demonstrated that WCOR410 protein prevents membrane injury and greatly improves freezing tolerance in leaves of transgenic strawberry. HSPs (Sect. 2.2.6.1) accumulate in response to low temperature. The HS response in plants has been extensively investigated (Waters et al. 1996). Plants synthesize predominantly small (15–30 kDa) HSPs (sHSPs) during the heat-shock response, and it has been suggested that the accumulation of sHSPs is correlated with thermotolerance (Vierling 1991). Guo et al. (2007), characterized a sweet pepper cDNA clone, CaHSP26 encoding the chloroplast (CP)-sHSP, with regard to its sequence, response to various temperatures, and function in transgenic tobacco plants. Expression of the CaHSP26 gene showed that the mRNA accumulation of CaHSP26 was induced by heat stress. Higher transcript levels were observed when sweet pepper leaves were treated at 42 C for 3 h. However, the expression of the CaHSP26 gene was not induced by chilling stress (4 C) in the absence of HS and the transcripts were detected at 48 h at 4 C after HS while not at 25 C. The photochemical efficiency of PSII (Fv/Fm) and the oxidizable P700 in transgenic tobacco overexpressing CaHSP26 were higher than that in WT tobacco during chilling stress under low irradiance. These results suggest that the CP sHSP protein plays an important role in the protection of PSII and PSI during chilling stress under low irradiance.

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Osmoprotectants/Compatible Solutes

Long-term acclimation to the cold and winter survival in herbaceous plants is strongly correlated with the recovery of photosynthesis at low temperature and the maintenance of soluble carbohydrate reserves (Stitt and Hurry 2002; Strand et al. 2003), namely, through upregulation of sucrose biosynthesis. Strand et al. (2003) tested this hypothesis by comparing the acclimation responses of WT Arabidopsis with transgenic plants overexpressing sucrose phosphate synthase (over-sps) or with antisense repression of either cytosolic fructose-1,6-bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants overexpressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT plants. The improved photosynthetic performance by the overexpressing sps plants was associated with an increase in freezing tolerance relative to WT (–9.1 and –7.2 C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (–5.2 and –5.8 C for antifbp and antisps, respectively). Similarly, metabolic engineering for the biosynthesis of fructans is a potential strategy for increasing waterstress tolerance. Fructans are a class of water-soluble fructose polymers based on sucrose, which accumulate in many bacterial and plant species serving as an important storage carbohydrate and protecting plants against water deficit caused by low matrix potential, salinity, or low temperatures (Spollen and Nelson 1994). In plants, fructans are synthesized in vacuoles from sucrose by the action of two or more different fructosyltransferases, including sucrose:sucrose 1-fructosyltransferase (1-SST), sucrose:fructan 6-fructosyltransferase (6-SFT), fructan:fructan 1-fructosyltransferase (1-FFT), and fructan:fructan 6G-fructosyltransferase (6G-FFT; Vijn and Smeekens 1999). Kawakami et al. (2008) used rice, which is highly sensitive to chilling temperatures and is not able to synthesize fructans, to study the effect of fructan biosynthesis against water stress. Two wheat fructan-synthesizing enzymes, 1-SST, encoded by wft2, or 6-SFT, encoded by wft1, were introduced into rice plants. The transgenic seedlings with wft2 accumulated significantly higher concentrations of oligo- and polysaccharides than nontransgenic rice seedlings, and exhibited enhanced chilling tolerance (11-day exposure to 5 C). The oligo- and polysaccharide concentrations of seedlings expressing wft1 were visibly lower than those of lines expressing wft2, and no correlation between oligo- and polysaccharide concentrations and chilling tolerance was detected in wft1-expressing rice lines. The results suggest that transgenic rice lines expressing wheat-derived fructosyltransferase genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced chilling tolerance at the seedling stage.

2.4.3.5

Transcription Factors

Cold acclimation in plants is a complex process involving changes in the expression of numerous cold-responsive (COR) genes (Chinnusamy et al. 2006). This results in

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modification of plant cell structural, biochemical, and photosynthetic properties that facilitate an increase in the plant’s freezing stress tolerance. Regulatory factors influencing expression of COR genes and/or freezing tolerance have been identified over the last decade (Chinnusamy et al. 2006). The cold-induced CBF transcriptional regulatory factor CBF1-3 controls the cold-responsive expression of a major regulon of COR genes that increases plant freezing tolerance (van Buskirk and Thomashow 2006). Ectopic expression of CBF transgenes under warm conditions activates a suite of COR genes without cold stimulus, increasing plant freezing tolerance and inducing biochemical and structural alterations normally observed during exposure to cold. Pino et al. (2008) studied the effect of ectopic AtCBF overexpression on physiological alterations that occur during cold exposure in frost-sensitive (Solanum tuberosum) and frost-tolerant (S. commersonii) potato. Relative to WT plants, ectopic AtCBF1 overexpression enhanced expression of COR genes without a cold stimulus in both species and imparted a significant increase in freezing tolerance gain in both species (2 C in S. tuberosum and up to 4 C in S. commersonii). Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of WT S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to WT, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest that an endogenous CBF pathway is involved in many of the structural, biochemical, and physiological alterations associated with cold acclimation in potato. The cuticle is one of the most important barriers for all terrestrial plants and mitigates damage to above-ground biomass caused by low humidity and other biotic and abiotic stresses (Jenks and Ashworth 1999). The important physiological and ecological functions of the cuticle (which include control of transpiration and leaching and facilitation of foliar penetration of pesticides) are related to the presence of cuticular waxes that are embedded in or deposited on the cutin matrix (Kunst and Samuels 2003). Increased accumulation of cuticular waxes in leaves has been achieved through the overexpression of TF genes such as WIN1/SHN1 in Arabidopsis (Broun et al. 2004) and WXP1 in alfalfa (Zhang et al. 2005). Elevated leaf cuticular wax deposition led to significant improvement in drought tolerance of the transgenic plants (Broun et al. 2004; Zhang et al. 2005). Zhang et al. (2007b) reports the functional characterization of two putative ERF TF genes WXP1 and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis led to significantly increased cuticular wax deposition on leaves of 4- and 6-week-old transgenic plants. Differences in the accumulation of various wax components, as well as their chain length distributions, were found in the WXP1 and WXP2 plants. Analysis of fresh weight loss from detached leaves

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revealed that the transgenic leaves retained more water than controls. Both WXP1 and WXP2 transgenic plants showed significantly enhanced whole-plant drought tolerance. Analysis of freezing tolerance at the whole-plant level and measurement of electrolyte leakage from detached leaves revealed that the WXP1 plants had increased freezing tolerance while the WXP2 plants were more sensitive to low temperature when compared to the control. Transgenic expression of WXP1 had no obvious effects on plant growth and development; however, the expression of WXP2 led to slower plant growth. These results indicate that WXP1 is a useful candidate gene for improving plant drought and freezing tolerance by genetic transformation.

2.5

Nutrient Deficiency and Nutrient Use Efficiency

In all, plants require 17 essential elements, 14 of which are taken up in inorganic forms by the roots. The absence or paucity of any one of these essential elements will commonly lead to plant death or inability to complete its life cycle. In the presence of nutrient deficiencies, even at asymptomatic levels, crop performance, yield, and quality are frequently compromised. Hence, fertilizer and other soil ameliorations are essential to ensuring food security and the sustainability of agriculture, and these add a major economic and potentially environmental burden to crop production. The ability to improve and enhance the efficiency of nutrient uptake and utilization in major crop plants has become a major objective in modern plant improvement. Interestingly, different species have evolved differential mechanisms for improving their ability to scavenge essential elements at low concentrations in the soil. In this section, we will discuss the ways in which biotechnological intervention can be applied to improve the ability of crop plants to survive and produce yield in nutrient-poor environments. The discussion will focus on the most limiting nutrient, nitrogen, with some discussion of phosphorus and iron, which are particularly susceptible to soil pH variation.

2.5.1

Nitrogen

Nitrogen (N) is quantitatively the most important nutrient that plants obtain from the soil (Paungfoo-Lonhienne et al. 2008), with an estimated 1011 tons of N fertilizer applied annually worldwide (Lea and Azevedo 2006). The total cost of this agricultural input exceeds US $50 billion. In general, however, plants are only able to acquire 30–40% of this applied N, with significant losses occurring through leaching, denitrification, and the volatilizationz of ammonium to the atmosphere (Lea and Azevedo 2006). Nitrogen assimilation is the process by which inorganic N forms (typically nitrate) are reduced to ammonium, and then converted into organic forms (amino

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acids) for transportation and use by the plant. Nitrate is the main form of N taken up by plants and is reduced in the plant through the combined action of the enzymes, nitrate reductase (NR) and nitrite reductase. It has long been believed that this step is the major control point in nitrate assimilation and it has been shown that expressing a constitutive NR as opposed to a fully regulated NR demonstrates accumulation of high concentrations of asparagine and glutamine (Good et al. 2004; Lea and Azevedo 2006). Inorganic N is then converted into organic amino acids by the GS-GOGAT cycle, and these amino acids are used to transport N around the plant. The major N transport amino acids are glutamate, glutamine, aspartate, and asparagine, with differences in importance among different phyla and also environmental conditions. Ammonium is incorporated into glutamine through the enzyme glutamine synthetase (GS). This places the ammonium molecule initially into the amide position of the glutamine molecule from where the N is relocated to the a-amino position of glutamate through the action of glutamate synthase (GOGAT) (Glass et al. 2002; Good et al. 2004; Lea and Azevedo 2006). The GS-GOGAT has been considered a primary target for the manipulation of nitrogen use efficiency (NUE). NUE is usually described as unit biomass per unit of N present in the plant or the yield of N in the plant per unit of available N in the soil (Bushoven and Hull 2001; Lea and Azevedo 2006). NUE is dependent on physiological traits such as uptake and assimilation of N (Good et al. 2004). While there are other processes that use N in plants, it is these two traits that control NUE. The ability to uptake and transport N throughout the plant is, along with photosynthetic efficiency and WUE, one of the major determinants of plant survival, growth, and yield. In addition, the mobilization of organic N into fruits and seeds is a major determinant of yield and product quality, especially in cereal and legume grains, where the protein content determines the nutritional and end-use qualities. N nutrients are powerful signaling molecules within the plant (Vidal and Gutierrez 2008). Nitrate controls the expression of thousands of genes involved in many plant processes, with some genes responding to nanomolar concentrations (Wang et al. 2003a, 2007b). This includes the nitrate transporter molecules (for an introduction to transporter genes, see the description in Sect. 2.3.4.2). The nitrate transporters, NRT1.2 and NRT2.1, are not only involved with N uptake, but they are also sensor molecules, and have been shown to increase N uptake rate at low rhizosphere concentration. It is known that root branching occurs at higher frequency in regions of the rhizosphere with local N-enrichment (Scott Russell 1977). The transporter NRT1.1 is involved in signaling, which leads to colonization of nitrate-rich patches in the soil by promoting localized root proliferation in concert with ANR1, a MADS-box TF gene not yet fully functionally characterized (Remans et al. 2006). Both these genes have regulatory sequences which direct reporter gene expression in root primordia and root tips. Amino acid synthesis and transport genes have been manipulated to alter the NUE in numerous plants. For example, the overexpression of a bacterial asparagine synthetase gene in the leaves of lettuce was shown to affect N status (Giannino

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et al. 2008). Early vegetative growth rate of these transgenic lettuce plants was approximately 1.3-fold higher for the first 35 days post-germination, accompanied by higher leaf number and leaf area. However, these plants also attained reproductive phase earlier, which was not advantageous for a leaf vegetable. Similar effects have been reported in transgenic oilseed rape (Seiffert et al. 2004) and Lotus (Vincent et al. 1997). The transgenics had approximately twofold more aspartate and asparagine than WT, and interestingly also had elevated levels of glutamine with no effect on glutamate concentration. Leaf assays also revealed a lower level of free nitrate in the transgenic lettuce lines, a phenomenon previously reported in lettuce overexpressing a nitrate reductase gene (Curtis et al. 1999). The authors suggested that these plants may be suitable for breeding new quick-harvest lettuce varieties with lower N fertilizer requirement, or enhanced NUE.

2.5.2

Phosphorus

Worldwide, it is estimated that 5.7 billion hectares of land lack sufficient quantities of plant-available Phosphorus (P) (Batjes 1997). P deprivation in plants has been widely studied, as P becomes limiting to plant productivity with falling soil pH, and tends to bind very tightly with soil. In mildly acid soils with pH < 6, soil P rapidly becomes immobile and forms insoluble compounds, commonly with Fe and Al. Even in soils with abundant P, usually only about 1% of the soil P is actually in a readily available, soluble form, and over 90% is generally bound tightly to soil particles in organic and inorganic forms, which require mineralization before they become plant available. P uptake and transporter genes are generally regarded as high or low affinity, with the high affinity genes becoming more important for scavenging nutrient from the soil as P becomes limiting. Many Australian soils are particularly P-deficient and, as a result, many native Australian plants have evolved different mechanisms to overcome this with high-affinity P transporters, association with vesiculararbuscular mycorrhiza, the secretion of organic acids into the rhizosphere, and the formation of proteoid roots (Lambers et al. 2006). Attempts to overexpress high affinity P transporters have met with mixed success. Overexpression of high-affinity transporter genes in barley did not have any effect on P uptake or plant productivity and growth (Rae et al. 2004), despite having demonstrated that these genes increased P uptake in transgenic rice callus cultures. However, the overexpression of homologous high-affinity P transporter genes in rice enabled the plants to accumulate twice as much P as WT rice, and the resulting phenotype produced more tillers (Seo et al. 2008). Further research has also demonstrated that many plant phosphate transporters have complex interactions with vesicular arbuscular mycorrhizae (Glassop et al. 2007). Plants have been demonstrated to alter the rhizosphere with specific exudates, commonly organic acids or enzymes, to improve the availability of nutrients such as phosphate. The exudation into the rhizosphere of acid phoaphatase has led to

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improved biomass and P accumulation in Arabidopis (Xiao et al. 2007) and rice (Park et al. 2007). Similar results have been achieved with the ectopic expression of phytase in root tissues of potato (Hong et al. 2008).

2.5.3

Iron

Iron is not only an essential plant nutrient, but its deficiency, known as anemia, also represents the world’s most frequent and debilitating human mineral nutrient deficiency (Wintergerst et al. 2007). Hence, improving the ability of plants to uptake, translocate and store iron in bioavailable forms is not only a plant health and productivity issue, it is a major human and animal nutritional target. Plants have the ability to sense low-iron conditions, which induces a coordinated response. The predominant form of iron in aerobic soils is Fe(III), and plants must possess strategies to utilize this form. Most plants use what has become widely known as Strategy I, whereby they acidify the rhizosphere with the induction of a specific proton pump which solubilizes more Fe, and then, by the production of Fe chelate reductase, convert Fe(III) to Fe(II), which is then transported into the cell by a membrane-bound Fe(II) transporter. Grasses and cereals, however, utilize Strategy II, which involves the production and secretion of a specialized group of chemicals, known as phytosiderophores (PS) into the rhizosphere. PS are a form of mugineic acids, which form strong chelates with Fe(III), and help to solubilize them for plant uptake by specialized protein forms, known as the Yellow Stripe proteins (von Wiren et al. 1994; Curie et al. 2001), which are actually Fe(III) transporter genes. Interestingly, rice is a special case and appears to be able to utlilize both strategies of iron uptake. Unusually, among the grasses rice has an efficient Fe(II) uptake system and does not produce much PS (Mori et al. 1991). Rice has been engineered to produce larger quantities of PS, and the transgenic lines were more tolerant to Fe-deficient soils (Suzuki et al. 2008). Other successful approaches to improve Fe uptake of transgenic rice in alkaline soils have included the overexpression of Fe chelate reductase genes (Ishimaru et al. 2007) and barley nicotianamine amino transferase genes (Takahashi et al. 2001).

2.6 2.6.1

Engineering Metal Toxicity Tolerance in Plants Heavy Metal Contaminated Soil

Soils naturally consist of varying high levels of heavy metals; however, concentrations in soils are greatly increased where humans have extracted them and used them for industrial purposes (Greger 1999; Harmsen 2002; UN-Oceans 2008).

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Heavy metals, especially those that are present from weathering of parent rock, are usually bound or immobile in soils and their removal via leaching or plant accumulation is slow and inefficient. As a consequence, metals accumulate in soils (Haygarth and Jones 1992). Heavy metals usually exist as cations under biological conditions and form complexes with soil sediments and colloids, which are made up of negatively charged organic substances and inorganic clay particles. Formation of this complex is a slow process and, because unbound metals are bioavailable and may deleteriously affect agricultural products or leach into groundwater, areas where human activities have led to elevated soil metal content are a socioeconomic as well as an environmental concern (Greger 1999; Harmsen 2002). Common heavy metal soil pollutants include arsenic, cadmium, chromium, copper, nickel, lead, and mercury (UN-Oceans 2008). Cd and Hg are particularly concerning because they are widespread and have no known function in human metabolism (Nordberg et al. 2002; UN-Oceans 2008).

2.6.2

Engineering Heavy Metal Tolerance in Plants

Few known studies have focused on engineering heavy metal tolerance in plants. It is assumed that this is due to the complex interactions of stress factors, which mean that many studies focusing on improving other types of abiotic stress, such as osmotic and low temperature stress, include analysis of improved tolerance to one or a few heavy metals. For example, Zhang et al. (2008c) isolated an aquaporin gene BjPIP1 from the heavy metal hyperaccumulator Indian mustard, which is upregulated in leaves under drought, salt, low temperature, and heavy metal stress. Constitutive expression of BjPIP1 in tobacco decreased water loss rate, transpiration rate, and stomatal conductance of transgenic plants compared to WT under osmotic stress. On exposure to Cd, transgenic plants displayed enhanced Cd resistance of root growth and lowered transpiration rates and stomatal conductance, increased activities of antioxidative enzymes, lower levels of electrolyte leakage, and lower malondialdehyde content. The study suggested that the increased heavy metal resistance was conferred by maintaining reasonable water status in transgenic plants. Other proteins that have been implicated in conferring heavy metal tolerance include LEA proteins and cation-efflux transport proteins (Zhang et al. 2007c). When Koh et al. (2006) introduced a yeast Cd factor (YCF1), which sequesters glutathione chelates of heavy metals and xenobiotics into vacuoles, into Arabidopsis in order to improve heavy metal tolerance, transgenic plants were found to have improved salt tolerance as well (Zhang et al. 2007c). While the use of genetic engineering to develop crops with improved heavy metal tolerance remains relatively unexplored, a related field is the use of genetic engineering to develop plants with increased ability to uptake and accumulate metals in order to remove the soil contamination in a process known as phytoremediation. Many of the plant characteristics required to confer metal accumulation ability also confer heavy metal tolerance and, as such, plants engineered with enhanced phytormediation

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capabilities inevitably possess enhanced heavy metal tolerance as well. Unlike engineering plant heavy metal tolerance, this field has been the focus of many studies over the last decade. Therefore, the remainder of this chapter will briefly discuss the use of transgenic technology to improve the phytoremediation capabilities of plants.

2.6.3

Soil Heavy Metal Management and Remediation Options

There are many management and remediation options available to prevent entry of soil heavy metals into the human food chain via agricultural crops. These include: prevention, agronomic management, and physical remediation. These options, while contributing to reductions in human exposure to some heavy metals, can be unrealistic, labor intensive, and prohibitively expensive (McLaughlin et al. 1999; Ensley 2000; Glass 2000; Clarkson 2002; Madden et al. 2002; Prasad 2002; Robinson et al. 2003; Song et al. 2003).

2.6.3.1

Phytoremediation: Engineering Crops to Increase Heavy Metal Uptake

Phytoremediation is a biological technology, which utilizes the physiology of plants to extract and detoxify soil and water pollutants (Kramer and Chardonnens 2000; Rosselli et al. 2003). The use of plants for contaminated land reclamation is based on the existence of metal hyperaccumulator plants, which are capable of growing on metal contaminated soils and accumulating extremely high levels of heavy metals in their harvestable tissue (Robinson et al. 2003). The major metal tolerance mechanisms identified in heavy metal accumulator plants include: (1) cell wall metal ion binding; (2) inhibition of metal ion transport across plasma membranes; (3) active metal ion efflux from cells; (4) chelation and detoxification of metal ions; and (5) compartmentalisation of metal ions in organelles (Bargagli 1998). The several types of phytoremediation include: phytostabilization (the use of plants to immobilize metals in the environment to prevent them from leaching into groundwater (Prasad 2002)), phytodegradation (the use of plants to degrade contaminants in the root zone), phytoextraction, and phytovolatization. Phytoextraction and phytovolatilisation are particularly important for metal phytoremediation. Phytoextractor plants remove metal ions from soils and store them in above-ground tissue using the photosynthetically driven processes of soil-ion extraction and translocation. Normal harvesting processes enable easy removal of contaminants and their responsible disposal. Phytovolatilization is similar to phytoextraction but, rather than be stored in biomass, extracted elements are converted into less toxic, volatile forms via plant metabolic pathways and released into the atmosphere (Kramer and Chardonnens 2000; Robinson et al. 2003).

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Phytovolatilization of Soils Contaminated with Hg

Relatively low exposure to Hg and mercurial compounds can cause severe detrimental physiological effects in all biological organisms. Atmospheric Hg increased from approximately 2  106 kg to 4  106 kg over the twentieth century because of Hg use in the chemical, medical, paper, mining, and defense industries (Bizily et al. 2000). Environmental Hg is present as Hg(II), elemental Hg (Hg(0)), and organomercurial compounds (R-Hg+) such as CH3–Hg+. Toxicity symptoms between these forms differ; CH3–Hg+ is the most dangerous because it is lethal at doses two to three orders of magnitude lower than Hg(0) or Hg(II), diffuses passively through biological membranes, is highly reactive with biological compounds, has a long retention time in the body, and is biomagnified through the food chain (Rugh et al. 2000). There are no known naturally occurring plants that are able to detoxify or hyperaccumulate Hg (Pilon-Smits and Pilon 2002). Therefore, a bacterial Hg volatilization pathway has been used in the development of Hg phytoremediators (Pilon-Smits and Pilon 2002). Bacterial colonies that metabolically convert Hg(II) and R-Hg+ compounds into the less toxic Hg(0) have been discovered inhabiting Hg-contaminated sites. The volatilization pathway is conferred by the presence of the mer-operon, which encodes a set of genes involved in the detection, mobilization, and enzymatic detoxification of R-Hg+ compounds via two main reactions, which are catalyzed by organomercurial lyase (MerB) and mercuric ion reductase (MerA) (Rugh et al. 1998). A modified form of the MerA gene, merA9, was transferred into Arabidopsis and tobacco and transgenic plants were found to be resistant to 50 mm Hg(II) (Rugh et al. 2000). Expression of the same gene in the forest species, Liriodendron tulipifera (yellow poplar) resulted in transgenic plants, which thrived in solution containing 50 mm Hg(II) and volatized Hg(0) at a rate tenfold higher than WT controls (Kramer and Chardonnens 2000). MerA has also been expressed in the eastern cottonwood (Populus deltoids) with similar success. The transformation of Arabidopsis with constructs of both the merA and merB genes resulted in transgenic plants able to tolerate media containing 40-fold higher CH3– Hg+ levels than WT plants and were able to volatilize Hg at a rate severalfold higher than WTs (Bizily et al. 2000). Tobacco was also transformed with MerA and MerB via the chloroplast genome and transgenic plants grew well with root Hg concentrations up to 2,000 mg g–1, accumulated R-Hg+ compounds and inorganic mercurials to levels surpassing soil concentrations and displayed a 100-fold increase in the efficiency of shoot Hg accumulation over WT plants (Hussein et al. 2007). MerA and MerB have also been expressed simultaneously in transgenic poplars and eastern cottonwood trees (Lyyra et al. 2007; Young et al. 2007). Transgenic poplars were tolerant to 50 mM HgCl2 and 2 mM CH3HgCl in culture; however, a high level of variation in Hg-tolerance was observed in transgenic plants (Young et al. 2007). Transgenic eastern cottonwood trees were highly resistant to R-Hg+ compounds and detoxified them two to three times more rapidly than controls (Lyyra et al. 2007).

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Phytoextraction of Soils Contaminated with Cd

Plants able to tolerate high Cd levels do so via exclusion (reducing metal uptake by cell wall binding) or intracellular compartmentalization (binding of metals with detoxifying ligands within the cell and the sequestration of these complexes in organelles where the metals cannot interact with the plants metabolic processes (Robinson et al. 1994)). The two major heavy metal detoxifying ligands in plant cells are the metallothioneins (MTs) and the phytochelatins (PCs; Cobbett and Goldsborough 2000). Class I and II MTs are low molecular weight metal-binding products of mRNA translation. PCs are represented by the class III MTs and are products of enzymatic reactions (Cobbett and Goldsborough 2002).

2.6.5.1

Metallothioneins

It has been difficult to analyze plant metallothionein (MT) proteins because they are unstable under aerobic conditions; however, isolated and cloned mammalian MT genes have been assessed for their ability to increase metal tolerance when expressed in transgenic organisms. Table 2.2 lists the findings of some of these studies. These results, obtained in laboratory trials, demonstrate that expression of mammalian MTs in plants can provide protection against toxic effects of heavy metal ions such as Zn2+, Cd2+, and Hg(II); but it has not yet been possible to achieve similar results in the field (Kramer and Chardonnens 2000).

2.6.5.2

Phytochelatins

Phytochelatins (PCs) were first discovered in plants and are structurally related to GSH, which is thought to act as the substrate for PC synthesis by PC synthase (PCS). Various methods have been employed to enhance the effectiveness of PCassisted metal detoxification. Indian mustard was engineered to express the E. coli GSH synthesis gene, gsh2, and transgenic plants displayed increased GSH and PC levels and a 25% increase in shoot Cd concentration (Zhu et al. 1999). The enzyme that catalyzes synthesis of the GSH precursor, g-glutamylcysteine synthase (GCS), was also expressed in B. juncea and shoot Cd concentrations were increased by 40–90% in the transformed plants (Kramer and Chardonnens 2000). A variety of PC genes have been isolated from different species and overexpressed in endogenous or exogenous species to determine their potential for Cd phytoremediation. The most extensively studied PCS gene is AtPCS1 from Arabidopsis, which has been found to play roles in increased tolerance and/or accumulation of Cd and As (Gasic and Korban 2007b). However, depending on the expression level, transfer of PCS genes have also been associated with decreased Cd accumulation and Cd hypersensitivity (Lee et al. 2003b, c; Li et al.

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Table 2.2 Genetic transfer of MT genes for improved phytoremediation capabilities of plants Transformed Transgene(s) Findings References species Eschericia Human MT genes Increased metal adsorption Kramer and coli Chardonnens (2000) Ralstonia Mouse MTs genes Enhanced Cd immobilizing ability Valls et al. eutrophia when expressed on cell surface (2000) Tobacco Mammalian MT Root-shoot Cd transport is reduced Kramer and gene Chardonnens (2000) Brassica Yeast MT gene, Increased leaf Cd accumulating Kramer and oleracea CUP1 ability Chardonnens (2000) Tobacco Construct containing Up to 90% increased Cd Macek et al. CUP1 and an accumulation in harvestable (2002) additional metalparts without any visible binding domain difference in growth characteristics Sunflower CUP1 Enhanced Cd tolerance when Watanabe et al. expressed at the callus stage (2005) Tonkovska et al. Tobacco Arabidopsis MT Expression strongly induced by (2003) gene, MT2aI Cu2+, Zn2+, and Cd2+ suggesting the promoter has specificity for heavy metal stress Tobacco Construct encoding a 45–75% increase in Cd Pavlikova et al. poyhistidine accumulation in harvestable (2004a, b) cluster with a plant parts and increased yeast MT resistance to Cd-induced stress Arabidopsis Garlic MT gene Stronger Cd tolerance and higher Zhang et al. Cd accumulation (2006) Zhigang et al. Arabidopsis Brassica juncea MT Increased tolerance to Cu2+ and Cd2+ based on shoot growth and (2006) gene, BjMT2, chlorophyll content and decreased root growth in the absence of heavy metal exposure Tobacco Silene vulgaris L. Signigicantly increased Cd Gorinova et al. MT gene accumulation in roots and (2007) leaves Sun et al. (2007) Arabidopsis Brassica rapa MT Chloroplast target of gene resulted gene in detoxification of Cd and H2O2

2004, 2005b; Gasic and Korban 2007a). Other studies have shown that AtPCS1 can lead to increased Cd tolerance and accumulation in roots but not translocation of Cd into harvestable tissue (Pomponi et al. 2006). PCS genes from Cyndon dactylon (CdPCS1) and wheat (TaPCS1) have also been transformed into tobacco and have led to increases in leaf accumulation of Cd and other heavy metals (Li et al. 2006; Martinez et al. 2006). Tobacco transformed with TaPCS1 was grown in the field to

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assess its Cd-accumulating ability and it was found that transgenic plants could accumulate more heavy metals and 100 times more biomass on contaminated soils than the hyperaccumulator Thlaspi caerlescens (Martinez et al. 2006). Another approach was employed by Song et al. (2003) and was aimed at increasing the efficiency of metal transport across the tonoplast. In this study, YCF1 from S. cerevisiae was overexpressed in Arabidopsis and transformed plants displayed increased tolerance to Cd and Pb and improved vacuolar Cd sequestration. Many studies have also shown that dual- or multiple-gene expression using constructs with combinations of PC synthesis genes such as GCS, GSH synthase, ATP sulfurylase, and serine acetyltranferase may be the most promising route toward the development of a useful Cd phytoremediator and a phytoremediator useful for removal of mixtures of heavy metals from soils (Bennett et al. 2003; Wawrzynski et al. 2006; Guo et al. 2008; Reisinger et al. 2008).

2.7

Conclusion and Future Directions

This review has briefly summarized some of the stress resistance mechanisms that have been targeted for manipulation in the endeavor to develop crop plants with improved resistance to drought, salinity, cold, nutrient deficiencies, and metal toxicities. In the majority of cases, there are many knowledge gaps that need to be filled prior to development and release of these crops. Water-limiting environments are the most widespread form of stress, and also the least amenable to rapid advances in resistance. Reasons for this include the incomplete knowledge regarding plant drought responses, lack of field trials and drought stress treatments which are truly reflective of climatic conditions, the lack of site and crop specificity of drought tolerance studies, and the lack of integration of disciplines. The last century of breeding effort and crop physiology studies have led to increases in the economic yield of most major crop species and have elucidated many traits that are associated with plant adaptability to drought-prone environments (such as small plant size, reduced leaf area, early maturity, and prolonged stomatal closure). However, many attempts to improve drought tolerance through breeding have been associated with reduced yield potential (Cattivelli et al. 2008). Nevertheless, enormous advances should have been make in the understanding of the physiological and molecular responses of plants to water deficit through breeding and physiological studies, and scientists in these areas will continue to have a fundamental role in the development of transgenic drought-resistant crops. One of the greatest limitations in drought stress tolerance breeding has been the fact that drought takes many varied forms. Depending on the crop and the season, water stress may be experienced in early vegetative stages, during transition to flowering and even post-anthesis. In some cases, this may be a terminal stress or, more commonly, a recurring event broken by sporadic rainfall precipitating a recovery of the whole plant, sometimes with reduced yield potential as a result of conservative plant cell survival responses.

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It is entirely possible that the development of transgenic crops with improved abiotic stress tolerance may lead to unforeseen outcomes, both positive and negative. The development of “super varieties” that offer promising solutions to the problems of water deficit or salinity stress, may also give rise to problems associated with high-input agriculture in monoculture situations. Additionally, this review has shown that there are a plethora of genetic components that may be manipulated in order to confer improved abiotic stress tolerance of crops and that there is, therefore, unlikely to be a single gene that will result in a suitable “droughttolerant” or “salt-tolerant” variety for all conditions. Consequently, it may be beneficial to trial novel technologies such as overexpression of multiple genes known to be involved in the adaptation to stress and introgression of these genes in random combinations into many cultivars of the one crop prior to field release. This may increase the stability of these genes and allow specific environments to “naturally select” the transgenes that are most suitable for their particular climatic conditions in the same way that increased natural genetic diversity does so in natural populations. Salt tolerance is a physiologically complex trait and halophytes and less tolerant plants show a wide range of adaptations to salt stress. Attempts to enhance tolerance have involved conventional breeding programs, the use of in vitro selection, physiological trait pooling, interspecific hybridization, the use of halophytes as alternative crops, the use of marker-aided selection, and the use of transgenic plants. The assessment of salt tolerance in transgenic experiments as described above has mostly been carried out using a limited number of seedlings or mature plants in laboratory experiments. In most cases, experiments were carried out in greenhouse conditions where the plants were not exposed to conditions that prevail in high-salinity soils (e.g., alkaline soil pH, high diurnal temperatures, low humidity, presence of sodic salts, and elevated concentrations of selenium and/or boron (Yamaguchi and Blumwald 2005)). Therefore, the salt tolerance of plants needs to be evaluated under field conditions and, more importantly, salt tolerance needs to be evaluated as a function of yield. The evaluation of field performance under salt stress is difficult because of the variability of salt levels in field conditions (Daniells et al. 2001) and the potential for interactions with other environmental factors, including soil fertility, temperature, light intensity and water loss due to transpiration. Evaluating tolerance is further complicated because of variation in sensitivity to salt during plant life cycles. According to Flowers and Flowers (2005), conventional breeding programs have rarely delivered enhanced salt tolerance, while wide crossing to achieve salt tolerance generally reduces yield to unacceptably low levels (Flowers and Flowers 2005). There has been success using physiological criteria as the basis of selection for rice (Dedolph and Hettel 1997) and such an approach has been advocated for wheat (Munns et al. 2002). According to Flowers and Flowers (2005) recent analysis has shown that, while it is possible to produce a wide range of transgenic plants where some aspect of a trait relating to salt tolerance is altered, none or few transgenic plants have been tested in the field and few claims for success meet minimal criteria required to demonstrate enhanced tolerance (Flowers 2004).

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While adaptation to stress under natural conditions has some ecological advantages, the metabolic and energy costs may sometimes mask and limit its benefit to agriculture and result in yield penalty. An ideal genetically modified crop should possess a highly regulated stress-response capability that does not affect crop performance when stress is absent. In this respect, conventional breeding and selection techniques will continue to make a contribution (Wang et al. 2001). As a result of this, transgenic approaches to plant improvement are best regarded as a means of widening genetic variation. Transgenic plants will nevertheless continue to be extremely useful tools in basic plant science research, and will lead to improved understanding of the gene networks and molecular physiology of plant responses to abiotic stresses.

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Chapter 3

Transgenic Crops for Herbicide Resistance Stephen O. Duke and Antonio L. Cerdeira

3.1

Introduction

A year after the introduction of the first commercial transgenic crop (Flavr Savr™ tomato with a longer shelf life) in 1994, transgenic, herbicide-resistant crops (HRCs) were introduced (Table 3.1) with the introduction of bromoxynil- (3,5dibromo-4-hydroxybenzonitrile) resistant cotton and glufosinate- [2-amino-4(hydroxymethylphosphinyl)butanoic acid] resistant canola. Bromoxynil resistance had little market penetration during the years when it was available. The next year, 1996, marked the introduction of the first glyphosate- [N-(phosphonomethyl) glycine] resistant (GR) crop (soybean). Other GR and glufosinate-resistant crops were introduced in the subsequent years. GR crops now represent well over 80% of all transgenic crops grown worldwide (James 2008). Accordingly, this chapter will deal primarily with GR crops. Several reviews (e.g., Duke 2005; Duke and Cerdeira 2005; Cerdeira and Duke 2006) and two books (McClean and Evans 1995; Duke 1996) are available on the topic of HRCs, but this rapidly evolving topic requires timely updates. Before the advent of transgenic crops, there was both controversy and optimism about their potential impact on farming, human health, and the environment (e.g., Goldberg et al. 1990; Duke et al. 1991). We have now (early 2009) had 14 years of experience with HRCs over vast areas in many parts of the world, providing a wealth of information on the utilization of this technology, as well as the impacts of HRCs on the environment. This review will deal, in part, with these topics as they apply to HRCs

S.O. Duke (*) Agricultural Research Service, United States Department of Agriculture, University of Mississippi, MS 38677, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_3, # Springer‐Verlag Berlin Heidelberg 2010

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3.2 3.2.1

Present HRCS and Their Impact Commercially Available HRCs

In 2008, after 14 years of HRCs, there are only nine different HRCs being grown in the US (Table 3.1), and only a few of these are grown in other countries. From 1995 until 2000, one or two new HRCs were introduced to the market every year, after which the number of introductions has dwindled, with only a single new HRC in occasional subsequent years. The adoption rate of GR soybean was rapid in the US (Fig. 3.1), currently representing more than 90% of the area planted in soybean. The adoption rate of GR soybean in Argentina was even more rapid, reaching almost 90% adoption within 4 years of introduction (Penna and Lema 2003). Adoption of this HRC has also been rapid in other parts of South America. Both cotton and maize have varieties that are either stand-alone GR varieties or varieties that combine GR and transgenic Bt (Bacillus thuriengensis toxin) traits for insect resistance. In both the crops, there are also stand-alone Bt toxin varieties. To generate the data in Fig. 3.1, adoption rates of the two types of GR varieties must be added. GR cotton adoption was initially similar to that of soybean, but it has stabilized at about 70% (Fig. 3.1), partly because of the adoption of glufosinateresistant cotton in places where it fits the weed problems better than GR cotton. The economics for GR maize was not quite as good as with existing weed management methods when it was first introduced, but its adoption in the US is now rising rapidly and has almost caught up with that of cotton (Fig. 3.1). Relatively little GR canola is grown in the US, but about 90% of the canola grown in Canada was GR canola in 2006 (Dill et al. 2008). Of the canola grown in the US, 62% was GR

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Fig. 3.1 US adoption rate of glyphosate-resistant crops. Data from: http://www.ers.usda.gov/Data/ BiotechCrops

and 31% was glufosinate-resistant in 2005 (Sankula 2006). After a false start in 1999, GR sugar beet was reintroduced in 2008, with an unprecedented adoption rate of about 60% for the initial year of availability and an anticipated 95% adoption rate in 2009 (Thomas Schwarz, Beet Sugar Development Foundation, pers. comm). The adoption rate was limited by the availability of transgenic seed. GR alfalfa was introduced and well accepted by farmers in 2005, but deregulation was challenged in court by organic alfalfa growers in 2007, resulting in the removal of the product from the market. Glufosinate-resistant crops are also available (Table 3.1), but they have garnered a much smaller fraction of the HRC market. Their biggest market penetration is with canola in the US. Glufosinate-resistant cotton has been adopted at high rates in the US state of Texas. Partly owing to the evolution of GR weeds, glufosinateresistant crop adoption is increasing. Crops with both GR and glufosinate resistance are being made available. The economics for the biotechnology industry with HRCs is also good. HRCs offer profits from both a “technology fee” added to seed costs and for the purchase of the herbicide. No other type of transgenic trait offers this opportunity for dual profits from the seed and a chemical upon which the value of the gene is dependent. There has been some consideration of linking expression of transgenic traits to a chemical inducer of transgene expression (e.g., Jepson et al. 1998), but farmers would be unlikely to pay much for such a chemical, and the cost of applying the

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inducer would probably skew the economics away from such a strategy, unless the value of the trait was large.

3.2.2

Herbicide Resistance in HRCs

Plants can be made resistant to herbicides or other phytotoxins by a number of mechanisms. The molecular target site of the herbicide can be modified so that it no longer binds it and is thereby resistant. One or more herbicide-inactivating or herbicide-degrading enzymes can be introduced to or increased in a plant. The plant can be altered to have a mechanism that prevents the herbicide from reaching the molecular target site (increased sequestration, or decreased uptake or translocation). All three mechanisms have been described in weeds that have evolved resistance to herbicides. Metabolic inactivation or degradation is the principle mechanism in most cases of natural crop resistance to selective herbicides. The first two approaches have been useful in producing commercial HRCs. Glyphosate’s molecular target site is an enzyme of the aromatic amino acid pathway (the shikimate pathway). This enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), is highly sensitive to glyphosate (Duke 1988), and there are apparently no other good inhibitors of the enzyme. All plant EPSPS is sensitive to glyphosate, making it a nonselective herbicide that can be used to kill almost all weed species. Some fungi and bacteria also have EPSPS, and there are bacterial versions of the enzyme that are very resistant to glyphosate. A gene encoding Agrobacterium sp. EPSPS, the CP4 gene, has been used as a transgene for almost all GR crops (Cerderia and Duke 2007). In some maize varieties, a maize EPSPS gene modified by site-directed mutagenesis has been used as the transgene in GA21 varieties (Dill 2005). In canola, a gene from the soil bacterium Ochrobactrum antropi that encodes a glyphosate-degrading enzyme (glyphosate oxidoreductase, GOX) has been used with the CP4 EPSPS. This enzyme catalyzes the degradation of glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate, both relatively innocuous compounds. The CP4 EPSPS alone makes soybean approximately 50-fold less sensitive to glyphosate (Nandula et al. 2007) (Fig. 3.2). CP4 and GOX genes together provide about the same level of resistance to canola (Nandula et al. 2007). Despite a generally high resistance factor, there can be problems if the transgene promoter is not sufficiently active in reproductive tissue, as appeared to be case in some of the first GR cotton varieties (Pline et al. 2002a,b; Pline-Srnic 2005; Yasuor et al. 2006; Dill et al. 2008). This problem was solved by using a promoter that is stronger in reproductive tissues. A similar, but lesser problem, has been reported in some maize varieties (Thomas et al. 2004). Under some environmental circumstances, glyphosate can cause leaf damage in GR soybean (e.g., Reddy and Zablotowicz 2003), although this is a rare occurrence from which the crop eventually recovers. The degradation product of glyphosate, AMPA, is a weak phytotoxin (Hoagland 1980). Reddy et al. (2004) correlated

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Fig. 3.2 Response of glyphosate-resistant (GR) Asgrow 4603RR and nonGR HBKC 5025 soybean in the one- to two-trifoliolate leaf (22-day-old, 45-cm tall) growth stage to glyphosatepotassium 3 weeks after treatment. Mean values of nine replications are plotted. From Nandula et al. (2007)

mild phytotoxicity in glyphosate-resistant (GR) soybeans to high levels of AMPA formed from high-glyphosate-dose applications. Applications of AMPA alone that resulted in the same endogenous AMPA concentrations caused the same level of phytotoxicity. Papers have been published supporting the view that glyphosate impairs growth of GR crops under some conditions because of effects on micronutrient status (e.g., Jolley et al. 2004; Bott et al. 2008). Glyphosate chelates divalent metal cations (Duke 1988), which could limit their availability when the glyphosate levels are very high and the divalent metal cation concentration is very low. Conversely, binding of Ca2+, Mn2+, or Fe2+ can reduce the efficacy of glyphosate (e.g., Schonherr and Schreiber 2004; Bernards et al. 2005). The pH can strongly influence how tightly each cation species is chelated to glyphosate. There have been exceedingly few reports of nutrient deficiencies in the huge areas in which GR crops are grown. Considering the very disproportionate abundance between the number of molecules of glyphosate that would reach the soil and the number of divalent cations available for binding, it seems implausible that glyphosate would have a significant effect on the availability of essential minerals in GR crops. However, if a plant had a borderline mineral deficiency, chelation in vivo might cause a problem. The proper experiments have not been conducted to determine if this possibility could cause a significant problem in the field. Ozturk et al. (2007) reported that glyphosate inhibits ferric reductase in plant roots, suggesting that this effect would cause

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iron deficiency in GR crops. However, this work was carried out with glyphosatesensitive sunflower and the assay was an in vivo assay performed hours after treatment with high concentrations of glyphosate so that there was no way to determine if the effects were direct or indirect. These studies should be repeated with GR crops to answer this question. Root-associated microbes are involved in plant mineral nutrition (e.g., Jakobsen et al. 2002). Some of these microbes are glyphosate-sensitive (e.g., Moorman et al. 1992). Some of the glyphosate applied to foliage is exuded by roots (e.g., Coupland and Caseley 1979; Laitinen et al. 2007). Thus, mineral nutrition could be altered by adverse effects on root-associated microflora. However, considering that nutrient deficiency problems have not been a commonly reported problem with GR crops after more than 10 years of adoption over vast areas, the potential mineral nutrition problems reported by only very few laboratories are of questionable impact in the field. Glyphosate is toxic to Bradyrhizobium japonicum (Moorman et al. 1992). Several studies have indicated that there is potential for reduced nitrogen fixation in GR soybeans, but yield reductions due to such an effect have not been documented in the field when glyphosate is used at the label rate (Zablotowicz and Reddy 2004, 2007). The no longer used bromoxynil-resistant crops owed their resistance to a transgene of microbial origin (Klebsiella ozaenae) that converts the benzonitrile to a nonphytotoxic benzoic acid derivative (Stalker et al. 1996). This gave the crops a more than tenfold level of resistance. Bromoxynil is an older category of selective herbicide that inhibits photosynthesis by binding the D1 protein of photosystem II (Devine et al. 1993). Glufosinate is a synthetic version of the natural product, phosphinothricin. It is not accepted by organic farmers, partly because it is chemically synthesized as a racemic mixture, and the D enantiomer of the racemic mixture is not a natural compound. It acts by inhibition of glutamine synthetase, thereby causing accumulation of toxic levels of ammonium ion and indirectly stopping photosynthesis (Lydon and Duke 1999). It is considered a broad-spectrum herbicide, but is not quite as effective in some situations as glyphosate. One of the microbes that produce phosphinothricin, Streptomyces hygroscopicus, has an enzyme that inactivates phosphinothricin and glufosinate by acylating it. The gene (bar) encoding this enzyme, phosphinothricin N-acetyltransferase (PAT), is used as a transgene for glufosinate-resistant crops (Vasil 1996). In addition to the HRC use, the bar gene has been used extensively as a selectable marker.

3.2.3

Impacts on Weed Management

Nothing has had more impact on weed management in such a short time period as GR crops, except perhaps the introduction of synthetic, selective herbicides. Several factors have contributed to the strong acceptance of GR technology. A strong

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argument can be made that glyphosate is the most effective and useful herbicide available (Duke and Powles 2008b). It is a slow-acting, highly translocated, foliarly applied product that kills weeds by inhibiting a molecular target site that is ubiquitous to all plants, EPSPS (Duke et al. 2003a). Its slow action allows translocation to meristematic tissues, ensuring that all growing points, including subterranean ones, are killed. It has very good environmental safety characteristics (see Sect. 3.2.5) in that it is highly nontoxic, does not easily move to ground or surface water, and has a relatively short half-life in soil. Before GR crops, it could only be used in places without crops or with methods that avoided crop contact. GR crops opened the door to widespread utilization of this exceptional herbicide directly on field crops. In addition to the generally profound economic advantages to using GR crop/ glyphosate technology (Gianessi 2005, 2008; Clewis and Wilcut 2007), this technology simplifies weed management (Bonny 2008). The farmer can often rely on only glyphosate applied once or twice during a growing season for weed control, rather than using a complicated strategy of both soil-incorporated and foliarly applied herbicides, involving multiple herbicides with different molecular target sites. Since glyphosate is used only as a postemergence herbicide, the farmer can wait to see what kind of weed problem emergences before spraying. Tillage can often be reduced or eliminated, creating both environmental (see below) and economic benefits. The benefits of this technology are not farm size dependent. A small farmer who farms only on weekends might derive more benefits, as this technology is more forgiving than traditional methods of weed management. Thus, one does not have to hire pest management consultants for prescription recommendations in order to obtain excellent weed control at a reasonable cost. This technology is also useful to farmers who grow multiple GRCs, in that they can apply one herbicide to multiple crops. Prior to GRCs, a particular herbicide could rarely be used on more than one crop. Initially, weed management with GR crop technology was excellent, as indicated by the rapid adoption. But, the specter of the evolution of GR weeds is jeopardizing reliance on this weed control method (Powles 2008a). Although Bradshaw et al. (1997) gave reasons why the evolution of GR resistance was implausible, the first report of an evolved GR weed occurred the same year as their paper (Heap 1997). Since then, the number of cases of evolved GR resistance has grown at a steady pace all over the world (Table 3.2, Fig. 3.3), and about half of the cases have occurred in GR crops, where the selection pressure is intense (Fig. 3.3). In most of the other cases, resistance evolved in vineyards or orchards in which glyphosate was sprayed several times a year for several consecutive years. The levels of evolved resistance of weeds to glyphosate are much lower than that of GR crops, usually in the range of two- to tenfold. The most common mechanism of resistance in these evolved biotypes is reduced translocation (e.g., Lolium spp., Conyza spp.) (Preston and Wakelin 2008), although mutations in EPSPS that provide marginal resistance have also occurred in Eleucine indica (Baerson et al. 2002) and some populations of Lolium spp. (Wakelin and Preston 2006; Perez-Jones et al. 2007). In at least some evolved GR Conyza, increased numbers of EPSPS transcripts may also contribute to resistance (Dinelli et al. 2008).

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S.O. Duke and A.L. Cerdeira Table 3.2 Occurrence of evolved GR weeds by species, year, and country Species Year Location Amaranthus palmeri 2005 USA Amaranthus rudis 2005 USA Ambrosia artemisifolia 2004 USA Ambrosia trifida 2004 USA. Conyza bonariensis 2003 South Africa 2004 Spain 2005 Brazil 2006 Columbia 2007 USA Conyza canadensis 2000 USA 2005 Brazil 2006 China, Spain 2007 Czech Republic Digitaria insularia 2006 Paraguay 2008 Brazil Echinochloa colona 2007 Australia Eleusine indica 1997 Malaysia 2006 Colombia Euphorbia heterophylla 2006 Brazil Lolium multiflorum 2001 Chile 2003 Brazil 2004 USA 2006 Spain 2007 Argentina Lolium rigidum 1996 Australia 1998 USA 2001 South Africa 2005 France 2006 Spain Parthenium hysterophorus 2005 Columbia Plantago lanceolata 2003 South Africa Sorghum halepense 2005 Argentina 2007 USA Urochloa panicoides 2008 Australia Source: From the International Survey of Herbicide-Resistant Weeds: http:// www.weedscience.org/In.asp and Vila-Aiub et al. (2008)

Nature abhors a vacuum. In addition to the evolution of GR weeds, when farmers rely on glyphosate year after year, other species of weeds can fill the ecological niches vacated by the species that are easily managed with glyphosate. The process of weed species shifts in GR crop fields has been documented (e.g., Reddy 2004; Owen 2008). Some of these species have a low level of natural (not evolved) resistance, and others can avoid glyphosate by germinating later in the growing season or having a broad germination pattern throughout the season. One potential mechanism of some species that are naturally resistant is enhanced conversion of glyphosate to AMPA (Reddy et al. 2008). The advent of evolved GR weeds and shifts to naturally GR or glyphosateavoiding weeds has made the GR crop/glyphosate weed management package less

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Fig. 3.3 Incidence of species that have evolved resistance to glyphosate by year

effective in some locales. Application rates and number of applications of glyphosate in GR crops have increased. Herbicides, in addition to glyphosate, are being used with GR crops. New herbicide-resistant crops are being developed to have resistance to glyphosate, plus resistance to another herbicide or class of herbicides that might be effective on weeds that are not controlled by glyphosate. Other options for coping with GR weeds and weed shifts are resistance management strategies that involve approaches such as alternation of herbicides, utilization of alternative/or residual herbicides in conjunction with glyphosate, and use of cultivation (e.g., Gustafson 2008; Neve 2008; Werth et al. 2008). The effects of HRCs on weed management is in a constant state of flux because of weed dynamics, economics, and changes in the availability of both old and new weed management tools. GR crops have been a valuable asset for weed management and will probably continue to be, even as GR weeds evolve and weed species shift. The effective longevity of this technology will depend, in part, on how wisely it is used (Powles 2008a).

3.2.4

Impacts on Food and Feed

One of the concerns of those opposing transgenic crops is that the transgene will alter the quality and/or safety of the consumable part of the plant. This could occur through the protein from the transgene being toxic, through a metabolic product of

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the enzyme encoded by the transgene being toxic, through pleiotropic effects of the transgene, through alteration of expression of nontransgenic genes by the position of the transgene in the genome or through more indirect effects. For regulatory approval, transgenic crops are scrutinized to a far greater level than conventional crops using analytical, nutritional, and toxicological methods (Atherton 2002; Malarkey 2003; Ko¨nig et al. 2004), although some have proposed that even more extensive tests be done by metabolomic, proteomic, and transcriptomic analysis to detect potential unintended effects of the transgene and its insertion on food safety and quality (Kuiper et al. 2002; Cellini et al. 2004). In a safety evaluation for the CP4 EPSPS enzyme introduced into soybean to provide glyphosate resistance, Harrison et al. (1996) found the protein to be: (1) nontoxic to mice when consumed at doses thousands of times higher than potential human exposure; (2) readily degraded by digestive fluids; and (3) not structurally or functionally related to any known protein allergens or toxins, based on amino acid sequence homology searches. The potential allergenic properties of the protein products of transgenes must be determined before approval. These data are provided to regulatory agencies, but publications on this topic are scarce. However, there are a few published studies showing no allergenic properties of transgene products associated with HRCs. Sten et al. (2004) in a study with soybean-sensitized patients, found that the allergenicity of 10 GR and eight nonGR soybean cultivars were not different. Chang et al. (2003) found no significant allergenicity to rats of the CP4 EPSPS gene product conferring glyphosate resistance. On the basis of both an analysis of published literature and experimental studies, Herouet et al. (2005) concluded that there is a reasonable certainty of no harm resulting from the inclusion of the gene for glufosinate resistance in human food or in animal feed. Most of the tests have simply examined HRCs for equivalence in food quality to nonHRCs. Health Canada’s review of the information, presented in support of the food use of refined oil from glufosinate-resistant canola line HCN92, concluded that such refined oil does not raise concerns related to safety. Health Canada is of the opinion that refined oil from canola line HCN92 is as safe and nutritious as refined oil from the current commercial varieties (http://www.hc-sc.gc.ca/foodaliment/mh-dm/ofb-bba/nfi-ani/e_nf7web00.html). The nutritional properties of glufosinate-resistant sugar beets and maize grains were found to be essentially equivalent to nontransgenic cultivars in feeding studies with swine and ruminants (Daenicke et al. 2000; Bohme et al. 2001); similar results have been produced with glufosinate-resistant rice in swine feeding studies (Cromwell et al. 2005). Studies with GR maize line GA21 evaluated the compositional and nutritional safety of maize line GA21 compared to that of conventional maize (Sidhu et al. 2000). Compositional analyzes were conducted to measure proximate, fiber, amino acid, fatty acid, and mineral contents of grain and proximate, fiber, and mineral contents of forage collected from 16 field sites over two growing seasons. No significant differences were found. Similarly, Tutel’ian et al. (2001) found no compositional differences between conventional maize and maize line GA21. The nutritional safety of maize line GA21 was also evaluated by Sidhu et al. (2000) in a

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poultry feeding study. Results from the poultry feeding study showed that there were no differences in growth, feed efficiency, adjusted feed efficiency, and fat pad weights among chickens fed with GA21 grain or with parental control grain. These data taken together demonstrate that GR GA21 maize is as safe and nutritious as conventional maize for food and feed use. The occurrence of mycotoxins, specifically aflatoxin, contamination is of concern with maize cultivation in warm climates subject to preharvest moisture stress. Studies by Reddy et al. (2007) indicated increased incidence of aflatoxin contamination in GR compared to conventional maize in 1 year out of four. But, in 1 year out of four, fumonisin levels were higher in nonGR maize than in GR maize. The observed effects were thought to be due to differences in how the crops were grown, rather than due to glyphosate or the transgene. Several other studies have found no substantial difference in the nutrient content of GR and nontransgenic crops. These studies include maize (Ridley et al. 2002; Autran et al. 2003), soybean (Padgette et al. 1996), wheat (Obert et al. 2004), and cotton (Nida et al. 1996). In the Autran et al. (2003) study, the characteristics of glyphosate- and glufosinate-resistant maize in different foods (e.g., beer, hominy, oil, grits) were compared and found not to be substantially different from the respective, nontransgenic parental lines. Lappe et al. (1999) reported reductions of isoflavone levels on GR soybean varieties in the absence of glyphosate (i.e., a pleiotropic effect of the CP4 gene). However, this study was not done by comparing isogenic lines. Padgette et al. (1996) found no effects of the transgene on isoflavone content of soybean. Glyphosate targets the shikimate pathway (Duke et al. 2003a), and the estrogenic isoflavones of soybeans are products of this pathway. Glyphosate resistance from the CP4 EPSPS gene is not always complete (Pline et al. 2002a,b), and glyphosate preferentially translocates to metabolic sinks such as seeds (Duke 1988). Therefore, we reasoned that at relatively high and late applications of glyphosate to GR soybeans, a reduction of the content of these compounds could occur. In a wellreplicated field study at two sites, hundreds of kilometers apart, no significant effects of glyphosate on isoflavones were found at the highest and latest legal application rates (Duke et al. 2003b). Table 3.3 summarizes most of the published results of animal feeding studies with GR crops. All the studies support the view that food from GR crops is substantially equivalent to nontransgenic crops. In addition to these studies, no evidence of the CP4 gene or its protein product could be detected in pork from swine fed with GR soybean meal (Jennings et al. 2003). No effects on GR soybeans could be found on the immune system of mice (Teshima et al. 2000). HRCs, being highly resistant to the herbicide to which they have been made resistant, could also contain levels of the herbicide or its metabolite(s) that exceed the legal tolerance levels. Surprisingly, little has been published on herbicide residues in HRC foods. Most of what we know is from studies with nonHRC crops. However, herbicide residue data must be supplied for regulatory approval of HRCs. Glyphosate acid and its salts are moderately toxic compounds in EPA toxicity class II. Glyphosate (either the anion or the isopropylamine salt) is practically

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Table 3.3 Some results of animal feeding studies with glyphosate-resistant crops Crop Animal Result Reference Maize Rat No effect Hammond et al. (2004) No effect Healy et al. (2008) Maize Swine No effect Hyun et al. (2004) Maize Cattle No effect Erickson et al. (2003) Maize Dairy cattle No effect Donkin et al. (2003) No effect Ipharraguerre et al. (2003) No effect Grant et al. (2003) Maize Poultry No effect Sidhu et al. 2000 Soybean Rat No effect Zhu et al. 2004 No effect Hammond et al. (1996) No effect Appenzeller et al. (2008) Soybean Mice No effect Brake and Evenson (2004) Soybean Swine No effect Cromwell et al. (2002) Soybean Dairy cattle No effect Hammond et al. (1996) Soybean Catfish No effect Hammond et al. 1996 Soybean Poultry No effect Hammond et al. (1996) No effect Taylor et al. (2007) Canola Rainbow trout No effect Brown et al. (2003) Canola Poultry No effect Taylor et al. (2004) Alfalfa cattle No effect Combs and Hartnell (2008) Sugarbeet Sheep No effect Hartnell et al. (2005)

nontoxic by ingestion, with a reported acute oral LD50 of >5,000 mg kg
1 in the rat (Vencill 2002). The trimethylsulfonium salt of glyphosate is more toxic, with an oral LD50 of about 705 mg kg
1. It is not a restricted use pesticide and is a bestselling weed killer for home use. Animals do not contain the herbicide molecular target site (EPSPS) of glyphosate. Occasional reports of severe effects of ingestion of formulated glyphosate occur (e.g., Stella and Ryan 2004); however, the glyphosate molecule itself is considered one of the most toxicologically benign herbicides available. Williams et al. (2000) extensively reviewed the toxicology literature on glyphosate and its metabolites and concluded that under present and expected conditions of use, glyphosate does not pose a significant health risk to humans. In a testing program to detect whether GR soybeans had been sprayed with glyphosate or not, Lorenzatti et al. (2004) found glyphosate and AMPA in green, immature seeds. Both glyphosate and its degradation product, AMPA, were found in mature, harvested seeds of different GR soybean varieties grown in widely separated geographical regions (Duke et al. 2003b). Even though the glyphosate applications were at legal, but at relatively high rates and late timing, the residues were within the established tolerance levels. We were surprised to find higher AMPA than glyphosate levels since at that time plants were thought to degrade glyphosate very little, if at all (Duke 1988; Duke et al. 2003a). Subsequently, we found that some legume plant species readily convert glyphosate to AMPA (Reddy et al. 2008). In a study conducted earlier, but published later (Arregui et al. 2004), similar levels of glyphosate were found in harvested seed of GR soybeans, but these

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scientists found much lower AMPA levels. We have found no publications on glyphosate residues in GR crops other than soybean. Glufosinate is not a restricted use pesticide and is sold for home weed control in the USA. Its acute oral toxicity in rats is an LD50 of ca. 2.2 g kg
1. Glufosinate chemically resembles glutamine, a molecule used to transmit nerve impulses in the brain. Ebert et al. (1990) concluded in an extensive review that glufosinate is safe under conditions of recommended use. Similarly, Hack et al. (1994) also concluded from their studies that glufosinate is unlikely to cause health effects of either users or consumers when used as directed, although the herbicide target site, glutamine synthetase, is also found in animals. In a study to determine if glufosinate applied to glufosinate-resistant maize and canola could lead to an increase in herbicide residues or to the formation of new metabolites, Ruhland et al. (2004) found that L-glufosinate was in the form of known metabolites and the parent compound in both maize and canola. The highest content was in the leaves, and the lowest in the grains. No levels were found above the established tolerance levels. A last, but understudied, aspect of food quality and HRCs is their influence on contamination of food with poisonous weed seeds. Weed seeds can be the sources of toxic compounds (e.g., Powell et al. 1990). HRCs are generally more weed-free than conventional crops, resulting in less foreign matter, including weed seeds, in the harvested product (Canola Council of Canada 2001; Shaw and Bray 2003). Therefore, there is less likelihood of significant contamination of harvested food with toxic weed seeds in HRCs than with conventional crops.

3.2.5

Environmental Impacts

There are numerous possible environmental effects of HRCs. These can be either positive or negative. They can be associated with the transgene or with the herbicide to which the transgene is linked. But, ultimately, potential environmental impacts of HRCs must be compared with the impacts of the technologies that they replace. All the published studies and analyses of this type have found that the environmental benefits of substituting HRCs for conventional crops are usually substantial (e.g., Wauchope et al. 2002; Nelson and Bullock 2003; Bennett et al. 2004; Amman 2005; Brimner et al. 2005; Brookes and Barfoot 2006; Cerdeira and Duke 2007; Cerdeira et al. 2007; Kleter et al. 2007, 2008; Devos et al. 2008; Gardner and Nelson 2008; Shiptalo et al. 2008). Of course, the potential benefits vary with the HRC, the geographic location of use, the way the farmer uses the HRC, and different components of environmental impact. Since nature is not static, the environmental impact will change with time as farmers using HRC technology adjust their methods to deal with changing weed and other problems. Whether HRCs have increased or decreased herbicide use in terms of the amount of material used per hectare of crop can be argued either way, depending on many factors. However, in terms of environmental impact this factor is not useful, as the

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relative toxicity and environmental fate of glyphosate or glufosinate compared with the herbicides that they replace in HRCs can be large. Glyphosate and glufosinate have relatively low mammalian toxicity (Ebert et al. 1990; Giesy et al. 2000; Williams et al. 2000), compared to many of the herbicides that they can replace when used with transgenic crops. Glyphosate, in particular, has lower acute toxicity than aspirin or many other commonly ingested compounds. In a study, using acute mammalian toxicity data, Gardner and Nelson (2008) compared the number of LD50 doses per unit area that were decreased by GR crops in the United States. Depending on the crop and the location, they calculated that conventional weed management with other synthetic herbicides could result in as much as 3,000 more LC50 doses per hectare with maize, more than 375 with cotton, and more than 90 with soybean. An example of some of their data with cotton is provided in Fig. 3.4. They concluded that GR technology has a positive environmental effect. In a European study, Devos et al. (2008) found that use of GR maize and glufosinate-resistant maize would lower the pesticide occupational and environmental risk or weed management. The main benefits were due to the lower potential of these chemicals to contaminate groundwater and their lower acute toxicity. Glyphosate gave slightly greater reductions than glufosinate. Their calculations were done with the assumption that other herbicides would not be used with glyphosate nor glufosinate. However, glyphosate is increasingly used with other

Fig. 3.4 Increase in LD50 doses per hectare of herbicides needed if GR cotton were not available in the US. From Gardner and Nelson (2008)

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herbicides in GR crops because of evolved glyphosate resistance and shifts to naturally resistant weed species. Neither glyphosate nor glufosinate are problematic pesticides in the context of surface and groundwater contamination of the herbicides that they replace (reviewed by Duke and Cerdeira 2005; Cerdeira and Duke 2006; Borggaard and Gimsing 2008). Glyphosate does not move well in soil because of its strong sorption to soil minerals (e.g., Mamy and Barriuso 2005). Furthermore, in most soils, it degrades more rapidly than the herbicides that it replaces (e.g., Mamy et al. 2005). In addition to microbial degradation, both glyphosate and AMPA can undergo nonbiological degradation in soil (Barrett and McBride 2005), although the relative contribution of the two types of processes is unknown. Concern over AMPA contamination of groundwater has recently been expressed (Mamy et al. 2005, 2008). AMPA is more persistent than glyphosate in the environment, and it more readily leaches in soil (Mamy et al. 2008; Simonsen et al. 2008). Both glyphosate and AMPA are leached from soil containing high phosphate levels than soil with low levels (Simonsen et al. 2008). The relative toxicities of AMPA versus contaminants from conventional herbicide use have not been adequately analyzed. Fuel utilization associated with weed management has been reduced by GR crops because of reduced tillage and fewer trips across the field to spray herbicides (reviewed by Cerdeira and Duke 2006). Bennett et al. (2004) estimated that there would be a 50% fossil fuel savings in growing sugar beet in Europe by switching to GR crops. Brookes and Barfoot (2006) estimated that GR crop use in 2005 worldwide reduced carbon emissions approximately the same as the removal of four million family automobiles from the road. Effects of the herbicides used with HRCs on nontarget organisms are a concern. Obviously, since glyphosate is a herbicide, glyphosate drift to nontarget plants can be harmful (e.g., Bellaloui et al. 2006). This is not a new problem, as there have been problems with herbicide drift since the advent of herbicides that are sprayed. Concentrations reaching nontarget plants are generally only a small fraction of the recommended dose for weed control. At very low doses that one might expect with drift, herbicides can often stimulate growth, activate host defense systems against pathogens, or enhance nitrogen utilization, depending on the herbicide (reviewed by Duke et al. 2006). The phenomenon of a stimulatory effect at subtoxic concentration of a toxin is termed hormesis. Glyphosate clearly enhances growth of plants at very subtoxic application rates (e.g., Schabenberger et al. 1999; Wagner et al. 2003; Cedergreen et al. 2007; Velini et al. 2008). But, at least in barley, the effect is not sustained over time (Cedergreen 2008). The mechanism(s) of herbicide-caused hormesis is poorly understood and may well differ between herbicides with different molecular target sites. Perhaps, the greatest damage done by conventional agriculture, other than taking land out of its natural state, has been caused by tillage. The primary reason for tillage has been weed management. Glyphosate use in GR crops has resulted in significantly reduced tillage, especially in soybean and cotton (Fig. 3.5) (American Soybean Association 2001; Penna and Lema 2003; Dill et al. 2008; Locke et al. 2008;

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Powles 2008b). Even in some nonGR crops, reductions in the price of glyphosate and the increases in the cost of diesel fuel have favored conservation tillage rather than traditional tillage (Nail et al. 2006). Reduced tillage sometimes reduces soil compaction (Koch et al. 2003; Shukla et al. 2003). To our knowledge, this aspect of the influence of HRCs has not been studied. However, as both evolved and naturally resistant GR weeds increase, some farmers of GRCs are returning to occasional tillage for more complete weed management. There is concern about the potential of HRCs to create new weed problems, either themselves becoming a weed or the HR transgene escaping to relatives, either feral crops or related species, to create new weed problems. Gene flow to native populations of species with which the HRC can crossbreed could result in unwanted agricultural and/or environmental effects. The HR transgene confers no advantage where the matching herbicide is not sprayed, so the HR crop is no more likely to invade a natural habitat than the nonHR crop. None of the crops that have been made herbicide resistant with transgenes are crops that become weeds outside of agricultural fields. However, HRCs are sometimes problems in agricultural fields in which the same herbicide is used in

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subsequent years with a different HRC. This has already been a problem with GR crops (e.g., York et al. 2004; Soltani et al. 2006; Beckie and Owen 2007), requiring the use of herbicides other than glyphosate to control the “volunteer” GR crop. Gene flow to nontransgenic crops of the same species has been a commercial and political problem, but not an environmental threat. Organic farmers cannot retain the organic status of their crops if transgene presence is above a set limit, nor can crops be sold to markets that require the product to be nontransgenic if transgenic occurrence is above the level set the regulatory jurisdiction. For crops like soybean, outcrossing is not a problem, but considerable outcrossing can occur with maize, rice, sugar beet, and canola. Substantial gene flow can occur between HR and nonHR canola (e.g., Hall et al. 2000; Reiger et al. 2002; Mallory-Smith and Zapiola 2008). Outcrossing may account for the contamination of nontransgenic rice with the glufosinate-resistant gene (Vermij 2006), even though glufosinate-resistant rice has only been grown experimentally. To our knowledge, no herbicide-resistant transgenes have been found to be a problem in nonherbicide-resistant maize, although there is significant potential for this to happen (Allnutt et al. 2008). There has been considerable controversy about whether Mexican maize landraces are contaminated with transgenes (reviewed by Cerdeira and Duke 2006). No gene flow from transgenic to nontransgenic cotton has been reported in cultivated fields, but it should occur because of insect pollination. Gene flow from GR alfalfa to organic alfalfa was the ostensible reason for its re-regulation. Flow of a GR transgene from bentgras being evaluated for commercial use to nontrangenic bent grass occurred rapidly, and mitigation efforts have not been effective (MallorySmith and Zapiola 2008; Zapiola et al. 2008). A much bigger concern is the potential effect of gene flow from HRCs to weedy relatives. The only environmental aspects of transgenic crops that are not also associated with nontransgenic crops are those associated with the transgenes. HR transgenes offer no advantage in natural ecosystems where the herbicide is not used, but when coupled with transgenes imparting traits that would improve fitness in a natural ecosystem (e.g., insect or drought resistance), the HR trait would improve the likelihood of introgression of the gene into the unintended recipient species (see below). Once a transgene escapes to another species, it is unlikely that it could be eliminated in the population by human efforts. Indeed, removal of a GR transgene from a nontransgenic population of bentgrass has not been accomplished with the mitigation methods used (Zapiola et al. 2008). Gene flow or introgression involves the movement of a gene or genes into a sexually compatible species. This type of gene flow is also termed vertical gene flow. The first generation is generally not very fit (e.g., Scheffler and Dale 1994), but subsequent backcrosses to the noncrop species will eventually fully introgress the transgene into this species. Yearly spraying of the herbicide to which the transgene confers resistance should facilitate the process by selecting only crosses with the transgene and removing competing weeds to give the unfit F1 and usually the F2 generations a significant survival advantage. Transmission of HR transgenes could make weedy relatives of the HRC much more problematic for the farmer. This has not happened with soybean, cotton, and

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maize, presumably because there are few or no weedy species with which they are sexually compatible in the places in which they are grown. Canola outcrosses with several weedy related Brassica species, including related Brassica crops (e.g., cabbage, cauliflower, broccoli, etc.). These crosses are generally quite unfit, but introgression of a GR transgene from canola to its weedy relative, bird rape (Brassia rapa) has occurred in field situation, and the introgressed gene appears to be stable in the population, even in the absence of spraying glyphosate (Warwick et al. 2008). A potential exists for a transgene to move from through compatible relative (a bridge species) to a third Brassica species to increase the means by which a GR gene could spread (Brown and Brown 1996). Work by Darmency et al. (2007) indicates that gene flow from sugar beets to weedy beets is sufficient to cause problems for the farmers growing HR beets within a few years if herbicides are not rotated. However, the degree of outcrossing that they measured varied considerably between lines of transgenic beets, years, and field locations. Rice outcrosses readily with feral rice (e.g., red rice), as well as weedy related species. Nontransgenic, imidazolinone (IMI) herbicide-resistant rice created by selection in cell culture (Tan et al. 2005) was commercialized in the USA in 2002. Outcrossing to produce IMI-resistant weeds occurs (e.g., Shivrain et al. 2007). The incidence of IMI-resistant weeds in rice is growing in locations where IMI-resistant rice is grown (Valverde 2007), although whether this is due to evolved resistance or gene flow is unclear in most cases, because IMI resistance evolves quickly in some species when exposed to this herbicide class (Heap 2008). We can expect that gene flow from rice to feral rices and to sexually compatible species will occur with any transgene, including those imparting herbicide resistance. Keeping HRCs out of areas in which sexually compatible species exist is a daunting task. For example, transgenic, GR and glufosinate-resistant canola are not approved as crops in Japan, but the seed can be imported for processing. Saji et al. (2005) found these HRCs growing along routes to processing plants in Japan. There is considerable literature on strategies and technologies for mitigating or eliminating vertical gene flow (reviewed by Gressel 2002; Cerdeira and Duke 2006). Much of this literature deals with simply reducing movement of the genes to plants other than the transgenic crop (e.g., Devos et al. 2005). However, if the gene would pose a potential problem in a natural environment, a fail-safe approach would be preferable. Strategies coupling one or more technologies such as genetic use restriction technology genes to make transmission of a functional transgene impossible (Oliver et al. 1998) or linking the HR transgene to one that would confer unfitness to a wild plant (e.g., genes that prevent shattering or dormancy; Al-Ahmad et al. 2004; Gressel and Al-Ahmad 2004) have the potential for fulfilling this need. In the latter approach, the HR gene(s) should be in the same construct as the unfitness gene(s) to keep the genes from segregating. Lastly, some have voiced concern that there could be gene flow from HRCs and other transgenic crops to totally unrelated organisms (horizontal gene transfer), especially soil or gut microbes (e.g., Bertolla and Simonet 1999; Giovannetti 2003), from degraded HRCs in the field or through ingestion of food composed of GRCs.

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Almost all of the transgenes currently used for HRCs are from soil microbes. These genes are much more likely to be transferred to unrelated microbes from the natural, microbial sources than from HRCs (Kim et al. 2005). Levy-Booth et al. (2008) found that degradation of the cp4-epsps transgene from GR soybean leaf material in the soil was rapid, but could still be detected in soil after 30 days. Degradation of the transgene and a natural soybean gene in soil were similar. Dale et al. (2002) concluded that there is no compelling argument that there would be any more likelihood of such gene transfer transgenic crops than from nontransgenic crops. There has so far been no credible evidence of gene transfer from transgenic crops to microbes (Dunfield and Germida 2004), although some have criticized detection methods (e.g., Nielsen and Townsend 2004). Nevertheless, after 14 years of growing these crops on huge areas, there have been no reports of transgenes being transferred to microbes of any type.

3.2.6

Herbicide-Resistant Crops and Crop Disease

There are some untended benefits of GR crops and perhaps glufosinate-resistant crops. Both glyphosate and glufosinate are fungitoxic (reviewed by Duke et al. 2007). Thus, when used at full application rates, these herbicides may, under some circumstances, be providing sufficient protection from plant pathogens to prevent crop damage or to preclude spraying with a fungicide. Perhaps, the most carefully studied example is that of glyphosate effects of Asian rust in GR soybeans (Feng et al. 2005, 2008), in which glyphosate applications to GR soybeans reduced rust infection and damage, both as a preventative and a curative (Fig. 3.6) treatment. However, in many field situations, the optimal timing of glyphosate application for effective weed management is unlikely to also be most effective for rust control (Bradley and Sweets 2008). The effects of glyphosate on reducing this disease will % ASR incidence (N=15)

a

b

Water 1DAI Fungicide A 1DAI Glyp1x 1DAI Glyp1x 3DAI Glyp1x 6DAI

60 40

Surf 1DAI Water 1DAI Glyp.5x 3DAI Glyp1x 3DAI Glyp2x 3DAI

60 40

20

20

0 0

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40

0

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10 20 30 Days After Inoculation

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Fig. 3.6 (a) Curative activity for Asian rust in glyphosate-resistant soybeans sprayed with glyphosate (Glyp, 1 at 0.84 kg AE ha
1) at 1, 3 or 6 days after inoculation (DAI), water and fungicide A (carbendazim + flusilazole) at 1 DAI. (b) Curative activity as a function of glyphosate dose (0.5, 1 or 2) from spray application at 3 DAI, surfactant (1 g L
1 MON 0818) or water alone at 1 DAI. From Feng et al. (2008)

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Table 3.4 Reports of glyphosate interactions and lack of interactions with plant disease in glyphosate-resistant crops Crop Disease Effect References Soybean Phakopsora pachyrhizi Reduces Feng et al. (2005, 2008) Fusarium spp Increases Kremer et al. (2005) S. sclerotiorum No effect Lee et al. (2003) Increases Nelson et al. (2002) F. solani Increases Sanogo et al. (2001), Nijiti et al. (2003) Cotton Rhizoctonia solani Reduces Pankey et al. (2005) Wheat Puccinia triticina Reduces Feng et al. (2008), Anderson and Kolmer (2005) Sugarbeet Rhizoctonia solani Increases Larson et al. (2005) Fusarium oxysporum Increases Larson et al. (2005)

probably increase as glyphosate use increases, both in application rates and number of applications, because of the evolution of GR weeds and weed species shifts to more naturally GR weeds. On the other hand, glyphosate treatment is known to predispose nontransgenic plants to plant disease by more than one mechanism associated with the mechanism of action of glyphosate as a herbicide (reviewed by Duke et al. 2007), although these mechanisms should not exist in GR crops. There are many reported cases of exacerbation of plant disease symptoms with glyphosate, but these studies have generally been done with glyphosate-susceptible plants (reviewed by Duke et al. 2007). Nevertheless, there are a few reports of this phenomenon in GR crops (Table 3.4). Likewise, there are reports of reduced crop disease with glyphosate (Table 3.4). Thus, the interaction of glyphosate, fungal plant diseases, and GR crops is variable, depending on the crop, the disease, and perhaps the timing of herbicide application and infection. Also, differences in cultural practices between HRCs and nontransgenic crops can influence plant disease (e.g., Lee et al. 2005). Glyphosate effects on root exudates and soil moisture can influence root-borne soybean diseases (Kremer et al. 2005; Means and Kremer 2007; Means et al. 2007). Larson et al. (2005) found the that several factors, including the disease isolate, whether or not the crop was sprayed with glyphosate, and the variety of GR sugar beet determined whether there was increased disease with the GR crop. In a recent review, Powell and Swanton (2008) concluded that there was insufficient information from realistic field studies to determine whether glyphosate influences Fusarium spp. diseases in GR crops.

3.3 3.3.1

Coming Herbicide-Resistant Crops Transgenes for Herbicide Resistance

Many transgenes have been reported and/or patented for the production of HRCs. Only a few of them are listed in Table 3.5. Technology is at a stage at which resistance to any herbicide can be imparted by transgene technology. Yet, only

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Table 3.5 Some of the transgenes that have been used for making crops resistant to herbicides or classes of herbicides Herbicide or herbicide class Gene source and gene product References 2,4-D Microbial degradation enzyme Llewellyn and Last (1996), Bisht et al. (2004) Asulam Resistant microbial dihydropteroate Surov et al. (1998) synthase Dalapon Microbial degradation enzyme Buchanan-Wollaston et al. (1992) Dicamba Microbial degradation enzyme Behrens et al. (2007), Herman et al. (2005) Hydroxyphenylpyruvate Microbial, herbicide-resistant HPPD Matringe et al. (2005) dioxidase (HPPD) inhibitors Paraquat Chloroplast superoxide dismutase Sen Gupta et al. (1993) Phenmedipham Microbial degradation enzyme Streber et al. (1994) Phytoene desaturase (PDS) Genes from microbes and the aquatic Sandmann et al. inhibitors weed Hydrilla encoding resistant PDS (1996), Arias et al. (2005) Protoporphyrinogen oxidase Resistant microbial PPO and resistant Li and Nicholl (2005) (PPO) inhibitors Arabidopsis thaliana PPO

three types of HRCs have been marketed; crops resistant to bromoxynil, glufosinate, and glyphosate, utilizing only five transgenes. Considering the huge economic impact of the GR crops, one would expect that other HRCs would have been marketed. Considerable effort and expense was put into development of HRCs that resist both hydroxyphenylpyruvate dioxygenase- and protoporphyrinogen oxidase-inhibiting herbicides (Li and Nicholl 2005; Matringe et al. 2005), yet the decision was apparently made to postpone or terminate plans to commercialize these HRCs. Devine (2005) attributed the few HRCs making it to the market place to the high cost of developing and getting regulatory approval of HRCs. Further complications are international trade issues and the impact on the existing registration of the herbicide to which they are made resistant. Realistically, none of the herbicides on the market to which crops can be made resistant offer the advantages of glyphosate (Duke and Powles 2008b) or perhaps even glufosinate, both relatively safe, broad-spectrum herbicides. In addition to the few number of herbicides used (only two), the number of crops represented among all the currently available HRCs is few (Table 3.1). This is partly because the cost of obtaining approval of HRCs is considered too great for minor crops (Devine 2005). However, this does not explain the absence of GR wheat and rice and glufosinate-resistant wheat, where the market could be very large, worldwide. GR wheat was almost marketed in North America, but concern over acceptance of wheat products from this source in Europe prevented it being marketed. A similar concern slowed the acceptance of GR sugar beets until 2008. The horrendous problems with outcrossing of rice with weedy feral and other species of may be related to GR rice being unavailable.

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Table 3.6 Recently deregulated HRCs (http://www.aphis.usda.gov/brs/not_reg.html) that are not yet on the market and HRCs that have recent approval for field-testing in the USA (http://www.isb. vt.edu/cfdocs/fieldtests1.cfm) Crop Herbicide(s) Company Resistance gene(s) Petition for deregulation Glyphosate acetyltransferase and a modified Soybeana Glyphosate and ALS Pioneer inhibitors soybean ALS Cotton Glyphosate and Bayer Crop Maize EPSPS modified by site-directed glufosinate Sci. mutagenesis and bar gene Maize Glyphosate and ALS Pioneer Glyphosate acetyltransferase and a modified inhibitors maize ALS Approval for field-testing Alfalfa Glyphosate/ sulfonylureas Soybean Glyphosate/dicamba Glufosinate/dicamba Glyphosate Glyphosate/isoxazole Not available Glufosinate Not avaiable Maize Glyphosate Bentgrass Glufosinate

Pioneer Monsanto Monsanto Pioneer MS

Glyphosate acetyltransferase and a modified plant ALS CP4 EPSPS and a demethylase CP4 EPSPS and a demethylase Glyphosate acetyltransferase Technologies

MS Pioneer HybriGene, LLC

Technologies Glyphosate acetyltransferase Phosphinothrichin acetyl transferase

a

Deregulation approved

Many types of HRCs have gotten to the point of being field-tested, but few have proceeded to be deregulated (approved for commercial use). Table 3.6 lists products for which deregulation has been applied, as well as recently approved applications for field-testing in the USA. This information on the USDA/APHIS website provides the best indication of what new products are in the offing. Considering that many of these crops have glyphosate resistance, we consider these products separately.

3.3.2

Glyphosate-Resistant Crops

Considering the enormous success of GR crops and the fact that glyphosate is now a generic herbicide, other companies have discovered or created new glyphosateresistant transgenes and, in at least one case, are going forward with the commercialization process. This crop uses an artificially evolved glyphosate-resistant gene (Castle et al. 2004; Siehl et al. 2005). A gene from the soil bacterium Bacillus licheniformis, which encoded a weak glyphosate N-acetyltransferase (GAT) was put through 11 iterations of gene shuffling to increase its activity by almost four orders of magnitude. Properties of the resultant GAT are described by Siehl et al. (2005, 2007). Plants made resistant to glyphosate with this transgene were

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ca. 100-fold more resistant to glyphosate than to nontransgenic lines (Green et al. 2008). Other glyphosate-inactivating enzymes are apparently encoded by genes of soil microbes, because there are other routes of degradation or inactivation. For example, a C-P lyase that converts glyphosate to inorganic phosphate and sarcosine is found in several bacteria, including Arthrobacter spp., Rhizobium spp., and Pseudomonas spp. (Kishore and Jacob 1987; Liu et al. 1991; Dick and Quinn 1995). Not all bacterial C-P lyases will degrade glyphosate (White and Metcalf 2004). A good C-P lyase transgene for use in GR crops has apparently not yet been developed. A microbial transgene-encoded EPSPS with some properties that might be superior to that used in commercialized GR crops is available (Vande Berg et al. 2008). Thus, transgenes for glyphosate resistance are available for companies that do not have GR crops.

3.3.3

Other Herbicide-Resistant Crops

Crops with both the GAT gene and a gene for resistance to acetolactate synthase inhibitors are in the final stage of development (Green et al. 2008), including testing for food safety (Table 3.6) (McNaughton et al. 2007). Monsanto is developing a dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance trait (Table 3.6) that detoxifies this herbicide by demethylating it (Herman et al. 2005; Behrens et al. 2007). Dow Agroscience has developed HRCs with resistance to broadleaf-killing auxinic herbicides like 2,4-D [(2,4-dichlorophenoxy)acetic acid] and grass-killing aryloxyphenoxypropionate herbicides such as diclofop (()-2-[4-(2,4-dichlorophenoxy) phenoxy]propanoic acid). These crops are apparently being field-tested, but there is no specific information on the APHIS website as to what genes are being used. Thus, we have left them out of Table 3.6. A recent abstract mentions that a transgene encoding an a-ketoglutarate-dependent dioxygenase is used to confer resistance to these two herbicide classes (Simpson et al. 2008). A patent had been filed for such a unique bacterial (Ralstonia eutropha)-derived transgene by Dow Agroscience in 2005 (Wright et al. 2005).

3.4

The Future of HRCs

The future of HRCs will be influenced by the development of other approaches to weed management. Availability of acceptable new technologies that are economically competitive with HRCs may eventually stem their rapidly increasing adoption. There are other strategies for using transgenes for weed management (Gressel 2002; Duke 2003, 2006) including enhancing crop competitiveness and allelopathy, and enhancement of weed biocontrol agents. There is little likelihood of these approaches having a significant effect in the next decade, but ultimately they could have substantive impacts.

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To some extent, regulatory requirements regarding environmental impacts of weed management methods will affect this scenario. The increasing costs of meeting the requirements for deregulation of transgenic crops and the conflicting regulatory processes in different parts of the world have hampered the adoption of HRCs. Eventual harmonization of this process could benefit this technology. There are many other factors that could have an impact on the future of HRCs, such as the amount of biofuel crops that will be grown (Baylis 2008). Since these crops are not meant for human consumption, and, in some cases, could not be used for human or animal consumption, the regulatory requirements for deregulation of HRC biofuel crops may be simplified and less costly to fulfill. Indeed, GR sugarcane is being developed in Brazil (http://monsanto.mediaroom.com/index. php?s = 43&item = 656). Still, the issue of herbicide resistance management and gene flow will be as important for these crops as for those meant for human and livestock consumption. Agriculture has clearly found HRCs to be of great value, but their utility is being eroded by the evolution of GR weeds and weed species shifts in GRCs. Both of these processes are the result of over reliance on this highly effective technology. Some of the new HRCs that are to be introduced will help cope with this situation, but we see overreliance on glyphosate continuing to undermine its effectiveness. The unique properties of glyphosate as a herbicide (Duke et al. 2003a; Duke and Powles 2008b), almost necessitate that its utility be preserved for future harvests (Duke and Powles 2008a; Powles 2008a). It will be shame to see the evolution of GR weeds continue unabated, as the strategies for preventing it are well known. Finally, the future of HRCs has been and may continue to be affected by public opinion. For example, transgenic crops have made meager inroads in Europe for this reason. Even though Europeans grow almost no transgenic crops, they buy substantial amounts of transgenic crops grown elsewhere for animal feed. At the time of this writing, this situation does not appear to be changing significantly. In countries such as Australia and those of South America, resistance to transgenic crops has waned considerably and adoption is increasing rapidly. We expect available HRCs to be almost universal outside of Europe and perhaps a few small areas of resistance within a few more years.

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modified corn line MON 810 resistant to European corn borer and line GA 21 resistant to glyphosate: a chemical study. Voprosy Pitaniia 70:25–27 Valverde BE (2007) Status and management of grass-weed herbicide resistance in Latin America. Weed Technol 21:310–323 Vande Berg BJ, Hammer PE, Chun BL, Schouten LC, Carr B, Guo R, Peters C, Hinson TK, Beilinson V, Shekita A, Deter R, Chen Z, Samoylov V, Bryant CT, Stauffer ME, Eberle T, Moellenbeck DJ, Carozzi NB, Koziel MG, Duck NB (2008) Characterization and plant expression of glyphosate-tolerant enolpyruvylshikimate phosphate synthase. Pest Manag Sci 64:340–345 Vasil IK (1996) Phosphinothricin-resistant crops. In: Duke SO (ed) Herbicide-resistant crops. CRC Press, Boca Raton, FL, pp 85–91 Velini ED, Alves E, Godoy MC, Meschede DK, Souza RT, Duke SO (2008) Glyphosate applied at low doses can stimulate plant growth. Pest Manag Sci 64:489–496 Vencill WK (ed) (2002) Herbicide Handbook, 8th edn. Weed Science Society of America, USA, p 493 Vermij P (2006) Liberty Link rice raises specter of tightened regulations. Nat Biotechnol 24:1301–1302 Vila-Aiub MM, Vidal RA, Balbi MC, Gundel PE, Trucco F, Ghersa CM (2008) Glyphosateresistant weeds of South American cropping systems. Pest Manag Sci 64:366–371 Wagner R, Kogan M, Parada AM (2003) Phytotoxic activity of root absorbed glyphosate in corn seedlings (Zea mays L.). Weed Biol Manag 3:228–232 Wakelin AM, Preston C (2006) A target-site mutation is present in a glyphosate-resistant Lolium rigidum population. Weed Res 46:703–705 Warwick SI, Legere A, Simard M-J, James T (2008) Do escaped transgenes persist in nature? the case of an herbicide resistant transgene in a weedy Brassica rapa population. Mol Ecol 17:1387–1395 Wauchope RD, Estes TL, Allen R, Baker JL, Hornsby AG, Jones RL, Richards RP, Gustosfson DI (2002) Predicted impact of transgenic, herbicide tolerant corn on drinking water quality in vulnerable watersheds of the mid-western USA. Pest Manag Sci 58:146–160 Werth JA, Preston C, Taylor IN, Charles GW, Robets GN, Baker J (2008) Managing the risk of glyphosate ressistance in Australian glyphosate-resistant cotton production systems. Pest Manag Sci 64:417–421 White AK, Metcalf WW (2004) Two C-P lyase operons in Pseudomonas stutzeri and their roles in the oxidation of phosphonates, phosphite, and hypophosphite. J Bacteriol 186:4730–4739 Williams GM, Kroes R, Munro IC (2000) Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regul Toxicol Pharmacol 31:117–165 Wright TR, Lira JM, Merlo DJ, Hopkins N (2005) A bacterial gene for an aryloxyalkanoate dioxygenase conferring resistance to phenoxy auxin and aryloxyphenoxypropionate herbicides. Patent Application WO2005US1437 20050502 Yasuor H, Abu-Abied M, Belausov E, Madmony A, Sadot E, Riov J, Rubin B (2006) Glyphosateinduced anther indehiscence in cotton is partially temperature dependent and involves cytoskeleton and secondary wall modifications and auxin accumulation. Plant Physiol 141:1306–1315 York AC, Steward AM, Vidrine PR, Culpepper AS (2004) Control of volunteer glyphosateresistant cotton in glyphosate-resistant soybean. Weed Technol 18:532–539 Zablotowicz RM, Reddy KN (2004) Impact of glyphosate on the Bradyrhizobium japonicum symbiosis with glyphosate-resistant transgenic soybean: a mini review. J Envron Qual 33:825–831 Zablotowicz RM, Reddy KN (2007) Nitrogenase activity, nitrogen content, and yield responses to glyphosate in glyphosate-resistant soybean. Crop Prot 26:370–376 Zapiola ML, Campbell CK, Butler MD, Mallory-Smith C (2008) Escape and establishment of transgenic glyphosate-resistant creeping bentgrass Agrostis stolonifera in Oregon, USA: a 4-year study. J Appl Ecol 45:486–494 Zhu Y, Li D, Wang F, Yin J, Jin H (2004) Nutritional assessment and fate of DNA of soybean meal from Roundup Ready or conventional soybeans using rats. Arch Anim Nutr 58:295–310

Chapter 4

Understanding and Manipulation of the Flowering Network and the Perfection of Seed Quality Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski, Paul Scott, Diaa Al-Abed, and Shobha D Potlakayala

4.1

Introduction

In spite of an impressive 2,263 metric tons reported for cereal production in 2004, sustainable output has not kept pace with global population increases. By 2020, the world’s population is expected to exceed 8 billion and the projected minimum annual 1.3% output increase needed to feed the population is not likely to be met. Complicating the problem further is that the projected expansions are not expected to be distributed evenly. Among the Asian Countries, India could become the most densely populated country in the world. Although India is second to the US in arable acreage, food is already being imported. As reported by Saritha Rai in the New York Times (2006), India has imported 2.2 million tons of wheat. This has been attributed to global climate change resulting in decreases in rainfall in the arid/subhumid zones of the country. Temperature increases and significant alterations in rainfall pattern are expected to acerbate in the absence of global enforceable environmental laws designed to mitigate sustainability while doing no harm to the biosphere. Similar problems are expected to plague the Philippines and Indonesia. The world’s governments can no longer look at agricultural production statistics on a country-by-country basis as though the resolution of hunger is confined within national borders. Although the largest population increases are expected in Asia, the highest percent rise falls in Africa. The number of people is expected to multiply at an annual rate of three percent accompanied by significant demographic changes as people move from rural areas to the city, thus decreasing farm output. By 2020, Africa alone is projected to require 60 million additional metric tons from import suppliers.

S. Rudrabhatla (*) Environmental Engineering, College of Science, Engineering and Technology, Pennsylvania State University, Middletown, PA 17057, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_4, # Springer-Verlag Berlin Heidelberg 2010

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Where are these food sources to be found? India is already importing cereal and its demand will increase over the next two decades. Moreover, China, the world’s most populous country cannot sustain its growing population, since it has only 7% of the globe’s arable land. Already America is experiencing the tension resulting from an insufficient amount of corn where the interests of energy production compete with the interests of the cereal’s use as food, forage, and commercial additive. This tension is predictable as energy demand has resulted in historic per bushel prices exceeding US$7.00, with significant increases in food cost. Placing more land in production has its drawbacks, especially if increased cultivation results in global loss with respect to biodiversity. In this connection, threats to deceases in biodiversity with respect to land-use change have already resulted in successful legal challenges. Given the aforementioned facts, it is likely that the most immediate relief to projected global food shortages will come not only as a function of increased seed quality (either through standard breeding or transgenic production), but also as a function of being able to manipulate flower development. Indeed, new plants will have to be developed whose time-to-flower cycle is altered, thus mitigating challenges to both pollen production and fertilization associated with increased temperature and diminished water supplies. The ability to alter development by changing flowering time, life cycle length or, for that matter, where a plant is cultivated should result not only in significant increases in the availability of land usage per se, but also in production. If these alterations are likewise accompanied by improvements in seed quality as measured by protein or fat content, for example, positive movement is made towards the requisite goal of feeding people globally. All aspects of flowering must be dissected and understood if its mechanism(s) is(are) to be manipulated toward this end, and it is to this understanding that this review is dedicated.

4.2 4.2.1

The Regulation of Flowering The Floral Transition

The production of grain crops depends on the successful union of gametes at an optimal time to ensure plentiful generation of seed for harvest. The production and union of fertile gametes occur at flowering, which is a critical event in the life cycle of all higher plants. In order to flower, plants must first transition from vegetative to reproductive growth. During vegetative development, the shoot apical meristem (SAM), a population of totipotent cells at the growing point of the plant, gives rise to leaves and other above-ground organs. To switch to reproductive growth, the SAM ceases leaf production and becomes committed to the production of reproductive structures, such as an inflorescence bearing flowers (Fig. 4.1). The period when the SAM is reprogrammed is called the floral

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Fig. 4.1 The floral transition in maize, when the vegetative shoot apical meristem (left) ceases leaf production and adopts a reproductive fate (right). Scale bar is 200 mM

transition and the timing of the transition largely determines when a plant flowers. Flowering time is often measured by counting the number of leaves produced prior to the transition of the SAM and, thus, is a direct measure of the timing of the transition. Accordingly, late flowering varieties produce more leaves than early flowering varieties, as the transition is delayed and the SAM remains in the vegetative stage of growth for a longer period of time. The timing of the floral transition has been manipulated by plant breeders for centuries. Elite varieties have been selected to balance the extent of vegetative growth with the duration of grain fill to maximize yield in their adapted geographic locations. Identifying key regulators of the floral transition and understanding their functions will allow for the development of varieties improved for productivity and yield within a rapidly changing environment. Higher plants have developed sophisticated genetic mechanisms to ensure that flowering coincides with an optimal time for reproductive success (Blazquez and Weigel 2000; Hayama and Coupland 2003; Izawa et al. 2003; Komeda 2004; Putterill et al. 2004; Simpson et al. 2004). Previous physiological studies revealed that a combination of various environmental inputs and endogenous cues affect the timing of the transition. For many species, day length is the predominant input, while other species are day-neutral and rely almost entirely on endogenous signals. These early studies also established several relevant points regarding the floral transition (Bernier and Perilleux 2005). First, the inductive flowering signal originates in leaves, whether the transition is triggered by external or endogenous cues. Second, the inductive signal, often referred to as florigen (F), is mobile and is transmitted from the leaves through the phloem to the shoot apex. Lastly, the SAM, the target of the inductive signal in the shoot apex, must be competent to perceive the signal in order to transition. Thus, we know that leaves and the shoot apex are key tissues involved in the initiation, propagation and perception of floral signaling.

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The Floral Transition Network

At present, the molecular mechanisms that regulate the floral transition have been primarily elaborated in the model plant Arabidopsis thaliana using the wealth of flowering time mutants available in that species. Proper timing of the transition is controlled by signaling through a complex network of floral inductive and repressive activities. These activities function through four main regulatory pathways: autonomous, photoperiod, gibberellin and vernalization (Koornneef et al. 1998; Mouradov et al. 2002; Simpson and Dean 2002). The four regulatory pathways converge on the following key floral integrators; Leafy (LFY), Suppressor of Overexpression of Constans (SOC1), Flowering Locus T (FT) and Flowering Locus D (FD) (Lee et al. 2000; Samach et al. 2000; Abe et al. 2005; Michaels et al. 2005; Parcy 2005; Corbesier et al. 2007). From these integrators, the flowering signals are transmitted to early-acting floral meristem identity MADS-box genes Apetala1 (AP1), Cauliflower (CAL) and Fruitful (FUL) that promote formation of inflorescence meristems which ultimately produce flowers (Fig. 4.2; Yanofsky 1995; Ferra´ndiz et al. 2000). Less is known about what controls the floral transition in the cereals, but a growing body of evidence suggests some of the floral regulators and their signaling modules are conserved in Oryza sativa (rice), Triticum aestivum (wheat) and Zea mays (maize) (Table 4.1; Danyluk et al. 2003; Hayama et al. 2003; Izawa et al. 2003; Izawa 2007; Yan et al. 2003, 2006; Muszynski et al. 2006; Adam et al. 2007; Chengxia and Dubcovsky 2008; Danilevskaya et al. 2008). The genes known or presumed to regulate flowering time in the major cereal crops are listed in Table 4.1. The core photoperiod regulatory module in Arabidopsis – GIGANTEA (GI), CONSTANS (CO) and FT – has been shown to be conserved in rice; although, under long-day photoperiods, rice FT expression is suppressed, leading to suppression of flowering, which is the opposite regulation of FT in Arabidopsis (Fig. 4.2; Yano et al. 2000; Hayama et al. 2002; Kojima et al. 2002). Signaling downstream of FT to the floral meristem identity genes also appears conserved among Arabidopsis, rice, wheat and maize (Lim et al. 2000; Lee et al. 2003). Although a number of studies show that diverse plant species share parts of the regulatory pathways defined in Arabidopsis, whether all the components of these regulatory pathways exist in other plants is not apparent. Furthermore, for those components that are conserved, how they integrate within the larger network is unknown. Further analysis of floral regulatory mechanisms in several species is required to define common pathways as well as uncover unique regulatory elements that may have evolved to accommodate particular environmental conditions and species-specific physiologies. For example, although both Arabidopsis and rice predominantly rely on photoperiod to regulate the timing of the transition flowering is promoted in Arabidopsis by long days and in rice by short days. Unlike rice, many temperate cereals, such as wheat, are long-day plants that also require vernalization, a period of prolonged cold temperature, to promote flowering. Conversely, although domesticated from teosinte, an obligate short-day species, temperate maize is largely photoperiod insensitive and relies primarily on endogenous

4

Understanding and Manipulation of the Flowering Network Autonomous

Photoperiod Response Arabidopsis (LD) circadian clock LEAF

Rice (SD)

Maize (DN) RID1

id1

GI

OsGI

CO

Hd1

(F)

FT

Hd3a

ZCN8(?)

FD + FT

OsDlf1(?) + Hd3a

LFY APEX

171

dlf1 + ZCN8(?) x

AP1/CAL/FUL

OsMADS14/18(?)

zfl1, zfl2(?) ZMM4, ZMM15

Fig. 4.2 The floral transition networks in Arabidopsis (left), rice (center) and maize (right). Under inductive photoperiods (long day for Arabidopsis and short day for rice), the circadian clock drives expression of key leaf-expressed flowering time genes (GI and CO or OsGI and Hd1). These activate the downstream mobile floral stimulus FT or Hd3a which moves from the leaves through the phloem to the shoot apex. In the shoot apex, the mobile floral stimulus interacts with the floral inductive gene FD or OsDlf1(?) to activate the floral meristem identity MADS-box genes AP1/ CAL/FUL or OsMADS14/18(?). In Arabidopsis, LFY integrates inductive signals from the leaf to the floral meristem identity genes in a parallel pathway. In rice, the LFY ortholog does not appear to participate in floral inductive signaling; but RID1, an id1 ortholog, has been shown to control initiation of floral induction. In temperate maize, which is relatively day-neutral, an autonomous signal induces expression of id1 in leaves that regulates a florigenic factor (F). Signals downstream of F (perhaps ZCN8, a possible maize FT ortholog) are transmitted to the shoot apex and regulate dlf1 expression or DLF1 activity. Interaction of DLF1 and ZCN8 presumably activates downstream targets x and the ZMM4 and ZMM15. Redundant inductive signaling through an id1independent alternate pathway converges downstream of dlf1. The position of genes marked by (?) is speculative, hypothetical interactions are marked by dotted lines and the alternate pathway in maize is colored blue

cues to flower. The molecular nature of the endogenous signaling pathways is not known but some parts of the maize flowering network are shared with other cereals and Arabidopsis (Table 4.1). The succeeding section describes what is known about the floral transition regulatory network in maize and how the network components relate to components from other species.

4.2.3

Flowering in Maize

Growth of maize (Zea mays) is largely determined by the activity of the SAM (Fig. 4.1). During the first 3–4 weeks after germination, the SAM produces vegetative organs, such as leaves and stem tissue. After about 4–5 weeks of growth, the

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Table 4.1 Genes regulating flowering time in the major cereal crops; maize, rice and wheat Similarity to Reference Gene/locus Activitya Protein homology Arabidopsisb Maize Constans of Zea mays1 UNK B-box zinc finger CONSTANS (CO) Miller et al. (conz1) (2008) Delayed flowering1 P Basic-leucine FLORAL LOCUS D Muszynski et al. (dlf1) zipper (bZIP) (FD) (2006) Gigantea of Zea mays1 UNK Nuclear protein GIGANTEA (GI) Miller et al. (gigz1A and B) (2008) Indeterminate1 (id1) P C2H2 zinc finger None Colasanti and Sundaresan (2000) Phytochrome B (phyb) R Phytochrome PHYTOCHROME B Sheehan et al. (2007) Zea FLORICAULA/ P DNA-binding LEAFY (LFY) Bomblies et al. LEAFY (zfl1 and protein (2003) zfl2) Zea mays UNK PhosphatidylFLORAL LOCUS T Danilevskaya CENTRORADIALIS ethanolamine (FT) and et al. (2008) (ZCN) binding TERMINAL protein FLOWER (TFL) Zea mays MADS-box4 P MIKC-type APETALA1 (AP1), Danilevskaya (ZMM4) MADS-box CAULIFLOWER et al. (2008) protein (CAL) and FRUITFUL (FUL) ZmRap2.7 R AP2/ethyleneTARGET OF EAT1 Salvi et al. (2007) responsive (TOE1) elementbinding protein Rice Early heading date1 (Ehd1) GRAIN number, plant height and heading date7 (Ghd7) Heading date1 (Hd1)

P R

P

Heading date3a (Hd3a) P

Heading date6 (Hd6)

R

OsGI

P

OsMADS14

P

OsMADS51

P

B-type response None regulator Zinc-finger CCT None domain protein B-box zinc finger CO PhosphatidylFT ethanolamine binding protein a-subunit protein CK2 kinase Nuclear protein GI MIKC-type MADS-box protein Type I MADSbox protein

AP1/CAL/FUL

Doi et al. (2004)

Yano et al. (2000) Kojima et al. (2002)

Takahashi et al. (2001) Hayama et al. (2002) Jeon et al. (2000)

None (continued )

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Table 4.1 (continued) Gene/locus OsSOC1 (OsMADS50)

Rice FLO-LFY (RFL) Rice Id1 (RID1) Photoperiodic sensitivity5 (se5)

Activitya Protein homology P MIKC-type MADS-box protein P DNA-binding protein P C2H2 zinc finger

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Similarity to Arabidopsisb SOC1

Lee et al. (2003)

LFY

Rao et al. (2008)

none

Wu et al. (2008a,b) Izawa et al. (2000)

R

Heme oxygenase HY1

Wheat TaFDL2

P

Basic-leucine FD zipper (bZIP)

TaFT (VRN3)

P

Reference

Chengxia and Dubcovsky (2008) Yan et al. (2006)

PhosphatidylFT ethanolamine binding protein TaVRN1 P MIKC-type AP1/CAL/FUL Yan et al. (2003) MADS-box protein TaVRN2 R Zinc-finger CCT None Yan et al. (2004) domain protein TaVRT2 R MIKC-type SHORT Kane et al. MADS-box VEGETATIVE (2007) protein STAGE3 (SVP3) a Gene activity has been shown to either promote (P) or repress (R) flowering or has not yet been determined (UNK) b Similarity to Arabidopsis flowering time genes

SAM switches from vegetative over to reproductive growth during the period of the floral transition. This period is distinguished in the SAM by the cessation of leaf initiation and its rapid increase in size (Shaver 1983; Irish and Nelson 1991). The SAM continues to increase in size as it progresses through the floral transition, which terminates with the SAM acquiring inflorescence identity to become the tassel primordium (Fig. 4.1). In maize, the tassel is the apical inflorescence, which bears the male flowers that produce pollen. A similar transition occurs later in time, leading to the conversion of several axillary meristems into ear primordia. In maize, the ear is the axillary inflorescence, which bears the female flowers that exsert silks (elongated pistils). Flowering in temperate maize is largely controlled by endogenous signals, possibly involving plant size or leaf number (Irish and Nelson 1991). Much of our understanding regarding the molecular regulation of flowering in maize comes from the analysis of genetic variants with altered flowering time. Three single gene late-flowering mutants are described in maize; the recessive indeterminate1 (id1) and delayed flowering1 (dlf1) mutations, and the dominant Leafy (Lfy) mutation.

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All three mutations delay the floral transition and therefore we expect functional id1 and dlf1 to promote flowering, while normal Lfy function is difficult to deduce (Shaver 1983; Neuffer et al. 1997; McSteen et al. 2000). The id1 mutants flower extremely late, producing many more leaves and flowering many weeks later than normal. Additionally, id1 mutants often have aberrant morphology, such as the absence of ears (Galinat and Naylor 1951). The id1 gene has been cloned and encodes a unique type of Zn-finger transcription factor, defined by the ID domain (IDD). id1 is specifically expressed in immature leaf tissue and has been suggested to control a florigenic signal (Colasanti et al. 1998; Colasanti and Sundaresan 2000). Subsequent analysis showed that the ID1 protein localizes to nuclei and binds DNA suggesting it regulates the transcription of downstream flowering-time genes (Kozaki et al. 2004). Furthermore, ID1 protein accumulates soon after germination and high protein levels are detected in discrete regions of immature leaves up to the time of the floral transition, after which time levels decline (Wong and Colasanti 2007). id1 is unique to grasses and a putative homolog was not found in Arabidopsis suggesting id1 participates in a monocot-specific pathway (Colasanti et al. 2006). Recently, a rice ortholog of id1, RID1, was identified that was proposed to control the initiation of floral induction and appears to regulate multiple inductive pathways (Wu et al. 2008a,b). Plants lacking RID1 activity never flower and remain in a perpetual vegetative state of growth. The other recessive late-flowering mutation, dlf1, has a more moderate late-flowering phenotype, with mutant plants producing a modest number of additional leaves and flowering 10–14 days later than normal (Neuffer et al. 1997; Muszynski et al. 2006). The dlf1 gene was cloned recently and encodes a basic leucine-zipper (bZIP) homologous protein that resembles the Arabidopsis floral integrator FD (Muszynski et al. 2006). Similar to FD, dlf1 is expressed in shoot apices, peaking in expression near the time of the floral transition. dlf1 was shown to function downstream of id1, thereby defining an id1-dlf1 floral inductive module (Fig. 4.2). To date, an id1-dlf1 homologous flowering module has not been described in rice or other grass species, although an apparent dlf1 ortholog was identified in wheat (Chengxia and Dubcovsky 2008). Leafy (unrelated to the Arabidopsis LEAFY gene or similar homologous genes) is a unique, dominant, late-flowering mutation, which increases the number of leaves specifically between the uppermost ear and tassel (Shaver 1983). Additionally, the Lfy mutation disrupts the coordination of inflorescence maturation, such that the ear matures before the tassel, resulting in mutant plants exserting silks prior to shedding pollen (M.G. Muszynski, unpub.). A mutation with a similar phenotype has not been identified in other grasses. Little is known about the morphological and developmental aspects of this dominant mutation and it has yet to be molecularly isolated. Although relatively uncharacterized from a genetic perspective, it has been used to a modest degree in maize breeding programs in Canada to increase leaf biomass as a means to improve yield (Costa et al. 2002; Subedi and Ma 2005; Subedi et al. 2006). Additional information regarding the molecular control of the floral transition in maize comes from the study of flowering time variants which display a quantitative mode of inheritance. Two quantitative trait loci (QTL) mediating early flowering

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were identified on chromosome 8 and named vegetative to generative transition1 (Vgt1) and Vgt2 (Vladutu et al. 1999; Salvi et al. 2002). Positional cloning and association mapping recently pinpointed the molecular position of Vgt1 to an intergenic region ~70 kb upstream of an APETALA2 (AP2)-like transcription factor, designated ZmRap2.7 (Salvi et al. 2007). Variation in flowering time in various inbred lines was associated with sequence changes in this putative cis-element that presumably controls the expression of ZmRap2.7. Transgenic analysis showed that overexpression of ZmRap2.7 cDNA caused late flowering, whereas downregulation in antisense maize plants caused early flowering, demonstrating ZmRAP2.7 is a repressor of the floral transition. The similarity of ZmRAP2.7 to members of a family of Arabidopsis AP2-like genes that also have a negative effect on flowering suggests a potential orthologous role for ZmRap2.7 in the regulation of the floral transition (Aukerman and Sakai 2003). How ZmRAP2.7 functions within the maize flowering network is currently unknown. Homology-based investigations have also informed our understanding of flowering in maize. A recent study has identified maize homologs of GI (gigantea of Zea mays1, gigz1A and B) and CO (constans of Zea mays1, conz1) that display different diurnal expression patterns in response to varied day lengths, suggesting temperate maize does sense and respond to photoperiod (Miller et al. 2008). Whether these homologs affect the floral transition was not determined. Associations with flowering time and inflorescence architecture were correlated with differences in gene copy number of duplicate maize genes homologous to FLORICAULA of Antirrhinum majus and LEAFY of Arabidopsis (Bomblies and Doebley 2006). It was also shown that mutation of both paralogous Zea FLO/LFY1 (zfl1) and zfl2 genes leads to very mild late flowering and altered inflorescence patterning (Bomblies et al. 2003). Additional maize floral regulatory candidates have been identified based on their sequence homology to FT, which was recently shown to be part of the mobile floral stimulus in several species (Jaeger and Wigge 2007; Lin et al. 2007; Mathieu et al. 2007; Tamaki et al. 2007). Maize has at least 25 FTlike and related paralogos TERMINAL FLOWER (TFL)-like genes, designated ZCN (for Zea mays CENTRORADIALIS) that could encode candidates for a conserved florigenic protein (Danilevskaya et al. 2008). Determining which family member(s) participate in florigenic signaling will require functional analysis of each gene but ZCN8 stands out as a likely candidate based on its expression pattern and ability to interact with the DLF1 protein in yeast two-hybrid assays (Danilevskaya et al. 2008). Comprehensive phylogenetic analyses have determined that for the meristem identity AP1/CAL/FUL MADS-box gene clade, only FULlike homologs exist within maize and other noneudicot species (Litt and Irish 2003; Malcomber et al. 2006). The grass FUL-like genes fall into three subgroups; FUL1 and FUL2 are sister clades that are both sister to the FUL3 clade. A recent study has identified two maize FUL1-like genes, ZMM4 and ZMM5, which are initially expressed near the time of the floral transition (Danilevskaya et al. 2008). Overexpression of ZMM4 promotes early flowering and is able to suppress the late-flowering phenotype of id1 and dlf1 mutants, suggesting ZMM4 has floral inductive activity.

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Collectively, these studies have enabled the configuration of a maize floral transition regulatory gene network (Fig. 4.2). Unlike Arabidopsis and rice, where signaling is photoperiod-responsive and linked to the circadian clock, signaling in maize is principally autonomous and not connected to day length. In the maize network, id1 sits at the top of an inductive signaling hierarchy initiated in leaves by unknown autonomous inputs. id1 is proposed to regulate the production or transmission of a florigenic signal (F) that moves from the leaves to the shoot apex (Fig. 4.2). The relationship between F and ZCN8, a possible maize FT ortholog, is not known. Signals downstream of F are transmitted to the shoot apex and regulate dlf1 expression or DLF1 protein activity. The model network predicts interaction of DLF1 and ZCN8, which presumably activates several targets downstream of dlf1, including an early target (x) which feedback regulates dlf1 expression and one or more ZMM MADS-box floral identity genes (Muszynski et al. 2006). Redundant inductive signaling through an id1-independent alternate pathway converges downstream of dlf1 to activate both x and the ZMM MADS genes. The positions of ZmRAP2.7 and the zfl genes are not known, although the zfl genes make attractive candidates for participating in the alternate inductive pathway. Modern maize inbreds are exquisitely adapted to a wide range of geographical and environmental conditions, with selection for specific maturities to flower at particular latitudes. In fact, most inbreds are selected to flower as late as possible, but early enough to assure that yield is maximized. Inbreds developed for one area of adaptation are rarely used in another because of limitations of flowering, grain fill or kernel maturation. Thus, breeding between inbreds with different maturities is uncommon, leading to a reduction in germplasm diversity and impeding the transfer of superior alleles to new lines. A comprehensive understanding of the genetic determinants regulating flowering would enable breeders to manipulate maturity through molecular breeding or transgenic methods and in this way increase the diversity of germplasm utilized in a selection program. This is especially significant at a time when climate change may dramatically alter an environmental component of a particular geography, thus rendering elite lines maladapted to these new conditions.

4.3

In Vitro Flowering: An Alternative Route to Rapid Flower Production

Plants differ with respect to the length of their life cycle and the timing of the floral transition, to say nothing of robustness with respect to gamete and seed production. Seeds likewise differ with respect to ease of germination. When these limitations are factored against challenges arising from both biotic and abiotic stressors, the accessibility of year round flower production has broad economic implications. This is especially true for secondary plant products whose biosynthesis is restricted either to the inflorescence or to the flower. Recently concentrated efforts have been directed towards producing fertile flowers in vitro whose seed may be harvested

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directly from the culture dish. The ideal in vitro flowering protocol couples a reduction in flowering and seed maturation, in effect shortening the entire life cycle, thus increasing the number of segregating populations that can be produced annually. Hence, the ability to manipulate in vitro flower production will have an incalculable impact on the introgression of transgenes into robust breeding lines to say nothing of plant breeding per se. In vitro flowers have already been produced in culture using a number of different hormone regimes being sensitive to particular cytokinins in a concentration-dependent fashion. In vitro flowers in plant species as diverse as legumes, pearl millet, ornamentals and bamboo (Abou-Alaiwi 2007) result following sequentially the formation of in vitro shoots that lead to flowers. While flowers have been produced, the technical problems associated with the development of a medium are complex. Specifically, from a population of explants, flowers may be produced through an intervening shoot or through the induction of vegetative shoots or infertile shoots where the inflorescence develops aberrantly leading either to the absence or the production of defective gametes. Most elusive of all to date has been the expression of direct flowering where the vegetative development is completely inhibited (Lin et al. 2005; Rudrabhatla and Goldman 2009, US Patent # 7, 547, 548). For the purpose of this discussion, a true in vitro flower is one that is fertile and therefore capable of developing seeds that are indistinguishable from those harvested from traditional crosses.

4.3.1

In vitro Flowering in Grasses

4.3.1.1

Pearl Millet

Devi et al. (2000) reported the production of in vitro flowers from cultured apical meristems of Pennisetum glaucum (L.) R. Br. that is hormone- and concentrationdependent. If the apices were cultured with low levels of 2,4-dichlorophenoxyacetic acid (2,4-D) and the concentration of benzyladenine (BA) varied, the shoot apical meristem enlarged and adventitious shoots developed. At higher concentration of BA, somatic embryos were also observed. In the absence of 2,4-D, shoot tips produced many leaves and in vitro flowers but only upon the inclusion of BA in the medium. Notably, the plants developed from in vitro shoots are fertile. The varied developmental profiles reported are indistinguishable among the four genotypes tested and determined solely as a function of media composition with inclusion of BA being an obligate requirement for in vitro flower development. 4.3.1.2

Bamboos

As a general rule, in vitro flowers have been derived from cultured shoots following a number of manipulations that require the addition of specific concentrations of cytokinin. Working with Dendroclamus strictus Nees, a bamboo, Singh et al. (2000)

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produced flowers from cultured shoots on a modified Murashige and Skoog medium containing thidiazuron (TDZ). While shoots were robustly produced over a wide concentration of cytokinin (0.01–1.0 mg/l), in vitro flower production was restricted only to medium containing TDZ between 0.5 and 1.0 mg/l. The formation of anthers did not necessarily specify the development of normal flowers. Not only were there problems with pollen dehiscence but also with pollen development. In fact, only 20% of the grains were normal, with the remaining 80% being empty. Lin et al. (2003) described in vitro flowering in another bamboo species, Bambusa edulis, albeit with significant shoot formation. Unlike Singh et al. (2000) the shoots were initiated from in vitro derived spikelets. Specifically, these were cultured on Murashige and Skoog (MS) medium containing 9.3 mM kinetin and kinetin þ 13.57 mM 2,4-D. The callus-derived somatic embryos were then germinated on media supplemented with 0.5 uM TDZ plus 3% sucrose. After several subcultures, shoots were recovered. The shoots then were transferred to media containing different cytokinins and the effects determined by testing with a number of different concentrations. Coincident with this transfer, cytokinin is not only sufficient but necessary for the induction of in vitro flowers. Following the production of proliferating intervening shoots, culture on different cytokinins altered the developmental fate of the cells of the axillary meristems. When BA is substituted for TDZ, the number of flowering shoots is profoundly reduced; an effect further complicated as a function of exogenous hormone concentration. Hence, the sensory apparatus of cytokinin signaling can discriminate not only differences among cytokinin molecules but also in concentration leading to profound alterations in morphogenesis. This is in keeping with the earlier observation of Singh et al. (2001) who demonstrated that differences among endogenous concentrations can have profound and antagonistic effects on root and shoot meristem development. Finally, auxins can also effect in vitro development. Although not sufficient in themselves to specify flower formation, the addition of auxin in the media modifies morphogenetic fate in bamboo. If NAA is included in the media during shoot proliferation at high auxin to cytokinin ratios, the number as well as the percentages of reproductive shoots was significantly reduced and, accordingly, in vitro flowering decreased.

4.3.1.3

Maize

Maize in vitro flower induction originating from split-seed was observed after 3–4 weeks in medium containing BA and kinetin (Fig. 4.3 a, b; Al-Abed 2007;

<

Fig. 4.3 (a) In vitro flowers originating from maize split seed explants. Close images of in vitro induced ears (a) and (b) and tassels (b) and (c). (b) Comparisons of maize hybrids and inbred lines for the induction of in vitro flowers on various concentrations of BAP and Kinetin. R 23, Hi-II, B73 and LH 198  LH 227 are different maize hybrid and inbred lines. Numbers 1–6 correspond to different concentration combinations of BAP and kinetin. All percentage data were transformed using arc-sin transformation before statistical analysis

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Al-Abed et al. 2006). Although both ears and tassels were formed, only the ears were fertile. The induction frequency rose as a function of cytokinin concentration with an optimum of 17.6 mM 6-benzylaminopurine (BAP) and 22 mM kinetin. Four genotypes were tested to determine genotype independence, and no significant differences were observed in the frequency of in vitro flowering among the varieties. Interestingly, although the incidence of inflorescence production among the varieties B73, Hi-II and LH198  LH227 proved to be cytokinin concentration dependent, there were no significant differences for in vitro flower induction frequency among the four genotypes when the concentration of BAP was stabilized at 17.6 mM and kinetin varied between 4.6 and 18.4 mM. Similarly, when the concentration of BAP was stabilized at 22 mM and kinetin concentration varied as before no differences in induction were observed. Of equal interest, TDZ could not successfully substitute for either BA or kinetin; an incorporation of TDZ in the culture media resulted in no in vitro inflorescences formed. When the media contained BAP alone there was a significant difference in flower induction frequency among the four micromolar concentrations tested (8.8, 13.2, 17.6 and 22 mM). The number of flowers increased as BAP concentrations increased up to 22 mM. Shoots from line R23 were induced to flower, although the number of ears induced was higher than the number of tassels. On the other hand, there was no significant difference between the number of ears and tassels induced from B73, Hi-II and LH198  LH227. With further increases in BAP concentration (26.4 mM and higher), the shoots were stunted and additional rounds of multiple shoot production began. From these results, it may be concluded that flower induction frequency is correlated with incremental changes of BAP and kinetin concentration. Moreover, prolonged culture in cytokinin-supplemented media affects the development of the ear and subsequent silk formation. If shoot-derived ears were kept on 17.6 mM BAP þ 9.2 mM kinetin for more than 4 weeks, none of the ears formed silks. In contrast, if the shoots were cultured on 17.6 mM BAP þ 9.2 mM kinetin and then transferred to MS lacking hormones, the silks developed with the ears growing larger and more robust.

4.3.2

In Vitro Flowering in Dicots

4.3.2.1

Tomato

Dielen et al. (2001) investigated the floral transition in the tomato mutant uniflora (uf). Under winter growth conditions, homozygous plants exhibit a prolonged vegetative phase and flower production is suppressed. Floral transition can be induced on a medium containing sucrose and a variety of cytokinins, in addition to nitrogenous nutrients. The inclusion of gibberellic acid (GA3) was found to inhibit transition.

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Sheeja and Mandal (2003) produced in vitro tomato flowers through an intervening callus on medium containing 2 mg/l BAP. Flowers, however, required continuous light (2.2 mM2/s) in order to open. The number of flowers produced was low, averaging approximately 11 per explant. The flowers were fertile and tomatoes set 160 days after pollination. Flower production capacity was limited with respect to variety. Of the seven varieties tested on MS þ 2 mg/l BAP, only one variety, KS118, was able to produce fully formed flowers from callus developed from leaf explants.

4.3.2.2

Roses

In vitro flowering was induced in six vegetatively produced rose cultivars in MSmodified media containing different phytohormone combinations. All contained the auxin a-naphthaleneacetic acid (NAA) at 0.1 mg/l and a cytokinin which was varied. Media containing 0.5 mg/l TDZ or 0.5 mg/l zeatin induced in vitro floral bud production at frequencies exceeding 40%. The phytohormone response of each rose cultivar was not uniform.

4.3.2.3

Ginseng

Lin et al. (2005) reported the direct formation of Panax gensing inflorescences. In this connection, plantlets derived from somatic embryos produced clusters of dormant buds when cultured on B5 medium containing BA (1 mg/l) and GA3(1 mg/l). The buds were subcultured again on the same medium and approximately 15% of the buds cultured produced inflorescences directly without a vegetative phase. In addition to direct production of flowers, a mixture of plant organs developed on the explant including vegetative shoots and indirect flowers in addition to dormant buds. If TDZ was substituted for BA, the number of flowers produced without an intervening shoot increased as did the number of dormant buds. These observations confirm the importance of cytokinin as a prerequisite for flower development (Chang and Hsing 1980; Lim et al. 1997). While no gross structural or morphological differences could be detected among flowers produced either by direct or indirect flowering, Lin et al. (2005) reported neither the production of functional gametes nor seed. Hence, the utility of a season-independent, direct in vitro flowering system remains a goal.

4.3.2.4

Soybean

To date, in vitro flowers have also been obtained from dicots through culturing an intervening shoot as shown by Franklin et al. (2000) using the legume Pisum sativum. Following germination on hormone-free MS media, cotyledonary node and shoot tip explants were transferred to an MS medium containing 2mg/l BAP.

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The resulting elongated shoots were removed 15 days post-implantation and incubated on medium containing either auxin alone (indole-3-butyric acid, IAA; or NAA) or in combination with (GA3). Following culture on this medium roots and mature pods were formed within 25 days. The number of shoots per cotyledonary node or shoot tip is BAP concentration dependent. The inclusion of BAP, either below 0.5 mg/l or above 5.0 mg/l, results in the complete absence of shoots derived from cotyledonary node explants. Indeed, shoot formation is maximized between 1 and 2.0 mg/l. Consistent with this observation is the fact that the number of shoots per shoot tip explant is also BAP concentration dependent, albeit over a much narrower range. While BAP results in shoots regardless of the explant, a component of shoot development is fine-tuned based on the tissue that is the donor cell source. The in vitro flowers formed on these shoots, however, are dependent on the subsequent inclusion of specific auxins at highly defined concentrations. While indole-3-butyric acid (IBA) can induce flowering over a wide range of cytokinin concentrations (0.1–2.0 mg/l with or without GA3), substitution with NAA cannot. Not only is GA3 required but the range over which it is effective is restricted to 0.1–0.5 mg/l. In contrast, Rudrabhatla and Goldman (2009), (patent pending) report that culture of soybean cotyledons or radicles on MSB5 medium supplemented with specific cytokinin combinations produced in vitro flowers either directly or indirectly through an intervening shoot. For example, after 7–15 days in MSB5 medium containing TDZ and BAP, the bulging of cotyledons is prominent and small greenish protuberances appear at the proximal end of the cotyledon (Fig. 4.4a). The cotyledons are transferred to a modified MS basal medium containing B5 vitamins but lacking hormones. The small greenish protuberances morph into bud-like structures (Fig. 4.4b). Both shoot buds and/or flower buds form at the proximal end of the cotyledon. The formation of soybean in vitro shoots and/or flowers is not only defined by the cytokinin molecule per se but is also concentration dependent. As will become apparent, soybean tissues have the capacity to discriminate differences among classes of cytokinins on the same explant and in doing so alter the developmental fate of that fixed cell population. If soybean cotyledons are challenged with BAP alone, only multiple shoots were observed (Barwale et al. 1986), while a low frequency of flower buds were observed with TDZ alone. The highest frequency of flower buds was observed in the combination treatment of TDZ (2.0 mg/l) and BAP (1.0 mg/l) (Fig. 4.4c). A decrease in the frequency of flower buds (8.75%) was observed when the concentration of TDZ was kept constant and the concentration of BAP increased to 2.0 mg/l (Fig. 4.4d). Further increase in BAP (3.0 mg/l) resulted in the complete suppression of flower buds with a concomitant increase in the production of multiple shoot buds. Hence, using the same cell population and manipulating the growth regulators, the meristem identity of the tissue was changed from flower buds to shoots. All the flower buds were clumped or grouped together, even in those cultures producing multiple shoots along with flowers suggesting the flower buds arise from a specific group of cells. Among all the combinations tested, 2.0 mg/l TDZ along

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Fig. 4.4 Effect of hormones on in vitro flowering and viable seed set in soybean. Cotyledons producing direct flowers (a) and multiple shoots (b) on MSB5. Direct pod formation from cotyledons without vegetative phase (c and d)

with 1.0 mg/l BAP induced 70–75 flower buds. As soybean is a self-pollinated species, most of the in vitro flowers produced pods and set seed. The in vitro pods fully matured within 35 days after anthesis, and had well-developed seeds. Those in vitro developed seeds, when cultured on MSB5 basal medium, readily germinated and formed fully fertile plants that were easily hardened and grown in the greenhouse. This developmental course can be predictably varied by changing the concentration of BAP and TDZ in the media. In this connection, cotyledons incubated on MSB5 containing 3 mg/l BAP and 1 mg/l TDZ suppressed direct flower formation leading directly to the development of in vitro soybean shoots. From these shoots, fertile flowers developed and seeds indistinguishable from those grown in the field or greenhouse were recovered. The elimination of TDZ from this media likewise produced shoots, which eventually gave rise to fertile flowers at a low frequency (Rudrabhatla and Goldman 2009, patent pending). Similar results have also been obtained using radicle explants as summarized in Fig. 4.5a–c. Three-day-old germinated seeds were collected, the seed coats

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Fig. 4.5 In vitro flowering from radical explants in soybean. Soybean in vitro flowering from radical explants through in vitro developed shoots (a), flower (b) and pod (c)

removed and segments of the radicle and plumule excised. These were placed on MSB5 supplemented with TDZ (2 mg/l) and BAP (1 mg/l) and directly formed clustered fertile in vitro flowers (Fig. 4.5a). If the concentration of BAP was raised to 3 mg/l and the concentration of TDZ remained either constant at 1 mg/l or was raised incrementally to 2 mg/l, direct flower formation was suppressed and shoots formed. Notably, these shoots too developed normal fertile flowers. Taken as a whole, the phenomenon of in vitro flowering supports the hypothesis that cytokinins are essential not only for meristem maintenance and integrity but also as a determinant of cell fate. This conclusion is based on the fact that attempts to induce in vitro flowering through manipulation of cytokinin has resulted in the formation of shoots lacking flowers, shoots producing sterile flowers, shoots producing fertile flowers, direct sterile in vitro flowers where the intervening shoot is absent and direct fertile in vitro flowers. Each of these outcomes may be attributed to tension that results when cytokinin is supplied exogenously thus challenging the plant’s ability to maintain phytohormone homeostasis in the meristem. The role of cytokinin and its obligate requirement for meristem stability, integrity, and function is now well established (Werner et al. 2001, 2003; Riefler et al. 2006;

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Kurokawa et al. 2007). Transgenic tobacco plants overexpressing any one of the cytokinin oxidase genes, AtCKX1, AtCKX2, AtCKX3 or AtCKX4, produce plants with significantly reduced cytokinin concentration. Such plants are not only stunted but show significant size reduction in the shoot apical meristem (Werner et al. 2001). In subsequent experiments using Arabidopsis, Werner et al. (2003) demonstrated that overexpression of any of the six members of the AtCKX oxidase/ hydrogenase gene family has profound effects on the aerial portions of the plant impacting both shoot and floral development. The effect on development is likely a function of perturbations in differential cytokinin activity that is normally associated with growth zones, which are reduced because of the expression of the AtCKX genes that shrink the size of the SAM. Key to the production of in vitro flowers and the route by which they are produced is likely to be, in part, a function of challenges to cytokinin homeostasis in the meristem. T-DNA insertion mutants into the cytokinin receptor genes Arabidopsis histidine kinase genes (AHK2, AHK3) and cytokinin response (CRE/ AHK4) result in profound developmental changes (Riefler et al. 2006). The effect of these mutated genes is to reduce signaling and lead not only to increases in endogenous cytokinin concentration but to do so in a defined manner leading to the accumulation of specific metabolites. The ahk2 and ahk3 double mutants affect leaf development reducing cell number and total chlorophyll content while showing reduced sensitivity to cytokinin-dependent inhibition of dark-induced chlorophyll loss. The effect of the triple mutants on flowering remains a matter for discussion. While Higuchi et al. (2004) and Nishimura et al. (2004) claimed complete plant sterility, Riefler et al. (2006) demonstrated that some plants may be rescued. The said effect of disturbing homeostasis on flower development is unmistakable and consistent with the observation that mutants express anywhere from 16- to 19-fold increases in zeatin metabolites. While reduced signaling increases cytokinin accumulation, it remains unclear as to whether the changes seen in the development result from increased biosynthesis or decreased degradation. A decreased signaling results in increased endogenous cytokinin activity diminishing homeostasis and resulting in disturbances in the development. Indeed, the source of the change in endogenous cytokinin concentration is unimportant. Recently, Kurokawa et al. (2007) have detailed the expression of lonely guy (LOG) gene in rice. Plants overexpressing LOG produce plants with diminished panicles, reductions in the number of floral organs and a flattened meristem resulting in anomalies in organ development. The wild-type gene is definitive for the final step of cytokinin biosynthesis in the shoot tip. The differential response to cytokinin on in vitro flowering described here substantiates the fact that the uptake of exogenous cytokinin disturbs the delicate internal homeostatic balance needed if plant development is to proceed normally. The parameters regulating in vitro flowering are complex and result by signaling appropriate developmental pathways and repressing others. While cytokinin signaling is initiated through a number of histidine kinase receptors followed by phosphor relay, it remains unknown how increases in hormone concentration effect

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differential gene expression as a result of perturbations in homeostasis. This said, while the precise genes that are either expressed or repressed are unknown at this time, their definition is made possible. Our results clearly indicate that the unique hormonal combination applied at appropriate stage of in vitro culture seems to be a key factor that defines whether floral meristems are formed or not. Hence, it should be possible to determine if any, of the sentinel cloned genes at which all floral pathways converge are expressed, as well as those that are unique. Indeed, the collapsing of the vegetative phase and the appearance of flower buds beginning the 3rd or 4th week after transfer to flower induction medium raises questions as to whether the four flower induction pathways previously described for Arabidopsis finds a parallel in soybean or, alternatively, whether the in vitro flowering system expresses a regulatory pathway previously unknown. Identification of the genes regulating in vitro production of soybean flowers may offer the possibility of transferring genes that are absent to complete the pathway or identifying those genes that are uniquely expressed in response to the specific combination of the floral induction medium.

4.4 4.4.1

Genes for Improving Grain Quality in Corn and Soybeans Key Issues

Grain crops form the basis of the global food supply, either through direct consumption or in animal feed to produce meat. A number of trends lead to an increasing global demand for grain. First, the global population is increasing. The current population is 6.5 billion and this is expected to increase to 9 billion by 2050. Second, global meat consumption is increasing in developing countries. In the past 25 years, meat consumption in developing countries has doubled (Speedy 2003). Meat production by nonruminant animals often depends on grain, and conversion of grain to meat is relatively inefficient. Thus, an increase in meat consumption creates a larger demand for grain. Third, increasing amounts of grains are used to produce renewable biofuels such as ethanol and biodiesel. This increasing demand is compounded by environmental factors such as reductions in fresh water and arable land. It is clear that crop yields will need to be increased substantially to keep pace with the demand for grain. In addition to increasing crop yields, it is important to ensure that our grain supplies are used efficiently. It may be possible to use less grain for a given purpose if that grain is particularly well suited to that purpose. For example, oil crops that contain more oil require consumption of less grain to meet the demand for oil. Thus, part of the demand for grain can be met by producing better grain. The definition of grain quality depends on the end use of the grain. This chapter will examine the major uses of grain and the genes that have been important in improving grain for these purposes.

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4.4.2

Protein

4.4.2.1

Amino Acid Balance

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Meeting protein nutrition needs is one of the greatest challenges because plants tend to be low in protein and this protein has poor nutritional quality. Animal protein sources have much higher nutritional value. The nutritional quality of a protein source is defined, in part, by its amino acid balance. Of the 20 common natural amino acids, nonruminant animals can produce 10 amino acids from other sources and the remaining 10 are required in the diet. While the exact amino acid requirement depends on the animal species, developmental stage and other factors, in general, monocot grains tend to be deficient in lysine and tryptophan while dicot grains tend to be deficient in the sulfur-containing amino acids, cysteine and methionine. These deficiencies are largely a consequence of the amino acid balance of the seed storage proteins that accumulate to high levels in the seed and support the seedling until it is capable of photoautotrophic growth. The major seed storage proteins of cereals tend to be prolamins, which are characterized by low levels of lysine and tryptophan, while the main seed storage proteins in legumes tend to be globulins, which have low levels of sulfur-containing amino acids. These deficiencies lead to poor utilization of plant protein in diets, so improving the balance of amino acids is an important grain quality objective. Several approaches have been used to improve the amino acid balance of grain. Because the seed storage proteins have such a large impact on determining the amino acid balance, one approach to solving this problem is to alter seed protein deposition. In cereals, several genes involved in deposition of the prolamin family of seed storage proteins called zeins have been found to impact amino acid balance. Recessive mutant alleles of the opaque 2 (o2) gene of maize cause an increase in the lysine and tryptophan content of the grain (Mertz et al. 1964). The o2 gene encodes a basic leucine zipper (bZIP) transcription factor (Hartings et al. 1989) that binds to promoter elements of some zeins (Schmidt et al. 1990). Unfortunately, the soft, lowdensity kernels produced on these mutant plants are not well suited to agronomic production. Development of nutritionally improved maize with acceptable agronomic properties has required an extensive breeding effort to overcome the unfavorable effects of the mutation. The resulting varieties are called Quality Protein Maize (QPM) and carry the o2 gene as well as a number of unidentified “modifier loci” that condition desirable agronomic properties (Prasanna et al. 2001). Mutation in the floury-2 (fl2) gene also improves the lysine and methionine content of maize (Mertz et al. 1965). This mutation encodes a zein with an altered signal sequence that interferes with zein deposition (Coleman et al. 1995). The delta zein regulator (dzr1) gene conditions elevated methionine levels in maize and was originally identified in a screening program aimed at identifying high lysine genotypes (Phillips et al. 1981). This gene regulates the deposition of a methionine-rich seed storage protein, the 10 kDa delta zein by altering the mRNA stability of the gene (Cruz-Alvarez et al. 1991) and has been used to produce a series of high methionine inbred lines (Olsen et al. 2003).

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Transgenic approaches to improving the amino acid balance have taken advantage of information gained from the naturally occurring mutants to achieve effects similar to those of the natural mutations. Downregulation of zein transcription results in improved amino acid balance (Segal et al. 2003; Huang et al. 2004) as would be predicted from studies of o2, and enhancing the stability of the methioninerich delta zein message improves methionine content (Lai and Messings 2002) as would be predicted from studies of dzr1. A different transgenic approach involves modifying genes encoding enzymes involved in metabolism of amino acids of interest. Introduction of feedbackinsensitive versions of an enzyme in the lysine biosynthetic pathway results in increased levels of total and free lysine when used in combination with a transgene designed to downregulate the zein storage proteins (Huang et al. 2005). Downregulation of degradative enzymes is also effective is increasing free lysine levels (Houmard et al. 2007). An approach that involved both expression of a feedbackinsensitive biosynthetic enzyme and downregulation of a degradative enzyme with a single transgene was effective in increasing the free lysine levels 40-fold over nontransgenic controls. Approaches to improving the amino acid balance of dicot grain have been recently reviewed (Krishnan 2005). Expression of methionine-rich proteins from other species has been widely used to address this problem. For example, the 2S albumin from Brazil nut is a methionine-rich seed storage protein that has been shown to increase methionine content when introduced into the seeds of transgenic plants (Altenbach et al. 1989). Analysis of transgenic soybeans containing this protein indicated that the allergenic properties of this protein were present in the transgenic grain (Nordlee et al. 1996) and this approach has not been pursued further. Maize methionine-rich zein genes have also been expressed in transgenic soybeans (Dinkins et al. 2001; Kim and Krishnan 2004). While the transgeneencoded proteins accumulated in soybean seeds, the change to methionine content was modest. 4.4.2.2

Antigenicity

Allergenicity limits the utility of plant proteins in diets. Genetic approaches have been used to produce varieties with lacking certain antigens. In soybean, for example, the P34 protein is a major allergen. A transgene was introduced that uses gene silencing to prevent accumulation of this protein (Herman et al. 2003). In addition, several null alleles that fail to accumulate this protein were identified through screening of a germplasm of the Glycine genus (Joseph et al. 2006). These resources may allow the development of varieties with reduced antigenicity. 4.4.2.3

Digestibility

The digestibility of soybeans is reduced by the presence of trypsin inhibitors in the seeds. A null allele for the Kunitz trypsin inhibitor allele has been identified and

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designated ti (Orf and Hymowitz 1979). A line containing this mutation was shown to have improved protein efficiency ratio when compared to a nonmutant isoline. This difference was attributed to improved digestibility due to reduced trypsin inhibitor activity.

4.4.2.4

Oil

The fatty acid composition of vegetable oil has a large impact on its functional properties and its dietary impact on health. The specific composition with the highest value depends on the end use of the oil. Given the wide range of uses of vegetable oils, many types of modifications have added value. The fatty acid biosynthetic pathway has been manipulated by mutagenesis or genetic engineering to produce oils with many potentially valuable compositions (reviewed in Fehr 2007). Manipulation of fatty acid levels is complicated by the fact that most steps in the biosynthetic pathway of these compounds are encoded by families of genes.

4.4.2.5

Oxidative Stability

Unsaturated fatty acids, such as linolenic acid, are susceptible to oxidation at the high temperatures used in frying. This oxidation results in undesirable flavors and odors, and therefore low-linolenic acid oils are desirable. Breeders have used EMS (ethyl methyl sulfonate) mutagenesis to produce lines with low linolenic acid (Hammond and Fehr 1983; Wilcox et al. 1984). Reduced linolenic acid levels in these lines have been attributed to lesions in the members of the omega-3 fatty acid desturase (FAD3) gene family (Byrum et al. 1997).

4.4.2.6

Health Benefits

High levels of saturated fatty acids are correlated with coronary heart disease. In soybean oil, palmitic acid is the most abundant saturated fatty acid. Alleles of several genetic loci have been used by breeders to reduce palmitic acid levels in soybean, but most have not been molecularly characterized. The exception is FATB, which encodes a 16:0 thioesterase.

4.4.2.7

Flavor

Lipoxygenases are enzymes that catalyze the oxidation of polyunsaturated fatty acids (reviewed in Feussner and Wasternack 2002). These reactions produce a number of aromatic compounds that contribute to undesirable beany flavors in bean products. Three major isoforms of soybean lipoxygenase are designated LOX1, LOX2 and LOX3. Soybeans lines containing null alleles of these isoforms

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have been identified (Kitamura et al. 1983; Davies and Nielsen 1986). Characterization of soybeans containing LOX null alleles has been shown to accumulate reduced levels of compounds responsible for undesirable flavors (Davies and Nielsen 1987; Hildebrand et al. 1990; Moreira et al. 1993; Nishiba et al. 1995) and that tofu and soymilk made from LOX null soybeans had a less beany flavor than similar products made from normal beans (Davies and Nielsen 1987; Torres-Penaranda et al. 1998).

4.4.3

Starch

Starch accumulates in maize kernels as insoluble granules. When heated in the presence of water, starch gelatinizes. This behavior makes starch useful for many purposes in food as an adhesive and thickening agent and industrial applications such as making paper, paint and cosmetics. With such a broad range of applications, nearly any modification to the physical properties of starch will make it better suited to one of its end use.

4.4.3.1

Industrial Applications

The physical properties of starch are determined by its chemical structure. Starch is a polymer of glucose, but it can be fractionated into two polymers with different degrees of branching. Amylose is a largely linear polymer, while amylopectin contains a higher degree of branches. The ratio of amylose to amylopectin has a large impact on the physical properties of starch. Several genes control this ratio, and mutations in these genes are used in varieties that produce different industrial starches. It was shown that the waxy (wx1) gene lacks a glucosyl transferase activity (Nelson and Rines 1962), later determined to be due to the granule-bound starch synthase. Mutants of wx1 do not accumulate amylose (Sprague et al. 1943) and are particularly useful as adhesives. The amylase extender (ae1) gene of maize accumulates high levels of amylose, and encodes starch branching enzyme IIb (Stinard et al. 1993). The high amylose starches derived from this mutant forms strong gels well suited to confections and thickening agents.

4.4.3.2

Food Quality

In addition to mutants that change starch structure, mutants that change the amount of starch produced are valuable. Sweet corn that is used directly for human consumption is based on mutations that reduce the level of starch in the kernel. This reduction in starch is accompanied by a concomitant increase in the sugar content of the developing kernels, resulting in the sweetness and flavor desired by

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consumers. The sugary-1 (su1) gene encodes a starch debranching enzyme that is essential for the production of normal starch granules (James et al. 1995). Recently, the shrunken-2 (sh2) mutation has been employed to develop “supersweet” types of sweet corn (Tracy 1997). The sh2 gene encodes an ADP-glucose pyrophosphorylase (Bhave et al. 1990).

4.4.4

Other Components

Plants store phosphorus in the form of a phosphorylated sugar called phytic acid. Nonruminant animals including humans cannot efficiently digest phytate, leading to a deficiency in phosphorous in grain-based diets. In addition, phytate is an efficient chelator of several minerals, such as iron and zinc that are limiting in some diets, reducing their biological availability. In addition, undigested phosphate in animal manure contaminates ground water and contributes to eutrophication of streams and lakes. Thus, reduction of phytic acid is desirable for both nutritional and environmental reasons. Mutants of maize (Raboy et al. 2000) and soybean (Wilcox et al. 2000; Hitz et al. 2002) with reduced content of phytic acid identified, however, pleiotropic effects on seed weight in maize (Raboy et al. 2000) and seedling emergence in soybean (Oltmans et al. 2005) are a barrier to the development of commercial varieties with reduced phytate. A transgenic approach involving embryo-specific downregulation of the maize lysophosphatidic acid receptor (lpa1) gene may successfully overcome these problems (Shi et al. 2007).

4.5

Concluding Remarks and Future Prospects

Boosting global production of quality grain to feed our planet’s increasing population demands advanced understanding of plant biology and creative strategies to manipulate the inherent development and physiology of our food crops. One strategy is to couple altering the timing of the floral transition with in vitro flowering to change the length of the crop plant’s life cycle, thereby, allowing faster breeding, quicker adaptation to new environments and increased production of higher-quality grain. To be successful, a more complete understanding of the mechanistic interactions that regulate flowering in diverse crop plants is required. For example, the nature of the primary endogenous flowering stimulus in maize is unknown and represents a serious gap in our understanding of how the maize floral transition network functions. Identifying the primary floral stimulus is essential to predictably manipulate flowering in this grain crop. Further, how treatment with different hormone regimes impacts floral network function is not understood. Dissecting how hormone and floral network signaling intersect is an important area for future research. Increased investment in both basic and applied plant

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biology research will enhance our understanding of these key developmental processes and provide the components necessary to enable the modulation of a crop’s life cycle. In conjunction with improvements to grain quality, progress can be made towards the goal of feeding people globally.

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Chapter 5

Biotechnological Interventions to Improve Plant Developmental Traits Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe, Ajay Arora, Martı´n-Ernesto Tiznado-Herna´ndez, Ravinder K. Goyal, Anish Malladi, Pradeep S. Negi, and Autar K. Mattoo

5.1

Introduction

Unprecedented progress during the last three decades in our understanding of the principles of a living cell, particularly the identification of genes and signaling pathways involved in cell differentiation and organ development, has brought us a broader insight into plant biological processes. Technological advancements are revealing new and fundamental knowledge at the molecular and cellular levels, knowledge that is critical towards achieving the goal of precision-based crop improvement. Modernday genetic engineering has emerged as a promising precision-based technology for boosting up food production in the world and introducing desirable traits such as nutritional enhancement and disease and pest resistance, both important components of agricultural sustainability (Chrispeels et al. 2002; Fatima et al. 2008; Negi and Handa 2008). Achieving results that benefit the world will depend on the success of applying new knowledge to real-world field scenarios. The challenge, therefore, is also to simultaneously obtain knowledge on agroecosystem structure and function to understand how manipulation and control of specific gene expression will translate into directing processes at the ecological scale (Mattoo and Teasdale 2009). Developmental traits are coordinated at various levels in a plant and involve organ-to-organ communications via long-distance signaling processes that integrate transcription, hormonal action and environmental cues. Thus, plant architecture, root–soil–microbe interactions, flowering, fruit (and seed) development, and fruit ripening (and seed germination) are highly regulated genetic programs that are also impacted by processes such as organ abscission, organ senescence (and ripening), and programmed cell death (PCD). We note that belowground processes provide the anchor for a healthy and robust plant (Mattoo and Teasdale 2009) but in this

A.K. Handa (*) Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907-2010, USA e-mail: [email protected]

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chapter we have focused on the aboveground plant processes, bringing together information on the genes and biotechnological applications that can potentially affect production and quality of crop plants.

5.2

Shoot Branching

Shoot branching (tillering in monocots) involves the initiation and development of axillary buds of a leaf, which form new branches or flowers. It is one of the most important traits of plant architecture, affecting plants’ ability to intercept light energy for photosynthesis and adaptation to grow in different environmental conditions. Both branching number and branch angle are traits that control crop yield. Branching is regulated by endogenous plant hormones and environmental signals. Plant hormones such as auxin, cytokinin, and strigolactones or their precursors, have major roles in controling branching. Auxin is actively transported basipetally down the shoot to inhibit shoot branching (Ongaro and Leyser 2008), while cytokinin and strigolactones are transported acropetally to promote and inhibit bud growth, respectively. Modification of genes involved in the biosynthesis, transport, or perception of these three hormones is expected to affect branching. For example, three rice mutants, d3 (Ishikawa et al. 2005), htd1 (Zou et al. 2006) and dwarf10 (Arite et al. 2007), which are either deficient in strigolactone perception or biosynthesis, display increased branch numbers. D3, a leucine-rich repeat F-box protein, is required for strigolactone perception, while HTD1 and DWARF10, which encode different members of carotenoid cleavage dioxygenases, are required for strigolactone biosynthesis. Domestication of major cereal crops involved modification of shoot branching, allowing for denser planting and less shelf-shading. Cultivated varieties usually have fewer branches compared to their wild ancestors (Doust 2007). Some of the genes affecting branching number or angle have been cloned from cereal crops (Table 5.1). For example, teosinte, wild ancestor of maize, displays many tillers with slender ears, while maize shows strong apical dominance and typically develops a few tillers with thicker ears (Fig. 5.1). The apical dominance in maize is mainly controlled by teosinte branched1 (tb1) (Doebley et al. 1997). This gene was cloned by transposon tagging and shown to repress growth of axillary buds and promote female inflorescences. Overexpression of a rice TB1 gene under the constitutive rice actin promoter reduced lateral branching, whereas a rice line with nonfunctional TB1 exhibited enhanced lateral branching (Takeda et al. 2003). Similarly, overexpression of a maize TB1 gene in wheat suppressed tiller development (Lewis et al. 2008), while a loss of function of the Arabidopsis TB1 homolog, Branched1, in the T-DNA knockout lines or RNAi lines (Chap. 1-6), resulted in increased branching (Aguilar-Martı´nez et al. 2007). These results indicate that TB1mediated control of branching is conserved between monocots and dicots. Since branching is also affected by environmental and growth conditions, it remains to be seen whether fine-tuning of expression levels of branching-related genes via bioengineering (Chap. 1-7) will improve crop yield.

Tiller Angle Control 1 (TAC1) LAZY1 (LA1)

PROSTRATE GROWTH1 (PROG1) MONOCULM1 (MOC1) Promotes bud initiation and development Increases tiller angle

Promotes erect growth Loss of function in rice promotes prostrate growth (wider angle)

GRAS family protein, putative transcription factor Unknown function

Unknown function

Overexpression in rice increases tiller number, and loss of function in rice reduces tillering capacity Overexpression in rice causes wider tiller angle

Increases branching and tiller angles

Li et al. (2007a), Yoshihara and Iino (2007)

Yu et al. (2007)

Li et al. (2003)

Phenotypes Reference Overexpression in rice and maize Lewis et al. (2008), Takeda et al. decreases branching, loss of function (2003), Aguilar-Martı´nez et al. in Arabidopsis increases lateral (2007) branching Overexpression in rice causes prostrate Jin et al. (2008), Tan et al. (2008) growth

Zinc finger transcription factor

Table 5.1 Genes with potential to modify plant branching Gene Function Effect TCP transcription Inhibits shoot TEOSINTE factor branching BRANCHED1 (TB1)

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Fig. 5.1 Mature plants and inflorescence of teosinte branched 1 mutant and wildtype maize. a, b, the mutant; c, d, an inbred maize line. Modified from Doebley et al. (1997)

5.3

Male Sterility

Male sterility in higher plants is characterized by a failure to produce functional anthers, pollen or male gametes. It is a useful trait, both for studying the male developmental pathway and for exploiting heterosis to obtain high-yielding crop plants. Kaul (1988) classified male sterile types as structural, sporogenous

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or functional. Structural male sterile plants may show absence of stamens with anthers or presence of anthers that are devoid of sporogenous tissues. Sporogenous male sterile plants show normal anther development but the development of pollen is impaired because of irregularities in the division of sporogenous cells. Plants that show anthers with viable pollen development but are still incapable of affecting fertilization because of anther dehiscence failure, abnormal exine morphology or failure of pollen tube germination, are classified as functional male sterile types. Additionally, Taylor and Jorgensen (1992) have proposed the term “conditional male fertility” for the type of male sterility in which viable pollen is unable to germinate and shows pollen tube growth in self-crosses of the chalcone synthase-deficient progeny. At the genetic level, three types of plant male sterilities have been identified, namely, genic male sterility (GMS), cytoplasmic male sterility (CMS) and gene-cytoplasmic male sterility.

5.3.1

Genic Male Sterility

GMS is caused by spontaneous or induced mutations in nuclear genes. It is ordinarily governed by ms, a single recessive gene. GMS is inherited in Mendelian fashion and has been reported in approximately 175 species of angiosperms, including crop plants such as barley, maize, pea, tomato, rice and pepper. These plants can be propagated when pollinated by wild type, male fertile plants. Several male sterile plants exhibit structurally malformed male sexual organs. For example, genes such as apetalla-3 (ap-3), pistillata (pi) and antherless (at) in Arabidopsis affect the development of male sexual organs resulting in sterility (Chaudhury 1993). Mutations also affect genes participating in the differentiation and function of such anther cell types as the stomium, tapetum, endothecium and the archesporial and sporogenous layers of the anther primordium. Arabidopsis mutant ms2 shows defective tapetal layer development resulting in pollen abortion shortly after release from the tetrads (Aarts et al. 1993). Defective meiosis in Arabidopsis (msW and msY; Dawson et al. 1993) and tomato mutants (ms3, ms15 and ms29; Rick 1948) also resulted in male sterile plants. Furthermore, mutations in genes controlling metabolic pathways may also result in sterile/nonviable pollens. Induced petunia white anther (wha) mutant is characterized by the inability of the pollen grains to germinate in vitro. This phenotype is caused by a lesion in chs (chalcone synthase) gene that encodes the first enzyme in the flavonoid biosynthesis pathway (Napoli et al. 1999). Such examples of spontaneous or induced GMS are limited in their use for plant breeding purposes by the availability of a fertility restoration gene. Genetic engineering approaches to introduce a dominant male sterile gene into plant cells (Chap. 1–3) have overcome such barriers and resulted in male sterile transgenic lines in many crop plants. These approaches have been discussed in greater detail under biotechnological advances in Sect. 5.3.3.

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Cytoplasmic and Gene-Cytoplasmic Male Sterility

CMS, in most cases, is determined by mutation in mitochondrial genes (Hanson and Bentolila 2004; Pelletier and Budar 2007). CMS has been reported in approximately 150 species, comprising both wild and cultivated plants (Raghavan 1997). It is maternally inherited, easier to maintain in crop plants and therefore of much value to plant breeders. When the vegetative plant part is the harvested product, CMS alone can be used to obtain higher-yielding hybrid lines. However, when seed or fruit is the desired product, the heterotic hybrid lines should be self-pollinating and able to set seeds. A fertility restorer (Fr) gene must be introduced in such hybrids. Restorer genes, also known as Restorer of fertility (Rf) or Fr, are nuclear genes that mask the male sterility effect of CMS, such that the plant is rendered male fertile in their presence (Schnable and Wise 1998). Rf and CMS together form a binary system (genic-cytoplasmic or nucleo-cytoplasmic) that regulates the mode of reproduction of individuals in populations (Pelletier and Budar 2007). It is possible to remove the effect of Rf genes through interspecific crosses or somatic hybridization such that the nuclear genome of one plant species is present in the male sterility-inducing cytoplasmic background of another plant species (Chase 2007). Male sterility arising as a result of interspecific nuclear-cytoplasmic combination is known as alloplasmic male sterility (Hanson and Bentolila 2004). The absence of Rf gene from the alloplasmic nuclear-cytoplasmic combination unmasks the male sterility-inducing effect of the mitochondrial genes. However, in some cases, hybridization itself has proven mutagenic causing novel cytoplasmic genome configuration that arises because of disturbances in cytoplasmic genome replication and organization (Hanson and Conde 1985). CMS systems have been extensively used to generate high-yielding hybrid varieties in crop plants. In maize, several types of male sterility-inducing cytoplasms have been identified of which CMS-T, -S and -C are the most widespread and well characterized. These CMS systems are differentiated by their reaction to restorers; CMS-T is restored by Rf1 and Rf2, CMS-S is restored by Rf3, and CMS-C is restored by Rf4 (Sofi et al. 2007). Many CMS systems have been found in Brassica crops including the well-studied ogura (ogu), polima (pol) and napus (nap) CMS systems. The pol and nap CMS systems are restored by Rfp and Rfn genes, respectively. Both Rfp and Rfn map to the same chromosomal position and they are allelic (Li et al. 1998). In rice, CMS-BT (Boro type), CMS-WA (wild abortive) and CMS-HL (Honglian) are well established: Rf1a and Rf1b that confer fertility to CMS-BT and Rf5 and Rf6(t) that confer fertility to CMS-HL are all located on chromosome 10 (Fuji et al. 2008). The restorer genes, Rf3 and Rf4, which confer fertility in CMS-WA are located on chromosomes 1 and 10, respectively. CMS systems and their restorer genes in other crop plants are listed in Table 5.2. Many male sterility-conferring cytoplasmic genes have now been identified as open reading frames (ORFs, Table 5.2). For example, the male sterility-inducing CMS systems T-urf13 in maize, pvs-orf239 in bean and orf138, orf224 in Brassica are ORFs formed as a result of complex mitochondrial DNA (mtDNA)

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Table 5.2 Plant cytoplasmic male sterility systems Restorer locus Plant CMS system Mt ORFa/gene Radish Ogura orf 138 and orfB Rfo Kosena orf125 Rf K Rice CMS-BT Orf 79/B-atp6 Rf1a, Rf1b CMS-WA Unknown Rf3, Rf4 CMS-HL orf H79/atp6 Rf5, Rf6 (t) Petunia Pcf Rf1 Maize CMS –T T-urf13 Rf2, Rf1, Rf8, Rf* orf355-orf77,atp6, CMS-S Cob Rf3 CMS-C atp6-C Rf4, Rf5, Rf-I Brassica pol orf224/atp6 Rfp nap orf222 Rfn(Mmt) Sunflower PET1 orfB,orf552 Rf1 Sorghum A1 Rf1 A3 Orf107 Rf3, Rf4 a Mitochondrial open reading frames associated with CMS

205

Reference Brown et al. (2003) Koizuka et al. (2003) Wang et al. (2006a, b) Li et al. (2007a) Zhang et al. (2007a) Bentolila et al. (2002) Wise et al. (1999) Wen and Chase (1999) Dewey et al. (1991) Singh et al. (1996) Homme et al. (1997) Horn et al. (2003) Klein et al. (2005) Kuhlman et al. (2006)

rearrangement (Hanson and Bentolila 2004). These ORFs often comprise a mix of conventional mitochondrial genes and sequences of unknown origins. They are co-transcribed with essential mitochondrial genes and encode proteins with at least two shared features: lower molecular size (<30 kDa) and at least one hydrophobic domain. Studies comparing the respiratory activities of CMS and fertile lines have shown differences in the activity level of the respiratory complexes (Conley and Hanson 1995; Sabar et al. 2000). ATP synthase as well as cytochrome oxidase are associated with the inner membrane of the mitochondria (Hanson and Bentolila 2004). The presence of ATP synthase subunits in the CMS-associated chimeric genes raises the possibility of an impaired ATP synthase activity being the cause of disrupted pollen development that results in male sterility. Premature PCD, in which mitochondria play a central role, has also been suggested to be a possible cause of CMS. According to this suggestion, PCD destroys the tapetal cells that provide nourishment to the developing pollen inside the anthers and thereby causing male sterility (Balk and Leaver 2001; Ku et al. 2003; Rogers 2006). Fr genes encode members of pentatricopeptide repeat protein (PPR) family. PPR genes have been found in the restorer loci of Petunia, Brassica, Raphanus and rice (Hanson and Bentolila 2004). These proteins, targeted to chloroplast and mitochondria, are involved in organelle biogenesis in plants (Lurin et al. 2004). Studies involving nuclear gene mutants with compromized plastid functions have revealed PPR proteins to be involved in RNA processing, intron splicing and translation (Stern et al. 2004; Shikanai 2006). These studies indicate probable functions for mitochondria-targeted PPR proteins including those encoded by restorer genes. In most fertility-restored CMS lines, the protein product for the CMS determining locus does not accumulate. This loss of protein accumulation is accompanied by lower accumulation of CMS-associated transcripts or truncated transcripts. Whether the decreased transcript level or transcript truncation results in the lack

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of protein accumulation or is a by-product of failed translation or imperfect editing of CMS-associated transcript in the presence of a restorer gene is not known (Hanson and Bentolila 2004; Newton et al. 2004; Linke and Borner 2005; Chase 2007).

5.3.3

Bioengineering Male Sterility for Hybrid Plants

Mariani et al. (1990) successfully engineered the first GMS plant, thereby paving the way for the development of more transgenic male sterile crop plants. The ablation of tapetal cells inside anthers results in failure of pollen development. The barnase gene product (barnase) is an extracellular RNase found in Bacillus amyloliquifaciens that protects the bacterium from microbial predators (Hartley 1989). The bacterium is protected by the cytotoxic effect of barnase by intracellular barstar, a barnase-specific RNase inhibitor. A chimeric gene comprising tapetumspecific gene promoter TA29 and barnase gene coding sequences was used to develop transgenic male sterile tobacco and oilseed rape plants. These plants showed ablation of tapetal cells and impaired pollen development. However, these transgenic plants did not show any adverse effect on tapetal cells. When male sterile plants containing TA29/barnase genes were crossed with male fertile plants containing TA29/barstar gene, the resultant progeny was male fertile (Mariani et al. 1992). The barnase/barstar system has been used extensively to obtain male sterile lines in oilseed rape, tomato, cotton, corn and wheat (De Block et al. 1997). Following the pioneering efforts of Mariani et al. (1990), several biotechnological strategies to generate genic male sterile plants have been tested (Perez-Prat and van Lookeren Campagne 2002), which include: 1. Tissue-specific expression of a gene encoding a protein that can disrupt the cell function and thereby development of tissues that are vital for pollen development – barnase/barstar is one such system. Chemical induction of male sterility by selective expression of genes encoding a protein that converts a pro-herbicide into a herbicide only in male reproductive tissues (O’Keefe et al. 1994; Dotson et al. 1996; Kriete et al. 1996). Alternatively, a male sterility gene engineered such that it is induced by the application of a chemical (Goff et al. 1990). Fertility restorer gene or a repressor of male sterility under the control of a chemically inducible promoter employed for fertility restoration (Ward et al. 1993). 2. Manipulation of metabolite levels essential for normal pollen development. Genetic alteration of the levels of amino acids, sugars (Goetz et al. 2001), flavonols (Derksen et al. 1999), jasmonic acids (McConn and Browse 1996; Browse 1997; Sanders et al. 2000), biotin (Albertsen and Howard 1999) and auxins (Spena et al. 1992) to develop sterile lines. In such systems, fertility is inducible by applying the altered metabolite (McConn and Browse 1996; Browse 1997; Albertsen and Howard 1999; Sanders et al. 2000). 3. Crossing two transgenic parental lines, each carrying a gene encoding for an inactive part of a toxin in the male reproductive tissue. When brought together in

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the progeny, these inactive parts combine together to form the active toxin, which results in male sterility (Fabijanski and Arinson 1995; Gutterson and Ralston 1998). 4. Use of transgenic maintainer lines for natural or induced GMS lines. These lines allow the propagation of natural or induced mutations in such a way that male sterile plants may be obtained or male fertile plants may be visually identified and separated. Such maintainer lines are isogenic to the genic male sterile lines except at the transgenic locus comprised two components: (a) A restorer gene that renders the maintainer lines male fertile; and (b) genes or set of genes, which upon crossing a maintainer line with an isogenic male sterile line, either prevent the maintainer locus from passing into the progeny (such plants are called pollen lethality maintainers) or reveal the maintainer locus by simultaneously expressing a gene that results in pigment accumulation (color maintainers). In the latter crosses, 50% plants are male fertile; however, these seeds can be identified and separated according to pigment accumulation (Perez-Prat and van Lookeren Campagne 2002). GMS is limited in its use for hybrid seed production as it is inherited in Mendelian fashion. The plants segregate for male fertility and sterility. This presents a practical problem of separating the male fertile and male sterile plants. CMS, on the other hand, is inherited maternally. Breeders have widely exploited CMS systems to develop male sterile lines. Engineering male sterility through somatic hybridization has resulted in improved CMS systems as well as expansion of the CMS systems to a larger group of crop plants (Pelletier and Budar 2007). Somatic hybridization involves isolation, culture and fusion of protoplasts of two distantly or closely related or even unrelated plants at interspecific, intergeneric, intraspecific or interfamily levels. The heterokaryons resulting from fused protoplasts are selected based on a marker gene and then cultured to develop into complete plants. In some of these heterokaryons, nucleo-cytoplasmic interactions result in formation of cytoplasmic hybrids (cybrids), which carry the nuclear genome of only one parent but the cytoplasmic genomes from both parents. The nuclear genome of the mitochondrial donor should carry a restorer gene (Pelletier and Budar 2007). Male sterile cybrids have been developed in Brassica species (Budar et al. 2004), rice (Brar and Khush 2006) and tomato (Melchers et al. 1992). Natural CMS system is not found in all crop plants and its use for breeding purposes is limited if an appropriate restorer system is not present. Although transgenic dominant genic male sterile plants can be developed, this approach is restricted by the Mendelian segregation of the progeny. Stable, maternally inherited male sterility can be induced in crop plants by organelle transformation (Chap. 1-8). Male sterility induction has been reported by plastid transformation using b-ketothiolase gene (phaA) in tobacco plants (Ruiz and Daniell 2005). The phaA gene, under the control of light-inducible psbA promoter, alters the course of fatty acid synthesis, which in turn leads to pollen grain collapse and male sterility. Male sterility was reversed by growing these plants under continuous illumination. Plastid transformation holds great promise for genetic transformation of crop

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plants. It will be possible to take better advantage of this technology for developing male sterile crop plants and their restorer systems as more plant species become amenable to plastid transformation (Kuchuk et al. 2006). Studies on GMS and CMS systems have not only revealed the fundamental aspects of male developmental pathways, but also the molecular players in male sterility and fertility restoration in plants. Also, genetic engineering was employed to inhibit flower development and introduce reproductive sterility. Expression of a ribosome-inactivating protein (DTA) gene under the control of PTD gene – homologous to the MADS box genes DEFICIENS and APETALA3 – resulted in sterile flowers and consequently impaired dissemination of the transgene and the confinement of specific trait (Skinner et al. 2003; Wei et al. 2006). Biotechnological advances have allowed the expansion of these male sterility systems in a wider range of crop plants. Identification of mitochondrial genes controlling male sterility and those that restore fertility and understanding their mode of function will allow a better control over these systems. Advances in organellar transformation will provide a better process for inducing male sterility/fertility restoration in plants. Transformation of mitochondria remains the next hurdle to overcome and will prove a breakthrough advance towards understanding the role of this organelle in male development and sterility aspects.

5.4

Flowering Induction

Flower induction is under the control of several interconnected pathways involving light irradiation, temperature, plant growth regulators and the physiological state of the plant (Jaeger et al. 2006; Kobayashi and Weigel 2007; Wilkie et al. 2008). The genetic basis of these pathways are the subject of intense research in Arabidopsis (Ba¨urle and Dean 2006), as well as in maize, rice (Chap. 4.2) (Izawa 2007), soybean (Wong et al. 2009) and other plants of economic interest. The control of flower induction is a major agronomical event in crop productivity, e.g., cereal (Cockram et al. 2007), fruit (Carmona et al. 2008), plant fibers (Abdurakhmonov et al. 2007) or wood industry (Yuceer et al. 2003; Bo¨hlenius et al. 2006). In potato, more or less the same genetic components regulate flowering and tuberization (Rodrı´guez-Falco´n et al. 2006). In cereal crops, regulation of flower induction is critical for seed set and development. The day length and temperature were found to be the key elements for the growth attributes of wheat and maize when initially grown in the Fertile Crescent and Central America, respectively. In recent times, however, the area for growing cereals has widely expanded during centuries of selection and improvement (Cockram et al. 2007). In Arabidopsis, promotion of flowering involves long-day photoperiod, gibberellins, an autonomous route, and vernalization (Jack 2004; Putterill et al. 2004). The photoperiod pathway perceives light quantity and circadian clock – long day promotes flowering in Arabidopsis. In order to induce flowering at an appropriate time, the day length has to be in sync with an internal time keeper (Ba¨urle and

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Biotechnological Interventions to Improve Plant Developmental Traits

Table 5.3 Genes regulating flowering time in plants Gene Function Phenotype CO

Transcription factor

Biotechnology Reference intervention Constitutive Martı´nezoverexpression Garcı´a et al. (2002) Overexpression Kardailsky et al. (1999)

Altered flowering time. Impaired potato tuberization FT Kinase inhibitor Early flowering Arabidopsis Forms CO/ FT regulatory module pTFT1 Kinase inhibitor Early flowering. Timing of Overexpression Populus FT ortholog flowering and seasonal growth cessation in trees Ehd 2 (rice) Zinc finger Promotes flowering Mutation protein ortholog of maize INDETER MINATE1 OsCO3 (rice) Transcription Inhibition of flowering factor Reduced juvenile phase Overexpression Leafy (LFY) Apetala1 (AP1) Arabidopsis

209

Bo¨hlenius et al. (2006) Matsubara et al. (2008)

Pen˜a et al. (2001)

Dean 2006). Gibberellins promote flowering, especially under short-day photoperiod. The autonomous pathway is independent of photoperiod control, and causes flowering in Arabidopsis under short days. Vernalization refers to promotion of flowering by extended cold exposure, as naturally happens in plants during winter. Different pathways of flower-regulation genes converge at a few defined floral pathway integrators that include Flowering Locus T (FT), Suppressor of Overexpression of Constans 1 (SOC1) and Leafy (LFY), which activate floral meristem identity genes and trigger the transition from vegetative to reproductive phase (Table 5.3) Details of the transition from vegetative meristem to floral transition are described in Chap. 4.2. Two genes, Constans (CO) and Flowering Transition (FT), whose expression is tuned to the change in day length and involved in flowering, were initially isolated from Arabidopsis (Kobayashi and Weigel 2007). CO is a transcription activator that belongs to the B Box Zinc finger protein family. FT is a 23-kDa protein from the RAF kinase inhibitor family involved in regulatory cascade and signal transduction (Turck et al. 2008). FT protein is synthesized in the companion cell of the leaf phloem and mobilized to the shoot apical meristem to initiate the flowering (Cockram et al. 2007; Turck et al. 2008). In Arabidopsis, FT protein accumulation is directly under the CO control.

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CO protein does not bind DNA by itself but interacts with transcriptional complex (Kobayashi and Weigel 2007). The biochemical mode of action of CO is still unclear. Orthologs of CO have been identified in rice (Izawa 2007), potato (Martı´nez-Garcı´a et al. 2002) and aspen tree (Bo¨hlenius et al. 2006). CO transcription is under the control of circadian regulation through CDF1 (cycling DOF Factor). Its transcripts accumulate from the afternoon to the middle of the night either in long day (LD) or short day (SD) but with a slight shoulder at the end of the afternoon during LD (Fig. 5.2; Imaizumi et al. 2005). CO transcripts are degraded in LD plants at the end of the day and during the night but in SD this takes place only during the night (Valverde et al. 2004). Proteosome-mediated degradation of CO protein occurs in the dark as well as under red light. During LD conditions, CO protein accumulates at the end of the light cycle and activates FT transcription but under SD it is rapidly degraded and therefore unable to activate FT transcription (Fig. 5.2). In rice, a CO ortholog Hd1, activates flowering only slightly under the SD condition but inhibits the accumulation of FT under LD. Patterns of CO and Hd1 transcript accumulation are the same (Izawa 2007). Transgenic potato expressing Arabidopsis CO exhibited reduced growth and delayed tuberization, indicating an inhibitory effect of AtCO on tuberization (Martı´nez-Garcı´a et al. 2002). CO-FT regulatory module also controls timing of flowering and seasonal growth cessation in aspen trees (Bo¨hlenius et al. 2006). Overexpression of FT ortholog, PtFT1 in aspen, reduced the flowering delay in transgenic tree from several years to 6 months. Unexpectedly, it also regulated the SD-induced growth cessation and bud setting in the fall. This may explain growth cessation displayed by aspen trees a

c CO CO CO CO CO CO CO CO

b

CO CO CO CO CO CO CO CO CO

d CO CO CO CO CO CO CO

CO

CO CO

DegradedCO

CO CO CO CO CO CO CO CO

FLOWERING

ActiveCO

Fig. 5.2 Diurnal regulation of CO transcripts (—) CO protein (▪ ▪ ▪) and FT protein (- - -) accumulation of in plants under long day and short day growth conditions. (a) Accumulation of CO transcripts is regulated by circadian clock but not by the light status, (b) whereas accumulation the CO protein is light dependent (adapted from Bau¨rle and Dean 2006). (c) Under the short day conditions CO protein is rapidly degraded and inactivated by the proteasome during the dark period, (d) but when plants are grown under long day conditions it accumulates at the end of the afternoon before the beginning of dark period. Active CO proteins induce the flowering (adapted form Valverde et al 2004)

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sampled across a latitudinal gradient spanning northern Europe. LFY or APETALA1 (AP1) are other genes that promote flower initiation in Arabidopsis. When these genes were overexpressed under CaMV 35S promoter in hybrid citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf.), juvenile phase was reduced from 7 years to about 15–20 months (Pen˜a et al. 2001). Expression of AP1 was as efficient as LFY in the initiation of flowers, and no severe developmental abnormality was observed. Modifying transition phase from juvenile to mature plants should greatly improve the possibilities for breeding of trees for agronomical and industrial use.

5.4.1

Flower Senescence

Senescence results in significant losses of cut flowers and offers challenge for postharvest researchers trying to delay this process. The process of flower senescence has been shown to be a genetically programmed event (Stead et al. 2006; Arora 2008). Flower petals are ideal tissues for cell death studies as they are relatively homogeneous, short-lived tissue that can be manipulated by exogenous chemical treatment without substantial wounding. Plant hormones, membrane stability, water availability, cellular proteolysis and carbohydrate metabolism act in concert to determine the differential rate of senescence for different floral organs. However, the death of floral tissues is affected by several factors including the developmental stage, pro-senescence signals (e.g., pollination-induced petal senescence), and stress-related metabolism in response to temperature, wounding, and nutrient starvation (Stead et al. 2006; Arora 2008). Floral senescence is a developmental continuum in the flower that is preceded by tissue differentiation, growth and maturation of the petal, growth and development of seeds. Genome-wide searches have resulted in isolation and identification of a number of genes associated with flower senescence from several species, including Alstroemeria (Breeze et al. 2004), carnations (Verlinden et al. 2002), Chrysanthemum (Narumi et al. 2005), daffodil (Hunter et al. 2002), daylily (Panavas et al. 1999), rose (Channeliere et al. 2002), iris (van Doorn et al. 2003), Sandersonia aurantiaca (Eason et al. 2002), Petunia (Jones et al. 2005) and Gladiolus (Arora and Singh 2004; Arora et al. 2006). Characterization of their function has provided insights into the roles played by ethylene signaling, proteolysis, nucleic acid and chlorophyll breakdown, and lipid and nitrogen remobilization in the progression of flower senescence (Gan and Amasino 1997; Stead et al. 2006). The future genetic analysis of floral senescence will likely identify molecular mechanisms that help maintain a non-senescent “juvenile” state and provide novel interventions to extend post-harvest life of not only cut flowers but also fruit and vegetable crops (Mattoo and Handa 2008). Ethylene is one of the plant hormones with profound effects on plant growth and development including senescence and ripening processes (Fluhr and Mattoo 1996; Mattoo and Handa 2004). Key genes regulating biosynthesis of ethylene

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include S-adenosylmethionine (SAM) synthase, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase (Fluhr and Mattoo 1996). Different plant organs show responses to ethylene to different degrees. Even cultivars within flower species exhibit a significant variability in sensitivity to ethylene. Most modern carnation cultivars exhibit much reduced sensitivity to ethylene indicating possibilities of genetically engineering flower vase life by reducing ethylene biosynthesis or perception. Impairing ethylene biosynthesis by expressing an antisense ACC oxidase gene resulted in dramatic reduction of ethylene production with a concomitant increase in the longevity of carnation and Torenia fournieri flowers (Savin et al. 1995; Aida et al. 1998). However, similar attempts in Begonia failed and transgenic flowers did not show prolonged flower life (Einset and Kopperud 1995). Increasing longevity of carnation cut flowers was also achieved by reducing expression of ACC synthase by co-suppression (Michael et al. 1993). Another avenue to control floral and leaf senescence is to manipulate perception of ethylene. Characterization of an ethylene receptor gene from Arabidopsis, AtETR1 (Chang et al. 1993), and tomato (Zhou et al. 1996a,b) led to the identification of a family of ethylene receptor genes in plants. Tomato genome contains six members, LeERT1–LeETR6, with each of the receptor genes having a distinct expression pattern during plant development and in response to external stimuli (Klee and Tieman 2002). Ethylene-insensitive 3 (EIN3) encodes a transcription factor that functions downstream from the ethylene receptors in the ethylene signal transduction pathway (Wang et al. 2002). EIN3 possesses two mitogen-activated protein kinase (MAPK) phosphorylation sites that have opposing effects on EIN3 stability. Homologs of the Arabidopsis EIN3 gene are present in tomato: LeEIL, LeEIL1, LeEIL2 and LeEIL3. EIN3-BINDING F-BOX1 and 2 (EBF1/2) coordinately control 26S proteasome degradation of the transcription factors, EIN3 and EIL1. ETHYLENE-INSENSITIVE5 (EIN5), which encodes the exoribonuclease XRN4, represses expression of EBF1/2. However, functional significance of each member of the ethylene receptor family is not fully understood (Kevany et al. 2007, 2008). A mutated ERS gene (ethylene response sensor, an Arabidopsis gene uncovered by cross-hybridization with the Arabidopsis ETR1 gene) conferred dominant ethylene insensitivity to wild-type Arabidopsis (Hua et al. 1995). Overexpression of a mutated dominant Arabidopsis ethylene resistance gene etr1-1 under the CaMV 35S promoter conferred ethylene insensitivity to Petunia (Wilkinson et al. 1997; Clark et al. 1999; Gubrium et al. 2000; Celvenger et al. 2004). Among the effects produced by the introduction of ethylene-related transgenes in plants were: enhanced ethylene production in pollinated flowers; slightly faster flowering; poor root development of cuttings; lower seed weight; less-efficient seed germination and rooting; delayed seedling growth; and reduced flower senescence, abscission and fruit ripening. The delayed flower senescence was seen both in the presence and absence of ethylene in pollinated and non-pollinated flowers. Most of these effects can be explained by the roles played by ethylene in different developmental processes. The enhanced ethylene production in flowers is probably due to the uncoupling of the feedback regulation from the plant’s response to pollination.

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213

However, the poor rooting ability of cuttings limits the use of this methodology. Overexpression of a mutated ers homolog from Brassica oleracea (Boers) also imparted ethylene insensitive to Petunia and induced prolonged flower longevity (Shaw et al. 2002). An unintended effect of this transformation, however, was increased susceptibility of the transgenic plant to fungal diseases, most likely because of an interference with the ethylene-induced defense activated by ethylene. The use of a flower-specific promoter from Petunia (fbp1) to express etr1-1 in carnation resulted in stronger insensitivity to ethylene without the undesired side effects as observed in earlier experiments (Bovy et al. 1999). Transformation of an ornamental Nemesia strumosa with a mutated etr1 melon homolog under a constitutive promoter also exhibited enhanced flower longevity by altering ethylene perception (Cui et al. 2004). Overexpression of isopentenyl phosphotransferase (ipt gene) from Agrobacterium tumefaciens under a senescence-regulated promoter Psag12 increased cytokinin levels with concomitant delay in ethylene production and enhanced ethylene tolerance of the flowers of the transgenic plants (Chang et al. 2003). The flower longevity was prolonged by 100% in non-pollinated flowers and approximately 450% in pollinated flowers. Interactions between ethylene, cytokinins, sugars and various hydrolytic enzymes are known to differentially mediate the progression of flower senescence. However, individual signals appear to be species-specific and vary among the plant variety and floral organs. The challenge for post-harvest scientists is to identify a hierarchy of regulators or specific patterns underlying the progression of flower senescence. The use of tissue-specific promoters (Chap. 1-5) is recommended since these may reduce undesired effects of the introduced gene, particularly in modifying plant hormone levels (Clark et al. 1999; Shaw et al. 2002). Conventional breeding has made significant advances in increasing the number of flowering buds, extending the longevity of inflorescence and improving post-harvest performance, as demonstrated in Lilium (Van der Meulen-Muisers et al. 1999). Coupling transgenic approach with molecular breeding might pave a way to greatly improve flowers, fruits and vegetables with desirable traits including shelf life.

5.5 5.5.1

Fruit Fruit Shape, Size and Mass

Domestication of almost all fruit species has invariably led to the selection for increased fruit size. In addition to size, fruit crops have been bred for shape, texture, flavor, shelf life and nutrient content. Genetic evaluation of various species, especially tomato, has provided a wealth of information about loci that control fruit size, weight and shape (Frary et al. 2000; Tanksley 2004; Cong et al. 2008). However, their quantitative nature has impeded characterization of individual genes regulating these fruit attributes (Grandillo et al. 1999). Table 5.4 lists some of the genetic

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Table 5.4 Transgenic genetics of fruit developmental attributes Enzyme/protein Transgene Phenotype Fruit shape, size and mass fw 2.2 Negative Overexpression Reduced fruit size, regulator of cell fruit weight division; possibly involved in cell-tocell communication SUN, IQ67 domain– Overexpression More elongated fruit containing family shape Ethylene regulated ripening ACC oxidase Underexpression Prolonged fruit shelf life ACC synthase Antisense Impaired ethylene production and fruit ripening. Extended shelf life Ethylene receptor LeETR1 Delayed abscission, antisense shorter internode length & reduced auxin transport. No effect on pigmentation and fruit softening SAM decarboxylase Overexpression Spermidine and spermine accumulation. High lycopene, improved juice quality and longer vine life Ripening mutations Ripening-inhibitor (rin) Overexpression Complements mutation MADS-box Regulates fruit transcription factor ripening Colorless non-ripening VIGS SPB-box transcription (Cnr) Factor Inhibits fruit ripening high-pigmentOverexpression Elevated carotenoid and 2 DETIOLATED1 flavonoid accumulation Fruit Texture Polygalacturonase (PG) Overexpression Increased pectin solubilization Antisense Slightly firmer fruit. Increased juice viscosity. Reduced pectin solubilization PG-b-subunit

Antisense

Reference Fray et al. (2000)

Xiao et al. (2008)

Xiong et al. (2005) Oeller et al. (1991)

Whitelaw et al. (2002)

Mehta et al. (2002)

Vrebalov et al. (2002)

Manning et al. (2006)

Davuluri et al. (2005)

Giovannoni et al. (1989)

Langley etal. (1994), Schuch et al. (1991), Kramer et al. (1992), Brummell and Labavitch (1997) Increased fruit softening, Watson et al. (1994), decreased middle Chun and Huber lamella cohesion. (1998) Reduced tissue integrity (continued)

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Biotechnological Interventions to Improve Plant Developmental Traits

Table 5.4 (continued) Enzyme/protein Pectin methylesterase

Pectate lyase b-galactosidase1

b-galactosidase3

b-galactosidase4

Expansin Expansin and PG

PG and PME Cellulase

endo-1,4-b –glucanase

Transgene Antisense

Phenotype No effect on softening but reduced tissue integrity in overripe fruit. Increased degree of methylesterification. Reduced pectin degradation. Increased soluble solids. Increased juice and paste viscosity. Increased serum viscosity Antisense Increased fruit firmness in strawberry. Antisense No effect on cell wall galactose. No effect on fruit softening. Antisense Reduction in TBG1 and TBG4 transcripts. Higher GalA in cell wall. Reduced fruit deterioration. Antisense Reduced tomato fruit softening. Higher GalA in cell wall. Antisense Increased firmness; reduced polymer. Both antisense Increased fruit firmness Modified juice rheology. Both antisense Modified juice rheology. Antisense No reduction in xyloglucan. depolymerization, pepper. Overexpression No increase in xyloglucan. depolymerization, pepper

215

Reference Tieman et al. (1992), Hall et al. (1993), Tieman and Handa (1994), Thakur et al. (1996a, b)

Jime´nez-Bermu´dez et al. (2002) Carey et al. (2001)

de Silva and Verhoeyen (1998)

Smith et al. (2002)

Brummell et al. (1999b) Kalamaki et al. (2003)

Errington et al. (1998) Harpster et al. (2002a)

Harpster et al. (2002b)

loci and genes that play a role in determining fruit shape, size and weight. Although so far these genes have not been used to develop cultivars with altered fruit physical attributes, they provide a resource to genetically engineer fruit crops for different phenotypic attributes. Fruit size and shape seem closely related as more extreme shapes were more confined to larger fruit phenotype than smaller fruits (van der Knaap and Tanksley 2003). Mutation in three genes (ovate, sun and fs8.1) affected fruit shape through

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modulation of early stages of carpel development (Gonzalo and van der Knaap 2008). About 30 quantitative trait loci (QTLs) have been identified regulating tomato fruit size and shape but less than 10 loci could account for majority of the changes associated with tomato domestication (Grandillo et al. 1999; Tanksley 2004). The effect of these loci appears to be largely confined to fruit mass except fw3.1 (van der Knaap et al. 2002; van der Knaap and Tanksley 2003). The fw2.2 has the strongest effect on fruit size and is associated with a hyper mitotic index during cell division stage just after anthesis. It encodes a negative repressor of cell division particularly during fruit development and has a sequence similarity to the human oncogene c-H-ras p21A (Frary et al. 2000; Cong et al. 2002). Introduction of fw2.2 into a large-fruited cultivar causes expected reduction in fruit size. fasciated locus on chromosome 11 and the locule number locus on chromosome 2 are reported to increase fruit size by increasing the number of carpels in the flower, which after fertilization develops into locules resulting in larger and wide fruits (Lippman and Tanksley 2001; Barrero et al. 2006). All the large-fruited, multilocular tomatoes carry mutations in either one or both of these loci while plants carrying mutated alleles of fasciated can produce more than 15 locules that affect the shape of the fruit. Although the exact nature of the genes at these loci is yet to be characterized, putative tomato homologs for fascinated locus have been identified in Arabidopsis. Thus far, a complete separation between the loci that control fruit size and those that control fruit shape has not been achieved. The organ-determining genes fasciated and locule number affect both the final size and shape of the fruit. However, the fruit size loci, fw1.1, fw2.2, fw3.1 and fw4.1 exert their effects largely on fruit growth resulting in changes in size with no or little change in shape. Three major loci, ovate (chromosome 2), sun (chromosome 8), and fs8.1 (chromosome 8), that modulate fruit shape have minimal effect on size. Ovate locus could account for both pear and elongated shape of tomato and the sequence comparisons of OVATE alleles indicated that all tested pear-shaped varieties of tomato share the same nonsense mutation that causes truncation of the predicted protein and could account for the loss-of-function (recessive) nature of these alleles (Ku et al. 1999; Liu et al. 2002). A single major locus, fs8.1, is responsible for both blocky and slightly elongated appearance of processing tomatoes (Grandillo et al. 1996). Changes in fruit shape caused by fs8.1 are initiated very early in floral/carpel development (Ku et al. 2000).

5.5.2

Ripening Mutant Genes

A large number of genes that regulate fruit development and ripening have been cloned and functionally characterized (Alba et al. 2005; Giovannoni 2007; Seymour et al. 2008). Genes responsible for attenuated ripening in ripeningimpaired tomato mutants have provided an attractive model to modify fruit ripening. Table 5.4 lists some of the genes that have been used to modify fruit ripening

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217

process. Several single gene tomato-ripening mutants that exhibit attenuated ripening have been known for quite some time. Identification and molecular characterization of these mutant genes have opened new avenues for modifying ripening process in fruit crops by transgenic approaches. RIPENING-INHIBITOR (Vrebalov et al. 2002) and COLOURLESS NON-RIPENING (CNR) mutants encode a MADS-box and SPB-box (Manning et al. 2006) genes, respectively. These genes are required for ripening and their study has begun to provide an insight into mechanisms that control ripening upstream of the plant ripening hormone, ethylene. Delving into the Never-ripe mutant led to the cloning of the first ethylene receptor gene from tomato (Wilkinson et al. 1995). Studies on Green-ripe identified a gene that encodes a fruit-specific, novel component that likely affects ethylene receptor-copper homeostasis during ethylene signaling (Barry and Giovannoni 2006). Gene mutations responsible for the high-pigment 1 (Damaged DNA Binding Protein 1 (DDB1)) and high-pigment 2 (Detiolated1 (DET1)) phenotypes should lead to dissection of regulatory pathways that impact metabolic content and associated fruit quality (Mustilli et al. 1999; Liu et al. 2004).

5.5.3

Ethylene-Regulated Fruit Ripening

Fruit ripening is a genetically regulated transitional period during which many dynamic processes occur which are manifested into perceivable and altered changes including firmness, pigmentation, and weight loss. Although these changes are desirable for human consumption, overripeness leads to wastage and discarding large portions of harvested fruit. Once initiated, ripening is a relatively irreversible process. Treatment with inhibitors of ethylene biosynthesis (e.g., aminoethoxyvinylglycine) or ethylene perception [e.g., silver salts or 1-methylcyclopropene (1-MCP)] results in delaying fruit ripening (Mattoo and Suttle 1991; Lurie and Paliyath 2008). However, the recognition of involvement of ethylene-dependent as well as ethylene-independent processes in ripening and fruit quality is impacted by preventing the ethylene responses in most fruits. Nevertheless, since ethylene plays a critical role in fruit ripening (Oeller et al. 1991), especially in climacteric fruits, approaches altering ethylene biosynthesis or its perception have been largely used to engineer ethylene insensitivity in several plant species. Fruit ripening control has been achieved by downregulating the expression of ACC synthase (Oeller et al. 1991) or ACC oxidase (Hamilton et al. 1990). Overexpression of a bacterial ACC deaminase (Klee et al. 1991) or SAM hydrolase (Good et al. 1994) also resulted in attenuated fruit ripening in tomato by reducing the levels of ethylene biosynthesis substrates ACC and SAM, respectively. Engineered suppression of a member of ethylene receptor family, LeETR4, in a fruit-specific manner was found to result in early fruit ripening without large effects on fruit size, flavor or yield (Kevany et al. 2008). These authors suggested that ethylene receptors might work as biological clocks to regulate the onset of fruit

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ripening. A progeny from a cross between two transgenic tomato lines impaired in the expression of LeEtr1 or LeERT2 using antisense RNA technology had a phenotype similar to that observed for LeERT1 antisense plants indicating its role in ethylene signaling during tomato growth and development (Wang et al. 2006a, b). The functional significance of members of ethylene-receptor family and downstream signaling is complex (Kendrick and Chang 2008). Degradation of ethylene receptor was found correlated with enhanced sensitivity to ethylene (Kevany et al. 2007).

5.5.4

Transgenic Interventions to Alter Fruit Texture

The economic value of post-harvest fruit softening has generated considerable interest in understanding the biochemical changes associated with fruit textural modifications (Brummell 2006; Vicente et al. 2007; Negi and Handa 2008). Ripening-associated fruit textural changes usually involve structural modification of the polysaccharide components of the primary cell wall and middle lamella. Structural proteins, such as expansins, also play an important role in this process (Brummell 2006; Vicente et al. 2007). The overall interplay between primary cell wall and middle lamella components is a very complex process. Loosening the structure of and cross-links between the cell wall and middle lamella have been implicated in the transition of a firm unripe fruit to a softer/crispy and juicy fruit (Brummell 2006). Genetic engineering approaches have led to the identification of the complexity of the cell wall chemistry and the role various cell wall modifying enzymes play in fruit softening and texture. Table 5.4 lists the phenotypes of transgenic plants obtained by decreasing the expression of genes encoding some of these enzymes. Antisense inhibition of polygalacturonase (PG) had only a slight effect on fruit softening but significantly impacted depolymerization of pectins, resulting in increased juice viscosity due to higher molecular size of the constituent pectins (Thakur et al. 1997). Similarly, transgenic plants expressing an antisense gene of pectin methylesterase (PME) showed >95% reduction in fruit PME activity concomitant with markedly improved juice viscosity and increased total soluble solids (Tieman et al. 1992; Gaffe et al. 1994; Thakur et al. 1996a,b). Although softening of transgenic fruits with reduced PME activity occurred to similar levels as the nontransgenic fruits, the transgenic fruits exhibited loss of tissue integrity after extended storage (Tieman and Handa 1994). Homology-dependent silencing of TBG1 resulted in about 90% reduction in its transcript accumulation but it did not affect the total exogalactanase activity, cell wall galactose content or fruit softening, indicating little, if any, role of this gene in fruit texture (Carey et al. 2001). Suppression of TBG3 by antisense RNA caused reduction in TBG1 and TBG4 transcript levels and ~75% loss in extractable exo-galactanase activity with retention of the cell wall galactose content but with little effect on fruit softening (de Silva and Verhoeyen 1998). However, reduction in TGB4 transcripts by antisense

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219

RNA caused about 90% reduction in extractable exogalactanase activity, reduced galactose levels in mature green fruits, and produced firmer fruits than the control (Smith et al. 2002). Specific members of the ß-galactosidase, expansin and pectate lyase (PL) gene families partially regulate softening in tomatoes and strawberry (Brummell et al. 1999b; Jime´nez-Bermu´dez et al. 2002; Smith et al. 2002). However, functional analyses of specific PGs, PMEs, xyloglucan endotransglucosylase/hydrolases (XTHs) and 1,4-endoglucanases (EGases) in tomatoes, strawberries and peppers have failed to establish their roles in softening (Lashbrook et al. 1998; Brummell et al. 1999a; Woolley et al. 2001; Harpster et al. 2002a,b). Fruit firmness was not significantly altered when PG or expansin was suppressed using transgenic tomatoes, but when genes encoding both proteins were simultaneously downregulated fruit firmness was retained for longer duration (Powell et al. 2003).

5.6

Cuticle Modification

Cuticle is the outermost layer of plants (Fig. 5.3), which plays important roles in preventing plant dehydration by limiting non-stomatal water loss, improving resistance of plants to biotic and abiotic stresses (See Chaps. 2-1 and 2-2), controlling plant morphology, and regulating organ fusion (Pollard et al. 2008; Samuels et al. 2008). As a barrier to pathogen attack, cuticle negates germination of pathogen spores (Gniwotta et al. 2005). Further, cuticle plays a role in osmotic adjustment as well as in protecting plants against the negative effects of light, temperature and pollution (Shepherd and Griffiths 2006). By regulating gas exchange, this layer likely plays a significant role in extending post-harvest shelf life of fruits and vegetables (Saladie et al. 2007). The cuticle layer is composed of two main lipophilic components: cutin and cuticular wax. Cutin is lipid-derived polyester composed mainly of a- and o-hydroxyl and epoxy C16 and C18 fatty acids and glycerol (Nawrath 2006). The wax component of cuticle is made up of aliphatic C24–C34 compounds made

Fig. 5.3 Section of a leaf showing the cuticle with respect to other tissues. Adapted from Purves et al. (1997)

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entirely of carbon and hydrogen or including functional groups like alcohol and ketone (Kunst and Samuels 2003). The pathways responsible for the formation of this component including its transport to the outside of a plant organ as well as the intercellular and extracellular assembly remain to be elucidated (Pollard et al. 2008). Microarray analysis of transcripts (Chaps. 2-10) from the top epidermis of Arabidopsis led to the isolation of 85 upregulated genes playing a role in lipid metabolism (Chung et al. 2005), whereas the same analysis in the wax deposition zone in barley leaf led to the isolation of five upregulated wax-deposition genes (Richardson et al. 2007). Although more experimental evidence is needed, these data suggest that the number of genes involved in cuticle assembly is rather few compared to the number of genes participating in the biosynthetic pathway of the cuticle components. Double knockouts of glycerol-3-phosphate acyltransferase (GPAT), gpat4/gpat8, caused significant reduction in cutin. The mutant plants were less resistant to desiccation and infection by the fungus Alternaria brassicicola. Also, overexpression of GPAT4 or GPAT8 led to about 80% increase in the levels of C16 and C18 cutin monomers in leaves and stems (Li et al. 2007b). In order to modify cutin composition, these authors overexpressed the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis. The simultaneous overexpression of both enzymes caused accumulation of new C20 and C22 o-hydroxyacids and a,odiacids typical of suberin with altered fine structure and water-barrier function of the cuticle (Li et al. 2007b). Substantial experimental evidence has accumulated on the roles that cuticle plays in various developmental and physiological processes. Modification of cuticle has been shown to result in a number of attributes such as: (a) Altered leaf and petal epidermal cell structure, trichome number and branching as well as density of stomata (Aharoni et al. 2004) (b) Reduced leaf size and plant growth, seed production and seed germination (Schnurr et al. 2004) (c) Abnormal bending of embryos, ectopic adhesion between cotyledons, a permeable epidermal structure and an altered distribution pattern of stomata in several tissues (Tsuwamoto et al. 2008) (d) Dwarf appearance due to amorphous and smaller cells and enhanced tendency to dehydrate (Cominelli et al. 2008) (e) Altered morphology of trichomes and pavement cells (Panikashvili et al. 2007) (f) Post-genital organ fusions and stunted growth (Sieber et al. 2000; Bird et al. 2007; Luo et al. 2007; Ukitsu et al. 2007) (g) Hypersensitivity to drought (Chen et al. 2004; Ukitsu et al. 2007) (h) Disruption of microspore normal development along with reduced pollen development (Jung et al. 2006) (i) Irregular shape of pollen (Ukitsu et al. 2007). In tomato fruits, the manipulation of cuticle by expressing the gene cwp1 (cuticular water permeability 1), which imparts the microfissure/dehydration phenotype in the wild species Solanum habrochaites, caused a microfissured fruit

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Table 5.5 Genes with potential to modify cuticles Gene Function Physiological effect CaCAF1 DQ672569

Transcription and mRNA decay

WXP1 TC107019

Ethylene response transcription factor Ethylene response transcription factor

WXP2 TC94548

GPAT5 Glycerol acyl At3g11430 transferase CUTE Cutinase protein M29759 SHN1 AP2/EREBP At1g15360 transcription SHN2 factors At5g11190 regulating lipid SHN3 biosynthesis At5g25390

Biotechnology Reference intervention Growth enhancement, Overexpression Sarowar thicker cell walls and pCaMV35S et al. cuticle layers. Increased (2007) resistance against Phytophtora infestans Enhanced drought and Overexpression Zhang freezing tolerance pCaMV35S et al. (2007b) Enhanced drought Overexpression tolerance. Increased pCaMV35S low-temperature sensitivity. Reduced growth. Increased drought tolerance Overexpression Li et al. pCaMV35S (2007b) Total resistance to Botrytis Overexpression Chassot cinerea pCaMV35S et al. (2007) Increased cuticular wax, Overexpresion Aharoni drought tolerance and pCaMV35S et al. recovery. (2004)

cuticle leading to a dehydrated fruit (Hovav et al. 2007). Table 5.5 lists some of the genes that have been manipulated to modify cuticle and their effects on plant growth and development. Transgenic tomato plants overexpressing CaCAF1, a CCR4-associated factor 1 protein belonging to the CCR4-NOT complex that plays an important role in the control of transcription and mRNA decay in yeast and mammals, exhibited significant growth enhancement, with thicker leaves with twofold enlarged cell size, thicker cell walls and cuticle layers, and enhanced resistance against Phytophthora infestans compared to the control plants (Sarowar et al. 2007). In the same study, virus-induced silencing (Chap. 1-2) of CaCAF1 in pepper resulted in significant growth retardation and enhanced susceptibility to bacterial pathogen Xanthomonas axonopodis pv. vesicatoria. Introduction of putative ethylene-responsive transcription factor (ERF) genes, WXP1 and WXP2, from Medicago truncatula into Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response to freezing tolerance (Zhang et al. 2007b). Both WXP1 and WXP2 transgenic plants showed increase in n-alkanes, the major wax component in Arabidopsis, but only the WXP1 transgenic plants exhibited increase in the amount of primary

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alcohols. The WXP1 transgenic plants showed no change, but the WXP2 plants exhibited increased chlorophyll bleaching. Both WXP1 and WXP2 transgenic plants retained more water than the control and exhibited significantly enhanced plant drought tolerance. On the basis of electrolyte leakage from detached leaves, the WXP1 plants showed increased freezing tolerance while the WXP2 plants were more sensitive to low-temperature stress than the control plants. WXP1 overexpressing plants showed no obvious effects on plant growth and development, but the expression of WXP2 resulted in slower plant growth. It is becoming increasing clear that the manipulation of cuticle will help designing plants with higher resistance to pathogens and abiotic stresses. Stress due to fungi and water loss are significant factors causing post-harvest losses of fruit and vegetable crops (Troncoso-Rojas and Tiznado-Herna´ndez 2007). The cuticle engineering has a high potential to reduce these losses.

5.7

Abscission

Abscission is a process by which plant organs (leaves, flowers and fruits) detach (are shed) from the parent plant. This process is under the cue of developmental signals as well as environmental stimuli. Thinning or reduction of crop load, a common practice in the production of fruit crops, involves manual or chemicalinduced detachment of excess fruits to optimize fruit size. Harvesting of fruit crops is one of the most demanding aspects of fruit production both in terms of labor as well as economic inputs. There is currently renewed interest in mechanical harvesting to circumvent these issues. Efficiency of mechanical harvesting systems can be greatly improved by facilitating abscission-related processes. However, untimely detachment of immature fruit can lead to crop loss and low profitability, and therefore needs to be avoided. In seed crops, pod shattering and seed loss can be prevented through better understanding of dehiscence, a process that is somewhat similar to abscission. Hence, knowledge of abscission and its manipulation can greatly benefit crop production. Abscission occurs at specialized regions, termed abscission zones (AZ), which are located proximal to the organ that will subsequently detach (Fig. 5.4). Cells within the AZs are often small and rounded, and usually undergo extensive cell division prior to organ separation (Sexton and Roberts 1982; Goren 1993). Cell separation in the AZ occurs within a group of cells called the separation layer. Activity of cell wall hydrolytic enzymes, in response to the abscission stimulus, leads to dissolution of the primary cell wall and the middle lamella within cells of the separation layer (Taylor and Whitelaw 2001; Roberts et al. 2002). Subsequent loss of adhesion or collapse of cells in the separation layer leads to organ detachment due to the weight of the subtending organ. Either during the process of separation or immediately following it, cells within the layer proximal to the separation layer expand to form a protective layer (Patterson 2001).

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Biotechnological Interventions to Improve Plant Developmental Traits

Abscission Zone Differentiation

JOINTLESS; BOP1; BOP2 (AZ)

SHATTERPROOF SHATTERPROOF2 (DZ)

223

FRUITFULL

AZ (Separation Layer) ETHYLENE (ACC SYNTHASE; ETR1; EIN2)

Abscission Signaling

AUXIN (ARF2) ETHYLENE INDEPENDENT (DAB; IDA)

Cell Separation

Cell Wall hydrolases IDA

Fig. 5.4 Progression of abscission is represented here in three phases involving: abscission zone (AZ) differentiation; abscission signaling; and cell separation. AZ differentiation is regulated by genes such as the JOINTLESS gene in tomato and the BLADE ON PETIOLE (BOP1; BOP2) genes in Arabidopsis. The dehiscence zone (DZ) differentiation is regulated by expression of the SHATTERPROOF genes which are in turn inhibited by the FRUITFULL gene in Arabidopsis siliques. Abscission signaling is antagonistically regulated by ethylene and auxin. Additionally, ethylene-independent mechanisms involved in Arabidopsis flower abscission have been reported in the delayed abscission (dab) and inflorescence deficient in abscission (ida) mutants. Cell separation is facilitated by the degradation of the middle lamella and the cell wall, activities that are largely facilitated by cell wall hydrolases. Additionally, the IDA gene may also be involved in regulating final stages of cell separation

5.7.1

Development of the Abscission Zone

Development and differentiation of AZs are under tight genetic control and usually occur at specific sites within the plant (Fig. 5.4). Several genes associated with the development of AZ and those that have an impact on the abscission process are

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Table 5.6 Genes with potential to modify abscission Gene Function Role in Biotechnology abscission intervention JOINTLESS MADS-box AZ Mutation; over Development expression; silencing SHATTERPROOF, MADS-box Dehiscence zone Mutation SHATTERPROOF2 development FRUITFULL MADS-box Dehiscence zone Mutation; development overexpression

AGL15

MADS-box

Progression of abscission

Mutation

CEL1, CEL2

Cell wall modification

Promote cell separation

Silencing

Polygalacturonase

Cell wall modification

Promote cell separation

Silencing

ACC synthase

Ethylene biosynthesis

Promotes floral organ abscission

Overexpression; silencing

ETR1; EIN2

Ethylene Ethyleneperception mediated and signaling abscission signaling

Mutation; silencing

DAB; IDA

Unknown

Delayed floral organ abscission

Mutation

ARF2

Auxin response Delayed floral factor organ (transcription abscission factor)

Mutation

Reference Mao et al. (2000) Liljegren et al. (2000) Ferrandiz et al. (2000), Østergaard et al. (2006) Fernandez et al. (2000) Lashbrook et al. (1998), Brummel et al. (1999a) GonzalezCarranza et al. (2007), Jiang et al. (2008) Lanahan et al. (1994), Ecker and Theologis (1994) Bleecker and Patterson (1997), Whitelaw et al. (2002) Patterson and Bleecker (2004), Butenko et al. (2006) Ellis et al. (2005)

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listed in Table 5.6. One of the key genes regulating AZ development was isolated using a spontaneous mutation in tomato, jointless. The jointless mutants do not possess the AZ that typically forms midway along the flower/fruit pedicel. The JOINTLESS gene was isolated using map-based cloning and found to encode a MADS-box transcription factor that is essential for the development of the pedicel AZ (Mao et al. 2000). Overexpression of the JOINTLESS gene in the mutant background restored AZ development albeit not in the same location as in the wild type, while antisense suppression of this gene resulted in loss of the flower pedicel AZ in tomato (Mao et al. 2000). Several other MADS-box genes have been implicated in dehiscence. Two such genes are SHATTERPROOF (SHP1) and SHATTERPROOF2 (SHP2) both of which redundantly control cell separation by affecting dehiscence zone formation and its lignification in Arabidopsis siliques (Liljegren et al. 2000; Lewis et al. 2006). In contrast, FRUITFULL, another MADSbox gene, negatively regulates dehiscence zone development by inhibiting expression of the SHATTERPROOF genes (Ferra´ndiz et al. 2000). Another MADS-box gene, AGL15, is also implicated in regulating abscission through delaying progression of the process (Fernandez et al. 2000). Recently, two redundant BLADE ON PETIOLE genes, BOP1 and BOP2, have been identified as essential regulators of abscission zone development in Arabidopsis. These genes belong to the nonexpressor of PR1 protein (NPR1) family and appear to be involved in the establishment of the floral AZ as well as the vestigial cauline leaf AZ in Arabidopsis (McKim et al. 2008). Such genes affecting AZ zone development have immense potential for genetic manipulation of organ abscission in crops. In fact, the jointless mutant background has been widely used for developing “stemless” processing tomatoes that are not physically damaged during storage (Mao et al. 2000). Also, ectopic expression of FRUITFULL has been used to prevent unwanted pod dehiscence in Brassica (Østergaard et al. 2006).

5.7.2

Hormonal Control of Abscission

Ethylene has long been known to be a primary natural regulator of abscission (Jackson and Osborne 1970). Increase in ethylene evolution is often associated with the abscission process (Goren 1993; Taylor and Whitelaw 2001). Experiments involving genetic manipulation of ethylene biosynthesis, perception and signaling support a role for ethylene in promoting abscission of various plant organs. Constitutive expression of ACC synthase resulted in enhanced ethylene production and earlier flower abscission in tomato (Lanahan et al. 1994), whereas antisense suppression of ACC synthase delayed abscission in Arabidopsis (Ecker and Theologis 1994). The ethylene receptor mutant, etr1, and the ethylene insensitive mutant, ein2, exhibit delayed abscission of floral organs in Arabidopsis and tomato (Bleecker and Patterson 1997; Whitelaw et al. 2002; Patterson and Bleecker 2004). One of the main mechanisms through which ethylene aids cell separation is by enhancing expression and activity of cell wall hydrolyzing enzymes in the AZ.

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However, recent work with the delayed abscission (dab) and inflorescence deficient in abscission (ida) mutants, which exhibit altered floral abscission responses in an ethylene-independent manner, have led to the suggestion that ethylene may not be absolutely required for abscission in Arabidopsis but may act as an accelerator of the abscission process (Patterson 2001; Patterson and Bleecker 2004; Butenko et al. 2006). Nonetheless, alteration of ethylene biosynthesis and/or perception continues to be a promising approach for genetically altering abscission in plants. Auxin plays a dual role in regulating abscission. While auxin generally inhibits progression of abscission during early stages of the process, application of auxin at later stages accelerates abscission by increasing ethylene biosynthesis (Brown 1997). In fact, the ratio of ethylene and auxin levels at the AZ is considered a key factor determining progression of abscission (Sexton and Roberts 1982; Brown 1997). A flux of auxin across the AZ is thought to be essential to prevent abscission (Sexton and Roberts 1982). Auxin inhibits cell separation by decreasing the activity of cell wall hydrolyzing enzymes (Taylor and Whitelaw 2001). While rapid progress has been made in understanding auxin biosynthesis, transport and signaling, its role in regulating abscission has not been adequately addressed at the molecular and genetic level. Evidence is accumulating for the involvement of auxin signaling genes in regulating abscission. Expression of several AUX/IAA genes is altered in an auxin-dependent manner at the leaf and stem AZs in Mirabilis (Meir et al. 2006). Auxin response factors (ARFs) are transcription factors that, in conjunction with AUX/IAA genes, either activate or inhibit downstream auxin responsive genes. Loss of ARF2 function in Arabidopsis delayed floral organ abscission supporting a role for auxin signaling in the progression of abscission (Ellis et al. 2005). Further analysis of the molecular machinery involved in auxin transport and signaling during abscission should lead to the identification of potential candidates that can be utilized to alter abscission in plants.

5.7.3

Cell Wall Degradation during Cell Separation in the Abscission Zone

Enzymes facilitating dissolution of the middle lamella and the cell wall, and degradation of the cell membrane are obvious candidates for manipulating abscission. These include cellulases (Cels), PGs, pectin methylesterases (PME), expansins and phospholipases (Cho and Cosgrove 2000; Roberts et al. 2002; Malladi and Burns 2008). AZ-specific Cels and PGs have been identified and isolated from many plants (Tucker et al. 1988; Kalaitzis et al. 1995; del Campillo and Bennett 1996; Kazokas and Burns 1998). Antisense inhibition of the cellulase gene, Cel1, reduced floral abscission in tomato (Lashbrook et al. 1998). Similarly, antisense inhibition of the Cel2 gene increased the force required for detachment at the pedicel AZ in tomato by almost 50% (Brummell et al. 1999a). Loss of a PG gene in Arabidopsis resulted in delayed floral organ abscission in air or in the presence

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of the abscission-promoting hormone, ethylene (Gonzalez-Carranza et al. 2007). Virus-induced gene silencing (Chap. 1–2) of a tomato abscission-related PG gene, TAPG1, delayed ethylene-regulated petiole abscission and increased petiole break strength (Jiang et al. 2008). An analysis of the ida mutant in Arabidopsis revealed a function for this gene in regulating the final steps of cell separation, possibly through the secretion of arabinogalactan proteins. Constitutive expression of IDA results in enhanced floral organ abscission as well as ectopic abscission at several locations in Arabidopsis (Stenvik et al. 2006). These studies demonstrate practical applicability of altering cell wall/membrane modifying enzymes to manipulate organ abscission.

5.8

Programmed Cell Death

PCD is a highly coordinated and complex process (Fig. 5.5) that involves dismantling of the cellular apparatus. It serves an important function in the development and defense of the organism. Plants use a well-defined and self-regulated program of cell death during vascular bundle formation, defense management and senescence of leaf, flower or fruit. Whether senescence in plants is a form of PCD or precedes PCD is being debated in the literature; one school of thought considers senescence as a complete overlap with PCD (van Doorn and Woltering 2005), while others view the latter as culmination of senescence events (Mattoo and Handa 2004; Reape et al. 2008). The molecular events during senescence and hypersensitive response (HR)-induced cell death share some common features but vary in the time frame of their completion. PCD is believed to be more spontaneous and has a shorter span than the senescence. Both plants and animals employ PCD to complete their developmental process and manage defense against pathogenic (biotic) or abiotic stresses. Advances in our understanding of PCD have provided opportunities for biotechnological improvements in the plants ranging from a markedly improved tolerance against stress to delayed senescence for higher biomass (Table 5.7) Cell death is fundamental to the life cycle of an organism. In nature, it forms a link in the evolutionary process that facilitates every form of life to adapt to the natural vagaries. In multicellular organisms, the cell death serves the purpose of removing damaged or redundant cells. In animals, PCD occurs in two morphologically distinct ways – apoptosis and autophagy. Another mode of cell death is necrosis whose initial events bear footprints of PCD. Autophagy is a highly conserved mechanism involving degradation of cytoplasmic contents by the lysosomal vacuoles. It helps in the mobilization of cell constituents before the cell death. An autophagic type of PCD is often observed in plants during growth and development of growing cells (tissues), e.g., in tracheary elements or root growth and expansion. Animal cell apoptosis is characterized by cell shrinkage, nuclear fragmentation, formation of apoptotic bodies and their engulfment by the lysosomic action of another cell (Adrain and Martin 2001). In plants, the engulfment of apoptotic bodies by another cell is unknown. Characteristics such as retraction of protoplast, DNA

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Fig. 5.5 A model of programmed cell death in plants. JA jasmonic acid; MAPK mitogen-activated protein kinase; NO nitric oxide; PAMPs pathogen-associated molecular patterns; PK protein kinase; PM plasma membrane; PRI pathogen R-gene protein interaction; RLK receptor-likekinase(s); ROS reactive oxygen species; SOD superoxide dismutase

fragmentation and involvement of caspase-like proteins are shared by plant apoptosis. However, owing to yet-to-be-found other animal apoptotic characteristics, some investigators have preferred to identify the phenomenon in plants as apoptotic-like PCD (AL-PCD) (van Doorn and Woltering 2005; Reape and McCabe 2008). In plants, HR-induced PCD is a key defense mechanism to restrict the spread of pathogens during a compatible host–pathogen interaction. Also, induction of HR takes place during abiotic stresses due to cold, heat, drought, salinity, high light intensity, or ultraviolet radiation to mitigate the stress effect. The core components

Table 5.7 Genes related to programmed cell death and their potential to modify plant development or agronomic traits Gene Product/Function Effect Biotechnology intervention Fungal or bacterial resistance CaPO2 Extracellular peroxidase Resistance to P. syringae pv. Overexpression of pepper gene in Arabidopsis tomato OsAOS2 Allene oxide synthase Resistance to Magnaporthe Overexpression of the gene in rice grisea (rice blast) RCT1 TIR-NBS-LRR - R gene protein Resistance to anthracnose Expression of M. truncatula gene in alfalfa disease Pto Serine/threonine protein kinase – Resistance to P. syringae Overexpression of tomato gene R-gene protein Rpi-blb1 CC-NBS-LRR-R gene protein Resistance to Phytophthora Expression of Solanum bulbocastanum in potato infestans (late blight) (Expression of wild species potato gene in cultivated potato) Rxo1 NBS-LRR-R gene protein Resistance to Xanthomonas Expression of maize gene in rice oryzae N1141-flaA Flagellin Resistance to M. grisea (rice Expression of bacterial gene in rice blast) AtNPR1 NPR1 protein Resistance to Fusarium Expression of Arabidopsis gene in wheat head blight Cf9 & Avr9 R-gene proteins Resistance to Leptosphaeria Expression of tomato genes in Brassica napus maculans Viral protection Bcl-xL Or Animal cell death suppressor protein Protection from virusExpression of animal gene(s) in tomato Ced-9 induced necrosis Insect resistance ProsysteA signal protein for JA synthesis Resistance to herbivores Overexpression in tomato min gene Tolerance to abiotic stress Antioxidant Removal of superoxide radical Enhanced salt, drought Overexpression genes tolerance See Ashraf (2008) (continued)

Li et al. (2002)

Xu et al. (2004)

Zhao et al. (2005) Takakura et al. (2008) Makandar et al. (2006) Hennin et al. (2001)

Choi et al. (2007) Mei et al. (2006) Yang et al. (2008) Tang et al. (1999) van der Vossen et al. (2003)

Reference

5 Biotechnological Interventions to Improve Plant Developmental Traits 229

Transcription factor

Poly (ADP-ribose) polymerase

DREB1

PARP

Plastid NDH gene

Delayed leaf senescence

Delayed leaf senescence

OsDOS

Nuclear-localized CCCH-type zinc protein – negative regulator of JA A component of chloroplast NDH complex

Delayed leaf senescence

Bcl-xL or Animal cell death suppressor protein Ced-9 Delayed senescence AtNAP Transcription factor

Improved tolerance to drought, cold, and salinity Tolerance to high light, heat, and drought Salinity, cold, and wound tolerance

Enhanced drought tolerance Expression of tobacco gene in maize

Protein kinase

NPK1

Suppression of the gene in tobacco

Overexpression in rice

Suppression of the gene

Overexpression of animal gene(s) in tobacco

Suppression of the gene

Overexpression of the gene

Expression of tobacco gene in maize

Freezing tolerance

Effect Biotechnology intervention Enhanced drought tolerance Expression of IPT gene

Table 5.7 (continued) Gene Product/Function IPT Isopentenyl transferase – in cytokinin biosythesis MAPKKK Phosphorylation of regulatory proteins

Guo and Gan (2006) Kong et al. (2006) Zapata et al. (2005)

De Block et al. (2005) Qiao et al. (2002)

Reference Rivero et al. (2007) Shou et al. (2004a) Shou et al. (2004b) Ito et al. (2006)

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of PCD include perception of stimulus (extrinsic or intrinsic), signaling cascade and execution of cell death. The mechanism of perception of biotic stimulus is relatively better understood than that of any abiotic stimulus. The pathogens are recognized by receptors at the cell surface through pathogen-associated molecular patterns (PAMPs), which are highly conserved structural components required to support the lifestyle of a pathogen. The host receptors are termed pattern-recognition receptors, which resemble in function with toll-like receptors in animals (Nurnberger et al. 2004; Zipfel et al. 2004). A receptor–pathogen interaction triggers HR that helps to contain the proliferation and spread of pathogens to neighboring cells. An endeavor to survive – a basic principle of evolution – allows certain pathogens to evade host resistance by blocking the interaction-driven HR through secretion of “effectors.” Interestingly, recognition of these effectors by “R” (resistance) gene products enables plants to become resistant (Chisholm et al. 2006). Characterization of R genes has become a potential tool in marker-assisted breeding and genetic engineering to develop disease-resistant plants. Once pathogen is recognized, a defense response is initiated and decision on the fate of cell is made. The available information suggests the involvement of a multicomponent mechanism, which is conserved in animals and plants in its basic form (Hoeberichts and Woltering 2003; Franklin-Tong and Gourlay 2008; Williams and Dickman 2008). Some regions of the R-gene product show significant similarity with apoptotic proteins from humans (APAF) and Caenorhabditis elegans (CED4). The apoptotic proteins recruit caspases, which are specific type of cysteine proteases capable of acting on large number of substrates to effect cell death. In plants, though true caspases are yet to be identified, caspase-like proteases called metacaspases have been detected (see Mattoo and Handa 2004). The animal cell apoptosis is regulated by pro-apoptotic (e.g., CED4, APAF, Bax) and anti-apoptotic (e.g., CED9, Bcl-2, Bcl-xL) proteins. Bioinformatics has allowed predicting the presence of antiapoptotic proteins in plants despite the lack of sequence similarity with the animal counterparts. An ectopic expression of animal anti-apoptotic proteins suppressed apoptosis and imparted tolerance against certain type of stresses in plants (Table 5.7). In HR-induced PCD, reactive oxygen species (ROS) and nitric oxide (NO) have emerged as early messengers in the signaling cascade leading to defense response. The most notable species are superoxide radical (O2-1) and hydrogen peroxide (H2O2). A superoxide radical is highly reactive and dismutates to H2O2 by superoxide dismutase (SOD). The perception of a stimulus due to a pathogen or an abiotic stress is marked by an oxidative burst resulting in the synthesis and accumulation of ROS in large amounts. A similar increase in NO has been observed in plants (Zeidler et al. 2004). It is thought that a fine-tuned NO/ROS balance is responsible for the induction of genes during HR (Grun et al. 2006). ROS act in concert with MAPK to activate a host of biochemical responses, such as cell wall cross-linking, lignification, synthesis of pathogenesis-related (PR) proteins and phytoalexins. They are also known to regulate the synthesis of hormones, such as jasmonic acid, which in turn may initiate a defense response. NO interacts with salicylic acid and jasmonic acid, particularly during wounding, which indicates its role in stress signaling (see Grun et al. 2006). Miller et al. (2008) have reviewed the

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mechanisms of ROS signaling. Besides mediating a stress signal, ROS can deleteriously affect pathogen or host cell through oxidative damage of its biochemical constituents. Further, recent studies suggest that both ROS and NO act as signaling molecules in the development of plants (Gapper and Dolan 2006; Grun et al. 2006). Because ROS play a crucial role in the cell function, their intracellular level is tightly controlled. Different mechanisms are known to operate in the production and removal of ROS. Following pathogen recognition, the accumulation of ROS in apoplast is attributed to the action of more than just one enzyme. Respiratory burst oxidase homologs (Rboh) with similarity in function to mammalian neutrophil oxidase are recognized to play a dominant role (see Apel and Hirt 2004). More recently, extracellular peroxidases have also been shown to participate in the synthesis process (Bindschedler et al. 2006; Choi et al. 2007). During PCD in mammalian cells, mitochondria serve as a source of ROS when there is a transient change in their permeability because of stress and cytochrome-c is released to the cytoplasm. In plants, chloroplasts seem to significantly contribute to ROS generation. Accordingly, the degeneration of chloroplast is observed prior to leaf senescence and necrosis. Several antioxidant enzymes – dismutases, catalases and peroxidases – are active during HR-induced PCD, and their suppression in transgenic plants has been observed to severely compromise the ability of these plants to withstand stress. On the other hand, the increased antioxidant activity through overexpression of the genes results in enhanced tolerance against a variety of environmental stress (Table 5.7). Even though progress has been made in our understanding of ROS role in plants, their signaling pathways and metabolic crosstalk with other components are yet to be fully elucidated. Similarly, as roles of NO in plant development and stress responses are revealed (Parani et al. 2004; Lindermayr et al. 2005; Abat et al. 2008), the manipulation of its intracellular levels can serve yet another tool for biotechnological improvements in crop plants.

5.9

Future Perspectives

Molecular and genetic dissection of plant developmental processes has provided a map of complex interactions that regulate desirable plant traits. This knowledge is enabling scientists to search for regulatory genes that can be manipulated to enhance desirable developmental traits in agronomical and horticultural crops (Rothstein 2007). Details on the interactions among hormones, environment and physiology are being unraveled and have begun to reveal how differentially mediated progression of various processes regulate the life span of plants. The challenge is to identify a hierarchy of regulators or a specific pattern of events that control desirable attributes and then genetically modify critical and beneficial processes without negatively impacting other beneficial attributes in crop plants. Thus far, studies on transgenic plants and effectiveness of a gene construct have reinforced the advantage of using tissue-specific and regulatable promoters to tightly control the expression of introduced transgene and avoid affecting non-targeted developmental

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processes. Thus, the need is for identifying and characterizing tissue-specific promoters and promoter elements that control both tissue specificity and the level of expression of the introduced transgene. We also need to develop transgenic plants particularly suited to grow well in ecofriendly, sustainable agricultural systems that have minimal reliance on chemical input and synergistically (positively) influence plant metabolism (Neelam et al. 2008; Mattoo and Teasdale 2009).

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Chapter 6

Transgenics for Biofuel Crops Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh

6.1

Introduction

Fossil fuels, like petroleum and coal, are fast-depleting, nonrenewable sources that were produced over million years ago, when the higher- and lower-order plants were buried under the surface of the earth by volcanic activities and sedimentation. These were further acted upon by microorganisms leading to the formation of nonrenewable fuels. There is an urgent need to find alternative sources of energy in order to cope with the energy demands of the human race and the much-debated global climate change in the coming years. So intense is the problem that the world has been divided into energy-independent producers and energy-dependent consumer economies, triggering trade war and political unrest, leading to monopoly over production and pricing issues. In such a scenario, there is a greater urge for the fossil-fuel-dependent economies to look for alternative sources of energy to become more self-reliant when it comes to their own energy needs (Hill et al. 2006). Several alternative energy sources, which exist and are being exploited today, include wind, solar, hydroelectric, nuclear energy and possibly methane gas reserves entrapped in the underground seabed. These may provide a part of the solution. There are practical problems associated with harnessing, storage and transport of these new renewable resources compared with other popular nonrenewable sources. Biofuels or agrofuels are basically carbon-derived fuels (solid, liquid or gaseous state) where the source of carbon is either plants or animals, and are therefore indirectly solar energy sources. They can be derived from animal fats, vegetable oils (biodiesel), or from agro-residues (bioethanol, biomethanol, or biobutanol), or can be derived from solid forms (like refuse fuels, pellets, wood, sewage, and

A. Bhattacharya (*) National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton, GA 31794, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_6, # Springer-Verlag Berlin Heidelberg 2010

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History 200 Liquids

150

Coal

100

Natural Gas

Renewables

50 Nuclear 0 1990

2000

2005

2010

2020

2030

Fig. 6.1 Present and projected global energy demand. Adapted from Energy Information Administration (EIA), Official Energy Statistics from US government site available at http://www.eia. doe.gov/oiaf/ieo/world.html. Original source: International energy annual report 2005 and EIA, World energy projection plus (2008).

briquettes) (Petrou and Pappis 2009), which can be used to produce energy. They are generally less efficient than fossil fuels but their usage results in less emission of greenhouse gasses (Vogt et al. 2009). Besides, biofuels are termed green fuels because they emit less carbon dioxide compared to the fossil fuels, contain no sulfur and thus decrease the global warming effect (Vogt et al. 2009). They are biodegradable and contribute to sustainability (Puppan 2002). The concept of biofuel usage in automobiles is in fact not new, but dates back to the early nineteenth century, when Rudolf Diesel used peanut oil to drive engines used in agriculture machinery (ijalwan et al. 2006; Murugesan et al. 2009). However, the availability of low-cost fossil fuels in practical abundance resulted in the neglect of biofuel research. Rapid industrialization and fast economic growth of several developing nations, including China and India, in the twentieth century resulted in greater consumption of once abundant resources, which led to investigations of the feasibility of alternative fuels, and biofuels in particular. The annual global fossil fuel consumption and biodiesel production is estimated to be about 4.018 and 0.107 billion tons, respectively (Demirbas 2008), and is increasing every year (Fig. 6.1). Initially, biofuel production came from food crops like sugarcane, sunflower, soybean, sugarbeet, rapeseed, and corn, which have been blamed for triggering a food crisis in recent years. This raises the following question: Which should be given a priority when it comes to making a choice between energy and food resources? The answer to this paradox lies in the recently identified potential biofuel crops, for example, poplar, hemp, members of the grass family (switch grass, burmuda grass, miscanthus, prairie and wheat) and oil-rich crops like Jatropha and Pongamia sp.

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Biofuels

Gaseous

Liquid

Solid

Briquettes Pellets Wood

Bioethanol Biomethanol Biobutanol

Biodiesel

Biogas

Municipal Agriculture & Forestry residue

Sugar and Starch rich crop plants

Oil rich crop plants

Decomposing Biomass

Fig. 6.2 Classification of biofuels

A prerequisite to using crops in efficient biofuel production is to design the most effective way to break down complex carbohydrates into simple sugars. One of the ways to circumvent this issue is to biotechnologically engineer transgenic crops with modified lignin, tannin and cellulosic biosynthetic pathway, which will make their decomposition into biofuels quicker and more efficient when acted upon by microbes. Plants provide us with food, feed and fiber. We can genetically engineer parts of plants often thrashed away, like the straw in wheat and paddy, stalks of cotton, corn and several other crop plants, to degrade more quickly than they do naturally. This will not only enhance the rate of production of biofuels but also improve efficiency of the whole production process. At the same time, we can also genetically engineer the microbial strains used in this process to increase their rate of action. Some biofuel-related terminology has been briefly explained in this section and illustrated in Fig. 6.2.

6.1.1

Bioalcohol

The simple and complex polysaccharide plant food reserves are broken down into simple sugars, and fermented to produce bioalcohol. They are specifically designated as Bioethanol and Biobutanol.

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1:6CO2 þ 6H2 O þ Solar energy

!

C6 H12 O6 þ 6O2

Glucose=simplesugar ðFermentationÞ

2: C6 H12 O6 ! 2C2 H5 OH þ 2CO2 þ energy Glucose

6.1.2

ðBioÞethanol

Biogas and Biohydrogen

Biogas is produced from decomposed organic substances by pyrolysis in the presence of anaerobic bacteria, releasing gaseous substances (generally a mixture of hydrogen, methane and carbon dioxide). Recently, scientists have developed hydrogen and nitrogen-fixing bacteria capable of producing biogas.

6.1.3

Biodiesel

Plant lipids are composed of long-chain fatty acids which are broken down into short-chain fatty acids by esterification to produce biodiesels. Typical source material for biofuels include: (a) Starch and sugars. The first generation of biofuel crops included food crops like sugarcane, corn, barley, wheat and cassava. (b) Lignocellulasic material. Unlocking the cellulose from plant cell walls and efficiently converting it to ethanol is the key to make ethanol a universal, inexpensive form of fuel for the future. Aquatic plants like lotus, lily, etc. are naturally low in lignin content, which is less than 10% (Gunnarsson and Petersen 2007), and they grow at a rapid pace (Ripley et al. 2006). (c) Plant lipids. Crop plants like soybean, corn, sunflower, oil palm, castor seed, rapeseed, and mustard, along with primitive photosynthetic microrganisms like algae (including microalgae, kelps and fucoids), cynobacteria and phytoplanktons, are naturally rich in oil or starch, which can be converted into biofuel in mutistage processing. Biodiesel is better than conventional diesel fuel with respect to sulfur content, flash point, aromatic content, and biodegradability (Hanna et al. 2005).

6.2

Transgenics Approach to Biofuels

Conventional breeding has been largely responsible for the introduction of novel traits and has played an important role in the development of new cultivars. This includes domestication of wild relatives and selection of novel sports from popular cultivated species. However, complex polygenic traits are difficult to manipulate using this method and can take considerable time (Zuker et al., 1995; Mishra and

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Srivastava 2004). Crop plants in the past were never selected on the basis of their amenability for biofuel production, and traits such as low lignin content or high cellulose were lost systematically from the gene pool. Thus, limited gene pool and failure of distant crosses in conventional breeding have lead to the exploitation of genetic transformation in generating plants of relevance for the rapidly growing biofuel industry. Growth and crop productivity are polygenic traits making their breeding difficult under natural conditions. Successful genetic manipulation depends upon the integration of transgene(s) into the host genome, the regeneration capacity of the transformed cells often via tissue culture, and subsequent expression and integration over several generations with exceptions in vegetatively propagated plants (Robinson 1998). However, a transgenic crop production approach of modifying plant architecture by making available the gene of interest (manipulating biofuel productivity traits or pathways) in a cloned form and robust plant regeneration and transformation system paves the way for integration of transgene(s) in a highly directed manner. This will ensure the development of new transgenic varieties previously difficult to obtain through the conventional breeding. With the advent of high-throughput, low-cost sequencing technology, genomes of many plant species have been sequenced and annotated. An example of the possible pathway, which could be adopted to genetically modify crop plants to yield biofuel is described in Fig. 6.3. Many genes of importance in relation to polysaccharide biosynthesis pathways have been identified using database information, and a number of expression studies have been conducted to verify such results (Ragauskas et al. 2006). For example, overexpression of expansin genes, which bring about the loosening of the cell walls of plants, may be considered for modification in biofuel crops (Cosgrove 2005). Also, emphasis must be given to identification of quantitative trait loci (QTLs) controlling lignification in plants, and breeding programs for biofuel production must emphasize selection of cultivars with low lignin content. RNA interference (RNAi) and antisense approaches can also be adopted to selectively silence the genes involved in complex carbohydrate synthesis.

6.2.1

Manipulating Genes and Pathways Involved in Starch and Sugar Metabolism

Starch is produced by all green plants for energy storage (Fig. 6.4a). It is a large polysaccharide made up of glucose linked via a-1,4 and a-1,6 glycosidic linkages (amylose and amylopectin). These are stored in plant in a form of dense semicrystalline structure. Gelatinization of starch is required for enzymatic digestion and fermentation, which can be achieved by heating starch at 60 C. Hydrolysis by glucoamylase enzyme converts the starch molecule into D-glucose. Sucrose, on the other hand, is a disaccharide of glucose and fructose. Enzymatic hydrolysis of sucrose is catalyzed by invertase, converting sucrose into glucose and fructose (both C6H12O6).

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Identify major genes and / QTL involved in complex polysaccharide biosynthesis by bioinformatics and subsequently find analogs or orthologs in other plant species.

Generate ectopic expressor /knockout (T-DNA insertion or by TILLING)/antisense/ RNAi induced silenced lines to hasten tissue degradation

Genetically engineer complex metabolic pathway like cellulose, lignin, tannin biosynthesis. Harvest and subsequent storage of the biomass

Pretreatment with genetically modified microorganism and other enhancer chemicals

Genetically manipulate microorganism / enzymes

Recovery of product and residue mix

Liquid Biofuel

Residue can be used as sillage or biofertilzer or solid / biohydrogen biofuels

Fig. 6.3 Possible pathway to genetically engineer crops to yield biofuel

C12 H22 O11 ! C6 H12 O6 þ C6 H12 O6 ðSucroseÞ

ðGlucoseÞ

ðFructoseÞ

Enzymatic hydrolysis is followed by a fermentation process for the production of bioethanol. Commercial yeast such as Saccharomyces cerevisiae converts glucose molecule into ethanol under anaerobic conditions. ðFermentationÞ

C6 H12 O6 ! ðGlucoseÞ

2 C2 H5 OH þ2CO2 ðEthanolÞ

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a Sucrose

Cytosol

Uridine diphosphate Glucose

Glucose 1phosphate

Adenosine glucose pyrophosphorylase

Adenosine diphosphate glucose Debranching enzymes

Plastid Amylose, Amylopectin

(Starch Granule)

b

Fig. 6.4 (Continued)

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Fig. 6.4 Strategies for manipulating (a) Starch (b) Cellulose (c) Lignin (d) Lipids

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Therefore, cloning genes responsible for rapid conversion to ethanol and then expression of such genes in plant system will enhance the production of ethanol. Myers et al. (2000) reported that transgenic plants with suppressed debranching enzyme produce soluble phytoglycogen, thus no gelatinization by pretreatment is required in this condition. Aside from genetic engineering of plants for reducing the production costs, specific genetic manipulation may be required depending on the source categories of biofuel. For example, in sugarcane, sugar content was increased by targeting sugar accumulated in the vacuoles and subsequently converted to isomaltulose by the enzymatic rearrangement of the glycosidic linkage from a (1–2)-fructoside in sucrose to a (1–6)-fructoside (Wu and Birch 2007). This new arrangement resulted in doubling the total sugar concentration in these transgenic lines (Gressel 2008). The increase in sugars in transgenic lines directly translates into higher yield of biofuel. Smith (2008) reported that manipulation of ADPglucose pyrophosphorylase (which plays a role in the sugar signaling pathway) to increase the yields of starch have been met with limited success. They also advocated partitioning of photosynthate in storage organs and increasing the flux of photoassimilate from source to sink. Earlier studies concentrated on manipulating carbohydrate composition of major food crops by altering the enzymes which act on photosynthesis.

6.2.2

Manipulating Genes and Pathways Involved in Cellulose and Lignin Biosynthesis

With the recent advent of increasing prices of food products and their shortage, the first generation of biofuel crops were discouraged. Instead, research was vastly concentrated on non-food crops and waste (nonutilizable biomass) from food crops, which could be converted into biofuels without compromising the food needs of mankind. This strategy could lead to potential sources of employment, and at the same time help in land reclamation with little or no subsequent maintenance. Here, we needed more advanced technologies to extract biofuels. Cellulose (long chains of glucose molecules bound by b-glycosidic bonds) is a component of plant cell walls, making up between a quarter and half of them in terms of mass. However, it is not easy to extract, as the cellulose in a plant is embedded in a cross-linked matrix with other components such as hemicellulose (branched polymers of xylose, arabinose, galactose, mannose, and glucose that bind bundles of cellulose strands together), lignin (a complex polymer into which the above-described bundles are matrixed and thus unavailable for conversion to fermentable sugar), pectins, xylene, and proline-rich proteins. Further, lignin limits the access of cellulase enzymes needed for fermentation of sugars. The area of ethanol production showing the most promise, both in terms of potential energy savings and in terms of national security interests, is ethanol derived from cellulose. Current processes for producing cellulosic ethanol are time-consuming, low-yielding, and, above all, expensive. The process of cellulose

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biochemistry is still poorly understood. Cellulose degradation pathways require multiple steps of varying temporal, environmental, and efficiency penalties. The major hurdle to producing biofuel is the high cost of conversion of cellulose and hemicellulose into ethanol. Therefore, the price of cellulosic ethanol is two- to threefold higher than bioethanol produced from corn grains. These production costs could be cut down by bioengineering crop plants in such a way that the necessary cell wall degrading enzymes, such as cellulase and hemicellulase, are produced within the plant system, thereby reducing the dependency on microbial bioreactors for enzymes. The concept of self-destructive plants could be coined to such transgenic varieties. Ziegler et al. (2000) advocated the restriction of the hydrolytic enzymes produced by the transgenic plants in the apoplast region of the cell to avoid reaction with other symplast elements. Further, Schillberg et al. (2003) advocated the potential of selectively degrading protein bodies in subcellular compartments of the endoplasmic reticulum (ER), thereby preventing interference with other cellular activities. Afterwards, the same enzyme will help in increasing the degradation process upon extraction from the transgenic plants. It is very important to ensure that enzymes produced for cell wall degradation should not interfere with other cellular activities. The other strategy could be directing the expression of these enzymes in the chloroplast of the plant cells. Chloroplast transformation with transgenes has been shown to be consistently predictable and less prone to silencing. The other strategy could involve genetically engineering plants in a tissue-specific manner, so that the transgene does not interfere with parts used for human consumption (Dai et al. 2000). Further cost reduction could also be achieved by genetically engineering the cellulose biosynthetic pathway in plants so that there is a minimal need for expensive pretreatment. Another strategy is to upregulate the cellulose biosynthesis pathway enzymes for increased polysaccharide production, which in turn will increase the yield of cellulosic biofuel. Good et al. (2005) showed that ectopic expression of glutamate dehydrogenate from bacteria resulted in increase in biomass of tobacco. Oxenboll Sorensen et al. (2000) reported that pectin arrangement in rice and potato plants was drastically changed (confirmed by Fourier transform infrared microspectroscopy, immune-gold labeling, and fermentable sugar accumulation studies) by the ectopic expression of endo-galactanase gene from fungus under the influence of a tissue-specific promoter without altering the plant architecture. Skjot et al. (2002) also reported decrease in arabinose content in the cell wall of potato plants. Sticklen (2008) reviewed the production of Acidothermus cellulolyticus E1 endo-1,4-b-glucanase in different crop plants for efficient conversion to biofuels, which is a stepping stone in the production of biofuel from cellulosic material. Attempts have been made to increase cellulose-binding molecules in plants, which results in increase in cellulose production without subsequent increase in lignin production. This may provide an opportunity to increase cellulose content in plants and hence increase proportion of fermentable sugars (Fig. 6.4b). Another strategy could be manipulating C3 mechanism of photosynthesis in favor of C4 mechanism, which is present in the grass family and can only be grown in tropical regions.

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Van Camp (2005) overexpressed two enzymes from cyanobacteria in tobacco, resulting in enhanced rate of photosynthesis that translated into increase in biomass. This could ensure equal distribution and usage of biomass over the globe. Jing et al. (2004) reported increase in tree height and hence biomass by ectopically expressing glutamine synthase gene (GS1).Also, manipulating genes for enhanced solar energy capture and conversion of photosynthate to sugars, and genes which provide endurance to plants growing under saline conditions, or land unutilizable for cultivation, should be emphasized (Antizar-Ladislao and Turrion-Gomez 2008). In plants, lignin (composed of phenylpropanoid groups) acts as a polymer around the hemicellulose microfibrils, binding the cellulose molecules together and protecting them against chemical degradation. Lignin cannot be converted into sugars. Thus, it is not practical in biofuel production. Their degradation is a high-energy process. A simplified illustration of genetic manipulation of lignin has been described in Fig. 6.4c. Vanden Wymelenberg et al. (2006) reported a large number of genes involved in breakdown of lignin from the fungus Phanerochaete chrysosporium genome. Such genes could be expressed in plants to induce cell wall breakdown in the latter stages of plant maturity. Ralph et al. (2006) reported genetic manipulation of monolignins in such a way that they continue to provide crosslinkage to cellulose and hemicellulose microfibrils required for the firm framework of the plant and at the same time increase biomass conversion efficiency to biofuels. Studies with the model plant Arabidopsis have shown that secondary wall thickening is lost when knockout lines (nst1, nst2) are produced, without affecting vascular bundle architecture (Mitsuda et al. 2007). This provides documented evidence that lignin biosynthesis can be selectively altered without rendering the plants prone to lodging by vascular collapse or increasing their susceptibility to insects, pests, and disease. The major share of the cost in biofuel production is the expense incurred in the production of cellulase enzymes in microbial bioreactors. Secondly, the cost of pretreating lignocellulosic matter for its breakdown to the intermediate components, and subsequent removal of the lignin, is very high. Three families of enzymes responsible for digesting lignin, laccases, manganese dependent peroxidases and lignin peroxidases (Kirk and Farrell 1987) could be used in transgenic plant production to produce plants with low lignin content amenable for biofuel production. Li et al. (2003) showed that by downregulating a single lignin biosynthetic gene, 4-hydroxycinnamoyl CoA ligase (4CL), a prominent reduction in lignin composition was observed along with an increase in biomass. Ralph et al. (2006) found that decreasing expression of 4-coumarate 3-hydroxylase (C3H) in alfalfa (Medicago sativa) drastically altered the lignin profile and structure. This could enhance the fermentation process in the production of biofuels. Gressel (2008) reported the isolation of brown midrib (bmr) mutations in sweet-stemmed sorghum and maize with low lignin content that were highly digestible by industrial carbohydrases. Besides the grain component, a large portion of biomass is left unutilized as lignocellulosic material. Lignin is a polyphenolic polymer (composed of monolignin units) and is highly resistant to degradation. In a typical study, downregulation of a lignin biosynthesis gene, cinnamyl alcohol dehydrogenase (CAD) in

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alfalfa resulted in altered lignin biosynthesis, thus increasing the processibility (Baucher et al. 1999). Chabannes et al. (2001) found that reduction in expression of cinnamoyl CoA reductase (CCR) in transgenic tobacco resulted in reduced lignin content with a corresponding increase in glucose in plant cells. Blaschke et al. (2004) reported that downregulation of O-methyl transferase (OMT) enzyme in Nicotiana tabacum enhanced biomass content, though proportion of lignin content was decreased. Ragauskas et al. (2006) suggested that often downregulation of a lignin biosynthesis gene results in altered lignin architecture in transgenic plants. Similarly, Chen and Dixon (2007) reported that downregulation of six different lignin biosynthetic pathway enzymes in alfalfa may eliminate the pretreatment process associated with extraction of fermentable sugars. Hu et al. (1999) reported alteration in lignin content and subsequent increase in cellulose content in quaking aspen (Populus tremuloides) by decreasing the enzyme hydroxycinnamate- CoA/5-hydroxyferuloyl-Co-A ligase. Bate et al. (1994) similarly advocated that suppression of phenylalanine ammonia lyase (PAL) may decrease lignin content in poplar plants. Ralph et al. (2006) found that flexible polymerization is possible in plants, whereby monoligins can be actively substituted by polyphenols. Similarly, Riboulet et al. (2009) identified lignin biosynthesis genes from microarray analysis in maize, which are differentially expressed during plant development. Such information will be vital in developing new transgenic crop programs and plant breeding. This in turn may not affect the development process of plants, but will facilitate the extraction of saccharides needed for biofuel production from the plants. Among several other renewable sources biomass (includes residual non-food parts of cultivated crop plants, garden and kitchen waste) is probably the most promising alternative to fossil fuel resources. It has several advantages, such as it will be sustainably available in the future and it is considered to be environmentally friendly, since it has low net release of carbon dioxide and sulfur content. This will also help us to mitigate the problem of landfilling in industrial and urban generated waste. Biomass is a complex material primarily made up of cellulose (30–50%), hemicellulose (20–40%) and lignins (15–30%). Complex carbohydrates like cellulose and hemicellulose are first converted into their component sugars through a hydrolysis process and then the sugars are anerobically fermented into biofuel such as bioethanol. At present, only the cellulose and hemicellulose components of plant biomass can be converted into biofuels by the action of anaerobic microbes. The major obstacle in increased biofuel production is to find an efficient way to break down complex plant polymers into simpler derivatives.

6.2.3

Manipulating Genes and Pathways Involved in Lipid Metabolism

Oils from crops like palm, castor, soybean and oilseed rape are presently being used to make biodiesel. The biosynthesis of oils in plants starts de novo in the plastids

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where photosynthesis provides the carbon source, which are assembled into short chains to produce palmatic (C16), stearic (C18), oleic and linoleic acids (C20). In general, a greater proportion of short-chain fatty acids is more desirable in biodiesel production (Fig. 6.4c). The higher proportion of short-chain fatty acids like oleic acid will render oxidative stability to the oils even at high temperature (Metzger and Bornscheuer 2006). The fatty acids are then transported to the endoplasmic reticulum (ER) by an energy-dependent pathway, and there long-chain fatty acids are assembled from the short-chain ones. The final products (oil or fatty acids) are transported out of the ER to be stored in vacuoles as oil bodies, which act as a source of reserve energy for the plants. Therefore, the pathway to synthesize fatty acids and triacylglycerols will need to be modified to facilitate the production of biodiesel from these crops (Durrett et al. 2008). All the genes acting on the lipid biosynthesis have been cloned by Zhang et al. (2005). They further suggested that improving the composition of the fatty acid components of plant oils will enhance the acceptability of biodiesel in temperate regions of the world. For example, Lardizabal et al. (2008) achieved increase in biodiesel production from soybean by ectopically expressing diacylglycerol acyltransferase 2A (DGAT2A) from Umbelopsis ramanniana fungus during the seed development stage. Similar results have been reported in rapeseed by overexpressing a laurate-specific acylACP thioesterase gene from California bay tree and a laurate-specific LPAAT gene from coconut (Knutzon et al. 1999; Wiberg et al. 2000). Wiberg et al. (2000) also described that an increase in the content of caprylic acid (C8) and capric acid (C10) in rapeseed had resulted in a 30% increase of short-chain fatty acid in the seed. Also, the production of short chain fatty acids or oils should be enhanced from the non-oil-producing tissues of the plant. Recent studies have shown that plant storage tissue can store high quantities of oil bodies without any drastic effect of cell membrane polarity and hence on plant growth (Napier 2007). Increasing the percentage of short-chain fatty acids by a transgenic approach will reduce the cost associated with the production of biodiesel. Most vegetable oils are large, branched molecules; therefore, could not be directly used in diesel engines. These large and branched molecules of bio-oils are converted into smaller and straight-chain molecules through transesterification, making them suitable for regular diesel combustion engines. Production of high-quality short-chain fatty acids by using advanced biotechnological techniques (TILLING, RNAi, antisense approach, T-DNA insertion lines) may come to our rescue. Targeted induced local lesion in genomes (TILLING) is a very popular technique used today to identify mutant lines for any gene of interest. For example, a mutation in fatty acid gene (FAD1 in peanut) will cause the plant to accumulate short-chain fatty acids, as its capability to convert the short-chain fatty acids into long-chain fatty acids will decrease. Again, RNAi and antisense expression of the fatty acid gene will reduce translation of the gene, and thus the amount of long fatty acids will be decreased. Random T-DNA insertion is a technique introduced to prevent the production of a gene of interest. Random T-DNA may get integrated in the exonic region of the gene and thus translation may come to a premature stop.

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Conclusion

The era of cheap fossil fuel is rapidly coming to an end. Therefore, the need of the hour is to look at the alternative forms of energy, a strategy that was hardly emphasized in the past. There are also concerns about global climate change and severe food shortage. Biomass is the least expensive and most globally available resource. Therefore, priority should be shifted towards utilizing biomass, leaving aside food for human consumption. Plant architecture have been naturally modified, in fact, ever since the evolution of land plants commenced several million years ago and through the selection of desirable phenotypes by crop improvement (around 10000 BC, when man started cultivating plants for food and shelter) to the more sophisticated methods of using genetic manipulation and plant biotechnology to produce modern-day transgenic biofuel crops. Modified carbohydrate and lignin biosynthetic pathway has been a major target for chemical or genetic intervention in recent years. The use of transgene genes could also underpin conventional breeding to modify biofuel production, in view of the growing public concern over the potential environment damage and health hazards by the use of fossil fuel, and the limited availability of suitable genes to modify synthesis of sugars, lignin and lipids in plants, by adopting classical breeding approaches. Tissue-specific antisense expression of transgenes involved in biofuel production in vegetative parts will prevent unwanted assimilate partitioning and direct photosynthate towards storage organs (seeds or underground storage like bulbs, corms and rhizomes), increasing productivity besides contributing to biofuel processing and production. One of the aspects for biofuel production is synchronous maturity of the crops used for biofuel production. Many chemicals that promote synchronous maturity like NAA (napthalene acetic acid) could be useful. Also, application of gibberelic acid (GA) may result in high yield. Anti-GA substances like pacrobutrazol can be used to control plant height, or ectopic expression of GA2-oxidase will result in dwarf plants (Dijkstra et al. 2008). This will allow easy harvest of the produce with low cost on resources and saving time. Compact plants are also less prone to damage by biotic and abiotic stress. Genetic manipulation strategies should lay emphasis on downregulating lignin biosynthesis genes without drastically affecting plant architecture and making plants prone to infestation of disease, insects and pests. Therefore, production of transgenic plants with the above mentioned traits would help in developing a more robust plant phenotype that was not always possible through conventional or molecular breeding. This was because traits amenable for biofuel production did not mostly go hand in hand with increased food crop production. Thus, suitable traits for biofuels were lost in rounds of selection of superior genotypes responsible for increased crop production practiced over several thousand years. Now, crop breeders have been combining tissue culture approaches with traditional breeding in order to broaden the gene pool available for improvement of productivity and to introduce new, desirable traits. With the development of DNA microarray chips, it is now possible to identify novel genes and to generate plants with novel traits

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(in this case, traits amenable for biofuel production), and not just the development of varieties restricted to insect pests (Wang et al. 1996), disease (Marchant et al. 1998) and herbicide tolerance (Slater et al. 2003). Manipulating monolignins, and perhaps modifying three-dimensional lignin architecture, could also be an additional possibility. Therefore, modification of traits related to biofuel production may require manipulation of the multiple hormones, complex carbohydrate synthesis pathways, which may involve the introduction of multiple genes into plants. Such studies may also lead to a better understanding of the roles of the different hormone classes in biofuel crop development. Future research must be directed towards identifying tissue-specific or species-specific inducible promoters that could, for example, drive gene expression in the vegetative tissues, such as stem, internodes, and leaf petioles, thus ensuring that flowering and seed set, both qualitatively and quantitatively, are not affected. Furthermore, the possibility of using constructs carrying genes associated with enhanced biofuel production, but lacking the nptII selectable maker gene (Holn et al. 2001), should be explored in order to facilitate the acceptance of transformation technology to regulate plant biomass. This can be achieved by selecting plants directly, based on phenotype, such as with dwarf growth. It is also possible to use T-DNA related sequences, which have originated from plant genomes. These have been referred to as cisgenic plants, as their transformation vectors, including promoters and terminators, are derived from the same plant species, which is transformed with the gene of interest (Chandler and Tanaka 2007). Also, vectors from plant-originated selectable marker, recombinase recognition sequences (Cre-lox system) can also be beneficial (Zuo et al. 2001). The use of co-transformation (Komari et al. 1996) or the use of chimeric plants (De Vetten et al. 2003) may also be considered. Further, introduction of transgenes in male-sterile plants may be another viable option, or the use of terminator gene technology may be considered. The exact role of each lignin biosynthetic gene on plant development must be accessed to pinpoint genes (in turn enzymes) bringing about the greatest effect. The use of a specific promoter may be pivotal in driving gene expression under study. Low transformation efficiency can be correlated with a constitutive promoter, such as CaMV 35S (Zhu et al. 2008), which may result in poor regeneration and low recovery of transgenic plants (Petty et al. 2003; Zhu et al. 2004; Busov et al. 2006). The use of a viral promoter, like 35S, may result in gene silencing in plants as observed (Bhattacharya et al. unpub.). Furthermore, an ubiquitin-like promoter (Christensen and Quail 1996) may result in high efficiency of transformation compared to 35S promoter, and future genetic manipulation studies should include such ubiquitin-like promoters. Genetic modification paves the way for the development of new cultivars by gradually incorporating one or more genes (Miflin 2000). However, expression of genes across plant species is not always entirely predictable, leading to a large number of trial and error methods to reach a generalized conclusion. Another area of emphasis will be manipulating oilsynthesizing genes in lower primitive plants like algae, cynobacteria and phytoplankton. Exploiting everyday-generated crop waste will also help to lay the foundation of a carbon-balanced world.

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Antagonism to this technology has also restricted the development of the technology (Azevedo and Araujo 2003; Phillips 2004). There is no uniform system for international regulation on genetically modified crop plants with significant difference between countries (Halsberger 2006). Since transgenic biofuel crop plants are not anyway intended for human consumption, they should be theoretically more acceptable to the public. The Cartagena protocol (http://bch.biodiv.org) is the first attempt to bring uniformity in laws governing genetic modification, and many countries are adding these laws to their existing law in order to come to a unified attempt to regulate genetically regulated products. In conclusion, biofuel may not replace the use of fossil fuel in the foreseeable future; however, it can become a major component of alternative fuels, and may add towards the carbon economy in the age of rapid industrialization.

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Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270 Murugesan A, Umarani C, Subramanian R, Nedunchezhian N (2009) Bio-diesel as an alternative fuel for diesel engines-A review. Renew Sustain Energ Rev 13:653–662 Myers AM, Morell MK, James MG, Ball SG (2000) Recent progress toward understanding the biosynthesis of the amylopectin crystal. Plant Physiol 122:989–998 Napier J (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Plant Biol 58:295–319 Oxenboll Sorensen S, Pauly M, Bush M, Skjot M, McCann M, Borkhardt B, Ulvskov P (2000) Pectin engineering: modification of potato pectin by in vivo expression of an endo-1, 4-b-Dgalactanase. Proc Natl Acad Sci USA 97:7639–7644 Petrou EC, Pappis CP (2009) Biofuels: A Survey on Pros and Cons. Energ Fuel 23:1055–1066 Petty LM, Harberd NP, Carre IA, Thomas B, Jackson SD (2003) Expression of the Arabidopsis gai gene under its own promoter causes a reduction in plant height in chrysanthemum by attenuation of the gibberellin response. Plant Sci 164:175–182 Phillips AL (2004) Genetic and transgenic approaches to improving crop performance. In: Davies PJ (ed) Plant hormones: biosynthesis, signal transduction, action! Kluwer, Dordrecht, The Netherlands, pp 582–609 Puppan D (2002) Environmental evaluation of biofuels. Periodica Polytechnica Soc Manag Sci 10:95–116 Ragauskas A, Williams C, Davison B, Britovsek G, Cairney J, Eckert C, Frederick W, Hallett J, Leak D, Liotta C (2006) The path forward for biofuels and biomaterials. Science 311:484–489 Ralph J et al (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol Chem 281:8843–8853 Riboulet C, Guillaumie S, Mechin V, Bosio M, Pichon M, Goffner D, Lapierre C, Pollet B, Lefevre B, Martinant JP, Barriere Y (2009) Kinetics of Phenylpropanoid gene expression in maize growing internodes: relationships with cell wall deposition. Crop Sci 49:211–223 Ripley BS, Muller E, Behenna M, Whittington-Jones GM, Hill MP (2006) Biomass and photosynthetic productivity of water hyacinth (Eichhornia crassipes) as affected by nutrient supply and mirid (Eccritotarus catarinensis) biocontrol. Biol Con 39:392–400 Robinson A (1998) Developing sustainable global agriculture for the 21st century. In: Rice Reports, John Innes Centre, Norwich, UK, pp 1–8 Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants. Cell Mol Life Sci 60:433–445 Skjot M, Pauly M, Bush M, Borkhardt B, McCann M, Ulvskov P (2002) Direct interference with rhamnogalacturonan I biosynthesis in Golgi vesicles. Plant Physiol 129:95–102 Slater A, Scott N, Fowler M (2003) Plant biotechnology – the genetic manipulation of plants. Oxford University Press, Oxford, UK, pp 7–18 Smith AM (2008) Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54:546–558 Sticklen MB (2008) Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 9:433–443 Van Camp W (2005) Yield enhancement genes: seeds for growth. Curr Opin Biotechnol 16:147–153 Vanden Wymelenberg A, Sabat G, Mozuch M, Kersten PJ, Cullen D, Blanchette RA (2006) Structure, organization, and transcriptional regulation of a family of copper radical oxidase genes in the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl Environ Microbiol 72:4871–4877 Vogt KA, Vogt DJ, Patel-Weynand T, Upadhye R, Edlund D, Edmonds RL, Gordon JC, Suntana AS, Sigurdardottir R, Miller M, Roads PA, Andreu MG (2009) Bio-methanol: how energy choices in the western United States can help mitigate global climate change. Renew Energ 34:233–241

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Chapter 7

Plant-Produced Biopharmaceuticals Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown, Charles Spillane, and Lokesh Joshi

7.1

Introduction

The term “biopharmaceutical” is commonly used to denote a therapeutic protein produced by recombinant (genetic) engineering. In this process, genes encoding proteins or peptides of interest from humans or other organisms are identified, cloned, inserted into an expression vector and the protein or enzyme produced within a prokaryotic or eukaryotic expression host “production platform” organism. Such production platforms may typically be bacteria, yeasts, insects, mammals or plants. Batch cultures of cell suspensions are also commonly used for large-scale production of recombinant therapeutic proteins, a method often referred to as “fermentation culture.” Bacteria and yeast can be grown in closed-loop bioreactors, as can insect, mammal and plant cell-lines, which are generated from whole organisms. This ensures several generations of stable cell line propagation, allowing production of the protein or enzyme of interest. The use of animal cell lines and bacterial or yeast strains to produce therapeutic proteins has resulted in several successful products of medical interest (Walsh 2006). Such pharmaceutical products include glycosylated human erythropoietin (rhEPO), which acts as a red blood cell stimulating hormone, recombinant human clotting factor VIII (rhCFVIII), used for treating hemophilia, and recombinant insulin, used to treat diabetes mellitus. Sales of rhEPO were estimated at US $10.7 billion for 2005, while those of rhCFVIII reached an estimated US$1 billion by the same year (Walsh 2006). The total market for biopharmaceuticals is expected to reach approximately US$70 billion by 2010 (Walsh 2006), with insulin estimated to generate US$14 billion from 16,000 kg of protein (Moloney et al. 2003) and monoclonal antibodies contributing approximately US$20 billion (Hiatt and Pauly 2006). L. Joshi (*) Glycoscience and Glycotechnology Group, Martin Ryan Institute and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland e-mail: [email protected]

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Non-human mammalian cell lines, such as Chinese hamster ovary (CHO) cells, are currently considered to be the most desirable expression systems as they produce proteins similar to those from human cells, particularly with regard to glycosylation or other post translational modifications (Hamilton and Gerngross 2007). However, one of the major challenges associated with mammalian cell culture technology of particular public concern is that contaminating pathogens, which are capable of infecting other mammalian species (i.e., xenozoonosis), may be transferred to humans during product recovery. These include viruses, endogenous retroviruses and internalized bacteria and mycoplasmas, as well as prions. This inherent biological limitation poses a particular challenge to the development of pathogen-free Good Manufacturing Practice (GMP) procedures for mammalian cell culture systems. Furthermore, as complex growth media is required for mammalian cell bioreactors, minute variations in media components and fermentation conditions can lead to undesirable changes in the end product(s). Even under the most stringent conditions, maintaining consistency from batch to batch is challenging as the interactions of cells with each other and their environment may also create variations in end products (Butler 2006). Also, endogenously produced immunogenic agents, such as proteins or species-specific glycosylation, harvested as a co-extracted or incorporated component of biopharmaceuticals produced in CHO cells, may create unwanted effects in patients even at extremely low levels (Hamilton and Gerngross 2007). In addition to the above, there are a number of other key drivers for the development of improved production platforms for biopharmaceutical production. The costs associated with growing and maintaining mammalian cells for pharmaceutical production remain relatively high compared to yeast and bacteria (Fischer et al. 1999). Costs for establishing a pharmaceutical-grade mammalian cell culture facility may exceed €250 million (Sardana et al. 2007), and each requires extensive testing and certification with the result that only a relatively small number of such facilities worldwide can produce biopharmaceuticals on a large (kilograms per year) production scale. Hence, any approach that leads to a manufacturing cost-saving will gain competitive advantages over time. Increasing numbers of new protein therapeutics coming on stream are generating a production capacity crisis within the available mammalian cell culture systems. This situation is likely to worsen – as of 2006, there were 2,500 biotech drugs in discovery phase, 900 in preclinical trials and 1,600 in clinical trials (Walsh 2006). Moreover, the advent of high-volume protein therapeutics targeted at much larger numbers of people (e.g., monoclonal antibody-based therapeutics such as Herceptin, Enbrel, Mylotarg, Remicade, etc.) and the need to produce generics or biosimilars are placing strains on current biopharmaceutical production platforms to deliver at the scale and cost levels needed. To produce 300 kg of secretory IgA antibodies per year, plant-based production systems have the lowest capital costs (maize or tobacco; €0.5–2.0 million/300 kg per year) when compared to platforms based on transgenic goats or mammalian cell culture (~€6–7 million/300 kg per year) (Richard McClosky,

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Centocor). Finally, with escalating elderly populations in developed countries increasing product requirement, and patients and governments demanding affordability, there is a definite need to innovate and develop more cost-effective platforms for large-scale production of biopharmaceuticals.

7.2

Transgenic Plants as Production Systems for Biopharmaceuticals

Transgenic plants (or derived plant suspension cell lines) are considered very attractive alternatives to mammalian cell systems for the production of recombinant mammalian glycoproteins. Plants as multicellular eukaryotes have many advantages as production platforms for recombinant proteins or enzymes, including similar eukaryotic biosynthetic pathways, high protein yields, freedom from animal pathogens and bacterial endotoxin contamination and low production cost per unit protein overhead (Gomord et al. 2005). Indeed, cost savings associated with plant technologies and end-product purification may lead to a 10- to 100-fold advantage over currently used mammalian fermentation systems such as CHO cells (Vidi et al. 2007). Additionally, with the use of organ-specific promoters to drive protein expression, the seeds, stems, roots, fruits, and leaves of whole plants suitable for growth in fields or greenhouses can be targeted for transgenic protein production (Fischer et al. 2004). To date, the highest yields have generally been achieved by targeting recombinant proteins to chloroplasts, oil bodies and seed protein bodies of whole plants (see below). It has been estimated that 250 acres of greenhouse production of transgenic plants producing hepatitis B vaccine could meet Southeast Asia’s demand. Indeed, the scalability of plant platforms for biopharmaceutical production is one compelling advantage of this approach, e.g., in response to rapid demand for therapeutics for any future pandemics. The possibility of plant systems for facilitating freedom from refrigeration would be a tremendous advantage for economically challenged populations, where maintenance of a “cold chain” for handling and storage may simply not be possible (Rademacher et al. 2008). The capacity of plant storage organs (seeds or tubers) to act as protein storage systems is truly remarkable (Fischer et al. 1999) – antibodies in plant seeds stored at room temperature have been found to be stable and fully active after several years. The market potential of plant-based recombinant technology was first demonstrated in the production of pure, research-grade proteins. By 2005, three major recombinant proteins had been produced in corn (Zea mays) – the biotin-receptor, avidin, and the enzymes, trypsin and b-glucuronidase – and were commercially available (Ma et al. 2005). Seed storage protein bodies (PBs) are of particular interest as the recombinantly expressed proteins can be accumulated in a comparatively high

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proportion into a relatively stable, compact mass, greatly contributing to relative ease of harvest, storage and purification of products (Stoger et al. 2005). Despite the clear value of seed storage targeting, for many purposes it is more appropriate to make use of plant cells propagated as suspension cultures. Such techniques also aid containment (see below). Plant suspension cells are derived from calli, clusters of undifferentiated cells. Calli are first stimulated from mature plant tissues through direct contact with hormone-enriched solid growth media. After reaching a suitable size, the frangible calli are transferred to a liquid growth medium, agitated to shake the cells apart and cells continue to divide within the liquid growth medium (Hellwig et al. 2004). Plant suspension cells are grown using media that is much simpler in formulation than that used for insect and mammalian systems, typically containing just a few salts and minerals, sucrose and a plant hormone (Kolewe et al. 2008). A wide range of plant species are used commercially for molecular pharming purposes, including well-known biological models (e.g., Arabidopsis thaliana, Medicago truncatula), food crops, and non-food crop species specialized for particular purposes (Twyman et al. 2003; Fischer et al. 2004; Stoger et al. 2005). Amongst the seed crops, molecular pharming is underway in cereals (maize, wheat, rice, barley), legumes (peas, soybean) and oilseeds (safflower, rapeseed). The root and tuber crops, carrot and potato, are used for pharming, while lettuce, spinach, alfalfa, banana and tomato are examples of fruit and green leaf crops being used for pharming. Non-food crops in use for pharming include tobacco and falseflax, while non-cultivated species include duckweed (Lemna spp.) and moss (Physcomitrella patens), and also unicellular photosynthetic organisms, such as microalgae (Chlamydomonas reinhardtii). Importantly, all these eukaryotes share the common characteristics of eukaryotic protein machinery and being amenable to genetic transformation. Transformation of plants and plant cells is typically done using one of two species of Agrobacteria which are able to transfer transgenes (encoding recombinant biopharmaceuticals) into the host plant genome. Agrobacterium tumefaciens is competent to transform foliar and floral tissues and cultured cells (Broothaerts et al. 2005), while A. rhizogenes is competent to transform root tissue or cultures (Shi and Lindemann 2006). First, the Agrobacteria are transformed with DNA plasmids containing the gene of biopharmaceutical interest, under the control of a suitable plant promoter. In response to the patent landscape (Dunwell 2005) and the need to develop complementary approaches for transferring desirable genes and associated traits to plants, a range of other techniques for plant transformation have been developed (Broothaerts et al. 2005; Vain 2007). These include plant transformation by particle bombardment, otherwise known as the gene gun method (Brereton et al. 2007). Plasmid vectors adhered to minute particles of a carrier agent, often gold particles, are accelerated by compressed air to penetrate and transform the nuclear genome of the target cells. Vectors for transformation can be rapidly assembled using high-throughput cloning techniques, such as Invitrogen’s Gateway system, which allows combination of specific features (Curtis and Grossniklaus 2003). Plasmid vectors may be constructed to allow

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for selection of successfully transformed cells by transformation with genes conferring antibiotic or herbicide resistance and subsequent growth in media containing these agents or encoding reporter enzymes or fluorescent molecules (Sala et al. 2003). Promoters may be chosen for constitutive high expression throughout the plant, or tailored for expression in cell culture (i.e., inducible systems) or a particular organ, such as the seed or tuber. These transformation techniques can also be targeted for chloroplast expression, a relatively new alternative (Hou et al. 2003). Directing genes for recombinant proteins to chloroplasts in whole plants may allow yields of up to approximately 45% of total leaf protein (Arlen et al. 2008). The chloroplast is an endosymbiotic organelle of prokaryotic origins with its own genome. Hence, chloroplast genes and translational machinery differ from those of the nucleus, although there is regulatory interaction and protein trafficking between these two cellular compartments (Pogson et al. 2008; Woodson and Chory 2008). An important attribute of transgenic chloroplast systems is that chloroplasts are maternally inherited and therefore rarely transmitted via pollen (there are some species exceptions) and therefore transgenes located in chloroplast genomes are typically biologically contained (Ruf et al. 2007). Lipoprotein particles found within chloroplasts (plastoglobules) may be specifically targeted as depositories for recombinant proteins. Vidi and colleagues fused recombinant yellow fluorescent protein (YFP) to a naturally occurring plastoglobulin protein to study the viability of the process (Vidi et al. 2007) and found that plants expressing the YFP fusion protein did not have a significantly altered phenotype. Moreover, such plants had similar growth characteristics to those expressing recombinant proteins targeted to oil bodies. One disadvantage of chloroplast expression systems is a lack of access to the post translational machinery of the secretory pathway (see below). Hence, proteins produced by chloroplasts cannot be endogenously glycosylated, deacylated or phosphorylated (Anisimov et al. 2007). Some recombinant proteins may be improperly folded or even rendered biologically inactive without these modifications (Gomord and Faye 2004; Byrne et al. 2006; Eichler et al. 2006; Mitra et al. 2006). High-level production of recombinant proteins within cytoplasmic compartments of plant cells depends on promoters to drive the expression of the gene of interest. Many promoters have been used in both monocot and dicot species to date, and the sophistication of such promoters is increasing as understanding of plant gene regulation grows (Venter 2007, see also Vol. 1, Chap. 5). Low-level expression using dicot promoters in cereals has highlighted the necessity of developing monocot-specific promoters (Stoger et al. 2005). Additionally, the use of an engineered plant virus-based transient expression systems (e.g., magnICON from Icon Genetics; Gleba et al. 2005) can allow a significantly faster turnaround time and a much higher protein yield than other stable plant transformation techniques (Mallory et al. 2002; Gleba et al. 2005), although the higher mutation rate of RNA-based viral expression systems is a consideration for the generation of homogeneous protein product (Domingo et al. 2006).

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Challenges Regulatory Challenges

Despite the distinct advantages transgenic plants offer, some specific challenges remain. As transgenic plants will be producing protein compounds with known pharmaceutical effects, it is imperative for the success and acceptability of the molecular pharming field that such production operates in a biologically contained system and is also compliant with current GMP (cGMP) procedures for the production of therapeutic grade proteins. Given the issues with controlling the abiotic and biotic environment, open-field production of food crops or other cultivated species producing high-potency pharmaceutical compounds may not be desirable for molecular pharming industry development. Closed-loop production systems which ensure that pharma-producing seeds or plant materials are not used for any other purposes are essential. This would include ensuring that any biotic predators or pests are not adversely impacted by the pharmaceutical-producing plants. However, pharmaceutical-producing plants have been deliberately cultivated since the first pharmacopeias were compiled for apothecaries in the Middle Ages. Physic gardens, which trained the earliest physicians in the use of medicinal plants, were the forerunners of today’s botanic gardens, many of which were originally associated with the faculties and departments of pharmacy in universities. Today, there are multiple examples of highly potent (non-transgenic) medicinal or toxic plants (e.g., opium poppy, high erucic acid rapeseed), which are cultivated for the production of pharmaceuticals without any adverse effects on humans or environment. Nonetheless, to further bolster public trust and to avoid any unforeseen secondary consequences of plant-based recombinant technologies, it will be prudent to apply efficient measures of biological containment so that pharma-producing seeds or transgenes are not used outside of the closed-loop production systems for which they were developed. Hence, methods for the containment of reproductive material (e.g., male sterility, maternal inheritance systems such as chloroplasts or autonomous apomixis) in conjunction with the use of genetically-linked, visible phenotypic markers for any transgenic seeds/plants expressing pharma proteins will be advisable. Other approaches worth considering are inducible expression systems. In general, the most prudent containment will be to use the best greenhouse-controlled environment conditions (for whole transgenic plant systems) or photobioreactors (for cell culture suspensions) for commercial production of the first generation of plant-derived biopharmaceuticals. It is certain that plant-made pharmaceutical (PMP) production will be like any other pharmaceutical production system in terms of its strict regulatory control. The regulations for pharmaceutical production in plants are not currently harmonized internationally with different governing bodies operating in the USA and European Union (EU). In the USA, regulatory oversight for PMPs is under the aegis of the following bodies: the US Department of Agriculture

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(USDA: http://www.usda.gov/wps/portal/usdahome) regulates growth of plant and defines the required safeguards for the production and transport of the product; the Food and Drug Administration (FDA) Centre for Biologics Evaluation and Research (CBER: http://www.fda.gov/%20BiologicsBloodVaccines/default.htm) regulates biologic products, their manufacture and distribution; the FDA Centre for Food Safety and Applied Nutrition (CFSAN: http://www.foodsafety.gov/list. html) and Centre for Veterinary Medicine (CVM: http://www.fda.gov/cvm/default. html) are involved in consultations on food and feed safety, and finally the PMP developer is responsible for safety assessments, drug master files, chemistry manufacturing controls and biologics license application for the client’s drug registration (Streatfield 2005). In the European Union, regulatory procedures for PMPs are in flux due to a need to tailor the existing regulations to deal with PMPs. Any field-grown plant pharming would require notification under EU Directive 2001/18/EC (http:// eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32001L0018:EN:HTML), which covers both food and non-food transgenic crops. Containment-grown plant pharming would fall under EU Directives 90/219/EEC (http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=CELEX:31990L0219:EN:HTML) and 98/81/EC (http://ec.europa.eu/environment/biotechnology/pdf/dir98_81.pdf). For any commercial release, the European Food Standards Agency (EFSA) would be involved in the review process under 1829/2003/EU Food and Feed Guidelines (http:// eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:268:0001:0023:EN: PDF). All pharma products produced from plants within the EU must adhere to the 2309/93/EU regulations (http://www.biosafety.be/EMEA/2309_93en.pdf). All decisions at the clinical trial stage for the PMP would be dealt with by the relevant national authorities in each EU Member State. At the commercial evaluation stage, the PMP would be regulated by the European Agency for Evaluation of Medicinal Products (EMEA). See also Vol. 2, Chap. 11 for discussion.

7.3.2

Biological Challenges

The potential allergenicity of any co-extracted endogenous protein is a further consideration. For example, targeting expression in plant seeds, which naturally produce high levels of storage proteins, is a means of generating high yields of recombinant products (Reggi et al. 2005; Nykiforuk et al. 2006). However, species such as certain legumes contain endogenous proteins which can induce allergic reactions. Several potent protein allergens have been identified in peanut (Arachis hypogeae) including storage proteins Arah1, Arah2 and Arah3 (Barre et al. 2005), and soybean (Glycine max) such as the vicilin Bd 28k (Tsuji et al. 2001). In plant species and organs where there are known allergens, isolation of recombinant protein products from the endogenous components requires stringent and exhaustive purification, generally requiring multiple steps, to minimize risk of allergic reaction.

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Glycosylation and the Production of Clinical Grade Biopharmaceuticals

One of the major roadblocks in the use of plants as recombinant protein production platforms is the absence of human-type glycosylation (Wilson 2002; Gomord and Faye 2004; Paccalet et al. 2007; Bakker et al. 2008). Protein glycosylation is an issue that has to be taken into account for all protein-production platforms for biopharmaceuticals that are glycosylated for parenteral administration by injection or diffusion, whether mammalian, yeast, plant, insect or E. coli (Fig. 7.1). Typically, the objective will be to replicate the glycosylation profile of the protein generated when the protein is expressed in the cells of the species in which the particular protein or gene variant is found. However, in some instances differences in glycosylation profiles may have no functional consequences in terms of therapeutic efficacy (Ko et al. 2003). Plants are capable of assembling oligosaccharides with linkages not found in humans and these moieties are immunogenic in humans (Fo¨tisch and Vieths 2001; Sourrouille et al. 2008). These motifs include b(1!2)-linked xylose (Xyl), a(1!3)-linked fucose (Fuc) and b(1!3)-linked galactose (Gal). The latter contrasts with the b(1!4)-Gal extension typically found at the distal ends of mammalian N-linked oligosaccharides which allows the correct attachment of terminal sialic acid structures (Sourrouille et al. 2008). There are a number of strategies

Bacteria

Yeast

xylose galactose mannose

Insect

Plant

N-acetylneuraminic acid N-acetylglucosamine fucose polypeptide

Fig. 7.1 Typical N-linked oligosaccharides from bacterial to mammalian

Mammalian

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being developed to “humanize” the plant glycosylation machinery (Lerouge et al. 2000; Bardor et al. 2006) to better refine the efficacy of their products, boost useable production and ultimately to lower costs (Castilho et al. 2008). These strategies are based on (a) inhibition of plant-specific glycosylation enzymes and pathways (Strasser et al. 2004) and/or (b) introduction of human-specific glycosylation enzymes and pathways (Bakker et al. 2001). On the other hand, it should be noted that topical or orally administered recombinant therapeutics may often contain plant glyco-epitopes with no negative consequences to test subjects. While plants do have many advantages, there are also competing (and complementary) technology platforms such as yeast where there are parallel efforts to develop humanized glycosylation pathways (Chiba and Jigami 2007; Hamilton and Gerngross 2007). Transformation of plants with a gene for human b(1!4) galactosyltransferase (b1,4GalT) is one way to combat the formation of natural plant glyco-epitopes on recombinant proteins (Sourrouille et al. 2008). Human b1,4GalT in its native state and fused with the Golgi targeting domain of a natural plant GlcNAc transferase were found to be effective at a1!3Fuc and b(1!2)-Xyl linkage-containing structures on plant proteins when cloned into alfalfa. The same study also found that siRNA-mediated silencing of sequences encoding alfalfa XylTs and FucTs was effective at decreasing the occurrence of plant-specific glyco-motifs on recombinant protein products (Sourrouille et al. 2008). The moss P. patens has been engineered to reduce endogenous synthesis of a(1!3)-Fuc and b(1!2)-Xyl linkages within N-linked oligosaccharides (Weise et al. 2007). P. patens plants were transformed with the gene encoding human erythropoietin (hEPO) and produced a highly glycosylated protein which still contained the essential N-linked oligosaccharides, but lacked the immunogenic a(1!3)-Fuc and b(1!2)-Xyl motifs. In small scale bioreactors (maximum 10 L), the moss plants secreted a maximum accumulation of rhEPO (250 mg g–1 dry plant weight) after 6 days of growth. In humans, a predominant terminal sugar structure is the nine-carbon sialic acid, 5-N-acetyl-D-neuraminic acid (Neu5Ac), which is most often found in either an a(2!3) or a(2!6) linkage to Gal or N-acetylgalactosamine (GalNAc), although polysialic acid termination with a(2!8) linkages also occurs. The efficacy of recombinant proteins can be greatly enhanced by the inclusion of complete human-type oligosaccharides which allow them to mimic naturally occurring blood-borne proteins and evade rapid removal by the body, thereby increasing the half-life (Goochee et al. 1991; Paccalet et al. 2007). hEPO is reported to have only a 2 min half-life without sialylation, but this is extended to 3 h when sialylated (Ngantung et al. 2006). Clotting Factor VIII has an unsialylated half-life of just 5 min while that of the fully sialylated version is 4 h (Ngantung et al. 2006). The presence of sialic acids has been reported in A. thaliana suspension cells, buckwheat and other plant species (Mayer et al. 1964; Onodera et al. 1966; Bourbouze et al. 1982; Shah et al. 2003). These findings have been subject to much debate in the literature, partly based on the identification methods used and partly due to the lack of plant homologs corresponding to mammalian or bacterial enzymes in their complete sialic acid biosynthetic pathways (Fig. 7.2; Lerouge et al. 2000;

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CYTOSOL GOLGI

UDP-GlcNAc

Sialylated glycoconjugate

ATP GNE

ManNAc-6-P

STr

PEP NANS

glycoconjugate

Neu5Ac-9-P

ST

NANP CTP Neu5Ac

CMP-Neu5Ac CMAS NUCLEUS

Fig. 7.2 Mammlian biosynthetic pathway of sialic acid after Castilho et al. (2008). Enzyme abbreviations are GNE, UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase; NANS, N-acetylneuraminic acid phosphate synthase; NANP, Neu5Ac-9-phosphate phosphatase; CMAS, CMP-Neu5Ac synthetase; ST, CMP-Neu5Ac transporter; STr, sialyltransferase

Faye et al. 2005; Zeleny et al. 2006). Recently, it has been reported that plants may be able to transport sialic acid (Bakker et al. 2008) and some species may have transferases capable of attaching sialic acid structures (Takashima et al. 2006). However, present or not, based on the quantities of sialic acid reported, plants would not produce sufficient sialylation to be of use in recombinant technologies regardless of the endogenous mechanism and, thus, molecular engineering to augment any endogenous pathways would be required (Paccalet et al. 2007). It would be useful to discover and use plant genes whose products perform the same function as those from mammals (Lerouge et al. 2000; Bakker et al. 2001; Bakker et al. 2008). However, this goal comes with the caveat that genes for enzymes of similar function in plants and mammals may not display high sequence homology. For example, the discovery of a typically mammalian Gal-b-(1!3) GalNAc-a-O-Ser/Thr structure on proteins from rice seeds (Kishimoto et al. 1999) implies the presence of a GalNAc transferase (GalNAcT) gene encoding the enzyme responsible for mucin-type (short oligosaccharide chains with GalNAc-aSer/Thr linkage to the protein backbone) O-glycosylation initiation. Despite the functional homology, no gene with significant sequence homology could be found

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in the rice genome via an in silico search (Kilcoyne et al., 2009). This could imply that certain enzymes of homologous function can differ widely from plant to mammals, or that the same function is fulfilled by other enzymes in planta. While the basic mechanisms of asparagine-linked core glycosylation in plants are now considered to be well understood (Lerouge et al. 2000; Wilson 2002; Faye et al. 2005), many questions still remain concerning exactly how plants perform fully extended N- and O-linked glycosylation of proteins.

7.4

Plant-Produced Biopharmaceuticals

Biopharmaceuticals have already had an enormous global impact from both health and economic perspectives. As demand is expected to exceed current supply capabilities, less expensive alternative production platforms are under intense investigation with plants firmly at the forefront in terms of biological and costefficiency (Giddings et al. 2000; Daniell et al. 2001; Goldstein and Thomas 2004; Yano and Takekoshi 2004). To date, many experimental products produced using plant recombinant technologies have been explored and validated. These will be discussed as three different groups of biopharmaceuticals: vaccines, antibodies and protein therapeutics (Table 7.1).

7.4.1

Vaccines

Vaccines comprise the largest class of recombinant biopharmaceuticals (Carter 2006). Vaccinations, either therapeutic or prophylactic, are a cost-effective method of reducing the occurrence of infectious disease in animals and humans. In general, vaccines are composed of an antigenic agent from the bacterium or virus containing an epitope known to induce an immunogenic response in the host, or else are based on an inactive or attenuated pathogen (e.g., Sabin polio vaccine). Effective vaccines have been developed and produced for a wide range of diseases, such as hepatitis B (Keating and Noble 2003; Sojikul et al. 2003; Greco et al. 2007) and rabies (see below), and are in development for many other diseases, such as HIV (see below). However, there are large populations vulnerable to major disease burdens, especially in developing nations, who remain unprotected due to the prohibitive costs of accessing even traditional or existing vaccines. Oral delivery of vaccines could address some of the issues that increase vaccine cost. Elimination of needles and reduced levels of training, expertise and equipment for delivery of oral vaccines would be beneficial for reducing the cost of vaccine delivery. Oral delivery can target the antigen to the mucosal surface of the intestinal lining, which can prime other mucosal surfaces, and this is especially relevant for gastrointestinal or sexually transmitted diseases. However, this delivery method also typically leads to degradation of the vaccination agent in the gastrointestinal

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Table 7.1 Three groups of biopharmaceuticals: vaccines, antibodies and protein therapeutics with examples and references Product Expression platform References Vaccines NV-VLP Tobacco, potato Mason et al. (1996), Zhang et al. (2006) HBsAg Tobacco, potato, tomatillo Mason et al. (1992), Richter et al. (2000), Kong et al. (2001), Gao et al. (2003), Huang et al. (2005), Youm et al. (2007), Sojikul et al. (2003) HIV-1/HBV fusion Arabidopsis, tobacco Greco et al. (2007), Guetard et al. (2008) protein FI-V fusion protein Tobacco, tomato Jones et al. (2003), Williamson et al. (2005), Mett et al. (2007), Alvarez et al. (2006), Santi et al. (2006), Arlen et al. (2008) LTB Tobacco, corn, potato, Wagner et al. (2004), Lamphear et al. soybean, carrot (2002), Moravec et al. (2007), Mason et al. (1998) PyMSP4/5 Tobacco, tomato Wang et al. (2008), Chowdhury and Bagasra (2007) Antibodies Anti-CD4/28 receptor scFv Anti-HBsAg Mab Anti-ErbB-2 scFv 2G12 Mab

Wheat

Brereton et al. (2007)

Tobacco Tobacco Corn

hsv81sc IgA Anti-HBsAg scFv

Micro algae Tobacco

Yano and Takekoshi (2004) Galeffi et al. (2006) Rademacher et al. (2008), Ramessar et al. (2008) Mayfield and Franklin (2005) Pujol et al. (2007)

Therapeutics MuIL-12 hGM-CSF rh-insulin

Tobacco Tobacco, rice, sugarcane Safflower, Arabidopsis

IFN-a Gcase Novokinin

Rice Tobacco Soybeans

Liu et al. (2008) Wang et al. (2005) Moloney et al (2003), Markley et al. (2006), Nykiforuk et al. (2006) Shirono et al. (2006) Reggi et al. (2005) Yamada et al. (2008)

tract, and to overcome this, large quantities of antigen have to be administered to ensure that enough will survive to induce immunogenicity. The amount of vaccine required for delivery of oral vaccines will place strains on the ability of current mammalian systems to cost-effectively deliver the quantities required. Ideally, a plant-produced vaccine would require minimal downstream processing. In this case, an edible (or orally ingested) plant vaccine would deliver the antigen to the intestinal mucosa in sufficient quantities and could protect the antigen from degradation in the harsh stomach environment as it is encapsulated within plant cells. A current drawback for making an edible vaccine a viable option is the

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low levels of accumulation of the recombinant protein in plant tissues, but advances in gene expression systems for plants will allow such issues to be overcome. Biosafety concerns about transgenic foods (i.e., ensuring production and delivery is within a strictly controlled closed-loop production and regulatory system) and consistency (cGMP-related) concerns regarding batch-to-batch consistency are also barriers to the implementation of edible vaccines. Biosafety issues for the production of medicinal plants (e.g., opium poppies) or toxic organs of food plants (e.g., poisonous leaves of Solanaceous species, such as potatoes) have been dealt with for decades and, based on these previous experiences, should be possible to address. In terms of the consistency issues, low-cost downstream processing approaches involving concentration or partial purification step(s) could make plant-produced vaccines feasible for commercial development. Plant-produced vaccines for livestock have made more progress in getting to market than those for humans, as they face fewer regulatory issues. Dow Agro Sciences LLC received the first regulatory approval in January 2006 for a plantmade injectable poultry vaccine against Newcastle Disease virus (NDV) from the USDA Center for Veterinary Biologics. The antigen is a haemagglutinin neuraminidase protein from NDV that was produced in a transgenic tobacco cell culture system. Diseases targeted for animal vaccine trials include foot and mouth disease virus, canine parvovirus, rabies virus, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus (Ma et al. 2005). Diseases and antigens currently targeted for human vaccinations are discussed below. Noroviruses (NoV) belong to the Caliciviridae family and consist of one species, Norwalk virus (NV; Xi et al. 1990). The species is genetically diverse and genotypes are distributed over five genogroups (GGI–GGV; Ramirez et al. 2008). NoVs are a group of non-enveloped, single-stranded RNA viruses and are the leading cause of viral gastroenteritis in humans worldwide (Xi et al. 1990; Vinje et al. 1997; Fankhauser et al. 1998). They are transmitted directly or indirectly by the fecal-oral route but may also become airborne and are highly contagious. There is currently no commercially available vaccine for norovirus but the economic and public health impact of gastroenteritis epidemics make NoV an ideal candidate for vaccine development. Previously, when the capsid protein was expressed in insect cells by recombinant baculovirus, it self-assembled into empty 38-nm virus-like particles (VLPs), which were similar to the native virus in morphology and antigenicity (Jiang et al. 1992; Prasad et al. 1994). These VLPs were immunogenic in CD1 and BALB/c mice when orally administered (Ball et al. 1998) and the intranasal route induced higher serum IgG and fecal IgA responses (Guerrero et al. 2001). Additionally, recombinant Norwalk (rNV) VLPs orally administered without an adjuvant to humans in a phase I study were found to be safe and produce a dose-dependent serum IgG response (Ball et al. 1999). The potential of rNV VLPs for use as an oral immunogen for a mucosal vaccine has made it a popular target for production in plants. Partially purified rNV VLPs from transgenic tobacco plants elicited humoral and mucosal antibody responses specific for rNV in mice, as did feeding with

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transformed potato tubers (Mason et al. 1996). In another study by this group, the plant optimized gene for rNV capsid protein was expressed in tomato and potato plants and the product successfully assembled VLPs. Lyophilized tomato fruit induced dose-dependent NV-specific serum IgG and mucosal IgA production in mice. However, larger quantities of freeze-dried potato tuber (1 g) were required to elicit the same response. It was found that rehydrated potato tuber was less immunogenic due to VLP instability caused by phenolic compound oxidation and that air-dried tomato fruit was more immunogenic than lyophilized tomato fruit and dried potato tubers (Zhang et al. 2006). Furthermore, when raw transgenic potato expressing rNV capsid protein was fed to human volunteers, 19 out of 20 volunteers developed an immune response, although the increase in serum antibody level was of limited magnitude (Tacket et al. 2000). These VLPs have also been produced in Nicotiana benthamiana leaves using an engineered plant virus-based transient expression system (magnICON). Oral immunization with partially purified rNV VLPs without adjuvant induced serum IgG and fecal and vaginal IgA response in mice (Santi et al. 2008). Coupled with a low-cost concentration and partial purification step, as demonstrated in this study (Santi et al. 2008), the goal of manufacturing oral vaccines in plant tissue for humans is becoming more achievable. Over two billion people worldwide are infected with hepatitis B virus (HBV), which is associated with chronic liver disease and liver cancer (WHO 1998). Hence, there is a need for an inexpensive vaccine for widespread immunization, especially in the developing world. Engerix-B1 (GlaxoSmithKline) and Recombivax HB1 (Merck and Co.) are commercial vaccines that confer seroprotection and consist of recombinant hepatitis B surface antigen (HBsAg) produced in the yeast Saccharomyces cerevisiae (Adkins and Wagstaff 1998; Keating and Noble 2003). The surface envelope glycoprotein exists in three isoforms produced by alternative splicing and initiation – large (L), middle (M) and small (S) – of which the commercial vaccine is the recombinantly produced S form (Fig. 7.3). This envelope protein is reported to have an important role in attachment to the host cell surface and subsequent infection (Paran et al. 2003). HBsAgs assemble VLPs and, when expressed in tobacco leaves, the subviral particles formed were similar to those produced in yeast (Mason et al. 1992). SHBsAg MHBsAg

12

pre-

108 109

pre-

163 164

S

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LHBsAg

Fig. 7.3 The three isoforms of hepatitis B virus surface envelope glycoprotein (HBsAg) produced by alternative splicing and initiation – large (L), middle (M) and small (S)

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The partially purified antigen from tobacco elicited B- and T-cell responses when injected in a mouse model and the T-cell antigen epitope was found to be a partial sequence of the S region of HBsAg (Thanavala et al. 1995). HBsAg VLPs were then produced in potato tubers with some improvements to increase the yield, such as targeting the gene product for retention in the plant cell endoplasmic reticulum (ER; Richter et al. 2000). Mice fed HBsAg-transgenic potatoes with a cholera toxin adjuvant generated HBsAg-specific serum antibodies. A long-lasting secondary antibody response was obtained on parenteral boosting (Kong et al. 2001) and, conversely, by parenteral prime followed by an oral boost (Richter et al. 2000). HBsAg has also been produced in transgenic cherry tomatillo (Physalis ixocarpa) that was orally immunogenic in mice (Gao et al. 2003). Additionally, the serum anti-HBsAg titre increased in over half the group of immunized humans who were fed uncooked transgenic potatoes without a mucosal adjuvant or buffering for stomach pH (Thanavala et al. 2005). MHBsAg from transformed tobacco resulted in an enhanced antibody titre in mice after intraperitoneal (i.p.) injection (Huang et al. 2005). This same result was also observed when mice were fed potato-derived MHBsAg (Youm et al. 2007). In an attempt to overcome low accumulation of antigenic proteins in plant tissues, a fusion protein of a soybean signal protein with HBsAg was introduced into suspension-cultured tobacco cells which resulted in greater accumulation. The fusion protein was more immunogenic in mice than the unmodified HBsAg and this may have been due to greater antigen stability, improved presentation of the antigenic determinant in the S domain and increased oligomerization (Sojikul et al. 2003). The highly immunogenic hepatitis B core antigen (HBcAg) has also been recombinantly produced in transgenic tobacco and was found to assemble into spherical particles 25 to 30 nm in diameter. In the hemagglutination-inhibition test, partially purified VLPs demonstrated serologic properties comparable to those produced in E. coli (Tsuda et al. 1998). High levels of production of HBcAg (up to 7.14% of total soluble protein (TSP)) were reported 7 days post-infection using the magnICON system (Icon Genetics). The product also self-assembled into VLPs and the partially purified product evoked strong serum antibody response in mice when injected i.p. Moreover, mucosal immunization (oral and nasal) with no adjuvant gave HBcAg-specific serum IgG and intestinal IgA response (Huang et al. 2006). This rapid, high-level antigen production of strong immunogenicity makes a potential oral vaccine from plants more feasible. Enterotoxin produced by pathogenic strains of E. coli is the major cause of death in developing countries and claims 1.6 million lives per annum (Tacket 2007). The heat-labile toxin is similar to cholera toxin and is composed of the toxic A 27 kDa subunit and the non-toxic B subunit (LTB) which is a 55 kDa homopentamer of 11.6 kDa subunits. LTB is a strong immunogen that binds to the GM1 ganglioside on enterocytes and is a popular choice for a vaccine candidate. It has been expressed in potato (Mason et al. 1998), maize (Lamphear et al. 2002), tobacco (Wagner et al. 2004) and soybean (Moravec et al. 2007); volunteers who consumed transgenic potato developed serum and/or mucosal immune response (Tacket et al. 1998).

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Transgenic defatted corn meal was generally well tolerated and was immunogenic in volunteers (Tacket et al. 2004). However, as raw potato and uncooked corn meal can be unpalatable to many, LBT was expressed in transgenic carrot taproots (Rosales-Mendoza et al. 2008), which can be eaten raw. The product was found to be immunogenic and also protected against cholera toxin challenge in mice (Rosales-Mendoza et al. 2008). A bivalent vaccine of recombinant HIV-1/HBV fusion protein VLPs has been produced in A. thaliana and tobacco (Greco et al. 2007). The HIV-1 portion consisted of a polyepitope comprised of eight epitopes from five major HIV-1 proteins. These particular epitopes were expressed together as HIV-1 and HBV have similar transmission pathways and co-infection with HBV occurs in more than 30% of HIV-1 patients. Oral administration to mice elicited anti-HIV-1 specific CD8+ T-cell activation (Guetard et al. 2008). Malaria is another major effective vaccine target. Its prevalence in areas of extreme poverty that lack infrastructure also makes it an ideal candidate for an oral or edible vaccine. Malaria is caused by the parasite protozoan genus Plasmodium transmitted to humans through the bites of infected female Anopheles mosquitoes. The species Plasmodium vivax and P. falciparum cause most infections in humans resulting in death and morbidity and are thus the main targets of vaccine development (Chowdhury and Bagasra 2007). However, the parasite’s complexity, its ability to change through its life cycle in humans and mosquitoes, and its ability to evade the immune system make this a challenge (Chowdhury and Bagasra 2007). An edible vaccine using transgenic tomatoes of different sizes, shapes and colors to deliver multiple antigens for the various stages of malarial infection has been proposed (Chowdhury and Bagasra 2007). In an attempt to immunize against one life stage, surface protein 4/5 (PyMSP4/5) of the murine P. yoelii merozoite, the homolog of P. falciparum merozoite surface proteins 4 and 5, has been selected. The merozoite life stage takes place in hepatocytes and PyMSP4/5 has been shown to provide protection for mice against lethal challenge (Kedzierski et al. 2001). PyMSP4/5 from tobacco parenterally delivered to mice induced antigen-specific antibodies but antibody levels were not high enough to provide protection against lethal challenge (Wang et al. 2008). The virulent Gram-negative bacterium, Yersinia pestis, is the cause of plague. The most common form, bubonic plague, is spread to humans by bites from fleas that have previously fed on infected animals and is then distributed systematically in the body. The disease results in swollen, tender lymph nodes (buboes), which can hemorrhage and become necrotic. The pneumonic form of the disease is almost always fatal and can also be transmitted by inhalation of infected aerosolized droplets, making plague a potential bioterrorism agent. Recently, there have been plague outbreaks in India, Algeria, Congo, Zambia and Malawi (WHO 2007) and over 2,000 cases are reported annually. Vaccines that are currently available include killed whole cells, which are not protective against pneumonic plague and have many undesirable side effects, and a live attenuated vaccine, which also suffers from unacceptable side effects and has not been approved for use in the United States (Anisimov and Amoako 2006). However, vaccines based on the

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antigenic subunits of F1 (fraction 1 anti-phagocytic capsular envelope glycoprotein), V (a secreted immunomodulatory protein) and an F1–V fusion protein have been intensely investigated and were successful immunogens in animal and human trials (Jones et al. 2003; Williamson et al. 2005; Mett et al. 2007). The F1–V fusion protein was expressed in tomato in an attempt to make a useable oral vaccine. The transgenic tomatoes were immunogenic in mice primed subcutaneously (s.c.) with bacterially produced F1–V and orally boosted by feeding with freeze-dried transgenic tomato fruit (Alvarez et al. 2006). In this case, no pathogen challenge was performed. The first report of a protective vaccination of animals using plant-produced antigenic material, which appeared in 2006, made use of the magnICON system to produce high levels of recombinant F1, V (2 mg g–1 fresh leaf weight) and F1–V (1 mg g–1 fresh leaf weight) antigens in tobacco leaves (Santi et al. 2006). Female guinea pigs were vaccinated s.c. with the partially purified antigens and alum adjuvant. After exposure to aerosolized virulent Y. pestis, V-vaccinated animals had the highest survival rate. The F1–V fusion antigen has been expressed in tobacco leaf chloroplasts, giving a maximum yield of 14.8% of product out of the total soluble protein. Mice were s.c. primed with enriched crude F1–V fusion protein from plant with alum adjuvant and boosted either s.c., with adjuvanted doses, or orally, with unadjuvanted doses. Oral F1–V mice had higher prechallenge serum IgG1 titers than s.c. injected F1–V mice. After exposure to an inhaled lethal dose of aerosolized Y. pestis, 33% of the adjuvanated F1–V s.c.-boosted mice and 88% of the orally boosted mice with unadjuvanted F1–V survived. Hence, it may be concluded that oral booster doses induce protective immune responses in vivo (Arlen et al. 2008).

7.4.2

Antibodies

Antibodies are the second largest class of biopharmaceuticals (Carter 2006). Serum from animals immune to particular infectious disease antigens have been used as therapeutics for at least a century (Yano and Takekoshi 2004). Orthoclone, a monoclonal antibody used to treat organ rejection after transplants, was approved for human use in 1986 (reviewed in Hiatt and Pauly 2006). The year 1987 marked the first studies on the feasibility of antibody production using plants as expression hosts, and the first report of recovered recombinant protein from plants followed in 1989 (Pujol et al. 2007). Monoclonal antibodies (MAbs) produced in plants are often referred to as “plantibodies,” though use of this designation remains arbitrary. Plants and plant cells may be engineered to produce antibodies by either stable or transient expression. A transient expression approach that has been used in plant suspension cell cultures has been reported to decrease the timescale for producing milligram quantities of antibodies from approximately a year (i.e., for whole transgenic plant systems) to under a month (Hiatt and Pauly 2006). One component of the process, termed magnifection (magnICON, Icon Genetics), is the result of adapting

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viral transcripts to the expression host machinery by including additional introns which better match those of mRNAs encoding endogenous plant proteins, and by shortening overall mRNA lengths (Gleba et al. 2005). When used with a single viral vector, magnifection is sufficient for the enhanced expression of individual light chains but not complex hetero-oligomeric antibodies. Complete MAbs (consisting of both light and heavy chains) were produced using magnifection and co-infection with two separate viral vectors (Giritch et al. 2006). In contrast to stable plant expression of antibodies, it is claimed that this approach offers more rapid development and better possibilities for efficient biological containment. Many examples of methods for producing plantibodies have appeared in the literature. Wheat (Triticum aestivum cv. Westonia), a monocot species, was transformed to produce single light chain antibody (scFv) fragments suitable for use in ocular disease treatment. The need to ensure very low levels of bacterial endotoxins, which may be present in bacterial expression systems, was cited as a major benefit of producing antibody fragments in plants. ScFv chains, which bound to CD4 or CD28 receptors on the surface of thymocytes, were produced using particle bombardment transformation of T. aestivum calli. The maximum expression was 180 mg g–1 in crude extracts made from mature seeds of T. aestivum (Brereton et al. 2007). The affinity of the plant-produced scFvs was approximately equivalent to that of single-chain fragments produced in bacteria. Hepatitis B infection may be treated by the administration of antibodies against the surface proteins of the viral envelope. Tobacco suspension cultures have been used to produce recombinant antibodies against HBV (Yano et al. 2004) and required a two-step transformation process. Cells derived from a patient carrying natural antibodies against HBV surface antigens were immortalized by transformation with Epstein–Barr virus (EBV). From these EBV cells, RNA coding for the MAbs was extracted and used to make cDNA copies which were cloned into plant suspension cells. Proteins harvested directly from EBV transformed cells cannot be used as human therapeutics due to the risk of infecting patients with EBV (Yano and Takekoshi 2004). Hence, plant suspension cultures were used to produce the monoclonal IgGs destined for use as human therapeutics. IgGs were purified and compared with their counterparts from EBV TAPC301-CL4 cells for efficacy. The plant-produced antibody was shown to produce a similar complement-dependent cytotoxicity (Yano et al. 2004). The Cuban Centre for Biotechnology and Genetic Engineering (CIGB) has been producing a monoclonal antibody (CB-Hep1) against HBV in transgenic tobacco plants since April 2006 when regulatory use approval was granted for this plantibody. The plant production platform for CB-Hep1 is used for large-scale purification of the active component of CIGB’s hepatitis B vaccine, sold under the trade name Heberbiovac-HB. Aggressive carcinomas have been experimentally treated with antibodies binding to ErbB-2, a surface receptor that is a member of the epidermal growth factor class of signaling proteins (Galeffi et al. 2006). A multi-platform approach was used to produce stable transgenic tobacco plants expressing anti-ErbB-2 scFv chains. scFv clones made from murine hybridomas were produced with and without peptide spacer arms and used to transform N. tabacum plants through

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A. tumefaciens infection. The resulting purified scFvs demonstrated a similar binding constant to the parental hybridoma-derived MAbs and were also shown to be useful in histochemical staining of tumor tissue (Galeffi et al. 2006). It may be possible to prevent human HIV infection in developing countries by using plant-produced antibodies as topical viruscides (Rademacher et al. 2008; Ramessar et al. 2008). The antibodies function to neutralize the virus particles, stopping them from entering hosts. Secretable forms of heavy and light antibodies (2G12) directed against the coat proteins of HIV-1 were made in transgenic corn (Zea mays L.). Antibodies accumulated in the endosperm of corn seeds, allowing them to be dried and handled without need for refrigeration. Furthermore, a threefold increase in the neutralization activity of the corn-produced antibodies was reported when compared to 2G12 antibodies derived from CHO cells (Ramessar et al. 2008). This increase in efficacy was suggested to be a result of multivalent presentation as a result of aggregation of the recombinant antibodies as they were deposited in the endosperm protein bodies (Ramessar et al. 2008). Microalgae (C. reinhardtii) have also been explored as a system for producing single scFv fragments of human A-type immunoglobulins (Mayfield and Franklin 2005). Chlamydomonas spp. are able to grow by phototrophy or heterotrophy and may take in carbon through acetate (Mayfield and Franklin 2005). Genomic and chloroplast DNA was transformed by the particle bombardment method. While the yield was conditional on a high degree of codon optimization specific both for the species and organelle transformed (nuclear genome or chloroplast), the authors reported a yield of up to 0.5% (w/w total soluble protein) for hsv81sc when optimized and used with atpA or rbcL promoters and accompanying 5’ untranslated regions (Mayfield and Franklin 2005). Apart from direct therapeutic uses, antibodies produced in plants may also have a role in the purification of co-expressed biopharmaceuticals. MAbs have been made in plants specifically to capture plant-manufactured antigens used in hepatitis vaccines (Pujol et al. 2007). Two transgene constructs, one for a scFv fragment and one encoding a complete copy of a mouse anti-hepatitis B surface protein antibody, have been produced in tobacco seeds and in suspension cultures. In the case of the suspension cultures, antibodies were transiently expressed after transformation with A. tumefaciens and samples collected five days after initial transformation. Recovery of whole “plantibody” structures was estimated to be on the order of 0.3 to 0.5 mg mL–1 of suspension culture (Pujol et al. 2007).

7.4.3

Therapeutic Proteins

Many therapeutic proteins can be produced in transgenic plants. For instance, cytokines are a class of bioactive therapeutic proteins with stimulatory and/or immune suppression functions. Cytokines have many possible therapeutic uses, including as pro-immune supplements co-administered during treatments for other disorders.

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Human interleukin 12 (IL-12) is a large, heterodimeric pro-inflammatory cytokine composed of the heavily glycosylated p35 subunit covalently linked by a disulphide bond to a lightly glycosylated p40 subunit. Mice are frequently used as an in vivo model for human immune characteristics and murine IL-12 (MuIL-12) is homologous in both amino acid sequence and function to that of humans. A single-chain construct of mouse cytokine MuIL-12 was expressed in N. tabacum whole plants and hairy roots (Liu et al. 2008). The p35/p40 fusion MuIL-12 consists of an IL-12p40 sequence joined by a triplet repeat of Gly–Gly–Gly–Gly–Ser to a p35 sequence (Mattner et al. 1993). A single chain was used instead of co-expression to ensure a 1:1 ratio of subunits and assembly of both IL-12 subunits. Mouse splenocyte proliferation was induced by the purified plant-produced MuIL-12 comparable to the effect of MuIL-12 produced in animal cells (Liu et al. 2008). Protein expression was up to 40 mg g–1 from fresh leaf tissue, while the hairy root cultures produced up to 33 mg g–1 of fresh tissue. Neutropenia, a condition characterized by a lack of granulocytes in the bloodstream, is often encountered as a side effect of treatments, which suppress immune function (Sardana et al. 2007). One of the therapies used to treat neutropenia relies on the replacement of granuloctyte-macrophage colony-stimulating factor (hGMCSF). hGM-CSF is a cytokine normally found in the human body which stimulates neutrophil and monocyte production as part of innate (antimicrobial) immune function. Production of recombinant hGM-CSF has been achieved in rice suspension culture cells, tobacco and rice seeds, and the vegetative tissue of sugarcane (Wang et al. 2005; Joo et al. 2006; Sardana et al. 2007). Seeds of Oryza sativa cv. Xiushui 11 accumulated 1.3% (w/w) of total soluble protein when transformed with the gene for hGM-CSF driven by a glutelin promoter, Gt1, which allowed protein body targeting of the recombinant protein (Wang et al. 2005; Sardana et al. 2007). Biologically active hGM-CSF has also been produced in sugarcane (Saccharum hybrid; Wang et al. 2005). The hGM-CSF gene was introduced by particle bombardment of embryogenic callus tissue and resulted in a maximum yield of 0.02% of total soluble protein (Wang et al. 2005). An ER-retention tag was necessary to achieve detectable accumulation levels in the transgenic plants and, over the course of 14 months, this accumulation rate remained stable. As sugarcane plants do not reach flowering under cultivation and are harvested prior to flower budding, no pollen-mediated gene transfer is possible (Wang et al. 2005). SemBioSys Genetics (Calgary, Alberta, Canada, http://www.sembiosys.ca), has begun efforts to produce human insulin at a very large scale in plants to meet the 16,000 kg demand expected by 2010 (Moloney et al. 2003). The innovative SemBioSys production system targets recombinant insulin to the oil bodies and allows traditional oil/water phase separation and purification to harvest the recombinant insulin from the seeds (Moloney et al. 2003; Nykiforuk et al. 2006). Oleosins, proteins which occupy the outer membrane of oil bodies, are used in the targeting of the recombinant oleosin fusion proteins to the oil bodies (Markley et al. 2006) by creating fusion constructs containing an oleosin-targeting peptide fused to the protein product of interest. Because of the lipid association of the oil

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body membranes and localized protein, liquid–liquid phase separation can be used to efficiently recover the biopharmaceuticals. Oil bodies in seeds of A. thaliana have also been targeted for the transgenic production of human insulin and shown to produce a maximum yield of 0.13% (w/w total soluble seed protein) with comparable activity to insulin produced in yeast expression systems (Nykiforuk et al. 2006). Shirono and co-workers have used transgenic rice suspension cells derived from dwarf rice plants (O. sativa L. cv. Hosetsu-dwarf) to produce active interferon-a (IFN-a). IFN-a is used to treat disorders resulting from the intrusion of some classes of chemicals and foreign bodies, including micoorganisms and viruses (Shirono et al. 2006). Transformation of the rice suspension cells with A. tumefaciens containing a transgene for IFN-a under the control of the constitutive 35S promoter resulted in stable production of active protein for at least ten generations. The transgene construct included the first intron of the rice cytosolic superoxide dismutase gene, a 10 kDa prolamin signal sequence, a GUS reporter sequence, and a thrombin cleavage sequence followed by the human IFN-a gene (Shirono et al. 2006). Current treatment for Gaucher disease requires replacement therapy utilizing human b-glucosyl-N-acylsphingosineglycohydrolase (also known as b-glucosidase, EC 3.2.1.45, abbreviated GCase), an enzyme that cleaves glucosylceramide into glucose and ceramide (Reggi et al. 2005). Gaucher disease is a fatal autosomal disorder manifesting itself in homozygous recessive infants in the general population at approximately 1:200,000 but approximately 1:640–10,000 in some Jewish subpopulations (Reggi et al. 2005; Weinstein 2007). While it is possible that functional, native GCase can be purified from human placental tissue, low yield and risk of contamination make the process less than desirable for therapeutic use. Recombinant GCase from tobacco plants was purified and found to be enzymatically active and readily taken up by human fibroblasts (Reggi et al. 2005). Furthermore, although the presence of an N-linked glycan at one site is required for the protein to be catalytically active, it was free from plant-specific glyco-epitopes containing Xyl and Fuc, thus greatly reducing the possibility of an immune response in patients. Novokinin is a hypotensive therapeutic hexapeptide with the sequence Arg– Pro–Leu–Lys–Pro–Trp, originally derived from the naturally occurring protein ovalbumin found in avian eggs (Yamada et al. 2008). A transgene vector coding for a modified form of naturally occurring a’ subunit of b-conglycinin soy protein, which incorporated four copies of the Arg–Pro–Leu–Lys–Pro–Trp peptide sequence, was introduced into soy plants and produced a yield of 0.5% of total soluble seed protein for the peptide of interest. Defatted flour from the transgenic soybeans administered orally reduced systolic blood pressure in spontaneously hypertensive rats (Yamada et al. 2008). Extracts from plants of the Digitalis genus have been used for centuries as cardiovascular therapeutics (Michael 2006). Lanatosides are secondary metabolites harvested from Digitalis lanata EHRH for the production of cardiotonic drugs (Shi and Lindemann 2006). After the leaves are processed to isolate the lanatosides,

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the enzyme cardenolide 16’-O-glucohydrolase (CGH-1) is recovered and used to remove glucosylation from the lanatosides. This enhances human resorption of the secondary metabolites and allows the formation of the active medicinal chemicals, digitoxin and digoxin. Purification of active CGH-1 from D. lanata leaves is a relatively expensive process. Therefore, recombinant expression of the enzyme has been explored to produce larger quantities of CGH-1 more economically. Since bacterially produced CGH-1 did not have comparable activity to that harvested from D. lanata leaves, a plant-based eukaryotic expression strategy was investigated. CGH-1 was produced in induced Cucumis sativis hairy root but it too was observed to have less activity than the foliar D. lanata enzyme (Shi and Lindemann 2006).

7.5

Plants as Model Systems for Biopharmaceutical Development for Humans and Other Mammals

Due to their limited foliar mass and small seeds, whole A. thaliana plants may not be a suitable volume expression system for most commercial biopharmaceutical production efforts. However, Cobento Biotech in Denmark is using A. thaliana for cGMP production of human intrinsic factor (rhIF), which is used for treatment of vitamin B12 deficiency. Nonetheless, A. thaliana has made a tremendous contribution to the understanding of plants, protein expression and the intricacies of molecular interaction within eukaryotes. To the biomedical and pharmaceutical research community, plants at first may seem unlikely candidates to study the effects of chemicals and pathogens on human health. However, from an evolutionary and comparative biochemistry perspective, plants can be used instead of animals to better understand many aspects of eukaryote gene regulation and biochemistry. Indeed, a recent study has highlighted that many significant discoveries with direct relevance to biomedical science and medicine have been achieved using the model plant A. thaliana, while many biological processes of relevance to human health are easier to study in this model plant than in mammalian or model animal (e.g., Drosophila, C. elegans) systems (Jones et al. 2008). Relative ease of care, rapid generation maturity in many model and crop plants and complete or near-complete genome sequence availability contribute to these uses (van Baarlen et al. 2007). The fully sequenced and extremely well-annotated genome of this model plant (in conjunction with its powerful genetics) allows the use of A. thaliana for studies directly linked to human drug metabolism, which may prove invaluable for the development of new drugs. The pharmacogenetic effects of small molecules on A. thaliana have been characterized to model individual organism sensitivity to drugs. Attributes that make this non-animal platform desirable for such studies include availability of a growing range of genetic and molecular tools including recombinant inbred lines (RILs), near-isogenic lines (NILs), a high degree of

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phenotype variation among accessions, and high polymorphism rates on a pernucleotide basis. Current efforts to re-sequence the genomes of 1,001 A. thaliana accessions (Ossowski et al. 2008) as well as the generation of extremely dense SNP maps of this model plant (Kim et al. 2007) will make it an even more powerful genetic model. These ongoing advances are making A. thaliana increasingly suited for “rapid molecular genetic characterization of alleles” (Zhao et al. 2007). For example, Zhao and co-workers (Zhao et al. 2007) elucidated the effects of glucosylation of the chemical hypostatin in subpopulations of A. thaliana plants, which would normally suffer growth retardation from this treatment. It was found that individuals which expressed large amounts of the enzyme HYR-1 produced glucosylated hypostatin which stunted growth of the plants. This led to a lower inhibitory concentration (IC50) value in connection with the stunted phenotype (Zhao et al. 2007). These kinds of data mirror the roles of various factors in drug efficacy and toxicity found in humans.

7.6

Conclusions

Clearly, transgenic PMPs have major promise for the efficient and cost-effective production of protein-based biopharmaceuticals. Many reports now demonstrate that transgenic plant systems can produce vaccines, antibodies and other proteinbased therapeutics cost-effectively and potential human pathogen-free. In the case of some vaccines, it may be possible using plant-based systems to develop approaches for these to be administered orally, which confers the potential to reduce vaccine delivery costs to poorer patients (e.g., in developing countries). One of the major barriers to the commercial realization of a PMP industry is the path-dependency and capital inertia in the current mammalian cell culture production paradigm. The concept of path-dependency is frequently used to analyze trends in innovation where path-dependency is associated with the idea of “lock-in” (Patel and Pavitt 1997). While a technology may be quite flexible when first developed, over time more fixed pathways become established which act as barriers to entry for new innovations. Examples of technology lock-in include the QWERTY keyboard or the VHS video format. The extremely high capital costs of producing therapeutic proteins in mammalian cell culture systems are inherently linked to an expensive and detailed regulatory approval system that has been developed specifically for mammalian, yeast and bacterial cell culture systems for production of protein-based biopharmaceuticals. In some jurisdictions (e.g., EU), the regulatory pathways for approval of recombinant protein production from disruptive innovations such as transgenic plantproduced pharmaceuticals are still under development and until such regulatory pathways are developed, a disincentive will remain for commercial development of plants as production platforms for biopharmaceuticals. While the regulations are catching up with the science, there is a need for continued research to improve further the efficiency, safety and cost-effectiveness

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of such systems. For instance, the glycoprotein biochemistry of plants differs from that of humans, requiring remodeling of biosynthetic pathways to avoid allergic or immunological response. Each plant system also has potential for further optimization of the expression system and yields, and both fundamental and applied research on understanding gene regulation, protein expression and post-translational biology in plants will drive further advances in this area. There is a growing acceptance in the PMPs community that the first wave of biopharmaceuticals to be commercially produced in plants will need to be produced in closed-loop systems ideally under biological containment (i.e., in reverse pressure controlled environment greenhouses or in photobioreactors). This will help address issues such as batch-to-batch variation and also any possible biosafety risks that could arise. As many of the biopharmaceutical products intended for production in transgenic plants are already under commercial production as recombinant (genetically engineered) products in mammalian cell culture, it is difficult to envisage logical objections to the commercial production of such recombinant proteins in transgenic plants under biological containment. Overall, it is clear that as the technology, regulatory systems and business models evolve for PMPs, a greater proportion of our therapeutics will be produced in plants in the future – hopefully, at a more competitive cost for society and public health than current therapeutic costs.

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Chapter 8

Biotech Crops for Ecology and Environment Saikat Kumar Basu, Franc¸ois Eudes, and Igor Kovalchuk

8.1

Introduction

Environmental pollution is a serious problem plaguing humanity, modern human society and the quality of human life all across the globe. Chemical pollution is a potent source of ecological and environmental degradation in recent times because of the extensive use of chemicals in our modern life (Gray 2006; Datta Banik et al. 2007). Environmentally toxic chemicals range from a wide diversity of different functional groups and species. They include: toxicants, irritants, mutagens, clastogens, carcinogens, teratogens, plastics, xenobiotics, pesticides and fertilizers, heavy metals, metalloids, pharmaceutical compounds, organic compounds, industrial effluents, untreated domestic and industrial wastes, different radioactive wastes, radionuclides, and abandoned military ammunition chemicals (Schnoor et al. 1995; Schnoor 1997; Salt et al. 1998; Thompson et al. 1998; Lucero et al. 1999; Hooker and Skeen 1999; Yoon et al. 2002). Aggressive industrialization and urbanization, rapid depletion of forest areas to extensive agriculture, mining, metal smelting, fuel production and energy generation, indiscriminate and unplanned dumping of industrial and domestic wastes (such as sludge dumping), and the enormous increase in human population in different parts of the world have all further aggravated issues and concerns about environmental pollution (Cunningham et al. 1995; Salt et al. 1998; Zayed 2004; Willey 2007). These factors have synergistically contributed towards an increase in inefficient and improper chemical waste disposal, seriously reducing the quality of land as well as human and animal life (Black 1995; Cunningham et al. 1995; Salt et al. 1998; Zayed 2004; Ghosh and Singh 2005).

I. Kovalchuk (*) Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4 e-mail: [email protected]

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Let us consider one of the most serious examples of chemical pollution in our recent history, the problem of mercury (Hg) contamination. According to Pacyna and Pacyna (2002), the global Hg emissions approximate to 1,900 tons, and ~75% of that originates from the use of fossil fuels, when coal is burnt for thermal power generation and related anthropogenic activities. The remaining 25% are supplied by waste disposal sites, cement manufacturers and waste incineration centers. The bulk of the pollution problem (about 50%) comes from countries in Asia while North American and European Union (EU) member countries share the other half of Hg emissions (Renneberg and Dudas 2001; Wagner-Dobler 2003). More than half of the total Hg emissions include elemental Hg, while the remaining half contains divalent and particulate forms (Rugh et al. 1996; Heaton et al. 1998; Pacyna and Pacyna 2002). The most serious concern regarding Hg emissions is related to the deposition of Hg in the form of snow and rainfall. In this case, it is converted into more toxic forms, such as ionic and organic Hg, thus posing a considerable health and environmental threat for arctic regions of Canada and the northeastern United States (Rugh et al. 1996; Boyajian and Carrieira 1997; Heaton et al. 1998; Renneberg and Dudas 2001; Pacyna and Pacyna 2002). It has been rightly pointed out that the majority of serious pollutants introduced into the natural environment and ecosystems are almost always anthropogenic in nature (Gratao et al. 2005; Datta Banik et al. 2007). Cleaning the polluted environment is a major task of our time. Plants having unique physiological and metabolic processes have always served as an excellent, handy, economical and eco-friendly source of natural remediation effort (Black 1995; Boyajian and Carrieira 1997; Salt et al. 1998). Nowadays, genetically engineered higher plants, called transgenic plants, popularly called Biotech Crops (BCs), play a very important role in the phytoremediation of toxic pollutants (Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Willey 2007). Also, different microbes (Liu and Suflita 1993; Pollard et al. 1994; Banat 1995; Shann 1995; Cassidy et al. 1996; White et al. 1998; Juhasz and Naidu 2000; Lovely 2003; Denton 2007; Mendez and Maier 2008), animals (Hammer 1996; Meier et al. 1997; Milanese et al. 2003; MacKenzie et al. 2004; Gifford et al. 2005, 2007; Giangrande et al. 2006; Stabili et al. 2006), algae (Olguin 2003), fungi (Singh 2006), and even lichens (McLean et al. 1998) have been reported to be involved in bioremediation of toxic pollutants. Using biotechnology innovations during the past few decades, researchers have exploited transgenic plants to reduce impacts of harmful pollutants in nature (Salt et al. 1998; Zayed 2004; Willey 2007; Aken 2008). In this review, we have provided a detailed historical overview of biotechnology progress in utilizing transgenic plants for phytoremediation of pollutants; we also discuss their applications, advantages and limitations. Possible outcomes and projected advances in genetic engineering of plants to be used for phytoremediation have also been provided. In addition, a very brief outline of other related applications of biotech crops in ecology and environment, such as biomonitoring and production of biopolymers and bioplastics, has been included.

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8.2 8.2.1

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Phytoremediation Definition

The word phytoremediation is derived from the Greek prefix phyto-, meaning “plant,” and the Latin word remedium, meaning “to cure, clean” (Gray 2006). Professor Ilya Raskin at the Rutgers University in New Jersey, US, is credited with coining this term (Black 1995). Other alternative terms for phytoremediation, as reported in primary literature, are “green remediation” and “botanical remediation” suggested by Chaney et al. (1997) to indicate environmental cleanup by green plants. Very recently, Hassinen et al. (2007) coined the term “green technology” for environmental detoxification based on phytoremediation. Phytoremediation is a natural, green-plant-mediated and solar-energy-driven process that is economically feasible and environment-friendly. It cleans up harmful and toxic pollutants from the environment by biodegrading, trapping and accumulating them in their specific organs, tissues and cells. Phytoremediation can also be defined as the use of plants to transform environmental contaminants into less toxic/non-bioavailable forms and even to stimulate soil microbial communities to either biodegrade or accumulate them, thereby restricting their movement or migration to nonpolluted sites and groundwater resources and protecting the environment (Salt et al. 1998; Reeves and Baker 2000; Suresh and Ravishankar 2004; Azevedo and Azavedo 2006; Gifford et al. 2007). As already mentioned, a large number of technical terms are associated with phytoremediation research and used by researchers. Several of them are used interchangeably or as alternative terms for almost the same applications (Black 1995; Salt et al. 1995, 1998; Datta and Sarkar 2004; Suresh and Ravishankar 2004; Zayed 2004; Azevedo and Azavedo 2006). To avoid unnecessary confusion over these widely used terms, we have provided a comprehensive table of terms and terminologies commonly used in phytoremediation research and in literature sources dealing with phytoremediation (Table 8.1). For further simplification of different types and activities related and/or associated with phytoremediation, a simple schematic representation of currently available techniques has been illustrated in Fig. 8.1.

8.2.2

Uses and Applications of Phytoremediation

Conventional procedures of eradicating contaminants and pollutants include expensive treatments such as excavation, dredging, electrolytic extraction processes, chemical and acid leaching, soil washing, solidification or stabilization, vitrification, chemical oxidation or reduction, electrokinetical treatment, use of incinerators, pumping and treating of contaminated water, vapor stripping, thermal desorption, and other expensive physical and chemical treatments (Raskin et al. 1994; Salt et al. 1995; Barcelo and Poschenrieder 2003; Gratao et al. 2005). Among

Table 8.1 Glossary of important terms and terminology associated with using plants for environment remediation Terminologies Definitions References Bioremediation Use of living organisms (predominantly microbes) for removal McGloughlin and Burke (2000), Reeves and Baker (2000), and/or detoxification of pollutants within a given Mendez and Maier (2008) environment Phytoremediation Application of green plants for degradation, removal and Raskin et al. (1994), Cunningham et al. (1995), Raskin detoxification of environmental pollutants. Also known as (1996), Chaney et al. (1997), Reeves and Baker (2000), “Green Remediation” or “Botanical Remediation” or Sursala et al. (2002), Eapen and D’Souza (2005), “Green Technology” Pilon-Smits (2005), Hassinen et al. (2007), Najmanova et al. (2007), Aken (2008) Phycoremediation Phytoremediation achieved by the use of different algal species Olguin (2003) Mycoremediation Phytoremediation achieved by the use of different mushrooms Singh (2006) and other fungal species Phytodecontamination Removal of contamination from the site using Cunningham et al. (1995), Sadowsky (1999) phytoremediation only Phytodetoxification Complete detoxification of contaminated sites by using green Cunningham et al. (1995), Salt et al. (1995), Bizily et al. plants (2000), Zayed (2004) Phytoextraction Harvest, extraction and subsequent treatment of above-ground Raskin et al. (1994), Nanda Kumar et al. (1995), Salt et al. plant biomass involved in phytoremediation, specifically by (1995), Trapp and Karlson (2001), Sursala et al. using hyperaccumulating plant species (2002), Gratao et al. (2005), Pilon-Smits (2005), Gifford et al. (2007) Phytoaccumulation Plant roots taking up metal contaminants and accumulating Raskin (1996), Blaylock et al. (1997), Salt et al. (1998), them in the stem and leaves. Also termed phytoextraction Suresh and Ravishankar (2004), Pilon-Smits (2005) and often used interchangeably Phytostabilization Application of plant roots for preventing further migration of Raskin et al. (1994), Cunningham et al. (1995), Chaney toxic pollutants in the soil, thereby regulating their et al. (1997), Salt et al. (1995), Reeves and Baker uncontrolled migration towards groundwater and converting (2000), Trapp and Karlson (2001), Zayed (2004), them into less bioavailable forms Pilon-Smits (2005), Gifford et al. (2007) Phytovolatilization Conversion of harmful and toxic pollutants to less toxic forms Cunningham et al. (1995), Salt et al. (1995), Raskin by green plants (1996), Chaney et al. (1997), Vroblesky et al. (1999), Trapp and Karlson (2001), Zayed (2004), Pilon-Smits (2005)

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Phytoindicators/ Bioindicators/Biological indicators

Phytosiderophores

Phytomining Phytotoxicity

Phytorestoration

Phytotolerance/ Phytoresistance/ Hypertolerance Phytostimulation

Plant hyperaccumulator/ Phytohyperaccumulators/ Hyperaccumulators

Phytodegradation

Phytotransformation/ Biotransformation

Green plants promoting the subsequent breakdown of environmental pollutants by microbial communities Complete remediation of a contaminated/polluted site to fully functional decontaminated soil Exploitation of sub-economic ore bodies using plants When a potentially harmful substance has accumulated in the plant tissue to a level affecting plant growth and development Siderophores (metallophores, chelating agents) that are nonsynthetic and are exclusively of a plant origin. They help in metal binding in the soil Plant assemblages associated with specific environmental conditions and/or ecosystems that are referred to as phytoindicators or plant indicators. When biological organisms (plants, animals) are meant, the term bioindicators are used. For animals. the term zooindicator is used; in case of microbes the terms microbial/bacterial indicators are used

Application of plants in biodegrading toxic organic compounds into less toxic chemical forms. The toxicity of several metals and metalloids can be reduced in plants by chemical reduction of the element because of its incorporation into available organic compounds (biotransformation) Enzymatic breakdown of organic pollutants both internally and externally by secreted enzymes. An alternate term for the above Plants species reported to accumulate toxic heavy metals in the ranges of >100 mg/kg for Cd, Cr, Co, Pb; or >1,000 mg/kg for Ni, Cu, Se, As, Al; or 10,000 mg/kg for Zn, Mn in their above-ground dry weight biomass Ability of plants to survive and thrive in heavily contaminated sites contaminated with heavy metals or metalloids

Biotech Crops for Ecology and Environment (continued )

Raskin et al. (1994), Simon et al. (1996), Mendez and Maier (2008)

Raskin et al. (1994), Chaney et al. (1997), Salt et al. (1998)

Brooks et al. (1998, 1999), Gratao et al. (2005) Beckett and Davis (1988), Naidu et al. (2003)

Cluis (2004), Suresh and Ravishankar (2004), Pilon-Smits (2005) Bradshaw (1997)

de Crombrugghe (1964), Chaney et al. (1997), Cluis (2004)

Newman et al. (1997), Salt et al. (1998), Trapp and Karlson (2001), Suresh and Ravishankar (2004), PilonSmits (2005), Gifford et al. (2007) Chaney et al. (1997), Salt et al. (1998), Reeves and Baker (2000), Sursala et al. (2002), Gifford et al. (2007)

Salt et al. (1995, 1998), Dietz and Schnoor (2001), Suresh and Ravishankar (2004), Gifford et al. (2007)

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Land farming

Organic pump/Tree pump

Mycofiltration Rhizosphere degradation/ Rhizodegradation

Rhizofiltration/Phytofiltration

Rhizosecretion

Table 8.1 (continued) Terminologies Plant biomonitor/ Phytomonitor Plant-assisted bioremediation

Definitions A plant providing complete quantitative information on environment quality Remediation of soils contaminated with organic pollutants by plant roots in association with rhizosphere-inhabiting microbial communities This is a subset of molecular farming designed to produce and secrete valuable natural products and recombinant proteins from roots Use of live plant roots for the removal of toxic heavy metals and other pollutants from water or any liquid source. Also referred to as phytofiltration Fungal mycelial mats used as biological filters Plant roots and/or root exudates provide a local environment rich in nutrients and enzymes in the rhizosphere that promotes degradation of soil contaminating pollutants by resident microbial communities. Microbial breakdown of organic pollutants in the rhizosphere Trees with dense root systems accumulate greater volume of water, thereby reducing possibilities of surface pollutants to migrate downwards towards the groundwater table and contaminate freshwater resources. Extensively used for regulating run off from agricultural fields and leaching of toxic pollutants from landfill sites Used for land affected by oil pollution. Sludge is ploughed onto topsoil, fertilizers are applied, and grasses mostly rye (Secale cereale) or alfalfa (Medicago sativa) are then sown on it Oil is degraded rapidly in the rooted, aerated and fertilized topsoil zone Trapp and Karlson (2001)

Salt et al. (1998), Trapp and Karlson (2001), Suresh and Ravishankar (2004)

Raskin et al. (1994), Dushekov et al. (1995), Raskin et al. (1997), Salt et al. (1998), Trapp and Karlson (2001), Zayed (2004), Pilon-Smits (2005) Stamets and Sumerlin (undated) Schnoor et al. (1995), Salt et al. (1998), Ramaswami et al. (2003), Suresh and Ravishankar (2004), Pilon-Smits (2005)

Gleba et al. (1999)

Salt et al. (1995)

References Kovalchuk and Kovalchuk (2001, 2003, 2008)

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CUMULATOR HYPER AC

PHYTOVOLATILIZATION PHYTOTRANSFORMATION PHYTODEGRADATION PHYTOCONVERSION

PHYTOREMEDIATION PHYTODECONTAMINATION PHYTODETOXIFICATION PHYTORESTORATION

Less toxic pollutant forms

PHYTOEXTRACTION PHYTOACCUMULATION

Highly toxic pollutant forms PHYTOSTABILIZATION RHIZOFILTRATION PHYTOMINING PHYTOSTIMULATION ORGANIC PUMP

RHIZOSPHERE DEGRADATION

GROUND WATER

Fig. 8.1 Schematic representation of different types of phytoremediation and relationships between them

biological soil treatments, the most common ones are landfarming (see Table 8.1) and ex situ techniques such as biopiles, slurry reactors and composting (Cunningham et al. 1995). Phytoremediation has been considered to be an environmentally compatible, sustainable, easily monitored, efficient and less expensive approach for the removal and detoxification of harmful environmental pollutants, compared to other chemical engineering alternatives (Baker and Brooks 1989; Baker et al. 1994; Nanda Kumar et al. 1995; Raskin et al. 1997; Datta and Sarkar 2004; Gray 2006). Reliable cost estimates associated with phytoremediation have been evaluated earlier by Cunningham et al. (1995) and recently by Pilon-Smits (2005). An important message as indicated by Gratao et al. (2005) is that phytoremediation processes are costeffective and safe alternatives to conventional physical and chemical treatments. Phytoremediation is a process with a lower impact on the surrounding environment and without any disruption of highly fragile and vulnerable ecosystems (Barcelo and Poschenrieder 2003; Zayed 2004). Although a large number of plants (known as hyperaccumulators) are capable of bioaccumulating high concentrations of toxic metals, they generally do not generate sufficient biomass and are not efficient for phytoremediation over a longer period of time. Hence, an alternative solution could be the creation of transgenic plants with greater biomass, faster growth rate, and better phytoremediation characters. Phytoremediation is an advantageous process in the sense that it helps remove toxic components in situ, and there are no direct risks of environmental contamination exposure during handling and transfer of pollutants from the contaminated

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Cost-effective, sustainable, non-intrusive, environment-friendly

Reduction of: Agricultural surface run-offs, loss of top soil, sediment run-offs, soil moisture

Soil stabilization, reclamation, amelioration & revegetation

Restoration of nutritional and biological qualities of contaminated soil

Reduction of soil erosion

Phytoremediation Benefits

Landscaping, increased value of remediated landfills

Wildlife habitat restoration

Immobilization and trapping of pollutants before reaching groundwater

Improvement of the local micro climate

Aesthetic and recreational values

Decontamination of contaminated sites

Biodegradation and detoxification of military munitions compounds Improving qualities of dump sites, landfills, agricultural lands, forests, wetlands, abandoned industrial sites, mining areas, marginal lands

Fig. 8.2 Benefits of phytoremediation

sites. Moreover, the process itself does not generate secondary waste products (Baker and Brooks 1989; Baker et al. 1994; Shann 1995; Chaney et al. 1997; Zayed 2004; Willey 2007). Benefits of phytoremediation have been compared to conventional physical and chemical remediation treatments (Fig. 8.2).

8.2.3

Historical Background

The basic and empirical studies of phytoremediation focused mainly on and around natural plant species capable of detoxifying harmful substances and on their natural properties of hyperaccumulation of toxic metals and environmentally detrimental toxic chemical compounds (Baker and Brooks 1989; Nanda Kumar et al. 1995; Salt et al. 1995; Shann 1995; Chaney et al. 1997; Raskin et al. 1997; Datta and Sarkar 2004). Over the past few decades, extensive investigations have been conducted on different phytoremediation strategies and techniques used by plants (Banuelos et al. 1993; Baker et al. 1994; Salt et al. 1998). The role of a number of phytoremediating species involved in complete and partial degradation of chemical explosives (as reviewed in Hannink et al. 2002), heavy metals and metalloids (Terry et al. 2003), and their biochemical pathways for uptakes has been extensively studied. Basic research associated with phytoremediation was primarily focused on identifying and screening plants capable of

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withstanding heavy metals and radionuclide pollution, such as natural hyperaccumulators. Plants capable of decontamination of polluted sites by rapid biosorption, bioaccumulation, biodegradation, and their conversion (biotransformation) to less toxic, non-bioavailable forms have also been extensively studied and reviewed in primary literature sources (Freeman et al. 2004; Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Pilon-Smits 2005; Denton 2007). A large number of chemicals such as heavy metals, xenobiotics, nitramines and nitraromatics, herbicides, pesticides, fertilizers, complex organic and inorganic salts and compounds, pharmaceutically important compounds, and radioactive chemicals have been reported to be successfully phytoremediated by different plant species and model plants used in the laboratories around the globe (Ellis et al. 2004; Freeman et al. 2004; Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Mezzari et al. 2005; Willey 2007). The promising potential roles played by different metallothionein (MT) and phytochelatin (PC) genes have been reviewed by Eapen and D’Souza (2005). Recently, Hassinen et al. (2007) reported that ~400 plant species have been identified as hyperaccumulators, and the majority of these plants are Brassicaceae (Arabidopsis family) members hyperaccumulating nickel (Ni). A large number of papers are also available on physiology, biochemistry, metabolism, transport mechanisms, sequestration patterns of toxic heavy metals and other pollutant compounds (Baker and Brooks 1989; Baker et al. 1994; Nanda Kumar et al. 1995; Shann 1995; Chaney et al. 1997; Pence et al. 2000; Ellis et al. 2004; Freeman et al. 2004; Denton 2007).

8.2.4

Transgenics Research on Herbs and Shrubs

Compared to natural and conventional phytoremediators, genetically engineered plants or transgenic plants (transgenics) have proved to be a rather handy tool in generating actively phytoremediating plants because of their enhanced ability to effectively reduce, degrade, transform and accumulate toxic pollutants from the environment; their growth rates are rapid, and they have better bioaccumulation characteristics (Cherian and Oliveira 2005; Willey 2007). Conventional plant breeding can only utilize available and rather limited genetic resources within species and genera of plants for phytoremediation. On the contrary, transgenic plants have specific pollutant-detoxifying or binding genes form widely divergent sources and are better equipped to address challenges associated with effective phytoremediation of contaminated sites and water bodies (Chaney et al. 1997). Another important reason for interest in producing transgenics in nondomesticated species is explained by the fact that growth rates of normal phytoremediating plants are slow and are often seasonally variable, and the amount of sequestration and degradation reported for such plants are often low (Terry et al. 2003). Hence, decontamination or detoxification does not always reach the required values accepted and set by regulatory agencies (Suresh and Ravishankar 2004).

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Even worse, at some contaminated sites, the level of pollutants could be at toxic concentration to plants or may be recalcitrant to degradation or bioaccumulation, rendering plant services completely ineffective (Terry et al. 2003). Hence, one of the most direct approaches for making phytoremediation by target plant species more successful is overexpression of genes involved in metabolism, uptake, transport, sequestration, or detoxification of harmful chemical pollutants (Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Willey 2007). Since the genetic diversity among natural phytoremediators is not very high, their ability to clean the environment is comparatively low (Fladung and Ewald 2006). Hence, the idea of transferring genes from bacterial, yeast, and animal members and even from other plants to produce target laboratory plants that are better equipped to be strong phytoremediator has recently gained enormous prominence (Oksman-Caldentey and Barz 2002; Fladung and Ewald 2006; Willey 2007). Terry et al. (2003) have discussed in details the significance of works on overexpression of enzymes catalyzing rate-limiting steps in sulfate assimilation and PC biosysnthetic pathways in transgenic plants exhibiting an increased resistance to selenium (Se) and cadmium (Cd). In addition, conventional contaminated site cleanup technologies have an extensive overhead cost that could only be addressed by promoting transgenic plants as phytoremediators, because they have been recently estimated to be more costeffective than traditional in situ or ex situ processes. They are easier to be used for cleaning up sites located in distant areas, difficult mountainous terrain, or other less inaccessible localities. Moreover, higher adaptability of transgenic phytoremediators to toxic compounds and their better survival rates make them a better choice for cleaning the environment (Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Fladung and Ewald 2006; Willey 2007). Till date, a large number of plants from 45 different plant families have been identified for phytoremediation abilities (Raskin 1996; Salt et al. 1998); the maximum number of phytoremediation species has been reported from the Brassicaceae family (Reeves and Baker 2000; Gratao et al. 2005). The first transgenic plants developed for bioremediation purposes were reported by Misra and Gedamu (1989). These plants expressed the human MT gene to develop tolerance to Cd toxicity. The next major breakthrough in phytoremediation research was reported by Rugh et al. (1996). To increase the tolerance of Arabidopsis thaliana to mercury, they generated transgenic plants overexpressing the mercuric reductase gene. The most important factors for considering plants for phytoremediation as suggested by Newman et al. (1997) and Tong et al. (2004) are: rapid plant growth, large biomass, easy multiple harvesting (3–4 times per year), better than average uptake. Although several plant breeding approaches have been targeted to develop plant varieties demonstrating better phytoremedaiation performance, the success has not been phenomenal. Hence, nowadays the emphasis is being made on genetic engineering and development of transgenic lines for producing better phytoremediating lines in target species (Salt et al. 1998; Clements et al. 2002; Willey 2007). However, it is important to note that transgenes procured from different sources need to be carefully tracked, and their expression needs to be targeted to appropriate

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cell compartments for maximizing the benefits of transgene incorporation (Salt et al. 1998; Clements et al. 2002; Bizily et al. 2003; Pilon et al. 2003). A comprehensive summary of diverse transgenes inserted into different target plant species used in phytoremediation and their corresponding responses after genetic engineering are presented in Table 8.2.

8.2.5

Transgenic Trees in Phytoremediation

Nowadays, trees are extensively used in phytoremediation (Sykes et al. 1999) because of their advantages over other plant species. These advantages are: greater biomass, larger size, strong extensive and proliferating root systems capable of spreading to a considerable depth for efficient phytoremediation, better ability to accumulate pollutants and prevent rapid soil erosion, better ability to withstand low water-stress conditions, and perennial growth habits. Other advantages include easier maintenance, no need for constant monitoring, easy harvesting, effective removal of pollutants from contaminated sites, and better ability to survive in diverse biogeographical and widely fluctuating climatic conditions. Both Clements et al. (2002) and Sykes et al. (1999) suggested introduction of “hyperaccumulation genes” into target tree species for making them more amenable to better phytoremediation in challenging sites and localities, where it was difficult to achieve success using other plant species. Recently, for the first time Doty et al. (2003) reported that the tropical leguminous tree Leucaena leucocephala is an excellent phytoremediating species that can take up and metabolize both toxic organic pollutants 2.4,6-trichloroethylene (TCE) and ethylene dibromide (EDB). The authors reported that this plant’s ability to debrominate makes it an interesting candidate to explore in terms of genetic engineering, if it is not recalcitrant to genetic modifications.

8.3

8.3.1

Phytoremediation of Inorganic Pollutants by Transgenic Plants Mercury

Hg pollution is a serious threat to a wide array of living organisms ranging from humans, animals, plants, and agriculturally important crops to beneficial microbes residing in the soil (Rugh 2001). Hg is commonly released into the environment in its ionic form (Hg+2), and then it is converted into methylmercury mostly by the process of methylation aided by methanogenic anaerobic microbes from aquatic sediment deposits (Meagher 2000). Methylmercury is approximately 200 times more toxic than the ionic form of Hg and is even considered to be more detrimental

A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana (Ac/Ds transposon tagging transformant) A. thaliana A. thaliana (cadl-3 mutant line) Atropa belladonna (hairy root culture) Brassica juncea B. juncea B. juncea B. juncea B. juncea

Increase intake of Fe and better tolerance to Cd Higher Cu accumulation Better tolerance to Cd and accumulation of Cd PCB degradation PCB degradation Detoxification of phenolic compounds like TCP Low CD accumulation; higher transport rate in leaves Rapid TCE metabolism Better Cd accumulation Increase in Zn and Cd uptake Increase in Zn and Cd uptake Increase in Cd tolerance and accumulation Increase in Se tolerance and accumulation

AtNramp3 PsMTA OASTL Lip, MnP Ds transposon + GUS LAC1 TaPCS1 P450 2E1 gshII g-GCS GC gshI SMT

Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae

Brassicaceae Brassicaceae

Solanaceae

Brassicaceae Brassicaceae Brassicaceae Brassicaceae Brassicaceae

Table 8.2 Summary of transgenes inserted into different target plant species used in phytoremediation and their corresponding engineering Target plant Family Transgene Phytoremediation response(s) reported Arabidopsis halleri Brassicaceae NAS Better Ni hyperaccumulation Arabidopsis thaliana Brassicaceae g-GCS, arsC Increased fresh weight and shoot accumulation of arsenates A. thaliana Brassicaceae merA, merB Better resistance to Hg toxicity A. thaliana Brassicaceae merB Better Hg volatilization A. thaliana Brassicaceae SAT Better tolerance to Ni A. thaliana Brassicaceae AtPCS1 Increase in PC concentration A. thaliana Brassicaceae YCF1 Bigger biomass and higher Cd uptake in leaves A. thaliana Brassicaceae merApe9 Better resistance to Hg contamination A. thaliana Brassicaceae SL Slightly increased Se accumulation and slightly lowered Se incorporation in proteins A. thaliana Brassicaceae SMT Increase in Se tolerance and accumulation A. thaliana Brassicaceae ZAT1 Higher tolerance to Zn

Liang et al. (1999) Bennett et al. (2003) Bennett et al. (2003) Zhu et al. (1999) LeDuc et al. (2004)

Banerjee et al. (2002)

Wang and Chen (2007) Gong et al. (2003)

Sonoki et al. (2007) Sonoki et al. (2007)

Ellis et al. (2004) Van der Zaal et al. (1999) Thomine et al. (2000) Evans et al. (1992)

Bizily et al. (2000) Bizily et al. (2003) Freeman et al. (2004) Lee et al. (2003) Gong et al. (2003) Rugh et al. (1996)

References Becher et al. (2004) Dhanker et al. (2002)

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312 S.K. Basu et al.

Brassicaceae Brassicaceae Brassicaceae

Magnoliaceae merApe9 Magnoliaceae gsh1 Solanaceae MT II

Brassica napa Brassica napus B. napus

Liriodendron tulipifera L. tulipifera Nicotiana benthamiana

AtMHX1 MT1 CUP1 bphC merA, merB

Solanaceae

Solanaceae

Solanaceae Solanaceae Solanaceae Solanaceae

Solanaceae

N. tabacum

N. tabacum

N. tabacum N. tabacum N. tabacum N. tabacum

NtCBP4

CUP1, GUS, HisCUP, HisGUS merA, merB

Solanaceae

N. tabacum

todC1, todC2 TaPCS1 MT merA merA nfs1 onr merA

Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae Solanaceae

N. benthamiana Nicotiana glauca Nicotiana glutinosa Nicotiana tabacum N. tabacum N. tabacum N. tabacum N. tabacum

ACC ACC MT II

GR

Brassicaceae

B. juncea

APS1

Brassicaceae

B. juncea

Better Hg phytovolatilization and phytoaccumulation Better tolerance to Ni and improved hyperaccumulation of Pb Reduce tolerance to Mg and Zn Higher tolerance to Cd Better tolerance to Cu Phytodegradation of PCBs

Increased phytoremediation of toluene Higher Pb and Cd accumulation Increased tolerance to Cd Better resistance to Hg toxicity Better resistance to Hg toxicity Biotransformation of TNTs Better detoxification of nitroglycerin compounds About a fivefold increase in Hg volatilization in roots compared to leaves and shoots Increased accumulation of Cd and Ni

Better phytovolatilization of Hg contaminants Increased accumulation of Cd, Cu, Zn Increased resistance to Cd toxicity

Increased accumulation and tolerance to arsenate Higher heavy metal tolerance (Ni, Zn, Cu, Pb) Increased resistance to Cd toxicity

Increased tolerance to Cd

High accumulation of Se in a plant body

Arazi et al. (1999), Sunkar et al. (2000) Shaul et al. (1999) Pan et al. (1994) Thomas et al. (2003) Chrastilova et al. (2007) Ruiz et al. (2003) (continued )

Bizily et al. (2003)

Pavlikova et al. (2004)

Pilon-Smits et al. (1999) Pilon-Smits et al. (2000) Nie et al. (2002) Stearns et al. (2007) Misra and Gedamu (1989) Rugh et al. (1998) Arisi et al. (2000) Misra and Gedamu (1989) Novakova et al. (2007) Gisbert et al. (2003) Liu et al. (2002) Heaton et al. (1998) Meagher et al. (2000) Hannik et al. (2001) French et al. (1999) He et al. (2001)

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CYP2E1 P450CYP1A1 CYP1A, CYP2B6, CYP2C

Poaceae

Salicaceae

Salicaceae

Solanaceae Solanaceae

Cyperaceae

Brassicaceae Brassicaceae

O. sativa

Populus deltoides

Populus tremula
Populus alba (Hybrid clone) Solanum tuberosum S. tuberosum

Spartina alteriniflora

Thlaspi caerulescens Thlaspi goesingense

ZNT1 TgMTP1

merA, merB

Enhanced ability to withstand Hg toxicity and better phytovolatilization Increase in Zn uptake and tolerance Efficient Ni hyperaccumulation

Better resistance to Hg toxicity and greater biomass generation Increased phytoremediation of different hydrocarbons Herbicide detoxification Herbicide detoxification

Higher Hg phytoremediation in wetland areas Biodegradation of chlorinated aromatic compounds Enhanced resistance to herbicides

merA cbnA

Poaceae Poaceae CYP1A1, CYP2B6, CYP2C9, CYP2C18, CYP2C19 merA9, merA18

Phytoremediation response(s) reported

Transgene

Family

Table 8.2 (continued) Target plant N. tabacum (Chloroplast genome engineering) Oryza sativa O. sativa

Pence et al. (2000) Persans et al. (2001)

Yamada et al. (2002) Ohkawa and Ohkawa (2002) Czako et al. (2006)

Doty et al. (2007)

Ohkawa and Ohkawa (2002) Che et al. (2003)

Heaton et al. (2003) Shimizu et al. (2002)

References

314 S.K. Basu et al.

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because of its enormous biomagnification property up in the ecological food chain (Ruiz et al. 2003). Hg, compared to other toxic heavy metals detected in the environment, is extremely detrimental even at considerably low concentrations (Meagher 2000). Methylmercury is exceptionally toxic because of its unique property of hydrophobicity that enables it to migrate inside the cell and then deactivate several key metabolically important enzyme systems (Castoldi et al. 2001; Rugh 2001). A number of bacterial species have been reported that can demethylate methylmercury and transform it back to its less toxic native forms (Rugh et al. 1996; Bizily et al. 2003). Elemental Hg0 is extremely volatile and is readily transformed from the liquid phase to the vapor phase. Bacterial species capable of converting methylmercury ! Hg+2 ! Hg0 are characterized by the presence of a Hg-responsive operon consisting of: (1) Hg-responsive regulatory proteins; (2) transport proteins that bind and carry Hg into the cell; (3) a specific enzyme known as an organomercuric lyase (merB gene) that catalyzes the removal of CH3 group from methylmercury and transforms it to ionic mercury (Hg+2); and (4) mercuric ion reductase (merA gene) transforming ionic mercury to elemental mercury Hg0 (Rugh et al. 1996, 1998). Gene products are expressed only when species is exposed to Hg (Rugh et al. 1996, 1998). In the past, researchers exploited these Hg-responsive operons in bacteria for effective phytoremediation of toxic Hg-contaminated sites (Meagher 2000; Rugh 2001; Suresh and Ravishankar 2004). Earlier attempts to express merA in plant systems were not successful, because the gene was reported to be G + C rich (about 67%), and hence it expressed itself only in bacterial systems (Rugh et al. 1996). In addition, it also had a higher number of CpG motifs (sites for methylation and gene silencing). Rugh et al. (1996) for the first time exploited the merA gene by replacing codons 287–336 and thereby developing a modified gene merApe9 that was efficiently expressed in the A. thailiana system. Transgenic lines with the merApe9 gene produced viable seeds, and the corresponding seedlings survived on agar plates containing 25–100 mM HgCl2 compared to their corresponding non-transformed controls. Hg vapor analysis also confirmed that transgenic lines successfully phytovolatilized ionic mercury to elemental forms with approximately 50 ng Hg0/mg fresh tissue weight. The authors detected that plants expressing merApe9 were also resistant to gold. In an attempt to further improve key technology and extend its application to other plant species in addition to model laboratory plants, Rugh et al. (1998) used bigger biomass generating plants. The researchers further modified the previously developed transgene merApe9 to include an additional 9% of the coding sequence DNA fragment for better codon optimization and effective expression in a particular species of yellow poplar (Liriodendron tulipifera). The gene was delivered into plant embryonic masses using a particle bombardment approach. The authors reported transgenic seedlings growing and surviving on agar plates incorporated with 25 and 50 mM HgCl2 compared to their non-transformed controls and phytovolatilization rates detected in the species were also appreciable. Bizily et al. (1999) reported A. thaliana lines containing the bacterial merB gene and successfully surviving on higher concentrations of HgCl2 and phenyl mercuric

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acetate compared to their controls. The original bacterial merB gene was modified using PCR techniques to contain flanking sites incorporated with consensus plant sequences and restriction sites. Using Western blot analysis, the authors provided substantial evidence that a sufficient amount of the gene product (organomercurial lyase) was synthesized by A. thaliana transgenic lines. Later, Bizily et al. (2000) reported production of A. thaliana lines with enhanced abilities of Hg phytoremediation. In this case, separate transgenic A. thaliana lines carrying merA and merB genes, respectively, were crossed and hybrid F2 plants and screened for expression of both gene products. Plants with double gene expression (merA/merB) survived better in methylmercury-incorporated agar plates containing the concentration of methylmercury higher than 10 mM; compared to plants that expressed either merA or merB separately, which could survive at concentrations up to 5 mM of methylmercury. Western blot analysis confirmed the simultaneous expression of gene products. However, Bizily et al. (2000) suggest that transgenic lines were less hardy compared to control plants in most experimental trials. The authors attributed this to the transgene capable of reducing a wide diversity of ions (particularly merA), many of which being vital for physiology and metabolism of plants. In another study, He et al. (2001) reported detection of an approximately fivefold increase in Hg volatilization in transgenic tobacco (Nicotiana tabacum) roots compared to the above-ground biomass, thus suggesting the organ-specific and species-specific plant response to Hg phytoremediation. An important limiting factor for Hg phytoremediation is the diffusion rate of methylmercury to the cytoplasmically expressed MErB proten (Bizily et al. 2000). It was addressed later by Bizily et al. (2003) in developing a specific merB cassette that targets MerB proteins on the cell wall to avoid the slower diffusion rate of methylmercury in the cytoplasm. The authors showed that transgenic lines having this specific merB construct along with the merA cassette were 10–70 times better than any other competing lines developed. Ruiz et al. (2003) reported for the first time integration of a native operon with merA and merB bacterial genes into the chloroplast genome of tobacco by a single transformation event. The authors detected high levels of tolerance to phenylmercuric acetate (100, 200 and 400 mM) in the stable transgenic lines compared to the controls. These plants were highly resistant to toxic levels of organomercurials and had higher chlorophyll per unit dry leaf weight. The major advantages of this innovative approach have been better transgene expression without the necessity of expensive and laborious codon optimization that is usually necessary for expression in higher plant systems and lower levels of unwanted gene silencing (Ruiz et al. 2003). In addition to extensive work on engineering model plants for Hg phytoremediation, in a fairly recent study, Heaton et al. (2003) reported development of transgenic rice (Oryza sativa) with merA construct delivered through the biolistic gene delivery process. This is the first report on developing a transgenic wetland phytoremediation plant capable of detoxifying toxic mercury form aquatic sediments. Che et al. (2003) reported introduction of the merA gene into another wetland species, eastern yellow poplar (Populus deltoids), and it is good news as they

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confirm that genetic engineering technology is not just restricted to laboratory model plants and actively pursue developing actively remediating plant lines or transgenic plants suitable for specific ecosystems. Future studies need to investigate divergent species representing different ecosystems and habitats, especially those with higher Hg phytoaccumulation and inefficient phytovolatilization of the accumulated Hg for a safer, cleaner and healthier environment. Recently, Czako et al. (2006) reported developing green leaf tissue-specific merA expression cassettes using the wheat rbcS promoter in mature green leaves of A. thaliana and tobacco transgenic lines for enhanced merA gene expression, increased tolerance to Hg toxicity and better phytovolatilization. The authors also reported transgenic lines of wetland species Spartina alteriniflora (salt-water cordgrass/smooth cordgrass) with merA and merB genes under the control of two constitutive promoters 35S and Ubi. These ransgenic plants had higher resistance to both ionic and organic Hg toxicity. Researchers are reported to have been working on a number of other wetland species such as Arundo spp. (giant reed), Phragmites spp. (common reed), Typha spp. (cattail) and Schoenoplectus spp. (common threesquare). If this group is successful in developing a diversity of wetland-specific transgenic plants, it would mean a big step in the decontamination of Hg toxicity in different wetland ecosystems. In a very recent work, Hussein et al. (2007) reported developing transgenic tobacco plants with merA and merB genes engineered through the chloroplast genome. Transgenic lines exhibited a steady growth in the concentration of about 200 mg/g of phenylmercuric acetate or HgCl2 compared to non-transformed lines; and they also showed the ability to phytoaccumulate both organic and inorganic forms of Hg, with better absorption rates of organic Hg over inorganic forms. The authors for the first time reported a 100-fold increase in Hg phytoaccumulation and very rapid rates of phytovolatilization, varying between 2 and 7 days depending upon the chemical form of Hg used under experimental conditions compared to control lines. This is an interesting study that shows high success rate with respect to both Hg phytoaccumulation and phytovolatilization and holds great promise for the development of future transgenics for Hg decontamination.

8.3.2

Cadmium, Lead, Nickel and Zinc

Heavy metals like Cd, Pb, Ni, Fe (iron), zinc (Zn), copper (Cu), and Mg (magnesium) are important sources of environmental pollution because of their toxic effects on human and animal health. They have been under constant investigations by different research groups for their effective phytoremediation (PilonSmits 2005). In the late 1990s, Misra and Gedamu (1989) transferred human metallothionein-II (MT II) into tobacco (N. tabacum) and Brassica napus. Metallothioneins (MTs) represent a broad family of low molecular weight cysteine-rich chelator proteins that bind a number of heavy metals via the thiol group of its cysteine residues and transport them for sequestration into the plant vacuole

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(Pilon-Smits 2005). The transgene expressed the MT protein constitutively, and seeds form self-fertilized transgenic plants were able to survive in agar plates containing up to 100 mM CdCl2. Later overexpressed the metallothionein (MT) gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter in tobacco (N. glutinosa) delivered via AMGT. The authors reported that transgenic lines expressing MT grew successfully in the presence of 200 mM CdSO4 compared to non-transgenic lines that failed to withstand concentrations higher than 50 mM CdSO4. PCR analysis of T2 transgenic and non-transgenic seedlings demonstrated a strong correlation between phytotolerance to Cd and the presence of the transgene. This is an interesting strategy of increasing phytoremediation abilities in plants by overexpressing MT genes in the genome of higher plants. Phytochelatins (PCs) represent a big family of heavy metal-inducible peptides that play an important role in intracellular heavy metal detoxification by chelation in different organisms such as microbes, yeasts (Schizosaccharomyces pombe) and higher plants (Arabidopsis); but it has not been reported in animals yet (Ha et al. 1999; Gong et al. 2003). In 2003, Lee et al. reported overexpression of A. thaliana phytochelatin synthase (AtPCS1) back into A. thaliana with an increase (1.3- and 2.1-fold) in PC concentration. However, the authors reported hypersensitivity of transgenic lines to Cd stress measured as related to the proportion of root growth of transgenic plants compared to their wild types. The authors postulated that this might be due to higher concentration of glutathione in transformed plants. On the contrary, Gong et al. (2003) targeted wheat TaPCS1 (involved in PC synthesis) cDNA expression in Arabidopsis under the Arabidopsis alcohol dehydrogenase promoter and also developed special PC-deficient Arabidopsis lines (cadl-3) under the influence of the CaMV 35S promoter. The researchers detected lower Cd accumulation in transgenic plants compared to the original cadl-3 mutant lines and observed a higher rate of Cd transport into leaves. In the same year, Song et al. (2003) reported using a yeast vacuolar glutathione Cd transporter (YCF1) in A. thaliana that resulted in greater biomass and an approximately twofold increase in Pb and Cd tolerance and uptake compared to non-transformed controls. This technology has the potential to be used in developing advanced phytoremediators that can pump heavy metals into safe compartments, requiring only a very low expression of transporters compared to greater quantity of chelating peptides (Tong et al. 2004). Similarly, Bennett et al. (2003) generated transgenic B. juncea lines using microbial g-GCS and glutathione synthetase (GC) (both associated with PC synthesis) separately and reported approximately a 1.5-fold increase in Zn and Cd uptake compared to their corresponding controls. Researchers detected that overexpression of both genes enhanced soil-borne Cd by 25%, and they expected to increase the phytoremediation potential of transgenic lines from 1.5- to 3-folds, compared to their non-transformed control plants. Working on shrub tobacco (N. glauca) in Eastern Spain, Gisbert et al. (2003) genetically modified the species through AMGT using a wheat gene encoding phytochelatin synthase (TaPCS1). The authors reported a significant phytotolerance to Pb and Cd and an increase in root length by approximately 160%. Another significant achievement was reported by Zhu et al. (1999) when they transferred the Escherichia coli gene gshI (involved in PC synthesis) and

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overexpressed it in B. juncea with a fivefold increase in ECS and GC activity and better Cd accumulation (approximately 40–90%). Liang et al. (1999) in their study on Cd phytoremediation overexpressed the gshII gene encoding GC into the cytosol of B. juncea and exhibited higher Cd accumulation compared to wild-type plants. Vacchina et al. (2003) first reported a possible role of nicotinamine (NA) in the process of detoxification of a microelement Ni by Thlaspi caerulescens as a phytoremediator. Another group of researchers (Becher et al. 2004) besides Vacchina et al. (2003) found consistent results by overexpressing the nicotinamine synthase (NAS) gene in Brassicaceae family members such as A. halleri and T. caerulescens. The Cation Diffusion Facilitator (CDF) family homologs such as ZTP1 in T. caerulescens and AhMTP1 in A. halleri have been reported to be constitutively expressed at higher concentrations (Assuncao et al. 2001; Becher et al. 2004). Pence et al. (2000) cloned ZNT1 cDNA (a heavy metal transporter) from T. caerulescens via functional complementation in the yeast strain ZHY3 and found higher and lower affinities for Zn+2 and Cd+2, respectively. The authors also reported that ZNT1 (a heavy metal transporter that forms a complex with heavy metals and carries them inside plant cells for sequestration into the plant vacuole) is heavily expressed in roots and shoots of target plants. A comparative analysis was made between ZNT1 and high-affinity Zn+2 accumulation in roots of T. caerulescens (a hyperaccumulator) and T. arvense (a non-accumulator), and it established an excellent correlation between alterations in ZNT1 expression and speciesspecific Zn accumulation status resulting in overexpression of the target gene in T. caerulescens. This study highlights the pattern of regulation and molecular control mechanisms of heavy metal uptake systems in plants. In another interesting study, Pavlikova et al. (2004) tested four transgenic tobacco lines carrying transgenes: CUP1 (encoding MTs), the GUS reporter gene, HisCUP (CUP combined with a polyhistidine tail) and HisGUS (GUS with a polyhistidine tail) under the constitutive CaMV 35S promoter for Cd, Zn and Ni phytoaccumulation. The GUS line accumulated all the three metals. The HisCUP line showed the best Cd accumulation with the shoot content of Cd enhanced by 90% and the subsequent reduction in root accumulation by 40%. The HisGUS line confirmed the best performance in Ni accumulation, while no significant accumulation was detected for Zn in any of the lines tested. Other lines exhibited higher phytoaccumulation of only one specific metal, as discussed above. Recently, Stearns et al. (2007) reported efficient tolerance to Ni, Zn, Cu, and Pb in canola (B. napus) by transferring to the genome the bacterial 1-aminocyclopropane1-caboxylate (ACC) gene associated with ethylene biosynthesis for inducing better environmental stress response to metal toxicity indirectly.

8.3.3

Arsenic

The first clear evidence of arsenic phytoremediation by transgenic canola (B. napus) was reported by Nie et al. (2002). The researchers expressed the

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Enterobacter cloacae UW4 1-aminocyclopropane-1-caboxylate (ACC) deaminase in canola. The reason for expressing this particular enzyme is that it is associated with lower levels of stress-induced ethylene. Hence, these transgenic lines are expected to tolerate a wide variety of environmental stresses including heavy metal stress and toxicity. Transgenic plants survived quite well while grown in the presence of 2 mM arsenate; they exhibited higher seed germination percentage, enhanced fresh and dry weight of both roots and shoots, and higher concentrations of protein and chlorophyll in plant cells and tissues. The most important improvement reported in transgenic plants compared to their corresponding controls was a fourfold increase in their ability to accumulate arsenate. The authors also explored the role of the plant growth-promoting bacterium E. cloacae CAL2 in both transgenic and control lines with no significant observation with respect to arsenate uptake. They proposed the combined use of transgenic lines along with the growthpromoting bacteria as a better strategy to deal with arsenic contaminated sites. In the same year, Dhanker et al. (2002) were successful in increasing As tolerance in the model plant A. thaliana using two different genes, a bacterial g-glutamyl cysteine synthetase (g-GCS) of the glutathione biosynthetic pathway (involved in PC synthesis) and the arsenate reductase (arsC) from E. coli (for transforming highly toxic arsenates to less toxic arsenites). Constitutive overexpression of the g-GCS gene along with leaf-specific expression of arsC allowed to achieve increased phytoremediation of arsenate by transgenic plants with respect to fresh weight (~fivefold) and shoot accumulation (~threefold), as compared to nontransformed controls. Such advances may play a significant role especially in the parts of world that are adversely affected by severe arsenic pollution like the IndoGangetic plain and the Ganges delta (Ruiz and Romero 2002).

8.3.4

Selenium

One of the most comprehensive and detailed study on Se toxicity in plants was conducted by Banuelos et al. (1997). The authors investigated selenium-induced growth reduction in two different Brassica species (B. juncea and B. carinata). Several other studies have been conducted on Se phytoremediation with particular emphasis on plant physiology and biochemistry, Se toxicity and Se hyperaccumulation (see Suresh and Ravishankar 2004; Cherian and Oliveira 2005). Pilon et al. (2003) studied overexpression of a mouse selenocysteine lyase (SL) that breaks down selenocysteine into elemental selenium and alanine in A. thaliana. The overexpression resulted in a minor increase in the rate of Se accumulation, slightly lowering the amount of Se incorporation in plant proteins. It is very important to note that the researchers reported that chloroplastic SL reduced tolerance to Se, while vacuolar SL enhanced tolerance to Se, indicating the importance of precise localizations of transgenic proteins at the subcellular level (Pilon et al. 2003). Overexpression of metallothionein expressing selenocysteine methyltransferase

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(SMT) from A. bisulcatus to A. thaliana (Ellis et al. 2004) and B. juncea (LeDuc et al. 2004) resulted in an approximately two- to threefold increase in Se tolerance and accumulation in transgenic plants compared to their untransformed controls. This specific enzyme (SMT) is capable of detoxification of selenocysteine to a nonprotein amino acid methylselenocysteine via methylation, thereby reducing chances of toxic incorporation of Se in plant proteins (LeDuc et al. 2004). Recently, Banuelos et al. (2007) reported a twofold increase in Se accumulation and 1.8-fold increase in Se leaf accumulation by transgenic Indian mustard (Brassica juncea (L.) Czern.) overexpressing SL.

8.4

8.4.1

Phytoremediation of Organic Pollutants by Transgenic Plants Organic Hydrocarbons

Organic pollutants are another significant group of environmental toxicants used for rapid phytoremediation (Gratao et al. 2005; Pilon-Smits 2005). In an interesting recent study, Doty et al. (2007) reported the development of a transgenic poplar line from an original base population of the hybrid poplar clone INRA 717-1B4 (P. tremula
P. alba) via overexpression of rabbit CYP2E1 under the control of CaMV 35S. Stable transgenic lines showed excellent phytoremediation of different hydrocarbons (trichloroethylene, vinyl chloride, carbon tetrachloride, benzene and chloroform) from hydroponic solutions, compared to their controls. The plants also exhibited better phytovolatilization for trichloroethylene, chloroform, and benzene. Recently, Novakova et al. (2007) successfully transferred the bacterial todC1 and todC2 genes into N. benthamiana. The todC1 C2 genes were cloned into the plant genome to synthesize ISPTOL (a bacterial component of toluene dioxygenese), causing rapid oxidation of toluene and other organic pollutants. The overall performances of transgenic lines are still in progress to record their phytoremediation ability to degrade toluene and other organic compounds.

8.4.2

Trichloroethylene (TCE)

Doty et al. (2000) for the first time developed a transgenic tobacco line capable of phytoremediating TCE. The authors introduced the mammalian cytochrome P450 E1 gene (CYP2E1) into tobacco leaf disks, and transgenic plants were regenerated. Oxidoreductases of plant and mammalian origins being substantially similar, the mammalian P450 could successfully interact with its tobacco counterpart. Introduction of this specific gene resulted in a significant increase in both TCE and

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ethylene dibromide metabolism. The authors reported a 600-fold increase in TCE metabolism in transgenic lines, compared to their non-transformed controls. This specific gene encodes enzymes that can successfully oxidize a wide diversity of organic pollutants such as TCE, ethylene dibromide (EDB), carbon tetrachloride, chloroform and vinyl chloride, and many others (Aken 2008). Later on, the same group of researchers reported another intriguing finding: they successfully expressed the P450 E1 gene (CYP2E1) in hairy root cultures of the medicinal plant Atropa belladonna (Banerjee et al. 2002).

8.4.3

Polychlorinated Biphenyls (PCB)

PCBs are considered to be a formidable source of environmental pollution, and they are extremely toxic in nature (Sonoki et al. 2007). Chrastilova et al. (2007) transferred the 35S-driven bphC gene derived from the bacterial PCB degradation pathway into tobacco. Significant PCBs degradation was observed in the transgenic lines. Sonoki et al. (2007) also reported PCB degrading transgenic lines of A. thaliana. One of the lines developed contains fungal lignin-degrading enzymes (Lip, MnP), while the other line is an enhancer-trap Ac/Ds transposon tagging transformant of A. thalina containing a nonautonomous mobile Ds transposon linked to the reporter gene GUS. According to the researchers, the minute promoter-driven GUS can only drive gene expression when the Ds transposon GUS is shifted towards the enhancer region of the gene in the Arabidopsis genome. Hence, efficient monitoring of the genes(s) involved in Polyhydroxy butyrate (PHB) degradation is indirectly achieved by monitoring the expression of the reporter gene. Both lines have been found to efficiently degrade PCBs under laboratory conditions.

8.4.4

Phenolic Compounds

Recently, Wang and Chen (2007) reported transferring the laccase enzyme (LAC1) from cotton plants (Gossypium arboreum) into A. thaliana. Transgenic plants were efficient in degrading 2,4,6-tricholorophenol (TCP) by simple oxidation. In another study, Floco and Giulietti (2007) reported developing hairy root cultures of Armoracia lapathifolia using A. rhizogenes for phytoremediation of aromatic compounds like phenol from Argentina. This particular plant species have high concentrations of the enzyme peroxidase (E.C. 1.11.1.7) that is capable of detoxifying phenolic compounds. The authors exposed 30-day-old hairy root cultures to aqueous solutions of phenols of different concentrations (25, 50 and 100 mg/mL). They reported 70% phenol removal in the cultures after 3 h of incubation at all concentrations in the presence of hydrogen peroxide, and approximately 30–55% in the absence of an

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oxidant and co-substrate, suggesting a significant role of a plant peroxidase in phytodetoxification of phenols.

8.4.5

Herbicides, Pesticides and Organic Solvents

Herbicides, pesticides and organic solvents are other significant sources of environmental toxicants that have a detrimental impact on our ecosystems (Gratao et al. 2005). Shimizu et al. (2002) reported transferring the bacterial cbn4 gene from Ralstonia eutropha NH9 into rice (O. sativa) under the constitutive CaMV 35S promoter. Transgenic rice calli were successful in converting 3-chlorocatechol to 2-chloromucote. Such techniques may be suitable for phytoremediation of chlorinated aromatic compounds represented by herbicides, pesticides and several organic solvents. In another related study, Ohkawa and Ohkawa (2002) reported producing transgenic rice and potato (Solanum tuberosum) lines with mammalian cytochrome p450 monooxigenase genes. The researchers introduced five P450 genes in rice, namely, CYP1A1, CYP2B6, CYP2C9, CYP2C18 and CYP2C19. All stable transgenic lines (with the exception of one line) exhibited enhanced tolerance to herbicides metachlor, alochlor and acetochlor (inhibiting protein biosynthesis) and trifluralin (inhibiting cell division). T1 seeds of CYP2C9 showed resistance to such herbicides like chlortoluron, mefenacet, phenylurea herbicide, pyridazinone herbicide etc; while CYP1A1 exhibited resistance to phenylurea herbicide, mefenacet and quizalofopethyl. Rice lines carrying the CYP2C9 gene were resistant to chlorosulfuron and imazosulfuron; however, the line with the CYP2C18 gene did not show any specific resistance to any herbicide. A similar approach was used for generating transgenic lines of potato. Four lines (S1965, S1972, S1974 and T1977), each with three transgenes (CYP1A, CYP2B6 and CYP2C), were selected for testing. The T1977 line showed tolerance to atrazine, chlortoluron, methabenzthiazuron, acetochlor and metolachlor; while the S1972 line had tolerance to chlortoluron and methabenzthiazuron and was susceptible to all these herbicides except acetochlor and metolachlor; the S1974 line was partially tolerant to atrazine and tolerant to acetochlor and metolachlor.

8.4.6

Explosive Chemicals

Bioremediation of explosives is not an innovation; several researchers successfully used different fungal and bacterial members to degrade explosive and ammunition chemicals like TNT (trinitrotoluene), RDX (hexahydro-1,3,5-trinitro-1,3,5 triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and TETRYL (N-methyl-N, 2, 4, 6-tetranitroaniline) (Hooker and Skeen 1999; Jhonston 2002).

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In the last few decades, phytoremediation of explosive chemicals has received great attention in a large number of laboratory studies by different research groups (French et al. 1998, 1999). An excellent review by Hannink et al. (2002) covered in details energetic, metabolic, biochemical and transformation mechanisms associated with phytoremediation of explosive chemicals. TNT is one of the most dangerous explosive chemicals that require considerable time for complete biodegradation (Jhonston 2002; Cluis 2004). However, a soil bacteria E. cloacae PB2 has been reported to be able to use TNTs as its primary nitrogen source for growth and metabolism because of the presence of two unique enzymes, pentaerythritol tetranitrate (PETN) reductase and nitroreductase (French et al. 1998). Both these enzymes utilize NADPH as an electron donor source, and thereby can easily reduce TNTs into less toxic compounds. French et al. (1999) introduced the PCR-modified gene encoding PETN reductase (onr) into the tobacco genome with a plant consensus start sequence for better expression in the plant system. The authors reported that seeds from transgenic lines germinated and grew successfully in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM TNT, compared to their non-transformed wild types. The resultant transgenic seedlings grown in liquid medium with 1 mM GTN exhibited faster and complete degradation (denitration) of GTN than non-transformed lines. In another related study, expressed the PCR-modified bacterial (E. cloacae NCIMB101011) gene encoding for nitroreductase (nfs1) with a consensus start sequence to facilitate translation in tobacco plants. The nitroreductase enzyme catalyzed the reduction of TNT to hydroxyaminodinitrotoluene, following the subsequent reduction to aminodinitrotoluene derivatives. Transgenic plants expressing nitroreductase exhibited a significant increase in TNT uptake, tolerance, and subsequent detoxification compared to wild-type plants. The ability of plants to metabolize xenobiotic nitrate ester and glycerol trinitrate (nitroglycerin) in sugar beet (Beta vulgaris) cells and cell extracts has been convincingly demonstrated by Goel et al. (1997). Here, it is important to note that the authors suggested that GTNs could not be completely denitrated, they could only be transformed to mono- or dinitrated glycerols. Hence, there are opportunities for future researchers to explore these data and develop new transgenic lines for complete denitration of nitroglycerin compounds. In Fig. 8.3, we have illustrated some of the common pathways of phytoremediation within a plant cell.

8.5

Immunological Approach for Phytoremediation

Immunological approaches for phytoremediation represent entirely new concepts in the field of phytoremediation research. This technology can be called plant immuno-remediation or phytoimmuno-remediation. Drake et al. (2002) for the first time demonstrated the importance of hydroponicaly grown transgenic tobacco plants used for phytoimmuno-remediation. These plants express a murine IgG1

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HMT

PC/MT HM 1

HM 2

C

L

M Tonoplast

C

Cell Wall

M

GC

Cell Membrane

PC/MT HM

ER

HM N Vacuole L

I GSH O GSH

O

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3 3 3 O

Cytoplasm C

C 4

O GSH M

L

M

I GSH

C 4

C

4

I I

Fig. 8.3 Schematic representation of a plant cell and several phytoremediation pathways of target toxic pollutants. C Chloroplast; ER Endoplasmic reticulum; GC Golgi complex; HM Heavy metal; HMT Heavy metal transporters; I Inorganic pollutant; L Lysosome; M Mitochondrion; MT Metallothioneins; N Nucleus and nucleolus; O Organic pollutant; PC Phytochelatins. Pathway 1: PCs and heavy metals form complexes are translocated across the tonoplast and finally sequestered in the vacuole (Gong et al. 2003). Pathway 2: HMTs detoxifies toxic heavy metals by transporting across the vacuole to less toxic forms (Song et al. 2003; Pilon-Smits 2005). Pathway 3: Organic contaminants are phytoremediated by either getting adsorbed on the cell wall during entry or moving into the cytoplasm depending on the nature of pollutants. Within the cell cytoplasm, they are either attacked by series of enzymes and get transformed and degraded or form conjugated complexes with glucose and GSH and get sequestered in the plant cell wall or the vacuole (Pilon-Smits 2005). Pathway 4: Inorganic pollutants may form complexes with nicotinamine and organic acids and get adsorbed on the cell wall; if they can enter the cell cytoplasm, they often form conjugates with PCs and GSH and are finally sequestered in the plant vacuole (PilonSmits 2005)

monoclonal antibody either to neutralize toxic bioactive molecules in the rhizosphere, or to accumulate and concentrate molecules in the above-ground biomass. In their experiment, two different types of transgenic tobacco plants were used. First, a functional antibody was subjected to rhizosecretion and directed to bind with antigen in the surrounding media to generate an immune complex. In the second case, a monoclonal antibody was retained in plant leaves via a

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transmembrane sequence. It is interesting to report that the antigen incorporated in the medium was actively transported to axial plant leaves within a day; there, it underwent sequestration from binding to the antibody on the cellular membrane. The immune complex density remained the same even 3 days following antigen removal form the media. Such innovative technology could empower researchers to develop transgenic species that could phytoremediate any pollutant for which it is possible to develop a monoclonal antibody. It could also contribute to phytodecontamination and phytorestoration of contaminated sites using transgenic plants.

8.6

Limitations of Phytoremediation Research

In spite of its relevance and importance, phytoremediation still has several problems such as reduced growth rate and poor biomass of phytoremediating plants, limited remediation, and high plant mortality rates (Barcelo and Poschenrieder 2003; Cluis 2004; Gray 2006). In addition, there is always a permanent risk of bioaccumulation of toxic elements and compounds by plants and their transmission initially to immediate secondary consumers and subsequently into higher orders of food chains and food webs (Raskin et al. 1994; Barcelo and Poschenrieder 2003; Cluis 2004; Gratao et al. 2005). There are a number of concerns and issues associated with future effectiveness and potential of transgenic phytoremediators from food and feed crops (Dietz and Schnoor 2001; Cluis 2004; Ghosh and Singh 2005; Gratao et al. 2005). Although many plant species have been reported to show uptake, biodegradation and sequestration of several explosive chemicals, such as TNT and RDX residuals, these activities were low. Some chemicals, such as RDX, were only partially degraded or transformed, leaving space for engineered plants to take over in this area in the not-so-distant future (Goel et al. 1997; Dietz and Schnoor 2001; Ghosh and Singh 2005). Among other technical factors associated with phytoremediation, a subtle one is that of a gap between the scientist and the lay person, and misconceptions about benefits of the process and its scientific management (Trapp and Karlson 2001). A list of concerns and challenges haunting the successful development of transgenic lines has been presented in Fig. 8.4. Developing efficient phytoremediators for contaminated sites lab has always been extremely challenging for researchers (Black 1995; Cunningham et al. 1995; Jhonston 2002; McIntyre 2003). Among these challenges are genotype
environment interactions of responding plant species and variability of performance across the years (Cunningham et al. 1995; McIntyre 2003; Zayed 2004; Ghosh and Singh 2005; Willey 2007). Moreover, phytoaccumulation of toxic pollutants is also dependent on root growth of plant species involved. Restricted root growth in natural contaminated sites may or may not allow plants to effectively accumulate toxicants in the above-ground biomass or to immobilize pollutants preventing them from leaching into the groundwater table (Black 1995; Pulford and Watson 2003; Gratao et al. 2005).

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1. Phytotoxic concentrationof pollutants often detrimental to plants 2. Phytoremediation affected by locality, seasonal and climatic variations 3. Phytoremediation success depends on plant species and varieties and specific plant communities 4. Often difficult to establish a successful plant colony for effective and economical remediation 5. Ecological risk associated with introduction of new species 6. Phytoremediation limited by root length and root growth rate in plants

CHALLENGING QUESTIONS CONFRONTING FUTURE AND PROGRESS OF TRANSGENIC LINE DEVELOPMENT FOR PHYTOREMEDIATION

7. Uncertainty regarding production of secondary pollutants and effective disposal of phytoremediating plants 8. Economic and ecological cost of generating transgenic plants

Fig. 8.4 Future of transgenic phytoremediating plants

According to Salt et al. (1998) metal-accumulating plants could be either disposed off or used for metal recovery depending on economics of the process. However, it is important to note that reliable data and information regarding such disposal and success are not easily available (Jhoanston 2002). Although composting and compaction have been suggested by Cunningham et al. (1995) as pretreatment for volume reduction before actual disposal, it is important to make sure that such practices do not promote accidental leaching from compaction (Ghosh and Singh 2005). Incineration of harvested plants from contaminated sites after remediation has been strongly advocated by Ghosh and Singh (2005) to avoid the possibility of secondary waste generation through the application of phytoremediation. However, the impact of such technologies on the environment and ecosystems has not been well documented yet. Lastly, another important question to answer is whether transgenic plants can eventually become a source of secondary pollutants in the process of phytoremediation, and whether their effective disposal becomes another challenge and threat to ecology and environment (Black 1995; McIntyre 2003; Gratao et al. 2005; Willey 2007). It is very important to look for easy recycling of plant biomass to retrieve toxic pollutants and for the opportunity to reuse them after recycling to reduce our ecological footprints on the nature. In addition, it is also very important to look for enhancing the quality of phytovolatilization or phytotransformation

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of toxic pollutants within phytoremediating plants to reduce the possibility of generating secondary pollutants in the long run. The interaction between the plant genome and hyperaccumulation of toxic heavy metals needs to be investigated in further details to understand molecular genetics and physiological aspects of heavy metal uptake in phytoremediating plants (Mejare and Bulow 2001).

8.7

Transgenics in Phytomonitoring and in Biopolymer and Bioplastic Productions

In addition to phytoremediation, transgenics have made a significant impact on some other areas too. Although a detailed discussion would be too voluminous and is beyond the scope of this review, however, we would like to point out some features very briefly in the following paragraphs. One of them deals with the development of transgenic plants for rapid detection of environmental pollution and contamination using efficient molecular screening techniques (plant biomonitoring or phytomonitoring), and the second thriving field deals with the synthesis of bioplastics and biopolymers in transgenic plants.

8.7.1

Transgenic Plants in Biomonitoring

In recent years, substantial progress has been achieved in the realm of transgenic biomonitoring plants (phytomonitors) (Lebel et al. 1993; Kovalchuk et al. 1998, 1999a,b, 2000a,b,c, 2001a,b; Ries et al. 2000; Besplug et al. 2004; Boyko et al. 2006; Li et al. 2006; van der Auwera et al. 2008). One of the most important aspects in the development of transgenic biosensors is the option to customize the assay according to specific biomonitoring requirements (Kovalchuk and Kovalchuk 2008). Two most important assays that are reportedly used in biomonitoring in recent times are the Recombination Reporter Assay and the Point Mutation Reporter Assay (Kovalchuk et al. 2000b, c). The biggest success attributed to the transgenic recombination assay has been its application in detecting radioactive pollution in soil and water (Kovalchuk et al. 2001a, b). Transgenic Arabidopsis and tobacco biomonitoring lines have been reported to be excellent tools for detecting genotoxicity of radioactively contaminated sites (Kovalchuk et al. 1998, 1999a, b). In case of the Point Mutation Reporter Assay, Kovalchuk et al. (2000b, c) has developed a system in which they introduced a stop codon at the 50 -end of the GUS (uidA) gene by means of a single nucleotide substitution that completely inactivated the transgene. Transgenic plants responded to mutagens (either physical or chemical agents) by increasing levels of point mutations. They led to the restoration of the uidA gene activity. Cells where such restorations occurred were visualized as blue sectors on white plants after histochemical staining. Further details of these works are beyond the scope of the current article and are available

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in a series of papers (Hohn et al. 1999; Kovalchuk et al. 1999a,b, 2000a,b,c; Kovalchuk and Kovalchuk 2001, 2003, 2008; Filkowski et al. 2003).

8.7.2

Biopolymer Production in Transgenic Plants

Transgenic plants are catching up rapidly in the areas of biopolymer production. Industrially important biopolymers include proteins, enzymes, recombinant antibodies, vaccines and other biopharmaceutical products (Torres et al. 1999; Doran 2000; Fischer and Emans 2000; Fischer et al. 2000; Giddings et al. 2000; Langridge 2000; Walmsley and Arntzen 2000). Several industrially important proteins have been produced in transgenic plants: human milk proteins in potato (Chong and Langridge 2000; Chong et al. 1997); the oleosin–hirudin fusion protein in canola (Parmenter et al. 1999); phytase in canola (Ponstein et al. 2002); collagens in tobacco (Ruggiero et al. 2000); spider silk protein (spidroins) in tobacco and potato (Gosline et al. 1999); bio-elastic proteins in tobacco and avidin in maize (Giddings et al. 2000); biopolymers in peas and rice and wheat (Frigerio et al. 2000; Perrin et al. 2000; Stoger et al. 2000). Transgenic plants offer the following advantages over microbial and animal expression systems: lower rates of contamination, low production cost, easier and economic protein extraction and purification steps. Lastly, rapid advances in the areas of plant proteomics and protein targeting are also promoting the development of transgenic lines producing different biopolymers (Doran 2000; Giddings et al. 2000; Fischer et al. 2000; Nawrath and Bonetta 2001).

8.7.3

Bioplastics from Transgenic Plants

Transgenic plants producing bioplastics (polyhydroxy butyrate/PHB) are still not a cheaper alternative compared to the microbial PHB production (Scheller and Conrad 2005). According to Scheller and Conrad (2005), PHB production in transgenic plants still needs to reach a comfortable yield target to be economically feasible. Nawrath et al. (1994) first reported PHB production in the model plant A. thaliana. The majority of works on PHB transgenics are restricted to Arabidopsis. However, there are reports of the PHB accumulation in other plants: 5.7% of dry weight has been reported in maize (Zea mays) by Moire et al. (2003); 7.7% in oilseed rape (B. napus) by Houmiel et al. (1999), and 5% in sugar beet (B. vulgaris) by Menzel et al. (2003). Bohmert et al. (2000) reported 4% PHB production in Arabidopsis; however, researchers detected a negative correlation between the PHB accumulation and plant growth. Analysis of T2 plants indicated the loss of PHB synthesis to a biologically significant amount over generations. Among other plants, tobacco plants producing PHB are reported to have stunted growth (Nakashita et al. 2001;

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Lossl et al. 2003). As to fiber-yielding crops, in cotton the amounts of PHB are small – 0.34% fiber weight (John and Keller 1996), in flax – 0.5% fiber weight (Wrobel et al. 2004). Most studies highlighted rapid depletion of other essential plant metabolites because of the increase in PHB production. Recent progress in bioplastic production in transgenic plants is aimed at developing lines with higher PHB production without impacting on growth qualities of targeted plant species (Scheller and Conrad 2005).

8.8

Conclusions and Future Directions of Phytoremediation Research

Overall, it is important to note that in the past few decades, phytoremediation research has moved from its initial emphasis on physiological, biochemical and genetic investigations and screening of phytoremediating species towards advances and applications in plant biotechnology and genetic engineering. The future success of phytoremediation as an emerging agro-industry will depend on how successful we are in acquiring knowledge regarding the plant genome of model plants and applying this knowledge to “real-life situations” and “real-life nondomesticated plant species” to tackle the challenges of complex environmental pollution induced by chemicals. Tree species will certainly have priority over herbs and shrubs because of their better phytoremediation abilities. It has been reported that 64% of contaminated sites contain both organic and inorganic pollutants; hence, it is important to attract our attention towards generating transgenics with multitasking abilities (MTTs). Some authors have been successful in introducing 13 different transgenes in rice using biolistics. Although these transgenes functioned separately, multiple gene insertion with synergistic functions was successful in other plants (Hooker and Skeen 1999). Such approach would be necessary to introduce multiple genes for phytoremediation into a target plant to make it suitable for phytoremediation under diverse conditions and at sites contaminated with mixed toxicants. This approach named as a “molecular tool box” approach by Raskin (1996) could be efficient in dealing with future phytoremediation. Plant omics will help regulate future phytoremediation technologies and strategies to guide us towards a platform where it could be established as a formidable future industry (Fig. 8.5). Phytoremediation research and applications have been previously restricted to North American and European continents only (Raskin 1996; Salt et al. 1998; Pilon-Smits 2005). Now they have been adopted and extensively pursued in emerging giant economies such as India, China, Brazil, Russia, and several smaller EU countries, as well as in Australia and New Zealand (Gratao et al. 2005; Willey 2007). Since the focus on agriculture has shifted back (Raskin 1996), newer initiatives like phytoremediation can open up opportunities as full-scale agroindustries in the long run. The future directions guiding phytoremediation research has been envisioned and highlighted into three separate sections below.

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Fig. 8.5 Phytoremediation and its relationships of with different disciplines

8.8.1

Transgenic Trees: A Better Solution for Phytoremediation

Trees with promising phytoremediation genes from other bacterial, yeast, human and animal sources, and even from other plants could possibly be an essential tool for future phytoremediation of contaminated sites and soils (Barcelo and Poschenrieder 2003; Zayed 2004). It may be a challenging but provocative idea to transfer multiple phytoremediation genes into candidate tree species for an efficient multiphytoremediation approach. This plant would be more desirable than phytoremediating species carrying a single transgene. Such transgenic species can work at two or more different polluted sites contaminated with totally different chemical pollutants, making the process more efficient and cost-effective.

8.8.2

Multidisciplinary Research Approach

Huge progress has been made in the characterization and modification of the chemical nature of soil to facilitate phytoremediation of contaminated sites (Datta and Sarkar 2004) and also in understanding the basic mechanics of pollutant uptake, translocation, detoxification, and storage mechanisms in plants (as reviewed in

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Suresh and Ravishankar 2004; Pilon-Smits 2005). However, there is much still to be investigated to completely identify all the factors that interact in the process of phytoremediation, including soil and site, nature and types of a pollutant affecting contamination, and plants involved in phytoremediation. This realm of research involves a serious multidisciplinary approach, and it needs collaboration among scientists working in different fields (Suresh and Ravishankar 2004; Willey 2007). Although phytoremediating species are slowly turning into crop plants in real life situations, a lab-to-land transition will involve a lot of support research in related disciplines to facilitate and speed up the process. For example, in addition to developing transgenic plant lines, it will also be necessary to engineer plant growthpromoting rhizobacteria and arbuscular mycorrhizal fungi residing in the same contaminated soil to further facilitate the efficiency in the natural cleanup of contaminated sites (Salt et al. 1998). In future, more comprehensive efforts will be necessary to deal with sites contaminated with complex pollutants, pollutants representing different chemical species and products generated by their interactions. A dynamic and integrative approach should be used to address future challenges of phytoremediation.

8.8.3

Applications of Plant Omics for Advancing Phytoremediation Techniques

Research progress in this field with respect to a proteomic and metabolomic approach, and metallomic investigations of metal bindings and metalloproteins are evidently lacking (Azevedo and Azavedo 2006; Garcia et al. 2006). According to Cobbett and Meagher (2002), approximately 80–90% of all phytoremediation genes and gene families have been detected in the genome of A. thaliana. This offers great potential for manipulating and genetic engineering of phytoremediating plant species. It has been estimated that about 5% of the Arabidopsis genome constitute membrane transport proteins involved in the transportation of different ions and metals across the plasma membrane and other organellar plant cell membranes. Several putative metal transporters have not been identified yet, and even a handful of those identified are not fully characterized (Maser et al. 2001; Zayed 2004). According to Heinekamp and Willey (2007), the whole-genome sequence of A. thaliana will give researchers a grand opportunity to identify and analyze all genes associated with phytoremediation. Vacuolar compartmentalization has been suggested to be a second-generation approach to genetic engineering of phytoremediating species (Heinekamp and Willey 2007). According to the authors, targeting the accumulation of toxic metabolites inside plant vacuoles makes it possible to enhance the ability of plants to withstand high toxic concentrations of different target pollutants, and at the same time it enables plants to increase uptake and accumulation within a restricted growth period, thereby maximizing the scale of phytoremediation.

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Van der Auwera G, Baute J, Bauwens M, Peck I, Piette D, Pycke M, Asselman P, Depicker A (2008) Development and application of novel constructs to score C:G-t0T: a transitions and homologous recombination in Arabidopsis. Plant J 45:908–916 Van der Zaal BJ, Neuteboom LW, Pinas JE, Chadonnens AN, Schat H, Verkleij JAC, Hooykaas PJJ (1999) Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animal can lead to enhanced zinc resistance and accumulation. Plant Physiol 119:1047–1055 Vroblesky DA, Nietch CT, Morris JT (1999) Chlorinated ethanes from groundwater in tree trunks. Environ Sci Technol 33:277–281 Wagner-Dobler I (2003) Pilot plant for bioremediation of mercury-containing industrial wastewater. Appl Microbiol Biotechnol 62:124–133 Walmsley AM, Arntzen CJ (2000) Plants for delivery of edible vaccines. Curr Opin Biotechnol 11:126–129 Wang G-D, Chen X-Y (2007) Detoxification of soil phenolic pollutants by plant secretory enzyme. In: Willey N (ed) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA, pp 49–57 White C, Shaman AK, Gadd GM (1998) An integrated microbial process for the bioremediation of soil contaminated with toxic metals. Nat Biotechnol 16:572–575 Willey N (2007) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA, pp 351–468 Wrobel M, Zebrowski J, Szopa J (2004) Polyhydroxybutyrate synthesis in transgenic flax. J Biotechnol 107:41–54 Yamada T, Ishige T, Shiota N, Inui H, Ohkawa H, Ohkawa Y (2002) Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene. Theor Appl Genet 105:515–520 Yoon JM, Oh B-T, Just CL, Schnoor JL (2002) Uptake and leaching of octahydro-1, 3, 5, 7-tetranitro-1, 3, 5, 7-tetrazocine by hybrid poplar trees. Environ Sci Technol 36:4649–4655 Zayed A (2004) Phytoremediation: advances toward a new clean up technology. In: Goodman RE (ed) Encyclopaedia of plant crop science. Taylor and Francis, Oxford, UK, pp 924–927: doi:10.1081/E-EPCS 120005584 Zhu YL, Pilon-Smits EA, Tarun AS, Weber SU, Jouanin L, Terry N (1999) Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase. Plant Physiol 121:1169–1178

Chapter 9

Algal Biotechnology: An Emerging Resource with Diverse Application and Potential Stephen Cunningham and Lokesh Joshi

9.1

Introduction to Algae

Algae include a wide variety of species that range from diatoms, which are microscopic unicellular organisms, to seaweeds extending over 30 m (Fig. 9.1). They constitute a group of approximately 40,000 species, a heterogeneous group that describes a life-form, not a systematic unit; hence, a broad spectrum of phenotypes exists in this grouping. Algae are grouped into six main classes, mainly on the basis of their color (Fogg 1953). Algae are found in fresh or salt water, with a few being terrestrial (e.g., Chrysophyta and Cyanophyta). The eukaryotic algae are placed in the kingdom Protista, classified as euglenoids (phylum Euglenophyta), dinoflagellates (phylum Pyrrophyta) and diatoms (phylum Bacillariophyta). All have chloroplasts and carry out photosynthesis similar to that of plants. Prokaryotic blue-green algae belong to the phlyum Cyanobacteria. Unlike land plants, algae do not have true roots, stems or leaves. Algae of different size and shape not only occupy aquatic ecosystems, but also occur in a number of different habitats, some of which are extreme environments (Hallmann 2007). The environmental conditions have led to the development of adaptive tolerances, such as temperature, salt and pressure selection; such adaptive selection is widely observed in bacteria. Large forms of algae are often referred to as seaweeds or microalgae, which are widely distributed in the ocean, occurring from the tide level to considerable depths, free-floating or anchored, holding an important role in providing marine primary productivity. Eukaryotic green (phylum Chlorophyta), red (phylum Rhodophyta) and brown algae (phylum Phaeophyta) are all grouped as seaweeds. They are often found to produce considerable biomass, evident from natural population

L. Joshi (*) Glycoscience and Glycotechnology Group and the Martin Ryan Institute National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_9, # Springer-Verlag Berlin Heidelberg 2010

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344 Fig. 9.1 Demonstration of size diversity among algae species

S. Cunningham and L. Joshi 1 μm

Osteococcus tauri Cyanidioschyzon merolae Thalassiosira pseudonana

10 μm

Chlamydomanas reinhardtii Phaeodactylum tricornutum

100 μm

1 mm

1 cm

Asterionella formosa

Volvox carteri

Neomeris annulata

10 cm Ulva lactuca

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10 m Macrocystis pyrifera 100 m

of brown algae Macrocystis in Northern America and the cultivated populations of artificially cultivated brown algae Laminaria and Undaria in East Asia. Geographically, algae have been used as a food source in the Asian countries, while in Europe algae has been traditionally used for the production of phycocolloids such as agar and carrageenan both from red algae and alginate from brown algae (McHugh 2003; Hallmann 2007). In most cases, algae are used in human and animal foods for their mineral contents or for the functional properties of their polysaccharides. As in the case of terrestrial plants, development of key techniques of genetic manipulation specific for algae was necessary to enable creation of good cultivars and to transform seaweed into multiple, functional marine bioreactors. This has led to the application of algae being maximized in: 1) Eliminating heavy metal pollutants, 2) Reducing factors of eutrophication (e.g., nitrogen and phosphorus),

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3) Improving the feed quality for maricultured animals using transgenic algae delivering/introducing genes encoding immunologically active peptides (molecular pharming), and 4) The production of high-value materials such as oral vaccines and drugs for humans Great strives have been made in the genetic engineering of plants and microorganisms over the last two decades. It is established that an effective transformation model consists of elements permitting an effective transformation methodology, that applicable vectors carry recognizable promoters, and that a screening mechanism to select transformants is in place to isolate the transformants from the endogenous mass. These are sufficient for single-celled organisms; however, multicellular organisms require further factors for successful recombination. This chapter describes the application, development and status of algae as a source of natural and recombinant molecules for industrial and human health applications.

9.2

The Application of Non-Transgenic Algae

9.2.1

Nutritional and Health-Related Properties

Algae have been utilized both historically and currently as food sources for human, animal and mariculture to date. In relation to animal feed, as with human feed, algae have been used to enhance the nutritional content of conventional feed preparations (Spolaore et al. 2006). Recognition as a source of fatty acids such as polyunsaturated fatty acid family (o3) and sterols, has increased their consumption in health-orientated diets (Cardozo et al. 2007). The protein and amino acid nutritional attributes of algae have been reviewed elsewhere by Fleurence (1999) and MacArtain et al. (2007). 9.2.1.1

Polyunsaturated Fatty Acids

Human beings and higher plants lack the required enzymes to synthesize long o3 polyunsaturated fatty acids; therefore, they need to obtain them from external dietary sources. Several o3 polyunsaturated fatty acids including eicosapentaenoic acid (EPA), an important dietary supplement (Yokoyama et al. 2007; Doughman et al. 2007), have been identified in a number of algae species. Although a number of algae species are cultivated as natural sources of these fatty acids, only a few species have demonstrated the potential to be capable of industrial scale production because of low growth rates and low cell number in culture (Wen and Chen 2003). The application of transgenic algae may act as an alternative, like transgenic oilseed crops, providing an alternative sustainable source of these essential oils for human consumption (Abbadi et al. 2001; Doughman et al. 2007).

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Sterols

Sterols are one of the most important chemical constituents of algae and a major nutritional component in the diet of aquacultured organisms. Microalgae are an important component in the diet of many hydrobionts, such as bivalves (Ponomarenko et al. 2004). The ability of bivalves to synthesize or bioconvert sterols de novo varies among different species, but is generally low and sometimes completely absent. This implies that a dietary supply of sterol is necessary for bivalve growth (Soudant et al. 1998). The type of algae used as a food source determines the quality and sterol composition; seasonal shifts also occur. Therefore, this is used as a criterion for species selection for the culture of bivalves (Park et al. 2002). Plant and algae sterols have been shown to reduce cholesterol by blocking absorption, resulting in reduced quantities of cholesterol reaching the liver (Plat and Mensink 2005; Charest et al. 2004). Despite the ability of these sterols to block cholesterol absorption, the human intestine poorly absorbs them (Cater and Grundy 1998).

9.2.1.3

Carotenoids

The natural pigments, carotenoids, are produced in bacteria, algae and plants (Polı´vka and Sundstro¨m 2004). These carotenoids have important biological functional roles including optimal photosynthesis and indeed protection from potential damage arising from UV light exposure. To date, over 600 different carotenoids exercising important biological functions in bacteria, algae, plants and animals have been identified (Polı´vka and Sundstro¨m 2004). Animals lack the ability to synthesize carotenoids endogenously and thus obtain these compounds by nutritional intake. For human nutritional purposes, a number of carotenoids offer provitamin A activity (Mayne 1996). Vitamin A deficiency is a major health risk that has surfaced in the developing countries as the leading cause of preventable blindness in children and also leads to the increased risk of disease and death from severe infections (WHO, Micronutrient deficiencies: http://www.who.int/nutrition/ topics/vad/en/: accessed July 20 2009). They are also biological antioxidants, protecting cells and tissues from free radicals and singlet oxygen. Carotenoids are utilized in pharmaceuticals, dietary supplements, cosmetics, and as food additives. Dunaliella salina and Spirulina maxima have been utilized for cartenoid astaxanthin, a red-orange pigment used widely in the food industry (Meyers and Latscha 1997). The green algae Haematococcus pluvialis has been the focus of biotechnology companies for the commercial development of this carotenoid (Hussein et al. 2006). The potent antioxidant property of astaxanthin has been implicated in its various biological activities demonstrated in both experimental animals and clinical studies, with potential beneficial roles in human health (Hussein et al. 2006).A second group, the phycobiliproteins, consists of proteins with covalently bound phycobilins. Phycobiliproteins, primarily composed of a- and b-polypeptides, are a brilliantly colored group of disc-shaped proteins (Samsonoff and MacColl 2001; Liu et al. 2005). These have been implemented within laboratories as labels for biomolecules (Spolaore et al. 2006).

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347

Polysaccharides

Phycocolloids are unique polysaccharides produced by several seaweed species. Carrageenan and agar are sulfated polysaccharides extracted from some Rhodophyceae. The principal feature distinguishing the highly sulfated carrageenans from the less-sulfated agars is the presence of anhydro-d-galactose in the former and l-galactose or anhydro-l-galactose in the latter (Craigie 1990). Agar and carrageenan are extracted from red algae, while alginates are extracted primarily from brown algae. Phycocolloids are used in a wide range of products including foods, pharmaceutics and medical devices (Walker et al. 2005). Carrageenan, extracted from red algae is subdivided into kappa carrageenan, iota carrageenan and lambda carrageenan, each of which has different characteristics. They are used as gelling agents, stabilizers, texturants, thickeners, and viscosifiers for a wide range of food products (Cardozo et al. 2007). Alginates, the salts of alginic acid and their derivatives, are extracted from the cell walls of brown macroalgae. These carboxylated polysaccharides are used for a wide variety of applications within the food industry. Alginates are required for production of dyes. The water absorbing properties of alginates are utilized in slimming aids and in the production of textiles and paper. Calcium alginate has been used in the production of medical products, including burn dressings (Cardozo et al. 2007). Owing to its biocompatibility and simple gelation with divalent cations, it is also used for cell immobilization and encapsulation. Additionally, alginates are widely used in prosthetics and for dental molds. Use within the food sector and as a source of phycocolloids has lead to a need for cultivation for sufficient global demand, with current estimates of global production greater than 6.5 million tons in terms of fresh weight (McHugh 2003). Such large-scale production places this in a similar economic and global importance as land crops (McHugh 2003), with abilities to fix high volumes of carbon dioxide, absorb and consume eutrophied nitrogen and phosphorus transforming them into large amounts of seaweed biomass. In doing so, they serve to protect the marine environment.

9.2.1.5

Lectins

Lectins or agglutinins, defined as proteins that bind to carbohydrates without initiating any further modifications (Weis and Drickamer 1996), have been found widely in all organisms. They are involved in numerous biological processes including host–pathogen interactions, cell–cell communication, cancer, metastasis, differentiation and immune regulation. The presence of lectins in marine algae was first reported by Boyd and colleagues in 1966 (Boyd et al. 1966). Marine algal lectins differ from higher plant lectins (agglutinins) typically in exhibiting (1) lower molecular mass ranging from 4,000 to 25,000 Da, (2) occurrence mainly in monomer form, and (3) independence from divalent cations to maintain their structure (Rogers and Hori 1993). The applications for algal lectins are at an early stage of

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development, though many have emerged as potential therapeutic agents deserving further elucidation. To date, a number of lectins have been discovered in algae, which demonstrate antiviral activity, among them are cyanovirin-N (CV-N), scytovirn (SVN) and Microcystis viridis lectin (MVL) (Zio´łkowska and Wlodawer 2006). Antiviral properties of lectins is due to their interaction with glycans, which in turn disturbs the interactions between proteins of the viral envelope and the cells of the host (Botos and Wlodawer 2005; Balzarini 2006). As a high proportion of current antiviral therapeutics act through inhibition of the viral life cycle, lectins can prevent viral penetration of host cells. Algae lectins with anti-HIV activity have been reviewed previously (Zio´łkowska and Wlodawer 2006). Seaweeds are rarely promoted for the nutritional value of their proteins. Their protein contents differ according to the species and seasonal conditions. To date, little information is available on the nutritional value of algae proteins and on the compounds that affect their digestibility.

9.2.1.6

Antibacterial and Antiviral Properties

A high number of marine algae produce antibiotic and antiviral substances capable of inhibiting bacteria, viruses, and fungi. The antimicrobial activity of aquatic microalgae was first reported for Chorella vulgaris in the 1940’s (Pratt and Fong 1940, 1944). This concept was strengthened with growing literature and reports of the antiviral properties of polysaccharides from marine algae towards mumps virus and influenza B virus in the 1960s. The antibiotic/antiviral properties are dependent on factors including the species of algae, the agent in question, the season, and the growth conditions (Pesando and Caram 1984; Centeno and Ballantine 1999). The antibacterial activity of marine algae has generally been assayed using extracts in various organic solvents (Liao et al. 2003). A number of these chemicals are toxic to microorganisms and therefore may be responsible for the antibiotic activity reported (Ohta 1979). However, this does not reflect the antibacterial activity of marine algae under natural conditions. Earlier investigations have demonstrated the effects of the release of phenolics as antifouling substances, the release of organic substances, which hold both an inhibitory effect on growth of adjacent diatoms or a stimulatory effect depending on source (Liao et al. 2003). Polysaccharide fractions from red algae were found to inhibit a number of viruses including herpes simplex virus (HSV). At that time, these findings did not generate much interest because the antiviral action of the compounds was considered to be largely nonspecific (Witvrouw and De Clercq 1997). Isolation from algae of polysaccharides and sulphated polysaccharides and other compounds with antiviral activity against enveloped viruses increased the interest in algae as a source of antiviral compounds (Schaeffer and Krylov 2000). Enveloped viruses include HIV, HSV type 1 and HSV type 2, influenza A virus, RSV, simian immunodeficiency virus (SIV), pseudorabies virus, bovine herpes virus, and human cytomegalovirus (HCMV) (Schaeffer and Krylov 2000; Zio´łkowska and Wlodawer 2006).

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Anticancer Agents Brown algae grown in the Black Sea, among other global regions such as Japanese coastline, have been demonstrated to be potential sources of antitumor agents (Apryshko et al. 2005). Carotenoid fucoxantine (Fx) extracted from these algae possess antitumor properties, which have been tested using prostate cancer performed in Russia. During these studies, patients typically received dried brown algae Laminaria daily. Dosage of Fx was estimated to be 10–15 mg daily. During treatment, there was gradual improvement of general state and blood indices. Disease course became stabilized and survival rate increased. Similarly, the rate of breast cancer is greatly reduced in populations consuming brown algae (Apryshko et al. 2005). Anti-HIV Activity Most of the research on the anti-HIV activity of marine algae has focused upon red and brown macroalgae (Schaeffer and Krylov 2000). The initial studies using these algae isolated sulfated polysaccharides with antiviral activity and later investigators continued interest in this class of compounds. However, other classes of compounds with anti-HIV activity have been identified including polysaccharides, fucoidan and carrageenans (Schaeffer and Krylov 2000). A number of natural polysulfates isolated from algae and synthetic polysulfates exhibit differential inhibitory activity against different HIV strains, which suggests differences in the target molecules with which these compounds interact (Witvrouw and De Clercq 1997; Table 9.1). They inhibit the cytopathic effect of HIV and also prevent HIV-induced syncytium formation (Zio´łkowska et al. 2006). Antiviral activity increases with increasing molecular weight and degree of sulfation (Witvrouw and De Clercq 1997). Anti-HIV polysaccharides and polyphenols have been isolated from brown algae, Fucus vesiculosus, inhibition of both HIV-induced syncytium and HIV reverse transcriptase (RT) activity at nontoxic levels (Be´ress et al. 1993). The mechanism of this effect remains to be further elucidated. A sulphated polysaccharide isolated Table 9.1 Anti-HIV activity of algal lectins

Lectin Jacalin Myrianthus holstii lectin Urtica diocia agglutinin Concanavalin A N. pseudonarcissus lectin P. tetragonolobus lectin MVL* SVN CV-N GRFT Data from Zio´łkowska et al. (2006) *IC50 rather than EC50 reported

EC50 (nM) >227 150 105 98 96 52 30 (IC50 used) 0.3 0.1 0.04

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from S. horneri demonstrated potent antiviral activity against HSV-I, cytomegalovirus and HIV. Other anti-HIV polysaccharides have been found in Agardhiella tenera, Cochlodinium polykrikoides, and Nothogenia fastigiata. Carrageenans and their cyclized derivatives isolated from G. skottsbergii are potent inhibitors of herpes viruses, and inhibit HIV to a lesser extent. A number of cyanobacteria (blue-green algae) species have been found to produce highly anti-HIV active sulfoglycolipids. In addition to their activity, the sulfoglycolipids constitute part of the chloroplast membrane and are therefore abundant. Their abundance and accessibility should be highlighted for potential future development. Extracts of cultured marine cyanobacteria, Lyngbya lagerheimii and Phormidium tenue, have been screened for anti-HIV compounds leading to the discovery of sulfonic acid-containing glycolipids as a novel class of HIV-1inhibitory compounds (Gustafson et al. 1989). A number of cyanobacteria extracts have been screened for antiviral compounds including Phormidium cebennse, Oscillatoria raciborskii, Scytonema burmanicum, Calothrix elenkinii, and Anabaena variabilis. Compounds in all were found to be anti-HIV and extracts found to contain sulfolipids. Cyanobacteria antiviral polysaccharides have also been demonstrated.

9.3

Application of Transgenic Algae

The ease of growth, biomass content and low cost of production of algae make them immensely attractive for both pharmaceutical and therapeutic compound discovery and for recombinant engineering (Fig. 9.2). In the absence of cell differentiation, algae would provide a much simpler system for genetic manipulations compared with higher plants. Manipulation of algae by metabolic and genetic methods would both permit (1) selection of beneficial pathways redirecting cellular function toward the synthesis of preferred products and (2) introduction of non-algae genes for the generation of algal recombinant protein. The selection of favorable pathways may include increased resistance to environmental or stress changes on the culturing/life cycle of the algae. The potential of this system remains to be optimized as an alternative protein expression system.

9.3.1

Transformation and Engineering

Reports of plant-produced recombinant proteins began to be published in the late 1980s when antibodies were produced in tobacco (Hiatt et al. 1989) and human serum albumin was expressed in tobacco and potato (Sijmons et al. 1990). Genetic transformation of unicellular algae began in the 1970s (Shestakov and Khuyen 1970) with cyanobacteria, followed by in the 1980s with the work in marine algae (Stevens and Parter 1980). Transformation of algae is still an area of research in its

9

Algal Biotechnology: An Emerging Resource with Diverse Application and Potential Potential Resources Produced

Oxygen

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Environmental and Culture Conditions

Recombinant proteins Environmental Conditions

Hydrogen

BioDiesel

Light, Water, CO2

Lipids Photosynthesis

Nutraceuticals

Calvin Cycle Ethanol Nutrition, Carotenoids, Aquaculture

Transgenic Engineering

Carbohydrates

Biomass Nucleus

Gasoline

Organic compounds Nutrients

Fig. 9.2 Downstream potential for algae production, applications and uses

infancy. With continuous expansion of genome information, the application of algal models for the production of proteins will become an attractive alternative to the existing systems of recombinant protein expression. Recombinant engineering technology has evolved globally into an industry sector encompassing food, agriculture, pharmaceutics, biofuel (see sect. 6.1.3), and environmental areas. Recombinant production of proteins now produces a myriad of protein-based industrial and biopharmaceutical products in crop plants, aquatic plants and algae (Giddings et al. 2000). Stability of transformation and efficiency have, to date, been the key determinants upon experimental success. These are relative to the size and complexity of the algae used in each case (Hallmann 2007). Transformation has been focused on transient expression of reported genes, typically antibiotic resistance genes as these confer traits regardless of genotype (Qin et al. 2005; Hallmann 2007).

9.3.1.1

Status of Alga Genome Information

For the development and application of engineered algae for scale-up of endogenous molecules and for their application as a recombinant tool for protein production, highly annotated genome data are required. Complete genome annotation and sequenced expressed sequence tag (EST) mapping is currently available for a number of species. Like other genome projects, data increase almost exponentially. Sequencing and annotation of the 16.5 Mb Cyanidioschyzon merolae genome

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(Matsuzaki et al. 2004), the 34 Mb Thalassiosira pseudonana genome (Armbrust et al. 2004) and the ~120 Mb genome of Chlamydomonas reinhardtii has been completed (Merchant et al. 2003). Genomes of over 50 species are all currently undergoing annotation or nearing sequencing completion, 30 of which are genomes of cyanobacteria (NCBI, Entrez Genome Project: www.ncbi.nlm.nih.gov/sites/ entrez: accessed 20 July 2009). Owing to their small-sized genomes, completed genome information is being produced at a faster rate for blue-green algae.

9.3.1.2

Current Status of Algae Engineering

The genetic engineering and modification of algae is an area, which has received a lot of attention over the last few years (Leo´n-Ban˜ares et al. 2004; Walker et al. 2005). Reports detailing the introduction of DNA into the diatom Phaeodactylum, the green algae Chlamydomonas, and the blue-green algae Synechococcus and Synechocystis have been circulated (Raja et al. 2008). Genetic engineering of the expression of mosquito larvicidal properties in blue-green algae has also been reported (Boussiba et al. 2000). However, at the time of publication there has not been a report on the commercial use of any transgenic algae for the application of a functional transformation system. Literature supports the development of such methodologies demonstrated with the manipulation of diatoms to enhance lipid production (Dunahay 1996), the expression of a functional glucose transporter in the obligate phototrophic Phaeodactylum enabling this diatom to grow on glucose in the dark (Zaslavskaia et al. 2001) and the advancements using genetically modified strains of Chlamydomonas for hydrogen production as an alternative biofuel (Melis et al. 2000). Chlamydomonas is particularly relevant as a model algae system for genetic manipulation and is detailed further below. The potential of algae to be genetically modified, permitting the synthesis of recombinant proteins, opens up alternatives to the current recombinant systems, and presents a simpler model with respect to minimal or removed system contaminants for the use in expression of human antibody and therapeutic proteins.

9.3.1.3

Maintenance in Bioreactors

The ability to culture single-celled plants and aquatic plants in bioreactors offers two advantages over the use of terrestrial plants: (1) the growth conditions can be controlled precisely, insuring optimal growth conditions and batch-to-batch product reproducibility (yield, activity); and (2) growth in bioreactors is contained in-house, thus removing environmental biosafety issues associated with “release” of transgenic terrestrial plants. With respect to their high protein levels and their amino acid composition the red seaweed appear to be an interesting potential source of food proteins. With large scale production in bioreactors possible, this is a developing area of research and industry. Algae may represent functional foods, which remain to be utilized. Use

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of algae, as a food source for mariculture permits the delivery of target genes, influencing both the organism feeding and also downstream consumers. Such an approach to molecular pharming would permit edible therapeutics and health management. The amino acid content is of nutritional value, however, their protein digestibility in vivo remains to be completely elucidated.

9.3.2

Models of Alga Engineering

9.3.2.1

Laminaria japonica

The brown algae, Laminaria japonica, commonly referred to as Kelp is a cultivated seaweed, which has been utilized as both a foodstuff and as a raw material for iodine, mannitol and alginate production (Tseng and Qin 1999). The production of L. japonica is a low-cost, high-yield crop in China. Extensive research has been carried out for the breeding and strain development for the establishment of improved traits. Transformation of L. japonica has been hindered by a lack of knowledge in relation to kelp viruses or symbiotic bacteria, which could be used for direct gene transfer. Therefore, until this point particle bombardment and ultrasound have been the transformation methodologies applied. Research on a virus (ESV-1) infecting Ectocarpus, a filamentous brown algae found as epiphytic to kelp has been performed, including viral genome annotation (Delaroque et al. 2001). This virus infects unicellular spores or gamates, transmitting its genome by mitosis to all cells of the growing host plant (Mu¨ller et al. 1998). The transmission of its genome presents this virus as a potential vector for gene delivery. As no usable promoter from kelp has yet been identified, a number of promoter regions from terrestrial plants, unicellular algae and algal viruses have been introduced without much success. Stable expression has been shown for transforants using two promoters, fcp (diatom fucoxanthin-chorophyll a/c binding protein gene) and the SV40 (simian virus) (Qin et al. 2005). The potential of L. japonica in the production of recombinant protein was further demonstrated by the introduction of the vaccine gene encoding human hepatitis B surface antigen gene (HBsAg) (Qin et al. 2005). Expression levels in transgenic kelp were compared to that of transgenic tobacco. Comparable levels of protein were expressed from both systems (Qin et al. 2005), demonstrating the potential for edible/direct delivery of proteins.

9.3.2.2

Chlamydomonas Reinhardtii

The green algae, Chlamydomonas reinhardtii, has been utilized as a model organism in the study of photosynthesis and light-regulated gene expression. Recently, it has been explored as a potential host for recombinant protein synthesis. Production of several forms of human IgA antibody directed against glycoprotein D and HSV have been reported to date in C. reinhardtii (Mayfield et al. 2003; Franklin and

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Mayfield 2005). The production of these monoclonal antibodies in algae demonstrated that the production is both possible and is comparable to terrestrial plant production. Algae offers several advantages over terrestrial plants including: (1) transgenic forms can be generated quickly (only weeks between generation of initial transformants and their scale-up for production); (2) both the chloroplast and nuclear genome of algae can be genetically transformed by standard methodologies, i.e. microprojectile particle bombardment or electroporation, providing scope for the production of several different proteins simultaneously; and (3) the ability to culture volumes ranging from a few milliliters to 500,000 liters makes such an expression system advantageous at an industrial level (Franklin and Mayfield 2004).

9.4

Summary

The demand for improved systems of production of nutraceuticals and cost-effective protein expression systems (both industrial and pharmaceutical applications) lend themselves to the potential useful capacities of algae. Currently, recombinant expression of proteins is performed in both mammalian and nonmammalian systems routinely. As a system, algae provide a simpler model than plants. Limitations of manufacturing are a bottleneck limited on size, cost, time and levels of purification, especially with reference to mammalian recombinant proteins. The progress being made in relation to sequencing of algal genomes will permit the cloning and manipulation of genes and allow “omics” technologies to be applied. This advancement will aid in the identification of key regulators of metabolism and enable the eventual manipulation of cellular pathways. Such advances in transformation techniques will permit in future more sophisticated forms of recombinant engineering to be performed. Ultimately, algal biotechnology offers the potential to have impact on the advancement of recombinant technologies. This is only the beginning of this field of research and much remains to be achieved to optimize the full potential of algae.

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Lucas S, Medina M, Montsant A, Obornik M, Parker MS, Palenik B, Pazour GJ, Richardson PM, Rynearson TA, Saito MA, Schwartz DC, Thamatrakoln K, Valentin K, Vardi A, Wilkerson FP, Rokhsar DS (2004) The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86 Balzarini J (2006) Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral Res 71:237–247 Be´ress A, Wassermann O, Tahhan S, Bruhn T, Be´ress L, Kraiselburd EN, Gonzalez LV, de Motta GE, Chavez PI (1993) A new procedure for the isolation of anti-HIV compounds (polysaccharides and polyphenols) from the marine algae Fucus vesiculosus. J Nat Prod 56:478–488 Botos I, Wlodawer A (2005) Proteins that bind high-mannose sugars of the HIV envelope. Progr Biophys Mol Biol 88:233–282 Boyd WC, Almodovar IR, Boyd IG (1966) Agglutinins in marine algae for human erythrocytes. Transfusion 6:82–83 Cardozo KH, Guaratini T, Barros MP, Falca˜o VR, Tonon AP, Lopes NP, Campos S, Torres MA, Souza AO, Colepicolo P, Pinto E (2007) Metabolites from algae with economical impact. Comp Biochem Physiol C Toxicol Pharmacol 146:60–78 Cater NB, Grundy SM (1998) Lowering serum cholesterol with plant sterols and stanols: historical perspectives. J Postgrad Med, 6-14 Centeno POR, Ballantine DL (1999) Effects of culture conditions on production of antibiotically active metabolites by the marine algae Spyridia filamentosa. I. Light. J Appl Phycol 11:217– 224 Charest A, Desroches S, Vanstone CA, Jones PJH, Lamarche B (2004) Unesterified plant sterols and stanols do not affect LDL electrophoretic characteristics in hypercholesterolemic subjects. J Nutr 134:592–595 Craigie JS (1990) Cell Walls. In: Cole KM, Sheath RG (eds) Biology of the Red Algae. Cambridge Univ Press, Cambridge, UK, pp 226–236 Delaroque N, Mu¨ller DG, Bothe G, Pohl T, Knippers R, Boland W (2001) The complete DNA sequence of the Ectocarpus siliculosus Virus EsV-1 genome. Virology 287:112–132 Doughman SD, Krupanidhi S, Sanjeevi CB (2007) Omega-3 fatty acids for nutrition and medicine: considering microalgae oil as a vegetarian source of EPA and DHA. Curr Diabet Rev 3(3):198–203 Dunahay TG (1996) Manipulation of microalgal lipid production using genetic engineering. Appl Biochem Biotechnol 57:223–231 Fleurence J (1999) Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Sci Technol 10:25–28 Fogg GE (1953) The Metabolism of Algae. Methuen, London, UK Franklin SE, Mayfield SP (2004) Prospects for molecular farming in the green algae Chlamydomonas. Curr Opin Plant Biol 7:159–165 Franklin SE, Mayfield SP (2005) Recent developments in the production of human therapeutic proteins in eukaryotic algae. Expert Opin Biol Ther 5:225–235 Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155 Gustafson KR, Cardellina JH, Fuller RW, Weislow OS, Kiser RF, Snader KM, Patterson GM, Boyd MR (1989). AIDS: antiviral sulfolipids from cyanobacteria. J. Natl. Cancer Inst. 81:1254–1258 Hallmann A (2007) Algal transgenics and biotechnology. Transgen Plant J 1:81–98 Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78 Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H (2006) Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 69:443–449 Leo´n-Ban˜ares R, Gonza´lez-Ballester D, Galva´n A, Ferna´ndez E (2004) Transgenic microalgae as green cell-factories. Trends Biotechnol 22:45–52

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Chapter 10

Biotech Crops and Functional Genomics Narayana M. Upadhyaya, Andy Pereira, and John M. Watson

10.1

Introduction

The increase in human population, poor performance of crop cultivars under increasingly adverse environmental conditions and a decline in the available land for sustainable crop production are contributing to a shortage of global food supply and increase in its demand. Conventional breeding efforts in crops such as rice over the last three decades have resulted in a doubling of agricultural productivity (Khush 1997). However, for sustained increase in the agricultural productivity, crops which can resist pests, pathogens and tolerate salinity, drought and temperature extremes need to be developed and deployed. The deployment of a handful of gene classes developed as transgenes for insect and/or herbicide resistance in crops such as maize, soybean, canola, cotton, squash, papaya, alfalfa, and sugar beet reduces yield losses and increases agricultural profitability. However, commercial application of transgenes conferring more complex traits such as abiotic stress tolerance, yield, vigor and nutritional quality are yet to be achieved because of the lack of adequate knowledge of the critical genes controlling such traits and the complexity of their behavior under different environmental conditions. Long development times from discovery to commercial release, intellectual property constraints, high development costs, and regulatory constraints and costs are also slowing down the process of commercial deployment (Birch 2000). With continual improvements in transgene delivery, integration and expression modulation, several other transgenes of agronomic importance are being deployed and tested for efficacy in various crop plants. Major crops such as rice, wheat, barley and sugarcane are bound to become biotech crops as more and more novel genes, with high potential to be deployed as transgenes, are discovered, and

N.M. Upadhyaya (*) CSIRO Plant Industry, GPO Box 1600, Canberra ACT 260, Australia e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_10, # Springer-Verlag Berlin Heidelberg 2010

359

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N.M. Upadhyaya et al.

the concerns over the biosafety of their large-scale deployment are addressed scientifically and politically. Mapping and DNA sequencing of plant genomes and analysis of the information content present in genomic sequences commonly termed as “genomics” have the potential to provide valuable insight into genes controlling some of the complex traits mentioned above. Various genome-wide high-throughput “functional genomics” tools and resources are being developed worldwide. The ultimate aim of these approaches is to define the structures of all genes, and the functions of all gene products, as well as all the processes that occur during plant growth and development. Such knowledge could be used effectively, not only in molecular markerassisted breeding, but also in transgenic breeding. Arabidopsis, a model dicot, and rice, a model cereal, have emerged as front-runners with near-complete sequencing of the Arabidopsis ecotype Columbia (TAGI 2000) and two rice genotypes, namely, japonica cv. Nipponbare (IRGSP 2005) and indica cv. 93-11 (Yu et al. 2002, 2005). Genome sequencing efforts are now underway for several other food grain and tuber crops (barley, cassava, maize, mungbean, potato, sorghum and wheat), vegetable crops (tomato, cabbage and field mustard), fruit crops (grape, papaya, orange and apple), oil crops (rapeseed, Indian mustard, black mustard, soybean and castor), forage crops (barrel medic), biofuel crops (jatropha, miscanthus, switchgrass, pine, madhuca, arundo and pongamia) and other commercial crops such as tobacco and cotton (Tables 10.1 and 10.2; http://www.arabidopsis.org/portals/gen Annotation/other_genomes/#sequence). In this chapter, we give a brief overview of transgenic crops, plant genomics and plant functional genomics and then discuss various transgenic strategies being used to establish gene–phenotype relationships and elaborate on how they are facilitating the functional characterization of genes and gene control sequences.

10.2

Transgenic Plants

Transgenic crops have great potential for alleviating some of the production constraints such as insect pests, pathogens, salinity and drought. Since the first demonstration of the introduction and expression of foreign genes in tobacco in 1984, more than 150 plant species in at least 50 plant families have been experimentally transformed and transgenic events reported. Today, 13 transgenic crops are grown commercially in 25 different countries, including 15 developing countries (James 2008). Regulatory approvals for 24 transgenic crops, for the importation for food and feed use and for release into the environment, have been granted in another 30 countries (James 2008). Worldwide acreages of transgenic crops are increasing at the rate of ~12% each year (James 2008). However, the current successes in transgenic crops still have a narrow base with respect to traits (>95% are insect resistance and/or herbicide tolerance traits), crops (>95% are soybean, corn, cotton and canola) and countries (>95% are grown in US, Argentina, Brazil, Canada, India, and China).

Food

Forage legume model

Hordeum vulgare

Medicago truncatula

Barley

Barrel medic

Future

Future

Future

Food

Oil, industrial

Manihot esculenta

Ricinus communis

Cassava

Castor bean

Future

Future

Future

Fruit

Musa acuminata

Banana

Model

Vegetable

Dicot model

Thellungiella halophila

Model

Model

Dicot model

Arabidopsis thaliana

Arabidopsis (thale cress) Arabidopsis relative

Future

Biotech status

Model cereal

Fruit

Malus domestica

Apple

Brachypodium Brachypodium distachyon Broccoli Brassica oleracea

Type

Plant

Common name

na

Yes

na

na

Yes

Yes

Yes

na

Yes

na*

In progress

Sequencing status

62,592

79,444

na

20,449

260,238

501,366

5,524

38,022

18

9

5

8

7

11

7

5

17

Chromosomes (n)

Draft assembly 10

In progress

In progress

In progress

Completed

In progress

In progress

In progress

1,526,124 Completed

256,249

Maps ESTs

16957

13628

12577

18703

9508

9511

15719

18765

9506

NCBI project ID 12882

Biotech Crops and Functional Genomics (continued)

Sequencing group/consortium Haploid genome size (Mb) 750 IASMA, Institoto Agrario S. Michele all’Adige (http://www.ismaa.it/) 120 The Arabidopsis Information resource (http://www.arabidopsis.org/) 260 DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/50029.html) 600 The Global Musa Genomics Consortium (http://www.musagenomics.org/) 5,000 International Barley Genome Sequencing Consortium (http://www.public.iastate.edu/ ~imagefpc/IBSC%20Webpage/ IBSC%20Template-home.html) 500 Medicago truncatula Sequencing Resources (http://www. medicago.org/genome/) 300 DOE Joint Genome Institute (http://www.brachypodium.org/) 600 TIGR Brassica oleracea Genome Project (http://www.tigr.org/ tdb/e2k1/bog1/) 760 DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/51283.html) 400 J. Craig Venter Institute (http://www.jcvi.org/cms/ research/projects/castorbean-database/overview/)

Table 10.1 Current and future biotech plants for which full-scale genomics studies have been initiated or completed

10 361

Vegetable

Biofuel, grass

Brassica rapa

Arundo donax

Field mustard

Giant cane

Model

Arabidopsis lyrata

Lyreleaf rockress

Dicot model for comparative genomics

Model

Lotus japonicus Model legume

Future

Future

Future

Future

Future

Lotus

Biofuel

Oil, vegetable

Biofuel, tree

Biofuel

Biofuel

Pinus taeda

Jatropha curcas Jatropha tanjorensis Pogamia pinnata Brassica juncea

Future

Future

Future

Future

Biotech status

Loblolly pine

Leaf mustard

Karanj

Jatropha

Jatropha

Model for citrus

Oil, tree

Eucalyptus globulus

Eucalyptus (blue gum)

Japanese bitter Poncirus orange trifoliata

Type

Plant

Common name

Table 10.1 (continued)

na

yes

na

na

na

na

na

na

na

na

na

561

157,951

328,628

193

na

na

1,012

62,344

na

33,398

10,003

Maps ESTs

12

18

11

11

11

9

12

10

11

Chromosomes (n)

In progress

8

Draft assembly 6

In progress

In progress

In progress

In progress

In progress

In progress

In progress

In progress

In progress

Sequencing status

Sequencing group/consortium Haploid genome size (Mb) 600 The International Eucalyptus Genome Network (http://www.fabinet.up.ac.za/ eucagen) 500 The Multinational Brassica Genome Project (MBGP) (http://www.brassica.info/ resource/sequencing.php) 2,744 Nandan Biometrix Ltd (http://www.nandan.biz/) 380 International Citrus Genome Consortium (http://www.citrusgenome. ucr.edu/) 416 Nandan Biometrix Ltd (http://www.nandan.biz/) 416 Nandan Biometrix Ltd (http://www.nandan.biz/) 1,700 Nandan Biometrix Ltd (http://www.nandan.biz/) 1,200 The Brassica Genome Gateway (http://www.brassica.bbsrc. ac.uk/) 30,000 The Pine Genome initiative (http://pinegenomeinitiative. org/) 470 Kazusa DNA Research Institute (http://www.kazusa.or.jp/ lotus/clonelist.html) 230 DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3066.html)

15628

10747

30775

18137

32643

32385

12948

32663

12578

NCBI project ID 12504

362 N.M. Upadhyaya et al.

Potato

Solanum tuberosum

Populus trichocarpa

Food crop

Dicot model, Arabidopsis relative Tree model

Capsella rubella

Pink Shepherd’s purse Poplar

Future

Current

Model

Current

Fruit

Carica papaya

Yes

na

na

na

na

na

Model

Papaya

Moss

Current

Yes

na

Yes

na

na

Model

Future

Current

Future

Future

Oilseed rape

Physcomitrella patens

Moss

Model

Food and fodder

Biofuel

Biofuel tree

Model for comparative genomics Model for Selaginella evolutionary moellen study dorffii Brassica napus Oil

Mimulus guttatus

Madhuca longifolia var. latifolia Miscanthus sinensis Zea mays

Monkey flower

Maize

Maiden grass

Mahwa

In progress

In progress

231,704

89,943

na

77,158

596,249

93,806

20,453

14,587

14

10

19

13

19 (2
)**

8

8

In progress

12

Draft assembly 19

In progress

Draft assembly 9

In progress

Completed

Draft assembly 27

In progress

2,018,337 In progress

na

na

840

480

686

370

1,100

100

510

430

2,400

na

na

The International Populus Genome Consortium (http://www.ornl.gov/ sci/ipgc/) The potato Genome Sequencing Consortium (http://www. potatogenome.net/)

12984

10770

15659

16103

12478

13079

12940

15658

9514

32661

32407

Biotech Crops and Functional Genomics (continued)

Nandan Biometrix Ltd (http://www.nandan.biz/) The Maize Genome Sequencing Project (http://www. maizesequence.org/ overview.html) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3062.html) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3141.html) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3153.html) The Brassica Genome Gateway (http://brassica.bbsrc.ac.uk/) The Hawaii Papaya Genome Project (http://asgpb. mhpcc.hawaii.edu/papaya/) DOE Joint Genome Institute (http:// www.jgi.doe.gov/sequencing/ why/3066.html)

Nandan Biometrix Ltd (http://www.nandan.biz/)

10 363

Current Future

Medicinal crop

Commercial crop

Fruit

Glycine max

Cucurbita pepo Vegetable

Biofuel

Chlorophytum borivilianum Sorghum bicolor

Panicum virgatum

Nicotiana tabacum

Solanum lycopersicum

Safed musli

Soybean

Squash

Switchgrass

Tobacco

Tomato

Oil, vegetable

Food crop

Food

Oryza sativa japonica

Rice

Sorghum

Food

Oriza sativa indica

Rice

Current

Future

Current

Future

Future

Future

Future

Future

Oil, vegetable

Brassica nigra

Rapeseed

Biotech status

Type

Plant

Common name

Table 10.1 (continued)

Yes

Yes

na

na

Yes

Yes

na

Yes

Yes

na

Completed

In progress

Completed

Completed

In progress

Sequencing status

258,830

240,440

436,535

27

In progress

In progress

In progress

In progress

1,386,618 In progress

209,814

na

na

na

1

Maps ESTs

760

Sequencing group/consortium Haploid genome size (Mb) 700 The Brassica Genome Gateway (http://www.brassica. bbsrc.ac.uk/) 375 BGI Rice Information System (http://rice.genomics.org. cn/rice/index2.jsp) 390 International Rice Genome Sequencing Project (http://rgp.dna.affrc.go.jp/IRGSP/) 540 Nandan Biometrix Ltd (http://www. nandan.biz/)

DOE Joint Genome Institute (http://www.phytozome. net/sorghum) 20 1,200 DOE Joint Genome Institute (http://www.phytozome. net/soybean) 20 539 Cucurbit Genomics Database (http://www.icugi.org) 18–36 1,911–2,303 DOE Joint Genome Institute (2–4
) (http://www.jgi.doe.gov/ sequencing/why/50008.html) 24 (2
) 4,500 Tobacco genome Initiative (http://www.tobaccogenome. org/) 12 950 International tomato sequencing Project (http://www.sgn. cornell.edu/about/tomato_ sequencing.pl)

10

14 (2
)

12

12

8

Chromosomes (n)

9509

13234

17453

na

9507

10785

32621

9512

9512

NCBI project ID 18141

364 N.M. Upadhyaya et al.

Fruit and wine

Parasitic weed

Vitis vinifera

Triphysaria versicolor

Wine grape

Yellow owl’s clover

Model

Future

Future

Model

Future

Current

na

Yes

Yes

na

na

Yes

Pending

In progress

In progress

49,006

353,688

21 (3
)

7

9

26 (2
)

Pending

11

Draft assembly 19

1,051,736 Pilot scale

na

207,500

268,779

1,200

500

16,000

350

380

2,100

International cotton genome initiative (http://icgi.tamu.edu/ developing.html) International Citrus Genome Consortium (http://www. citrusgenome.ucr.edu/) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/51280.html) International Wheat Genome Sequencing consortium (http://www.wheatgenome.org/) International Grape Genome Program (http://www. vitaceae.org/index.php/ International_Grape_Genome_ Program) DOE Joint Genome Institute (http://www.jgi.doe.gov/ sequencing/why/3116.html)

*na ¼ not available or not determined or not assigned ** ¼ x denotes ploidy level Data source: NCBI, relevant web pages and Plant DNA C-values database (Bennett and Leitch 2005, http://data.kew.org/cvalues/)

Food crop

Triticum aestivum

Wheat

Flower model

Fruit

Citrus sinensis

Valencia orange

Western Aquilegia columbine formosa

Fibre, oil

Gossypium hirsutum

Upland cotton

15684

12992

9513

18647

9597

12542

10 Biotech Crops and Functional Genomics 365

Biotech status Current Future Future Future Near future Near future Near future Current Current Near future Near future Near future Future Future Current Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Near future Future Current Near future Near future Near future

Plant

Medicago sativa Prunus dulcis Prunus armeniaca Asparagus officinalis Amomum sp, Elettaria sp Brassica oleracea var capitata Eschscholzia californica Capsicum annuum Dianthus caryophyllus Brassica oleracea var botrytis Cichorium intybus Brassica oleracea var. alboglabra Coffea arabica Phaseolus vulgaris Gossypium arboreum Vigna unguiculata Agrostis stolonifera Cucumis sativus Cuphea Solanum melongena Brassica carinata Linum usitatissimum Lactuca sativa Nuphar advena Vigna radiata Cucumis melo Picea abies Papaver somniferum Prunus persica Petunia axillaris subsp axillaris Ananas comosus Populus tremula Populus tremuloides

Common name

Alfalfa Almond Apricot Asparagus Cardamom Cabbage California poppy Capsicum Carnation Cauliflower Chicory Chinese broccoli Coffee Common bean Cotton Cowpea Creeping bentgrass Cucumber Cuphea hybrid Egg plant Ethiopian mustard Flax Lettuce Lilly Mung bean Musk mellon Norway spruce Opium poppy Peach Petunia Pineapple Poplar Poplar

11,090 3,864 15,105 8,422 na 26,692 9,083 33,311 387 202 53,973 30,759 1,577 83,448 41,768 183,751 9,020 6,662 na 3 2,482 7,929 80,781 20,589 829 5,943 10,217 20,340 79,023 1,696 5,649 37,313 12,813

ESTs Yes na Yes Yes na na na Yes na na na na na na na Yes na na na Yes na na na na na na na na Yes na na na na

Maps Forage Nut Fruit Vegetable Condiment Vegetable Medicinal Vegetable Flower Vegetable Beverage Vegetable Beverage Vegetable Fibre, oil Food Lawn grass Vegetable Ornamental, oil Vegetable Oil Fruit Vegetable Flower Food Fruit Timber tree Medicinal Fruit Flower Fruit Tree Tree

Type

Haploid genome size (Mb) 900 300 300 1,323 na 600 1,103 3,000 613 760 na 760 1,176 630 2,132 588 3,430 370 na 1,100 1,544 686 2,597 2,400 515 931 18,228 3,724 290 1,372 569 na na

Table 10.2 Current and future biotech plants for which full-scale genomics studies are yet to be initiated Chromosome number (n) 8 8 8 10 12 9 6 12 15 9 9 9 11 11 13 11 28 (2
)** 7 8 12 17 15 9 17 11 12 12 11 8 7 25 19 19 13214 12944 20685 16855 na 12577 na 12486 na na na na 10702 12933 12946 31169 na 36671 na 15625 na na 12869 na 17571 na na na 12949 na na 13278 13258

NCBI Project ID

366 N.M. Upadhyaya et al.

Rosa chinensis Rosa hybrid cultivar Rosa luciae Phaseolus coccineus Carthamus tinctorius Beta vulgaris Saccharum officinarum Helianthus annuus Festuca arundinacea Camellia sinensis Nicotiana benthamiana Torenia fournieri Citrullus lanatus Trifolium repens Picea glauca Near future Near future Near future Near future Near future Current Near future Near future Near future Near future Future Near future Near future Near future Near future

1,794 5,563 1,932 391,138 41,011 26,870 246,379 133,682 44,377 6,416 42,658 na 7,891 46 284,329

na na na na na Yes na na na na na na na na na

Flower Flower Flower Flower, food Oil Sugar and herb Food Oil Forage Beverage Narcotics Flower Fruit Forage Wood, ornamental

564 578 na 662 na 760 3,969 3,000 5,629 3,824 3,136 na 424 956 19,796

7 7 7 11 9 9 40 (4
) 17 21 (3
) 15 19 (2
) 17 11 16 (2
) 12

*na ¼ not available or not determined or not assigned ** ¼ x denotes ploidy level Data source: NCBI, relevant web pages and Plant DNA C-values database (Bennett and Leitch 2005, http://data.kew.org/cvalues/)

Rose Rose Rose Runner bean Safflower Sugar beet Sugarcane Sunflower Tall fescue Tea Tobacco relative Torenia Watermellon White clover White spruce

na na na 12925 na 12562 12961 12865 na 31167 12911 na na na 32251

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Long development times from discovery to commercial release, intellectual property and regulatory constraints, and high development and regulatory costs are partly responsible for this (Birch 2000). So far, only transgenic crops and traits with high commercial return potential have been commercialized by large multinational companies in countries which allow the deregulation of genetically modified (GM) crops or have regulatory frameworks in place for commercial releases. In such countries and with such crops, companies, farmers and the consumers (to some extent) have reaped the financial benefits. It is worth noting that 20 countries have banned the importation and/or commercial cultivation of GM crops with another five countries having a moratorium on the same (http://www.centerforfoodsafety. org/geneticall5.cfm). As such, the issue of GM food crops for human consumption is still contentious because of the perceived health and environmental risks associated with the widespread usage of certain transgenes. Commercial deployments of transgenes conferring more complex traits such as abiotic stress tolerance, yield, vigor and nutritional quality are yet to be achieved. This is largely due to the lack of adequate knowledge of the genes controlling such traits and to the complexity of their behavior under different environmental conditions and in different genetic backgrounds. Quite often “green house champion” transgenic lines fail miserably under field conditions. Although very high transformation efficiencies are now achievable in many crop species, they are still highly genotype dependent. This is mainly due to the genotype dependency of tissue culturability and transformability (mainly via Agrobacterium). It is important to note that the proportion of transformation events resulting in an acceptable commercial phenotype is very low. Even with an optimized gene construct (gene promoter-coding region-gene terminator), this frequency could be one in 400 events (Birch 2000). Somaclonal variations introduced during tissue culture, pleiotropic effects of transgene expression, insertional inactivation of endogenous genes, integration position effects and transgene silencing are likely to reduce the frequency of “useful transformation events” (Birch 2000). Recent progress and applications in genomics and functional genomics are not only increasing the number of potential transgenes and gene control sequences but are also improving our understanding of the complexity of gene expression under different environmental conditions and in different genetic backgrounds. Progress with transgenics has been more rapid with rice than with any other cereals due to the very efficient rice tissue culture and transformation systems developed over the past 20 years (Upadhyaya et al. 2000). The use of appropriate embryogenic target tissues, gene delivery methods, gene promoters, selectable marker genes, selection regimes and reporter genes have all contributed to this success. Protoplasts derived from embryogenic callus have been used for gene delivery by either electroporation or polyethylene glycol treatment. Embryo or embryogenic calli have been the target tissues for biolistics and Agrobacteriummediated gene delivery. Although the initial successes were restricted to reporter and selectable marker transgenes, genes of agronomic value such as herbicide, insect, bacterial, fungal and viral resistances have also been introduced into rice and transgenic lines have been studied under field conditions. Transgenic rice is now an

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ideal tool for elucidating various aspects of gene expression and regulation, especially of genes from other monocots, which are not as amenable as rice to genetic manipulation. Some of the challenges in producing sustainable transgenic rice currently being addressed include the removal of selectable markers, targeted gene delivery (gene replacement), stability of transgene expression over many generations and the spatial, temporal and developmental control of transgene expression.

10.3

Plant Genomics

As proposed by Hieter and Boguski (1997), genomics can be broadly classified into two disciplines: “structural genomics” and “functional genomics.” Structural genomics corresponds to the initial phase of genome analysis resulting ultimately in the definition of the complete DNA sequence of an organism, while functional genomics makes use of the genome sequence to assess, on a large-scale, the functions of genes as well as their expression and interaction. With the near completion of the sequencing of their genomes (Table 10.3), Arabidopsis and rice are generally accepted as model dicot and monocot species, respectively for genetic and genomic studies. This is because of their small genome sizes (~135 Mb and 430 Mb, respectively), the ease with which they can be grown, transformed and used in genetic experiments, and the similarity of their respective gene orders and gene Table 10.3 Arabidopsis and rice genome sequence assembly and annotation – current status Rice Arabidopsis Rice TIGRb BGIc TAIRa Genotype/ecotype Columbia Japonica indica Cultivar – Nipponbare 93-11 Genome version TAIR8 Release 5 Release 2 Chromosomes 5 12 12 Sequenced genome size (bp) 119,186,497 372,077,801 360,157,649d (374,545,499)e Estimated complete genome size (bp) 134,634,692 388,820,000 NA Unassigned sequences (bp) – – 104,840,190 Predicted genes including transposable 33,282 56,278 (66,710)f 59,660 element (TE) genes and noncoding RNAs (38,963) Predicted non-TE genes 27,235 41,046 (51,286)f 49,088 Mapped Full-length cDNA 13,066 32,775 25,645 ORF cDNA 24,235 – – a The Arabidopsis Information Resource (http://www.arabidopsis.org/) b TIGR = The Institute for Genomic Research. TIGR is now merged with The J. Craig Venter Institute (http://www.jcvi.org/) and TIGR’s Rice Annotation Project is moved to Michigan State University (http://rice.plantbiology.msu.edu/) c BGI (http://rice.genomics.org.cn/index2.jsp) d Genome sizes based on the sum total of assigned contigs e Figures in the parenthesis are the genome sizes as the sum total of genome assigned scaffolds f Figures in the parenthesis are the total genes including splice variants

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sequences with other dicots (e.g., crucifers) or monocot cereals, (e.g., barley, wheat and maize). Thanks to the recent rapid developments in high-throughput nucleic acid sequencing technologies, genome sequencing and/or expressed sequence tag (EST) sequencing efforts are underway for the majority of crop plants (Table 10.1).

10.4

Plant Functional Genomics

Functional genomics is a rapidly expanding field of biological science. Various high-throughput technologies are being employed in the large-scale profiling of genes, mRNAs, proteins and metabolites that participate in various cellular processes during plant growth and development. These include: (1) data mining tools for structural similarities; (2) RNA level expression profiling with ESTs, oligonucleotide or cDNA chips; (3) protein level expression profiling (proteomics); (4) metabolite level expression profiling (metabolomics); (5) gene knock-outs or loss-of-function studies with naturally occurring alleles, induced deletion and insertional mutants; (6) gene expression knock-down (gene silencing) studies; and (7) gain-of-function studies with overexpression or misexpression transgenes.

10.4.1

Gene Predictions Using DNA Sequence Comparison

The most straightforward way of predicting the function of an unknown gene from one organism is by comparison of its DNA sequence with known gene sequences from other organisms, as functionally similar genes normally have sequence similarities at both the DNA and the protein sequence levels. The precision with which sequences can be compared has increased tremendously with recent vast improvements in computing power, gene prediction programs and various other bioinformatics capabilities. Several laboratories have embarked on sequence annotation using this approach (Antonio et al. 2007; Itoh 2007). Computational gene predictions in rice suggest that there could be more than 50,000 rice genes with ~60% having some evidence of expression. For example, according to the International Rice Genome Sequencing Project (IRGSP)’s rice annotation project database (RAP-DB; http://rapdb.dna.affrc.go.jp/), among the 53,461 predicted rice genes 31,439 show evidence of expression and 25,012 are protein-coding loci with fulllength cDNA support. The remainder is based on computer predictions without any evidence of transcriptional activity. The validation of gene functions predicted by sequence comparison needs to be done by other methods to avoid the progressive build up of inaccurate gene function assignments in the genome sequence databases. With the available japonica and indica genome sequences, attempts are being made to unravel allelic variations between these two subspecies using various functional genomics approaches. Transgenic approaches could be used to unravel the function of an unknown gene by overexpression, knock-out or knock-down of that gene as detailed later in this chapter.

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371

Gene Expression Studies at the RNA Level

Although there could be more than 50,000 genes in a plant genome, not all of these are transcribed into RNA at any given time, in any given tissue or under any given environmental condition. Even some of the transcribed RNAs are suppressed, broken down or rendered non-translatable. The characterization of all the transcribed genes, referred to as the “transcriptome,” is normally attempted by collecting large numbers of ESTs from diverse cDNA libraries. Currently, there are more than 17 million plant ESTs in the public database (http://www.ncbi.nlm.nih.gov/dbEST/) with ~5 million coming from current transgenic crops and 7 million from future transgenic crops. To date, there are 1,220,876 rice ESTs in the public database. Recent advances in the technology for construction of full-length cDNA libraries have made it possible to produce more than 30,000 rice full-length cDNAs (Kikuchi et al. 2003; Satoh et al. 2007). This has helped in improving the rice genome annotation, gene organization and genome-wide expression profiling. One other significant EST and full-length cDNA collection from indica rice comes from the Beijing Genomics Institute (BGI), which can be viewed through BGI-RIS (http://rice.genomics.org.cn/index2.jsp). Genome-wide expression profiling (including differential expression) of genes in various crop species is being facilitated by high-throughput techniques, such as microarrays, serial analyses of gene expression (SAGE), massively parallel signature sequencing (MPSS) and more recently by ultra-deep sequencing (e.g., 454, Solexa and SOliD technologies). These procedures are typically used to compare two mRNA populations derived from tissues of different developmental stages or those subjected to different environmental stimuli, to yield information on the comparative changes in gene expression in each tissue. The conceptual basis of this method is that genes contributing to the same biological process are likely to exhibit similar expression patterns and thus allow the putative assignment of gene function. With all these new developments in deep sequencing technologies, we are seeing an explosive increase in the RNA expression tag and small RNA datasets from diverse plants under different environmental conditions and experimental treatments. This will help in unraveling the complexities of the transcriptome, including that of non-coding RNAs, in diverse biological systems. Thus, it is now possible to study spatial and temporal RNA expression patterns which could provide insights into their cellular and developmental functions. The regulatory and developmental functions of a transgene could also be studied using these techniques.

10.4.3

Gene Expression Studies at the Protein Level

With recent advances in high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), staining, detection, peptide micro-sequencing and associated computer software, “proteomics” is also emerging as a powerful functional

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genomics tool. Here, instead of looking at gene expression, an assessment is made on the gene product, i.e., the protein. In the 2D-PAGE-based approach, intact proteins are separated by 2D-PAGE and protein abundance is determined by the relative stain intensities of protein spots on the gel. The differential proteome is confirmed by image analysis. The identity of a specific protein is generally determined by mass-spectrometric (MS) analysis of peptides after proteolysis of the protein spot or by protein sequencing after blotting the gel to a membrane. Comparison of the amino acid sequences of fragments of proteins with those predicted from DNA sequences greatly facilitates not only the validation of gene predictions but also provides insight into the cellular and developmental regulation of gene expression. Several public databases of 2D-PAGE-derived plant proteins are already available, such as WORLD-2DPAGE (http://expasy.org/ch2d/2d-index.html), Rice Membrane Protein Library (http://wardlab.cbs.umn.edu/rice/) and the Rice Proteome Database (http://gene64.dna.affrc.go.jp/RPD/), that provide extensive information on the progress of rice proteome research. In addition, progress is being made in detecting post-translational modifications such as glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation and proteolytic cleavage. Differential proteome analyzes of a particular transgenic plant and an appropriate non-transgenic plant (i.e., a segregating null and/or wild type plant) could highlight any unintended or flow-through effect of the transgene on the expression of other genes.

10.4.4

Metabolomics

Metabolomics is the comprehensive analysis of low-molecular-weight compounds in biological samples and is emerging as a biochemical phenotyping tool along with transcriptomics and proteomics in functional genomics (Tarpley and Roessner 2007). Technologies used in metabolomics are normally based on the chromatographic separation of complex compound mixtures, using either liquid or gas chromatography and mass-spectrometric detection. Nuclear magnetic resonance (NMR) spectroscopy is also playing a major role in metabolomic approaches. Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS) can mass-resolve metabolites with a mass accuracy of <1 ppm (Dunn et al. 2005), and thus provides a high-throughput method for metabolite fingerprinting. This technique has been used in conjunction with proteomics for comparing leaves, panicles and calli of wild type rice (cv. Nipponbare) and a transgenic line over expressing the YK1 gene, which is a homolog of the maize HC-toxin reductase gene (Takahashi et al. 2005). The YK1 gene is known to confer increased tolerance to rice blast and multiple environmental stresses. Although the global composition of organ-specific metabolites did not differ significantly between the two lines, alterations in less than 10% of the metabolites were observed. The transgenic line expressed several previously reported stress-responsive proteins suggesting that ectopic overexpression of a single gene (YK1) can affect the expression of unrelated proteins and metabolites.

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The current limitations of routine metabolomic analyzes, as a part of plant functional genomics programs, include access to expertise in the techniques of plant metabolomics, the necessary instrumentation, the establishment of adequate sample preparation procedures, the need forcoordinated comprehensive cataloging and/or control of environmental variables, the availability of databases providing storage and access to metabolome-specific data (Tarpley and Roessner 2007).

10.5

Transgenic Plants in Functional Genomics

Transgenic plants are being used in several functional genomics strategies. These strategies can be broadly classified into two categories, namely forward genetics and reverse genetics. The forward genetics strategies include gene disruptions (knock-outs) with T-DNA and/or transposon insertions, gene/enhancer trap (with promoter-less reporter genes) insertions, gene activations with promoter/enhancer insertions and gene deletions with site-specific recombinases. The reverse genetics strategies include site-selected insertional mutagenesis and gene knock-downs with RNA-silencing transgenes and gene activity disruption with modified genes producing mutant proteins.

10.5.1

Forward Genetics Strategies

10.5.1.1

Insertional Gene Disruptions (Knock-Outs)

One of the most direct approaches to determine gene function is the production of insertion mutations and the study of their effects on the plant phenotype. Alterations in a plant phenotype, as a consequence of the mutation, may then provide insight into the gene’s function. As the inactivated gene in such plants contains a known DNA insertion sequence, it is a relatively simple task to isolate the gene as it has been effectively “tagged” by the inserted sequence. Such tagging can be achieved by employing both non-transgenic and transgenic strategies. Endogenous transposons or “jumping genes” (both autonomous elements and their non-autonomous counterpart elements) such as Activator (Ac)/Dissociation (Ds), Enhancer (En)/ Inhibitor (I) (also known as Suppressor-Mutator or Spm/dSpm) and Mutator (MuDR/Mu) in maize, or retrotransposons such as Tos17 in rice have been used to generate insertional mutants by non-transgenic means. Transgenic strategies include Agrobacterium-mediated T-DNA insertions and heterologous transposons delivered through T-DNA. In both cases, plants can be initially screened for changes in phenotype (Fig. 10.1). One can then clone the mutated gene using the inserted DNA tag as a reference point (commonly referred to as flanking sequence tags or FSTs) and compare its sequence to sequences in the genome databases, thus linking the mutant phenotype with a known gene sequence.

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Rice lines each with a tag disrupting a different gene

Any change due to gene disruption associates function with gene sequence

Screen lines for changes in plant form or function

FUNCTIONAL GENE TAG DISRUPTED GENE TAG

Clone

Compare to sequence database

GENE MACHINE Poor leaf development

No flower formation

Stunted roots

Library of tagged lines and gene sequence information

Fig. 10.1 A schematic diagram describing gene tagging and identification in rice

For crop plants, such as rice, having efficient tissue culture, generation and transformation systems, T-DNA has emerged as the preferred insertion mutagen for generating large random libraries of insertional mutant lines. Research groups in Korea, China, France and Taiwan are generating T-DNA insertion libraries, characterizing T-DNA flanking sequences at insertion points and gathering phenotypic information in web-accessible databases (Hirochika et al. 2004; Guiderdoni et al. 2007; Krishnan et al. 2009). To date, more than 460,000 T-DNA lines and ~118,000 FSTs have been produced. Although these resources will be useful for reverse genetics, there are limitations in obtaining T-DNA insertions in smaller genes such as single-exon genes, which may account for up to 40% of the genes in rice. Furthermore, a large proportion of mutant phenotypes could be due to tissue culture-induced, non-tagged “somaclonal” mutations. Non-tissue culture transformation techniques such as seed transformation (Feldmann 1991), vacuum infiltration (Bechtold and Pelletier 1998) or floral dipping (Clough and Bent 1998) have partly overcome this problem in Arabidopsis. However, these techniques are yet to be made workable in common transgenic crops. The T-DNA system can also be used to deliver jumping genes or transposons, which will cause many random insertions once activated. Although there are quite a

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few groups of transposons (most of them originating from maize) being used in the several plant systems, we will confine our discussion to the use of the maize transposon Ac and its derivative Ds. The Ac element can excise and integrate randomly throughout the plant genome (although it tends to transpose close to its original position in the chromosome). A protein called transposase produced by Ac mediates these transpositions. A deletion derivative of Ac called Dissociation (Ds) has the capacity to jump but only in the presence of Ac, as it lacks the capacity to produce its own transposase. By removing certain sequences from Ac it can be made immobile (iAc), however it can still produce transposase. Since the first report of the activity of the autonomous Ac element in transgenic rice 18 years ago, sophisticated transposon tagging systems have been developed to improve both tagging and screening efficiencies in rice (Zhu et al. 2007) and other crop plants, and are primarily based on the two-component iAc/Ds (Chin et al. 1999) or En/I (Greco et al. 2004) systems. Since the successful cloning of a gene (BFL1/ FZP), which mediates the transition from spikelet to floret meristem (Komatsu et al. 2003; Zhu et al. 2003) several genes have been cloned by transposon tagging in rice. The process of producing stable Ds insertion lines in the two-component Ac/Ds system is illustrated in Fig. 10.2. Essentially, this involves the production of transgenic Ac lines and Ds lines by Agrobacterium-mediated transformation, followed by the production of Ac/Ds mutagenic lines either by crossing or by cotransformation or super-transformation. In this mutagenic population, under the influence of a transposase produced by iAc, the normally-stable Ds element starts to jump or undergo transposition. This transposition will continue while the iAc is present. In the subsequent generations, the iAc can be segregated from Ds elements that have integrated into new regions of the genome. Plant lines containing these stable Ds insertions in new genomic locations are then analyzed. Regions flanking the Ds element are then cloned and sequenced to create a database of flanking sequences, or molecular flags, that represent disrupted genes. Public sequence databases are then searched for similar sequences. A wide variety of Ac/Ds gene constructs have been produced by several research groups (Zhu et al. 2007). The additional features of these Ac and Ds constructs are that they have improved tagging and screening efficiencies. These features include a negative selection gene or a visual marker gene for Ac, a herbicide-resistance gene for Ds selection or an antibiotic gene to detect Ds transposition, inducible or developmental-specific promoters to control transposase activity to maximize germinal transposition, and removal of Ac after Ds transposition by site-specific recombinases. These features greatly assist in the elimination of plants without Ds insertions and positively select for plants with stable Ds insertions in different genomic locations. A novel method of producing stable Ds insertion lines, using a transientlyexpressed transposase (TET) system, has been developed (Upadhyaya et al. 2006). Constructs suited for high-efficiency insertional mutagenesis in general, and the TET system in particular, have also been developed. By super-infecting callus tissue from single-copy Ds/T-DNA lines, having both Ds excision and

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(I)

N

P

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(II)

(III)

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P

(No mutant phenotype) Gene

P

Ac

R

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T

S

S

R

T

Ds (mutant phenotype)

(IV)

a

b

– GA3

+ GA3

Fig. 10.2 The two-component Ac/Ds based gene (DsG) and enhancer (DeE) trapping system for rice. (I) Ac and Ds constructs are first delivered to rice by Agrobacterium-mediated transformation with hph as the selectable marker (S). The Ac construct also contains the negative selector such as tms2 (N) while the Ds construct contains sgfpS65T (R) as the launching pad reporter gene. (II) Selected homozygous progeny (containing single-copy Ac and Ds) are crossed. Alternatively, this can be achieved by co- or super-transformation. (III) In the F1 progeny of the crosses or T0 plants of double transformants, Ds under the influence of the Ac transposase is excised from the original location (launching pad) and reinserts into a new genomic location. The insertion in a coding region can lead to gene expression knockout thus producing an insertional mutant. Progeny seedlings with stable Ds (unlinked to Ac), either linked to the original launching pad (GFP +ve) or unlinked to the Ds launching pad (GFP +ve) are identified using negative selection via the use of naphaleneacetamide (NAM) selection. (IV) An acute dwarf mutant obtained in the screening population was later identified as having an insertion in the ent-kaurene synthase gene, the second gene in the GA biosynthetic pathway (Margis-Pinheiro et al. 2005). Panel A contains homozygous dwarf plant and the normal looking heterozygous plants. Panel B contains mutant plants with or without GA3 treatment showing the GA3 responsiveness

re-insertion markers, with Agrobacterium harboring iAc constructs containing a visual marker, sgfpS65T, stable Ds insertion lines have been regenerated at a frequency of ~5%, in addition to iAc/Ds double transformants (Upadhyaya et al. 2006). Mapped single-copy Ds/T-DNA launch pads produced using these

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constructs are highly suitable for efficient chromosomal region-directed insertion mutagenesis (Upadhyaya et al. 2006). Insertional mutagenesis by transposons has distinct advantages over that by T-DNA insertion mutagenesis. Large-scale, transposon-mutagenized populations can be produced using a relatively-small number of starter lines, because many independent insertions can be generated among the progeny of a single parental line. The tagged gene can be confirmed by revertants resulting from excision of the transposon due to secondary transposition. Transposons can also be remobilized to produce new insertion lines in order to target closely-linked genes in a specific chromosomal region, i.e., corresponding to a mapped quantitative trait locus (QTL). The iAc/Ds-based systems in rice yield 5–10% unique stable insertion mutant lines in the progeny of mutagenic lines (Zhu et al. 2007). The frequency of Ds re-insertions linked to the original Ds/T-DNA launch pads vary from 36 to 67% with the majority being within one cM of the Ds launch pad (Zhu et al. 2007). One potential drawback is that quite often a secondary transposition of Ds could leave a DNA footprint (scar) at the initial transposition site which could lead to an untraceable mutation. To validate phenotype and gene sequence relationships, complementation experiments can be carried out by introducing the corresponding wild type sequence into the mutant line as a transgene. The availability of multiple mutant alleles will also facilitate the validation. Alternatively, an RNAi system can be employed to determine whether the mutant phenotype can be mimicked by a targeted gene expression knock-out (discussed later in this chapter).

10.5.1.2

Insertional Enhancer/Gene Traps

Under normal growth conditions at a given growth stage, less than 3% of the insertion lines have obvious phenotypes. Some of the subtle mutants require specialized screening to visualize the phenotype and other conditional phenotypes can be revealed only when challenged with appropriate environmental cues such as biotic and abiotic stresses. The other major cause of the “phenotype gap” is functional redundancy wherein two or more genes have the same function. The expression patterns of disrupted genes can however be visualized by placing a “reporter gene” in the tagging element (T-DNA or transposon) as illustrated in Fig. 10.3. When such a promoter trapping element is inserted downstream of the promoter of a given gene, it results in reporter gene expression with a pattern mimicking that of the disrupted gene. Similarly, it is possible to detect enhancer elements in the vicinity of a gene by using a tagging element with a reporter gene also containing a minimal promoter. Enhancers can activate genes from a distance in an orientation-independent manner and thus the frequency of enhancer detection is normally high. Quite often, expression patterns reveal more about the functional category of the disrupted gene. This would facilitate further characterization of the gene with subsequent specialized screening. However, it should be noted that the identity and location of the target gene is sometimes difficult to

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a

b Enhancer Trap Ds (DsE)

Gene Trap Ds (DsG)

Trap reporter (gus) Plasmid rescue (bla, ori) Trap tracer (nptII or bar) Ds

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Ds

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Ds gene::gus RNA intron splicing

GUS RNA Complete or partial gene disruption GUS protein c1

GUS expression c2

GENE::GUS protein c3

Fig. 10.3 Ds enhancer and gene trapping systems. The Ds enhancer (DsE) trap (a) or the Ds gene (DsG) trap (b). Both the constructs used contain uidA (gus) as a trap reporter, the ampicillin resistance gene bla, E. coli origin of replication (ori, for one step cloning of flanking sequences by plasmid rescue) and nptII or bar as a tracer. The DsE contains minimal transcriptional activator (TA) sequences in front of gus. When DsE is inserted downstream of the promoter/enhancer elements (P/E) of a particular gene, these elements along with the TA activate gus transcription resulting in GUS expression faithful to the trapped promoter or enhancer. DsG contains an intron with splice acceptors (in all three reading frames) in front of gus. DsG trapped insertion in an intronic region of a particular gene (G) may result in correct splicing of the RNA (between the splice donors of the intron disrupted and the splice acceptors in DsG), producing a GENE::GUS fusion. As seen with DsE, the GUS expression pattern mirrors the activity of the gene disrupted depicted in root tip (c1), leaf vascular (c2) and leaf non-vascular (c3) specific GUS expression in different lines. Such insertions in some cases result in loss of the gene product or produce disfunctional gene product may lead to mutant phenotypes. Flanking sequences are then cloned and sequenced for further characterization

determine, especially with poorly or incompletely sequenced and annotated genomes. Another commonly-used type of tagging is “gene trapping.” Here the reporter gene is carried within the tagging element along with an intron with splice acceptor sites in all three potential reading fames. Insertion of such a “gene trapping” element within an intronic region of an endogenous gene may result in the splicing

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of this hybrid intron (i.e., the region between the splice donor of the disrupted gene and the slice acceptor in front of the reporter gene) resulting in the production of a hybrid endogenous gene/reporter gene transcript. Under these circumstances, the reporter gene expression mirrors the expression of the disrupted gene. Most of the gene-trapping constructs contain b-glucuronidase (gus) as a reporter gene, the expression of which is visualized by the addition of a chromogenic substrate, which can be broken down by the reporter gene protein into an insoluble colored product (indigo). Most of the T-DNA and transposon (Ds or I) constructs used as insertional mutagens have been modified to act as gene traps or enhancer traps. Gene trapping efficiencies of ~6% have been reported for these constructs in rice (Hirochika et al. 2004). The efficiency of T-DNA gene trapping depends on the frequency of “clean” T-DNA insertions, i.e., insertions devoid of direct or inverted T-DNA repeats, or of vector backbone (VB) sequences derived from outside the T-DNA borders (Sallaud et al. 2004; Upadhyaya et al. 2006). A “clean” Ds-containing T-DNA is also essential for the satisfactory mobilization of Ds.

10.5.1.3

Insertional Gene Activations

Any plant genome, including that of rice, contains a large number of dormant gene sequences, pseudogenes or genes with suboptimal cellular, spatial and developmental expression patterns. Activation tagging systems allow the trapping of these cryptic genes. Activation tagging involves introducing foreign DNA containing specific gene promoters and control sequences throughout the genome using random T-DNA or transposon insertions. Some of the different activation tagging systems being used are represented schematically in Fig. 10.4. In the classical activation tagging approach, random insertions of a cauliflower mosaic virus (CaMV) 35S enhancer element into the genome can result in the overexpression of native genes (or even dormant genes) in all cell types of the plant. Such increased gene expression can create mutants of essential and redundant genes that are either not present, or have no phenotype in knock-out collections. This gain-of-function approach reveals dominant mutations affecting the transcriptional control of genes, without altering the functional gene product. A sizable number of T-DNA activation tagged lines have been produced by research groups in France, Korea, China and Taiwan (Guiderdoni et al. 2007). Further refinement of activation tagging comes from the development of extensive GAL4 enhancer trapping resources in rice, which enable transgene expression to be targeted to specific cell types (Johnson et al. 2007). In the first step of a two-step process known as “transactivation,” a large number of GAL4 enhancer trapping “driver” lines are generated and the patterns of reporter gene expression are characterized. “Responder” lines are then produced in which genes of interest are placed downstream of the upstream activator sequence (UAS) element to which GAL4 binds. In progeny plants of crosses between the driver and the responder lines, the target genes are transactivated by GAL4 revealing the specific expression profile

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of each individual driver. In addition, random deployment of the UAS element into the rice genome, followed by crosses to specific driver lines, should enable activation tagging to be carried out in specific cell types of the plant. Cell typespecific activation tagging has the potential to uncover novel mutations that are missed or “averaged out” by the classical activation tagging technique (Johnson et al. 2007).

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Gene Targeting and Site-Specific Deletions

Gene targeting refers to the in vivo modification of a specific endogenous gene sequence to alter its function. Homologous recombination-dependent gene targeting is being used routinely in bacteria, yeast and lately in mice (Evans et al. 2001) to create precise deletions, insertions or mutations of DNA sequences within their native chromosomal contexts. The routine use of gene targeting in plants, however, has not been achieved yet because of the very low frequency of homologous recombination in plant cells (0.01–0.1% of homology-independent random illegitimate recombination). Despite numerous efforts made over the years, there are only three reports of the production of fertile transgenic plants with reproducible endogenous gene targeting, including two genes in Arabidopsis and one gene in rice (Hanin et al. 2001; Terada et al. 2002; Shaked et al. 2005). A few of the approaches tried, in order to overcome the barrier imposed by illegitimate recombination, are: (1) positive (for integration events including true gene targeting) and negative (for T-DNA border-associated random integrations) selections (Terada et al. 2002), (2) enhancing homologous recombination relative to illegitimate recombination by manipulating the plant’s recombination machinery. Examples of the latter are the overexpressions of Escherichia coli recA or ruvC genes in plants and the disruption of plant RAD50 homologs (Shalev et al. 1999; Reiss et al. 2000; Gherbi et al. 2001). Chromosomal breaks at the target site have been shown to stimulate the cell’s DNA repair system and, if a homologous template is present, this repair is achieved through homologous recombination. For example, enzymatic cleavage or Ac/Ds excision have been shown to increase recombination frequencies by 100–1,000 fold in plants (Reiss 2003). Recently, chimeric zinc-finger nucleases (ZFNs) have been used to create sitespecific chromosome breaks in the absence of pre-engineered target sites (Bibikova et al. 2003). Zinc-finger nucleases have a DNA recognition domain composed of an array of Cys2–His2 zinc fingers. The zinc fingers recognize and bind to specific nucleotide triplets. Zinc fingers are available that recognize all GNN and ANN and some CNN and TNN triplets, and multiple zinc fingers can be joined together to generate DNA-binding arrays that recognize extended sequence patterns with great specificity and high affinity. Fused to the zinc-finger array is a nuclease, typically a nonspecific cleavage domain from a type IIS restriction endonuclease such as Fok I (Kim et al. 1996). Fok I functions only as a dimer, and this has been capitalized upon to enhance the target specificity of the ZFN. Each Fok I monomer is fused to a zinc-finger array that recognizes a different DNA sequence; only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme. By requiring DNA binding to activate the nuclease, a highly site-specific restriction enzyme is created. Using a tobacco test system Wright et al. (2005) have shown that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective gus:nptII reporter gene (also containing a ZFN recognition site) integrated at various chromosomal sites in different transgenic tobacco lines. Protoplasts from each transgenic line

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were electroporated with DNA encoding the nuclease and donor DNA to effect repair of the reporter gene. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter gene’s chromosomal position. Approximately 20% of the gus:nptII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end-joining. Using this strategy, it is possible to engineer the DNA-binding domain encoded by zinc-finger nucleases to recognize a variety of chromosomal target sequences in order to achieve high frequency gene targeting. Thus, such transgenic strategies are currently being aggressively employed in gene targeting studies in plants. If successful, gene targeting will have tremendous application in plant functional genomics as well as in controlled transgene “docking” or even transgene “upgrades” for the sustainable deployment of transgenes of agronomic importance. Specific genomic deletions are also useful in functional genomics. Heterologous site-specific recombination systems like the bacteriophage P1 Cre-lox and the yeast FLP–FRT systems have been shown to work in plants to generate specific deletions (van Haaren and Ow 1993). Here, recombination occurs between two lox or FRT sites, mediated respectively by the Cre- or FLP-recombinases. These systems can also be delivered through transposons. For example, constructs carrying lox recombinase sites, both within the transposon (Ds-lox) and in the adjacent T-DNA have been used to produce small deletions in lines with Ds-lox transposed to closelylinked positions (Osborne et al. 1995). A Cre-lox site-specific recombination system has also been used to remove Ac from Ds transposants by triggering cre recombinase expression with Ds excision (Shaohong et al. 2004). The elimination of the Ac transposase gene helps to stabilize the transposed Ds elements in the rice genome.

10.5.2

Reverse Genetics Strategies

10.5.2.1

Site-Selected Insertions

In plant populations saturated with multiple endogenous transposon copies, it is possible to identify knock-out mutations in a specific gene. For example, in maize, snapdragon and petunia (Coen et al. 1989; Gerats et al. 1990; Walbot 1992) such populations have provided genome saturation and knock-out mutations in specific genes (Das and Martienssen 1995; Koes et al. 1995). Similarly, deletion mutant populations generated by chemical and physical mutagens can be used to identify mutants in specific genes by employing high-throughput screening methods including PCR screening and targeting induced local lesions in genomes (TILLING). Using the transgenic approach with T-DNA or Ac/Ds, it is theoretically possible to achieve this saturation with substantial numbers of tagged lines, from which DNA pools can be prepared. It is then possible to screen for mutations in a

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particular gene by polymerase chain reaction (PCR) analysis, using pooled DNA as a template and a gene-specific primer in combination with an insertion sequencespecific primer. Recovered mutants can then be subjected to custom screening for visible/obvious phenotypes. This type of reverse genetics approach can be very powerful in identifying the functions encoded by unknown sequences which are predicted to be important by other methods. However, the chance of recovering an insert in a target gene is dependent on the population size of the insertion lines, the size of the genome and the size of the gene target. The two types of populations that are currently being created in different laboratories worldwide include: (1) those with single- or low-copy stable insertion elements such as T-DNA, Ds and I, and (2) those with multiple actively-transposing elements such as Ac or En. In a population of ~110,000 Arabidopsis random insertion mutants one would expect an insert every kb, with a ~99% chance of mutating a gene of 5 kb (Krysan et al. 1999). The number of insertion mutants needed to tag every gene in rice is estimated to be between 180,000 and 460,000 (Hirochika et al. 2004; Krishnan et al. 2009). Larger genomes such as barley, wheat and maize would require much larger numbers of insertion lines.

10.5.2.2

Gene Knock-Downs with Silencing Transgenes

RNA silencing or gene silencing is a broad term used to describe mechanisms that interfere with gene expression in most eukaryotic organisms. This interference occurs either by suppression of gene transcription (transcriptional silencing) or the initiation of sequence-specific mRNA degradation or inhibition of RNA translation (post-transcriptional gene silencing or PTGS). RNA silencing has evolved to a high level of sophistication in the plant kingdom and is intimately involved in viral defense, suppression of transposon activity, control of chromatin modification and regulation of expression of genes involved in plant development (Waterhouse et al. 2001). RNA silencing mechanisms may have several parallels with the immune system of animals and there is evidence to suggest that it is likely to have been a major factor in the evolution of eukaryotes from prokaryotes (Margis et al. 2006). Gene silencing has now become a powerful transgenic technology to selectively suppress gene activity. Through the PTGS process, it is possible to block the expression of endogenous genes by introducing synthetic gene constructs that cause RNA interference (RNAi). These transgenes produce double-stranded RNA transcripts having sequence identities with their target genes that specifically trigger the degradation of endogenous gene transcripts. Theoretically, a specific gene or a set of genes, of unknown function, can be selectively silenced and the consequence of such “expression knock-outs” in the form of a phenotype can be studied. However, quite often structurally-similar genes, having functions, which are spatially and developmentally controlled in different ways, may be silenced simultaneously. This makes the functional characterization of individual genes potentially more difficult. One way of circumventing this problem is to ensure that the

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Fig. 10.5 Types of transgene constructs for RNA silencing in plants. (a) A plasmid containing infectious Potato virus X (PVX) cDNA can be transcribed in vitro and inoculated onto the plant. A component of the PVX cassette contains an inserted region of sequence from the targeted gene (Helliwell and Waterhouse 2003). (b) A typical T-DNA plasmid that can express hairpin RNA in plants. This construct can be introduced into the plant by DNA bombardment or stably transformed by Agrobacterium-mediated transformation. The latter method requires a selectable marker. (c) A T-DNA plasmid similar to the one above can express RNA with the target gene sequence located upstream of the hairpin structure. This vector can potentially be used for high-throughput screening with a cDNA library. (d) The general structure of the pANDA construct with the Gateway vector conversion system cloned in an anti-sense and sense direction and separated by gus linker. PCR products corresponding to the targeted gene are cloned into the pENTRO/D-TOPO vector followed by a LR clonase reaction to produce the final construct for transformation into rice

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sequence selected for targeting is specific to an individual gene. On the other hand, sequence-specific knock-outs can be used to block the expression of a whole class of genes involved in a particular process by targeting a common conserved sequence present in such gene families. Perhaps the immediate use of RNAi technology is in elucidating the functions of genes, which otherwise would show lethal phenotypes with insertion mutants. This is because PTGS causes a reduced level of gene expression rather than a complete gene inactivation. The molecular basis of PTGS remains to be fully elucidated despite recent significant advances made in understanding the different silencing pathways namely, (1) microRNA and trans-acting siRNA, (2) repeat-associated siRNA and RNA-directed DNA methylation and the various key proteins involved in these pathways (Curtin et al. 2007). A number of gene-silencing platforms have been developed for delivering gene silencing through transgenes in plants including sense and antisense transgenes, amplicon transgenes, hairpin RNA transgenes, direct-repeat and 30 -inverted repeat transgenes, and artificial miRNA transgenes. These are described in Fig. 10.5. Several plant single-stranded RNA (ssRNA) viruses have also been effectively used as silencing vectors. Virus-induced gene silencing (VIGS) was first demonstrated in tobacco with an infectious tobacco mosaic virus (TMV) clone (Kumagai et al. 1995). In principle, VIGS is achieved by producing a recombinant infectious virus containing a 300–800 nucleotide plant target gene sequence. Viral infections can be established with purified viral RNA in the absence of viral coat proteins. Viral RNA transcripts, synthesized in vitro from a plasmid containing a cDNA encoding the recombinant virus genome, have been widely used to initiate virus infections. Alternatively, the recombinant viral cDNA can be cloned into T-DNA vectors (with appropriate promoters) and delivered to the plant via Agrobacterium infection. Inside the plant cell, virally encoded RNA-dependent RNA polymerase generates both sense and antisense recombinant viral RNA (containing the target gene sequences) that has the potential to hybridize to form dsRNA and thereby trigger PTGS mechanisms to induce target gene silencing. Several plant viruses such as potato virus X (PVX), tobacco rattle virus (TRV), cabbage leaf curl virus (cbLCV) and tobacco mosaic virus (TMV) can be used to induce PTGS in various plant species. There are several advantages to using the VIGS system over other methods such as insertional mutagenesis or transgene-derived hpRNA. VIGS is a rapid method

<

Fig. 10.5 (continued) (Miki and Shimamoto 2004). (e) Multiple direct repeats of chloramphenicol acetyltransferase (CAT) and gus gene sequences were shown to trigger efficient PTGS called direct repeat-induced PTGS (driPTGS). (f) Schematic representation of the SHUTR construct containing an inverted repeat of the 30 -untranslated region from the Agrobacterium nos gene. (g) Artificial miRNAs are constructed using overlapping PCR on an endogenous miRNA precursor. Primers are designed to replace the existing miRNA and miRNA* sequences with artificial sequences (gray). The artificial miRNA is generated by combing all three PCR products A-IV, II-III and I-B in a single reaction with primers A and B (Schwab et al. 2006). (h) VIGS vectors (reproduced from Curtin et al. 2007)

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for generating multiple mutants (in either related or unrelated genes) and can be applied to monocotyledonous plants such as barley and polyploids such as wheat (Scofield et al. 2005). VIGS can be applied to both mature and juvenile plants to induce the silencing of embryogenesis-related genes and genes required for germination, which may be intractable by other methods. VIGS can also be applied to plants, which are difficult to transform. Some of the limitations of VIGS are the non-availability of viral infectious clones for all crop plants (host range), masking of the phenotype by viral symptoms, quarantine issues and target sequence size restrictions (Watson et al. 2005).

10.5.2.3

Gene Activity Disruption with Mutant Transgenes

Transgenes encoding mutant proteins, especially regulatory proteins, have the potential to disrupt the activity of the endogenous wild type protein thus producing a dominant negative mutant phenotype (Herskowitz 1987). Such a transgenic approach to introduce dominant mutations was demonstrated for the Arabidopsis MADS box gene AG, with the overexpression of specific protein domains (Mizukami et al. 1995), thus providing an insight into protein regulatory mechanisms in flower development.

10.6

Conclusion and Future Prospects

The commercial successes with the first wave of genetic engineering involving herbicide and/or insect resistance have provided great impetus for transgenic research worldwide in a wide variety of traits and crops. The scientific community by and large is optimistic about the potential of transgenic crop plants, not only in alleviating major production constraints such as insect pests, pathogens, salinity and extremes of temperature and drought, but also in producing designer crops with high product value. Besides Intellectual property and regulatory constraints, and consumer perception issues, there are still some unresolved technical limitations associated with plant transgenic technology. For many plant species, efficient transformation is still highly genotype dependent and the “useful transformation” frequency is still very low. This could be due to the use of inadequate promotergene combinations, integration position effects, insertional inactivation of endogenous genes, somaclonal variation, transgene silencing or pleiotropic effects arising from expression of the introduced transgene. The expectation is that functional genomics will reveal novel genes and gene control sequences conferring more complex traits such as abiotic stress tolerance, yield, vigor and nutritional quality. As a foundation for functional genomics, genome sequencing has been completed for two model plants, Arabidopsis and rice. Sequencing (DNA and cDNA) efforts are now underway for several other so-called transgenic crop plants.

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Functional genomics has also expanded the scope of biological investigation from a one-gene approach to a more system-based holistic approach encompassing genome, transcriptome, proteome and metabolome. Such an approach will undoubtedly provide considerable, and possibly dramatic, insights into the epistatic interactions between seemingly unrelated genes, which account for optimal plant growth, performance and productivity. Because of the objective nature of these powerful new bioinformatics technologies, they will also reveal a number of novel, and potentially-valuable strategies for the design of future food crop plants designed to keep pace with the uncertainties of predicted climate change. Transgenics has become an integral part of the various functional genomics strategies being employed. For example, transgenically produced insertion lines and the flanking sequences of model plants (rice and Arabidopsis) will enable the scientific community to find one or more insertions in any given gene. Following the association of a phenotype with a specific gene, the level or pattern of expression of that gene can be altered to achieve the desired effect. This can be done using transgenic methods to either reduce gene expression by RNAi technology, or to overexpress the gene of interest using suitable promoters. These specifically altered lines can then be incorporated into breeding programs. These transgenic solutions may be used if public antipathy to GM food crops can be overcome. Until then, novel gene sequences can at least be used as useful molecular markers in classical breeding efforts. Acknowledgments The authors wish to thank Dr. Jake Jacobsen for critical reading of the manuscript.

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van Haaren MJ, Ow DW (1993) Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning. Plant Mol Biol 23:525–533 Walbot V (1992) Strategies for mutagenesis and gene cloning using transposon tagging and T-DNA insertional mutagenesis. Annu Rev Plant Physiol Plant Mol Biol 43:49–82 Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence against viruses. Nature 411:834–842 Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA silencing platforms in plants. FEBS Lett 579:5982–5987 Wright DA, Townsend JA, Winfrey RJ, Irwin PA, Rajagopal J et al (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705 Yu J, Hu S, Wang J, Wong G, Li S et al (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79–92 Yu J, Wang J, Lin W, Li S, Li H et al (2005) The genomes of Oryza sativa: a history of duplications. PLoS Biol 3:e38 Zhu QH, Hoque MS, Dennis ES, Upadhyaya NM (2003) Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L). BMC Plant Biol 3:6 Zhu Q-H, Eun MY, Han C-D, Kumar CS, Pereira A et al (2007) Transposon insertional mutants: a resource for rice functional genomics. In: Upadhyaya NM (ed) Rice functional genomics challenges, progress and prospects. Springer, New York, USA, pp 223–271

Chapter 11

Deployment: Regulations and Steps for Commercialization Kelly D. Chenault Chamberlin

11.1

Introduction

Genetically modified (GM) crops produced by genetic engineering continue to increase in production worldwide. In 2007, 23 countries produced 282.3 million acres of GM crops, up 30.3 million acres since 2006 [International Service of AgriBiotech Applications (ISAAA)]. The US ranks first in GM crop production, followed by Argentina, Brazil and Canada, in that order. The potential for economic and social gain from the production of GM crops is generally greater in developing countries/economies, since there is usually a higher incidence of disease/pests and larger potential for yield increase (Abdalla et al. 2003; Qaim 2005). According to Nossal et al. (2008), GM crop production in 2007 increased by 20% in countries with emerging economies versus 6% in developed countries. There is no denying that in today’s economy, there is a great demand to decrease production costs and increase yield, advantages that are offered by biotechnology. However, before any GM crop can enter the public domain, it must first be approved or “de-regulated” for commercial production and public consumption. The emergence of GM crops that offer clear economic and/or health advantages to producers and consumers has forced different countries to develop regulations governing the release of transgenic plants for experimental or commercial purposes. This chapter discusses the regulatory frameworks and policy principles governing GM crop research and production in those countries playing major roles in worldwide GM crop production.

K.D. Chenault Chamberlin Wheat, Peanut, and Other Field Crops Unit, USDA-ARS, 1301 N. Western, Stillwater, OK 74075, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_11, # Springer-Verlag Berlin Heidelberg 2010

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GM Crop Regulation in the United States History

Oversight of genetically modified organisms (GMOs), including transgenic crops, began in the US in the early 1970s with the advent of recombinant DNA (rDNA) technology. The scientific community was first to recognize the huge potential and possible risks associated with rDNA technology and outlined its own internal operation guidelines in a meeting among concerned scientists held in Asilomar, California, in 1975 (Berg et al. 1981). Shortly thereafter the US federal government followed with their own set of rules regulating rDNA research in projects funded by the government agencies such as the National Institutes of Health (NIH), United States Department of Agriculture (USDA), Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) (NIH 1976, 1978). As it became apparent that crops would be improved using rDNA technology, groups began to seriously examine the impact of the release of transgenic crops and the need for regulatory action. In the early 1980s, several reports were issued on the risks of transgenic plants by various organizations including the Organization for Economic Co-operation and Development (OECD) and the National Institute of Health (NIH) (OECD 1982; NIH 1983). These concerns coincided with the first official report of transgenic plants, which described transgenic tobacco, which was resistant to methotrexate and kanamycin (Herrera-Estrella et al. 1983; Schell et al. 1983). The rapid advances being made by rDNA research prompted the US federal government to form a committee at the Office of Science and Technology Policy (OSTP) to create regulatory policy regarding rDNA research and products. The result was a publication by the OSTP in 1986 (http://www.ostp.gov) outlining the approach to be taken in regulating rDNA products, the basic concept of which is still being used to regulate transgenic crops in the US today. The OSTP urged regulators to focus less on the rDNA process and more on the risks of the products of such research and thus assigned regulatory control to the relevant federal agencies of the USDA, FDA and EPA. The roles of each agency are outlined in Table 11.1. All three of these agencies have adopted this “product-based” approach

Table 11.1 US regulatory authorities for agricultural biotechnology products Agency Jurisdiction Laws USDA Plant pests, plant veterinary biologics Federal Plant Pest Act (FPPA) FDA Food, feed, food additives, veterinary Federal Food, Drug, and Cosmetic Act drugs, human drugs, medical devices (FFDCA) EPA Microbial and plant pesticides, new uses Federal Insecticide, Fungicide, and of existing pesticides, novel Rodenticide Act (FIFRA); FFDCA, Toxic microorganisms Substances Control Act (TSCA) Source: http://www.agbios.com

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of GM crop regulation. The US regulatory framework for agricultural biotechnology has been the subject of several reviews (Belson 2000; McHughen and Smyth 2008). For an overview of the US coordinated regulatory framework for biotechnology, visit the website: http://usbiotechreg.nbii.gov/index.asp.

11.2.2

US Regulatory Framework

11.2.2.1

The USDA

The USDA is composed of 17 government agencies, all dealing with various aspects of food, agriculture, natural resources, rural development and related issues based on sound public management. The USDA agency involved in GM crop regulation is the Animal and Plant Health Inspection Service (APHIS: http:// www.aphis.usda.gov). APHIS is responsible for protecting and promoting US agricultural health, administering the Animal Welfare Act, and carrying out wildlife damage management activities. Within APHIS is the office of Biotechnology Regulatory Services (BRS; http://www.aphis.usda.gov/biotechnology/brs_main. shtml), which is responsible for regulating GM plants at all levels, including research and development, import, interstate movement, field trials and commercial release and cultivation. BRS derives its authority to write regulations from provisions of the Plant Protection Act, which is a part of the larger Agriculture Risk Protection Act of 2000 (APHIS 2000). Congress authorizes various parts of USDA to regulate specified areas of US agriculture under these federal statutes. APHIS uses the term biotechnology to mean the use of rDNA technology, or genetic engineering (GE) to modify living organisms. APHIS regulates certain GMOs that may pose a risk to plant or animal health. In addition, APHIS participates in programs that use biotechnology to identify and control plant and animal pests. APHIS considers a plant to be a “regulated article” if the plant and/or its progeny arose from a specific (single) transformation event, and such plants would continue to be regulated under APHIS until such time that a petition for nonregulated status was approved. Once the GM plant achieves non-regulated status, it may be released commercially with no further USDA action required. APHIS exercises its regulatory authority through a system that includes both permits and notifications. The regulatory process generally begins as the regulated article approaches the field trial stage.

Notifications Most field trials are approved under the notification procedure. Notification is an administratively streamlined alternative to the permit. The goal of the notification procedure is the same as the permit system: preventing the unintended release of

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the regulated article. In order to use the notification procedure, the genetically engineered (GE) plant must first meet all of the six eligibility criteria listed below: 1. The recipient organism is not listed as a noxious weed or considered to be a weed in the area of release into the environment 2. Stable integration of genetic material has been achieved 3. The function of the introduced genetic material is known and does not cause plant disease 4. The characteristics of the introduced gene or gene product do not include the production of an infectious entity, encode substances that are known or likely to be toxic or hazardous to non-target organisms, or encode products intended for pharmaceutical or industrial use 5. The introduced genetic material does not pose significant risk of creating new plant viruses 6. The introduced genetic material does not contain nucleic acid or coding sequences from human or animal pathogens Next, the introduction (the importation, interstate movement, or environmental release) must meet all performance standards. The performance standards are a set of six conditions that must be met in order to ensure that the regulated article is introduced in such a way that it is not inadvertently released beyond the proposed introduction, allowing it to persist in the environment. Generally, performance standards are characteristics associated with the act of introduction. The six required performance standards are listed below: 1. Shipping and maintenance at destination must be done to ensure that plant material is unlikely to be disseminated into the environment 2. Inadvertent mixing of materials in environmental releases is prevented 3. Identity of material is known and maintained while in use and plant parts are devitalized after use 4. Viable vector agents are not associated with the regulated article 5. No persistence of the regulated article or its progeny in the environment is permitted 6. No viable material of the regulated article is allowed to volunteer in subsequent sessions Protocols must be designed by the applicant to meet all performance standards for regulated articles and must be implemented during field trials. Once an applicant has determined that their regulated article meets all required eligibility criteria and performance standards, a letter of notification may be submitted to the APHIS BRS for review. Introductions cannot proceed without an acknowledgement letter from APHIS. Once approved, field trials are subject to inspection by federal and/or state inspectors and a field test report must be submitted to APHIS within 6 months of termination of the release. Online application of notification is now possible on the APHIS website. More details on the requirements of the notification procedure can be found in the USDA-APHIS BRS User’s Guide (http://www.aphis.usda.gov/brs/ pdf/Notification_Guidance.pdf).

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Permits For those GM plants not eligible for notification approval, the APHIS BRS requires a permit to be issued. The permit procedure is much more involved than that of the notification, requiring more data and information from the applicant. In addition to the information required for notification, permit applicants must provide a detailed description of the problem or “need” being addressed by release of the regulated article, along with a detailed description of the article itself. The article description must include details on the donor and recipient of the gene being introduced, method of introduction, and post-transformation analysis of resulting regulated articles. Detailed information must also be provided regarding the field testing protocol and containment issues, including assessment of the article’s capability to escape containment and possible consequences of such actions. More information of the permit application process can be found at http://www.aphis.usda.gov/ biotechnology/permits.shtml.

Deregulation and Deployment of GM Crops by the USDA USDA-APHIS regulations provide a petition process for the determination of nonregulated status. If a petition is granted, that organism will no longer be considered a regulated article and will no longer be subject to oversight by USDA-APHIS. APHIS grants non-regulated status if the GE organism poses no more of a plant pest risk than an equivalent non-GE organism (substantial equivalence). If a regulated article is very similar to a GE organism that has already been granted non-regulated status, APHIS may extend non-regulated status to that organism. The petitioner must supply information such as the biology of the recipient plant, experimental data and publications, genotypic and phenotypic descriptions of the genetically engineered organism, and field test reports. The agency evaluates a variety of issues including the potential for plant pest risk; disease and pest susceptibilities; the expression of gene products, new enzymes, or changes to plant metabolism; weediness and impact on sexually compatible plants; agricultural or cultivation practices; effects on non-target organisms; and the potential for gene transfer to other types of organisms. A notice is filed in the Federal Register and public comments are considered on the environmental assessment and determination written for the decision on granting the petition. The National Environmental Policy Act (NEPA) of 1970 requires federal agencies to investigate environmental impacts prior to making decisions or taking actions that could pose environmental risks. In compliance with the NEPA, APHIS will respond to a petitioner by generating an Environmental Assessment (EA) of the regulated article which reviews the environmental consequences of releasing the article. If the EA is in favor of article release, a Finding of No Significant Impact (FONSI) statement will then be issued which justifies the EA and the agency’s decision for release.

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If the EA is inconclusive, a Notice of Intent (NOI) may be issued, requiring a more detailed analysis of the impact of article release and demand the preparation of an Environmental Impact Statement (EIS) before a FONSI can be issued. For a complete listing of the steps involved in deregulation of GM plants, visit the NEPA website (www.nepa.gov). Copies of the USDA-APHIS documents are available to the public.

Current Status of US Deployment of GM Crops After more than a decade since the introduction of GM crops, only four countries plant 99% of the world’s GM crops. The US represents 55% of the area grown, while Argentina, Canada and Brazil account for the balance. The first GM crop to achieve deregulated status in the US was the Flavr SavrTM tomato from Calgene in 1992, in which an antisense polygalacturonase transgene from tomato resulted in delayed fruit ripening. Since that initial event, 74 additional petitions for deregulation of GM crops have been granted, 10 are pending decision, and over 35 GM crops are in development for market (www.trufoodnow.org/crop/pipeline.html). Among the GM crops approved for deregulated commercial production in the US are soybean, corn, rice, cotton, sugar beet, rapeseed, tobacco, potato, flax, beet, plum, papaya and squash. Estimated percentage of crops that are GM produced in the US as of 2008 are at 91% for soybean, 73% of corn, 87% of cotton, 75% of canola and more than 50% of Hawaiian papaya. For a complete and updated listing of GM crops achieving nonregulated status in the US, visit http://www.aphis.usda. gov/brs/not_reg.html.

11.2.2.2

The US Food and Drug Administration

The Food and Drug Administration (http://www.fda.gov) is part of the US Department of Health and Human Services and consists of nine internal centers, of which the Center for Food Safety and Nutrition and the Center for Veterinary Medicine are responsible for the evaluation of GE foods and feeds. The FDA is responsible for protecting the public health by assuring the safety, efficacy, and security of human and veterinary drugs, biological products, medical devices, our nation’s food supply, cosmetics, and products that emit radiation. The FDA is also responsible for advancing the public health by helping to speed innovations that make medicines and foods more effective, safer, and more affordable; and helping the public get the accurate, science-based information they need to use medicines and foods to improve their health. The FDA receives its regulatory authority from the Federal Food, Drug and Cosmetic Act. In 1992, the FDA published a policy statement and testing guidelines for foods developed using all methods of plant breeding, including the use of genetic engineering. These guidelines explain the types of food safety questions that developers should address in evaluating the safety of all plant-derived foods.

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In general, the FDA evaluates GM crops to determine if they are substantially equivalent (SE) to their non-modified counterparts, with focus on changes in nutritional and anti-nutritional components such as allergens and toxins. GM crops that do not differ significantly from a non-GM counterpart do not require FDA review and approval, while those that have significant differences are considered “adulterated” and are subjected to FDA regulations. Review of GM foods by the FDA is not legally mandated, but all GM crops produced in the US have undergone FDA inspection. Most developers of GM crops perform extensive analysis of their GM product, comparing its composition to that of a non-modified version, before considering application for release. The developer then submits a synopsis of these data to the FDA for review. Such data will include an evaluation of the crop’s genetic stability, compositional and nutritional analysis of the product and changes in allergenicity and/or toxicity as compared to a non-GM counterpart. To date, the FDA has not identified any examples of GM foods or crops as “adulterated.” The FDA has no information that the use of biotechnology creates a class of food that is different in quality, safety or any other attribute from food developed using conventional breeding techniques and therefore does not require the disclosure of the inclusion of GM foods on food labels. Any significant differences between the bioengineered food and its conventional counterpart do have to be disclosed in labeling. These would include differences in nutritional properties, the presence of an allergen that consumers would not expect in the food, or any property that would require different handling, storage, cooking or preservation. For example, when a manufacturer produced a line of soybeans whose oil had higher levels of oleic acid than found in conventional soybean oil, the FDA agreed to name the product “higholeic soybean oil” to distinguish it from traditional soybean oil. The high-oleic oil can be used in frying without the need for the chemical process of hydrogenation, which produces trans-fat. Food processors may voluntarily label either the presence or absence of a genetically engineered food in their products as long as the information is truthful and not misleading to consumers. The FDA has produced guidance to the industry for this type of labeling.

11.2.2.3

The US Environmental Protection Agency

The mission of the Environmental Protection Agency (EPA: http://www.epa.gov) is to protect human health and the environment. The EPA is largely concerned with pesticides and their effect on the environment and human health and thus regulates not only pesticides but also their properties and usage. The EPA was given authority to regulate GM crops under the Federal Insecticide, Fungicide and Rodenticide Act and focuses mainly on the potential pesticidal properties of GM plants. Examples of such regulated plants include those that produce plant incorporated protectants (PiPs) like Bt or other insecticides and plants that are resistant to pesticides such as Roundup ReadyTM crops.

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The data required by the EPA are similar to that of the USDA and FDA and include a summary of the genetic manipulation performed, an analysis of any known pesticidal properties and their origin, analysis of genetic stability, details on allergenicity and/or toxicity and effect on non-target organisms. The EPA also requires a detailed analysis of the pesticidal protein itself, including its sequence (amino acid and entire nucleic acid sequence of the construct), source of origin, allergenicity profile and overall expression pattern. The sustainability of the PiP must also be determined with respect to the environment (i.e., decay rate, soil response, etc.). The EPA also requires that appropriate resistant management practices are in place so as to decrease or avoid the generation of resistant populations of insects and crop species.

11.3 11.3.1

GM Crop Regulation in Canada History

Unlike the US where the market place determines the success or failure of a traditionally developed crop variety, the Canadian government regulates the release of conventionally bred crop varieties. The regulatory system governing GM crops is an extension of the framework used for non-GM crop releases. The agency responsible for such regulation is the Canadian Food Inspection Agency (CFIA) (http:// www.inspection.gc.ca). In 1985, the Canadian government enacted the Seeds Act (Department of Justice Canada 1985b), which mandates the performance standards for new germplasm. The Seeds Act focuses on germplasm uniformity, stability and uniqueness but also established thresholds for environmental safety risks such as gene flow, weediness, invasiveness and the effect on non-target organisms. Two other Acts involved in the regulatory framework are the Food and Drugs act (Department of Justice Canada 1985a), which deals with rules set for human consumption, and the Feeds Act (Department of Justice Canada 1983), which sets maximum tolerances for nutrients in livestock feed. It is the intent of the Canadian government that the integration of all three Acts in the regulatory framework for new plant varieties will identify all potential risks and ensure that new varieties will pose no more of a threat to human and animal consumption than those already existing in the environment. For a new crop variety to be approved for release in Canada, it must be at least of equal quality (in set parameters) of existing commercial varieties. In Canada, a plant breeder is responsible for risk management of the research and development of a new variety until the potential cultivar is ready to be examined for registration, at which time the government system becomes heavily involved in the process. Even for conventionally bred varieties, field tests must be

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designed not only to examine the agronomic traits of the new cultivar, but also its environmental risks. Field trial data are submitted to a recommending committee organized by the CFIA, which will make a decision on the potential variety’s merit. If registration is recommended, an application is then submitted to the Variety Registration Office (VRO), also part of the CFIA, which retains the final authority to grant variety approval. Once approved, the Canadian Seed Trade Association manages the breeder’s seed increase and the Canadian Grain Commission is responsible for setting and monitoring the standards for seed trade. Much like the GM crop regulatory system of the US, Canada bases its regulatory policies on the end-product that is established, not the process by which it was created. However, within the Canadian system, a new and unique classification of regulated plants has been established. Plants with novel traits (Table 11.2; PNTs) are defined as any plant which has a new trait, not present or previously characterized among existing crop systems. By definition, a plant does not have to be produced by genetic engineering to be a PNT. Many varieties developed by conventional methods have been considered PNTs due to their novel nature (see “novel foods” and “major change,” Table 11.2). Furthermore, some, but not all GM plants are classified as PNTs. If a plant was developed using genetic engineering but is not expressing a novel trait, that plant is exempt from PNT regulation and must only be governed by conventional regulations.

11.3.2

PNT Regulatory Framework

No regulatory framework for the development of GM crops was in place at the time of the first GM crop field trials in Canada in the late 1980s, but soon after, the federal government began to require permits for such experiments. Currently in Canada, biotechnology products are overseen by three agencies: The Canadian Food Inspection Agency, Environment Canada and Health Canada. For a comprehensive review on the topic of regulating GM crops in Canada, see Smyth and McHughen (2008). The roles of each agency are outlined in Table 11.3. The CFIA is responsible for PNTs, novel fertilizers, novel livestock feed and veterinary biologics. Within the CFIA is the Office of Food Biotechnology (OFB), which coordinates the safety evaluation of novel foods produced from PNTs. It is not possible in Canada to obtain a split permit where the crop would be approved for animal feed but not human consumption. Thus all PNTs are also subject to the regulatory approval of the other two agencies involved. Environment Canada oversees the regulation of all animal products of biotechnology not covered under other federal legislation and derives its authority from the Canadian Environmental Protection Act (Department of Justice Canada 1999). Health Canada oversees the safety assessment of goods, drugs, cosmetics, medical devices and pest control products, much like the Food and Drug Administration of the US.

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Table 11.2 Definitions Plants with A plant variety/genotype possessing Includes plants produced using rDNA novel characteristics that demonstrate techniques, chemical mutagenesis, traits neither familiarity nor substantial cell fusion and conventional cross (PNTs) equivalence to those present in a breeding distinct, stable population of a cultivated seed in Canada and that have been intentionally selected, created or introduced into a population of that species through a specific genetic change Novel foods A substance, including a microorganism, Includes food products from genetically that does not have a history of safe engineered plants, but also any food use as a food product without a history of safe use (e.g., novel fibers, single-cell A food that has been manufactured, protein), or an existing food product prepared, preserved or packaged by a manufactured or packaged in a process that has not been previously manner that results in a major applied to that food and causes the change food to undergo a major change A food that is derived from a plant, animal, or microorganism that has been genetically modified such that the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism – one or more characteristics of the plant, animal or microorganism no longer fall within the anticipated range for that plant, animal or microorganism Major change A change in the food that, based on the manufacturer’s experience or generally accepted nutritional or food science theory, places the modified food outside the accepted limits of natural variations for that food with regard to: – The composition, structure or nutritional quality of the food or its generally recognized physiological effects – The manner in which the food is metabolized in the body – The microbiological safety, the chemical safety or the safe use of the food Source: http://www.agbios.com

11.3.2.1

The Canadian Food Inspection Agency

In Canada, most GM crops have been considered to be “novel” and have been subjected to rigorous regulation by the CFIA. All plants are evaluated on a

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Table 11.3 Canadian federal authorities responsible for agricultural biotechnology products Department/ Products regulated Relevant legislation Regulations agency Canadian Food Plants and seeds, including Consumer Feeds regulations Inspection those with novel traits; Packaging and Agency animals; animal Labeling Act (CFIA) vaccines and biologics; Feeds Act Fertilizers regulations fertilizers; livestock Fertilizers Act Health of animals feeds regulations Food and Drugs Act Food and drug regulations Health of Animals Act Seeds Act Plant Protection Act Environment Biotechnology products Canadian New substances notification Canada under CEPA such as Environmental regulations (these microorganisms used in Protection Act regulations apply to bioremediation; waste (CEPA) products not regulated disposal, mineral under other federal leaching or enhanced legislation) oil discovery Health Canada Foods; drugs; cosmetics; Foods and Drugs Act Cosmetics regulations medical devices; pest CEPA Food and drug regulations control products Pest Control Novel foods regulations Products Act Medical devices regulations New substances notification regulations Pest control products regulations Source: http://www.agbios.com

case-by-case basis so the information required for approval is not always the same. Environmental safety is of utmost concern to the CFIA. The PNT must be extensively described and characterized molecularly. Information generally required includes molecular characterization and comparison with its conventional counterpart. The Regulatory Directive Dir94-08 (AAFC 1994) details the information required for the environmental impact analysis which includes: 1. Taxonomy and pedigree analysis 2. Method of modification 3. Description of the novel trait(s), including gene product activity, decay, byproduct activity and any adverse effects on non-target organisms, including humans 4. Biology of the PNT, including reproductive cycle and survival biology 5. Agricultural practices or behavior, including release sites, habitat, cultivation practices and management 6. Potential gene flow of the PNT to related species and subsequent consequences

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Unlike the US where guidelines are suggested but not mandatory for the research and development stage of GM crops, the CFIA begins regulating PNT development at a very early stage, requiring confined use even before field trials. PNTs with commercial release potential are then selected for evaluation in field trials. Confined field trials may be conducted if approved by the CFIA, and must be directed to obtain the environmental safety assessment data, which analyze the PNT with regards to the five risk categories listed above. The risk assessment of GM crops in Canada has recently been critically reviewed (Barrett and Abergel 2000). Following a CFIA review of the risk assessment data, a decision is rendered as to whether the crop variety is eligible to apply to the CFIA for unconfined commercial production. This application process can be delayed if additional scientific data are requested. The CFIA has been criticized for requesting additional data too late in the process and delaying commercialization of GM crops, which has resulted in the loss of millions of dollars to producers.

11.3.2.2

Environment Canada and Health Canada

Environment Canada is responsible for the regulation of new substances that may pose a threat to the environment and receives its authority from the Canadian Environmental Protection Act (CEPA) (Department of Justice Canada 1999). A “substance” is defined as animate matter by the CEPA and a “new substance” is one that is not listed on the Domestic Substances List (DSL). All PNTs would logically have the potential to produce a new substance and are therefore regulated by this process. The assessment of the PNT or its new substance includes a determination that the substance is not toxic. If the substance is suspected of being toxic, Environment Canada is responsible for controlling and/or prohibiting its import or manufacture pending the submission and assessment of additional data. New substances are also subjected to regulation by Health Canada, which reviews data related to human exposure and potential human toxicity risks. PNTs may produce novel foods, defined as foods resulting from a process not previously used, to produce food, genetically engineered foods, or from products without a history as safe to be used as food. Any GM or novel food proposed for sale in Canada is regulated by Health Canada. Novel foods are further defined as a food having a major deviation from the accepted limits of (1) composition, structure or nutritional quality, (2) metabolic properties or (3) microbiological, chemical or food safety. Health Canada requires that the developers of novel foods must examine and describe: 1. 2. 3. 4. 5.

How the novel food was developed, including molecular characterization Composition of the novel food compared to the conventional counterpart Nutritional profile of the novel food compared to the conventional counterpart Toxicity potential/profile Allergenic potential/profile

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Unlike the CFIA and Environment Canada, Health Canada will accept a history of safe production and consumption elsewhere (outside Canada) as evidence for regulatory approval. Once the GM crop/food has been approved for release/human consumption by the Canadian regulatory framework, no post-market monitoring or long-term surveillance is performed, due to the assumption that the biotechnology product has been judged no more risky to consumers than the conventional counterpart. Since the Canadian regulatory system approved GM canola as the first GM crop released for production in Canada in 1995, several other GM crops have also passed through the system and acquired approval, including GM alfalfa, cotton, corn, flax, tomato, potato, rice, soybean, squash, sugar beet, sunflower and wheat. In 2007, there were 7.0 million hectares of GM crops grown in Canada.

11.4 11.4.1

Regulation in the European Union (EU) History

As of January 2007, the EU had 27 member states and was comprised of over 450 million consumers making it the largest developed market in the world. In the 1980s, member states realized the necessity for establishing a common food policy that would harmonize their standards and ensure easy trade. Resulting from this action was a number of fragmented laws that created a set of directives often described as opaque and incoherent. Included in these directives were regulations involving food additives, pesticide residues in foods and contaminants in foods. Differing social values and risk concerns between the US and EU consumers have probably resulted in the differential regulatory approaches taken regarding GM crops. Many opinion papers have been written comparing the two contrasting approaches to regulating GM products (Haniotis 2000; Kalaitzandonakes 2000; Dale 2002; Morris 2006; Ramjoue` 2007). In response to public fears about genetically modified organisms (GMOs) in food, the European Union (EU) adopted a regulation in 2001 governing GM plants and two more regulations establishing an EU-wide system to trace and label GMOs and to regulate the commercialization and labeling of food derived from GMOs in July 2003 (http://www.gmo-compass. org). Finally, in May of 2004, the European Commission put an end to the “de facto” moratorium on approving new GM products for the European market, which had been in place since 1998. The EU and the member states are now of the opinion that using genetic engineering in agriculture and food production is permissible. Unlike the US and Canada where GM crops are judged using an end product base, the EU regulatory system is representative of the “process-based” approach, meaning that all crops developed by genetic engineering must undergo rigorous regulatory oversight before commercial release.

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Individual GMOs must receive approval before they can be sold as seed or used in food and feed. Approval is granted only after satisfying conditions of safety, freedom of choice, labeling and traceability. The product must be safe and cannot pose threats to human or animal health or to the environment. Consumers, farmers, and businesses must be given the freedom to either use or to reject products made from GMOs. It must remain possible in the long term to produce foods without the use of genetic engineering. The term used for this is coexistence. Genetically modified plants must be grown and handled in such a way that prevents uncontrolled mixing with conventional products. Whenever GMOs are intentionally used in a food product, it must be clearly stated on the label. Every consumer is thereby entitled to make an “informed decision.” Labeling is required even if GM content cannot be detected in the final product. This is why all producers, suppliers, and retailers must inform their buyers if GMOs were used in their products. To do this, stakeholders must set up systems for keeping and sharing information and documentation.

11.4.2

EU Regulations/Directives for GM Crops

Current EU legislation on GMOs is regarded as the strictest in the world. It deals with a plethora of issues, including rules relating to the release of GMOs into the environment, the traceability and labeling of GMOs and GMOs in food and animal feed. The co-existence of GM and conventional crops is currently under discussion at EU level. There are two different set of rules governing the authorization of genetically modified products in the EU: one is for the use of GM plants, while the other is for food and feed made from them. The EU Governing bodies within the member states must abide by these regulations regarding the commercial production/release of GM crops.

11.4.2.1

Directive on the Deliberate Release into the Environment of Genetically Modified Organisms (2001/18)

In effect since April 17, 2001, Directive 2001/18/EC (Official Journal of the European Communities 2001) repeals former Directive 90/220/EEC and regulates the deliberate release and placing on the market of GMOs and the environmental release of GM crops. A guide to help in navigating Directive 2001/18/EC was published in 2002 by the Department of Environment, Food and Rural Affairs (DEFRA; http://www.defra.gov.uk). This directive defines the risk assessment and decision making process on the release of GMOs into the environment as well as the information required to be given to the public regarding GMO release, labeling and traceability at all stages. Authorizations granted under this directive will be good for 10 years and subject to post-market monitoring.

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GM crops can only be allowed on the market once they have received authorization. The authorization process is carried out by the EU, and the resulting decision applies to all EU Member States. Under the terms of the amended release directive (2001/18/EC), a post-release monitoring plan must accompany applications for cultivating GM plants. Authorization is contingent upon a parallel, general monitoring plan, which in some cases can include special stipulations addressing crop-specific areas of concern. The purpose of monitoring is to identify hidden effects of large-scale GMO production on the environment. It can also be useful for determining if potential negative effects noticed during safety assessments actually cause problems. To obtain authorization to release a GM crop for commercial production, an application must be submitted to federal authorities of the pertaining EU member state. Authorizations under EU Regulation 2001/18 are for a 10-year period. An initial assessment (scientific opinion) by national agencies is made and documents are then forwarded to the national authorities of the Member States and to the European Commission. Safety assessments are then made by the European Food Safety Authority (http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_ home.htm).

11.4.2.2

European Food Safety Authority (EFSA)

The EFSA was established in 2002 as the central authority for the scientific evaluation of food and feed safety in the EU. As of 2005, EFSA is permanently based in Parma, Italy. EFSA was established based on the legal mandate of regulation 178/2002/EC of the European Parliament and European Council addressing basic principles of food law. New food law legislation and hence the EFSA were created in response to a number of food scandals that shook consumer confidence. EFSA addresses two main areas: (1) Scientific risk evaluation for all questions related to food and feed safety and (2) informing the public of potential risks. To assist with safety evaluations, EFSA is supported by eight scientific panels composed of independent researchers from various EU Member States. The GMO Panel is responsible for GMOs (including GM crops) and genetically modified food and feed. EFSA offers a solid scientific basis for making informed political decisions. The decisions themselves, however, are the responsibility of political bodies such as the European Parliament, the European Commission, and the Council of Ministers. EFSA cooperates closely with national authorities of all Member States. The safety assessments for GM crops (Fig. 11.1.), food or feed required by the EFSA are similar in many ways to those in other countries. A detailed understanding of the modification process, the genes or constructs introduced and their products and the resulting alteration of the plant and how it differs from the conventional counterpart are all necessary for proper assessment. Comparative analysis of the agronomic and nutritional properties of the GM crop with those of

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GM Crop

molecular characteristics

Non-GM Crop

genotype

composition

phenotype

Identification of Differences

inserted genes

expressed proteins

metabolites

Decision on Further Testing

gene transfer

allergenicity/toxicity

toxicity

Estimation of Actual Consumers Exposure to Hazards

Risk Assessment of GM Food Crops

Fig. 11.1 EU risk assessment strategy (comparative analysis) for GM food crops Source: European Commission Report; genetically modified crops in the EU: food safety assessment, regulation and public concerns; 2000

the traditional counterpart is required. For those GM crops with no traditional counterpart, a product-specific safety assessment is carried out.

Regulation on Genetically Modified Food and Feed (1829/2003) Enacted on April 19, 2004, the EU Regulation 1829/2003 (Official Journal of the European Communities 2003) regulates food and/or feed that is made from or contains GM plants. This regulation is an extension of EU Regulation on novel foods (258/97), and basically requires that GM food or feed produces no harmful effects on human or animal health or on the environment and that consumers are not mislead in the labeling process of GM materials. Authorizations for release into the public market require a safety assessment, which involves the determination that the GM food or feed is as safe as the conventional counterpart. Regulation

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1929/2003 also governs labeling of, detections methods for and post-market monitoring of GM products. Applications for authorization of GM product release are submitted directly to the EFSA, where a scientific evaluation from expert committee takes place and results in a recommendation to the European Commission. As for GM crop release, the European Commission considers the EFSA recommendation and sends a draft for vote to the Standing Committee for the Food Chain and Food Safety. The European Commission’s draft may be accepted or rejected with a qualified majority. If no qualified majority can be reached, the European Commission submits its draft to the Council of Ministers. Finally, a vote in the Council of Ministers results in approval or rejection by qualified majority – without qualified majority the Commission’s draft takes effect. Authorizations under EU Regulation 1929/2003 are for a 10-year period. Currently, valid authorizations for production in the EU have been approved for GM cotton, flowers, maize, rapeseed, soybean and sugar beet. For a comprehensive list of the status of the application process for GM crops in the EU and more on the EU regulatory system, go to http://www.gmo-compass.org/eng/gmo/db/.

11.5 11.5.1

Regulation in Argentina and Brazil GM Crop Regulation in Argentina

Argentina is second only to the US in GM crop production, producing 19.1 million hectares of GM crops in 2007 (www.isaaa.org) and like the US, regulates GMOs on a product, not process basis. In Argentina, the regulatory framework for Genetically Modified Plant Organisms (GMPOs) consists of Regulations and Resolutions issued by the Secretariat of Agriculture, Livestock, Fisheries and Food (SAGPyA; http://www.sagpya.gov.ar). The permit to commercially release a GMPO is granted by the Secretary based on three independent reviews, each issued by advisory committees within the scope of the SAGPyA. The committees involved are (1) The National Advisory Commission on Agricultural Biotechnology (CONABIA; http://www.sagpya.mecon.gov.ar/new/0-0/programas/biotecnologia/index_en.php), (2) The Technical Advisory Committee on the Use of GMOs which belongs to the National Agri-food Health and Quality Service (SENASA; http://www.senasa.gov. ar) and (3) The Directorate of Agricultural Markets (DNMA). Regulatory requirements for GM crops have been incorporated into Argentina’s farming sector general regulatory system. CONABIA was established to provide advice and support on the supervision of activities related to agricultural biotechnology and bio-safety, especially those concerning authorizations for the environmental release and commercialization of genetically modified plants and animals (to be used in agricultural/livestock and aquaculture activities). It is also devoted to define policies, and to design specific regulations, and to aid in the public diffusion of the SAGPyA activities related to biotechnology issues.

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CONABIA assesses all Applications to release GMPOs into the environment and advises the Secretary of Agriculture, Livestock, Fisheries and Food on the convenience of approving or not such releases. The assessment has two phases: 1. Field trial (low scale) release assessment, which aims at determining that the probability of having any environmental impact is non-significant 2. Large-scale release assessment, which aims at determining that the environmental impact of the released GMPO will not significantly differ from that of its nonmodified counterpart Permits are required to conduct greenhouse or field trials and the application for permits requires extremely detailed information on the GMO itself, the greenhouse/ field location and conditions, and post-harvest treatment and monitoring. While CONABIA is responsible for the environmental safety assessment of GM products, SENASA is responsible for their food and feed safety assessment and the DNMA determines the effect of the GM product on local and international trade markets. The SAGPyA considers reports from all three agencies before making a final decision on whether or not to release the GM product for commercial production/consumption. GM crops currently approved for commercialization in Argentina include soybean, maize and cotton.

11.5.2

GM Crop Regulation in Brazil

Brazil ranked third in the world for GM crop production in 2007, producing 15 million hectares of GM soybean and cotton. The first bio-safety law in Brazil took effect in 1995 (law no. 8974/95), but was replaced in 2005 with bio-safety law number 11.105/05. Several federal departments have active roles in bio-safety regulation with the national authority being the National Technical Commission on Bio-safety (CTNBio; http://www.ctnbio.gov.br/index.php/content/view/4060. html). The CTNBio is composed of members from the public and private sectors, including scientists, ministerial representatives and specialists. The CTNBio has developed bio-safety policies and a Code of Ethics for genetic engineering and has determined the risk assessments to be performed on GMOs before experimental or commercial release. Before any GM plant is field tested, the environmental risks must be assessed, including potential harm to human health, other organisms and the environment. An application for approval, which includes a summary of this information, must be submitted to the CTNBio. If the GM plant is not considered a potential environmental risk, field release is granted, but otherwise the GM plant must undergo an environmental impact study before field testing can take place. If the GM plant or its products will enter the human food chain, the food and safety regulations of the National Agency for Health and Surveillance of the Ministry of Health (ANVISA; http://www.anvisa.gov.br) must be followed. Other important governing agencies include the National Surveillance System, which regulates research laboratories,

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field experiments and commercialization and the Inspection Agencies from the Ministries of Agriculture, Environment and Health which regulate inspection, registration, operating license, import license, and temporary field testing license. If the GM crop produces a pesticidal substance, it must also be regulated under the Pesticide law, which governs experimental research, production, packing and labeling, transportation, storage and commercialization. Commercial approval for GM crops in Brazil must also be granted by CTNBio. A commercial release dossier must be submitted, which details the GM crop’s molecular characterization, protein expression, nutritional composition and agronomic/environmental risk assessment. Currently, a 2/3 favorable majority vote by the CTNBio is required for GM crop release. Once approved for release by CTNBio, the application is reviewed by the Council of Ministries which also considers the effect of GM crop release on the economical and political interests. Thus far the Brazilian bio-safety regulatory system has approved the commercial release of four varieties of GM corn, two varieties of GM cotton and one variety of GM soybean (www.ctnbio.gov/br).

11.6

Summary

Most GM crop regulatory policies can be categorized as either (1) “process-based,” where any and all biotechnology research and its products are strictly regulated, or (2) “product-based,” where each product of biotechnology is evaluated on a case by case basis to determine if regulation is necessary. Thus far, the largest production of GM crops comes from those countries adopting a “product-based” approach to biotechnology regulation. Social acceptance of GM crops plays a large role in directing the type of regulatory policies a country will adopt. Public view on the need to regulate GM crops varies. Some believe that biotechnology is just an extension of conventional breeding practices, while others see the need for extensive detailed analysis of all possible consequences that GM crop release may have on human, animal and environmental systems. Regardless of contrasting viewpoints, there is an international agreement on the need for GM crop regulation. It is certain that regulations governing GM crop research and commercial release will continue to evolve as economic and social pressures change.

References AAFC, Agriculture and Agri-Food Canada (1994) Assessment criteria for determining environmental safety of plants with novel traits, regulatory directive 94-08. AAFC, Ottawa Abdalla A, Berry P, Connell P, Tran Q, Buetre B (2003) Agricultural biotechnology: potential for use in developing countries. ABARE eReport, vol 03.17. Canberra, Australia APHIS (2000) Agriculture Risk Protection Act of 2000. http://www.aphis.usda.gov/brs/pdf/ AgRiskProtAct2000.pdf

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Barrett K, Abergel E (2000) Genetically engineered crops. Breeding familiarity: environmental risk assessment for genetically engineered crops in Canada. Sci Public Policy 27:2–12 Belson N (2000) US regulation of agricultural biotechnology: an overview. AgBioForum 3:268–280 Berg P, Baltimore D, Brenner S, Roblin RO, Singer MF (1981) Summary statement of the Asilomar Conference on recombinant DNA molecules. Proc Natl Acad Sci USA 72: 1981–1984 Dale PJ (2002) The environmental impact of genetically modified (GM) crops: a review. J Agr Sci 138:245–248 Department of Justice Canada (1983) Feeds Act. http://laws.justice.gc.ca/en/ShowFullDoc/cs/ F-9///en Department of Justice Canada (1985a) Food and Drugs Act. http://laws.justice.gc.ca/en/ShowFullDoc/cs/F-27///en Department of Justice Canada (1985b) Seeds Act. http://laws.justice.gc.ca/en/ShowFullDoc/cs/ S-8///en Department of Justice Canada (1999) Canadian Environmental Protection Act. http://laws.justice. gc.ca/en/C-15.31/text.html Haniotis T (2000) Regulating agri-food production in the US and the EU. AgBioForum 3:84–86 Herrera-Estrella L, Depicker A, van Montagu M, Shell J (1983) Expression of chimaeric genes transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209–213 ISAAA (International Service for the Acquisition of Agri-Biotech Applications). http://www. isaaa.org Kalaitzandonakes N (2000) Why does biotech regulation differ so much between the EU and the US? AgBioForum 3:75–76 McHughen A, Smyth S (2008) US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars. Plant Biotechnol J 6:2–12 Morris S (2006) EU biotech crop regulations and environmental risk: a case of the emperor’s new clothes? Trends Biotechnol 25(1):2–6, Jan 2007 NIH (US National Institute of Health) (1983) Risk assessment in the federal government. Managing the process. National Academies Press, Washington, DC NIH (National Institutes of Health) (1976) Recombinant DNA research. Guidelines. Federal Register 41, 27902, 27911–27943 NIH (1978) Guidelines for research involving recombinant DNA molecules. Fed Regist 43 (60):108 Nossal K, Abdalla A, Curtotti R, Tran QT, Brown A (2008) GM crops in emerging economies. Research Report 08.3. www.abare.gov.au OECD (Organization for Economic Cooperation and Development) (1982) Biotechnology, international trends and perspectives. OECD, Paris, France Official Journal of the European Communities (2001) Directive 2001/18/ec of the European Parliament and of the Council. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ: L:2001:106:0001:0038:EN:PDF Official Journal of the European Communities (2003) Directive 1829/2003 of the European Parliament and of the Council. http://www.gmo-compass.org/pdf/law/1829-2003.pdf Qaim M (2005) Agricultural biotechnology adoption in developing countries. Am J Agr Econ 87:1317–1324 Ramjoue` C (2007) The transatlantic rift in genetically modified food policy. J Agr Environ Ethics 20:419–436 Schell J, van Montagu M, Holsters M, Zambryski P, Joos H, Inze D, Herrera-Estrella L, Depicker A, de Block M, Caplan A, Dhaese P, Van Haute E, Hernalsteens JP, de Greve H, Leemans J, Deblaere R, Willmitzer L, Schroder J, Otten L (1983) Ti plasmids as experimental gene vectors for plants. Advances in gene technology: molecular genetics of plants and animals. Miami Winter Symp 20:191–209 Smyth S, McHughen A (2008) Regulating innovative crop technologies in Canada: the case of regulating genetically modified crops. Plant Biotechnol J 6:213–225

Chapter 12

Patent and Intellectual Property Rights Issues Jim M. Dunwell

12.1

Introduction

The present status and future prospects of genetically modified (GM or transgenic) crops have been the subject of several recent reviews (Dunwell 2000, 2002, 2004, 2008). Although these reviews include some information extracted from patent databases in order to provide a commercial perspective, this analysis has been necessarily limited in extent. The present review will supplement the information published previously on the patent and intellectual property rights (IPR) (Johns 2006) aspects of transgenic methodology (Dunwell 2005, 2006), horticultural crops (Dixon and Ogier 2007; Clark and Jondle 2008; Dunwell 2009b) and haploid plants (Dunwell 2009a) and will extend to a discussion of IPR relevant to the research scientist (Shear and Kelley 2003) and of those interested in international development (Koo et al. 2004), globalization (Parayil 2003; Aerni 2007; Beatty 2008), and sociological (Cabanilla 2007) and ethical aspects of the public- and privatesector relationships (Graff et al. 2003; Donnenwirth et al. 2004; Karapinar and Temmerman 2008).

12.2

Historical Retrospective

The history of patents and plants extends back over a century. In July 1899, an international conference on the subject of hybridization was organized by the Royal Horticultural Society (RHS) and held in London. One of the many speeches given at the conference banquet was that by the leading British judge, Lord Justice Lindley (Anon 1900). In this presentation, he made the following prediction: “I have heard J.M. Dunwell University of Reading, Whiteknights, Reading RG6 6AS, UK e-mail: [email protected]

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something about hybridisation of which I know little. I have heard something which leads me to suppose that the development of that art may react with the profession to which I have the honor to belong. Without being a prophet, I seem to see before me a vista of patent hybrids! What a treat for the patent lawyers! And what an accession of work for her Majesty’s Judges!” He could clearly see the potential for litigation and legal conflict even at that early stage. In a prelude to later discussions, Assistant Secretary of Agriculture, Willet M. Hays (Troyer and Stoehr 2003), at a meeting of the American Breeders’ Association (Hays 1905; Kimmelman 1983; Allen 2000) in 1905, remarked: “Possibly laws or business can be devised which will give private individuals, animal breeders, seed firms and nursery firms practically a patent or a royalty on new blood lines.” By 1906, the emphasis on patents was demonstrated in a review of the relations between science and industry, particularly the chemical sector where it was reported that “the German company Baeyer (sic) had achieved a monopoly position in novel chemicals, with 1,000 patents at home and 1,200 overseas” (Anon 1906). However, the first detailed discussion of patents in relation to plant breeding is probably that from the subsequent Third International Conference on Genetics, organized by the RHS in 1906 and most famous for the first public coining of the term “genetics” by William Bateson (Dunwell 2007). During this meeting, there was a session entitled “Copyright” for Raisers of Novelties (Anon 1907). In the report of the session, it is stated that Mr. George Paul, whilst commenting on the absence of several well-known plant breeders, remarked: “The fact is, these gentlemen do not like to tell us, or to show, what they have done in their experiments, because once their knowledge becomes public, they have not the slightest chance of receiving any pecuniary reward for their labors. If they were properly protected from being deprived of the due reward of their labors, they would no doubt be much more willing to come forward and help us and place their experience at our disposal.” During the subsequent discussion in this session, Professor Hanson replied: “I believe, in law, a seedling is regarded as the gift of God, and it would be hard to patent that; but could we not hope to have some law fashioned that would give a bonus to the man who does such skilled and valuable work as that which has come before us over and over again during the sessions of this conference?” The chairman, although sympathizing with the Mr. Paul, concluded that it would be unwise to pass a resolution on the subject since the discussions had demonstrated “[w]hat very great difficulty there would be in enforcing such a law, because we have gentlemen from all parts of the world maintaining that a thing is new, and others, equally capable, maintaining that it is old.” In the following years, there were to be several cases of speculation about the possible consequences of patent coverage for plants. For example, David Fairchild produced an interspecific hybrid in Actinidia and stated: “If I could patent it – this hybrid – then, should some other breeder have been at work on the same problem and made the same pollination a few days later, I would have the prior claim to a patent. Unlike the invention, the hybrid is not a crude unfinished thing but a perfect working machine from the moment it is made, and claims entered would not come into interference as they do in inventions because when the date of the hybridization

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was once established the date of the first perfected machine would be automatically determined” (Fairchild 1927). Following these early prescient comments and debates, it took several years, after much encouragement and lobbying by Luther Burbank (Burbank 1914), before the first legal protection for plants, The Plant Patent Act 1930, was enacted in the USA, and then only for clonal material (e.g., rose, apple and pear, though not potato) (Fowler 2000). The first US plant patent (PP00001) was issued for a climbing rose in the following year, 1931 (Cook 1931a). This was soon followed by further examples (Cook 1931b, 1933a), although it should be noted that even in those days the topic was the subject of controversy from scientific, legal, and financial experts (Anon 1931; Allyn 1933a,b; Cook 1933b, 1936; Barrons 1936; Rossman 1931; Fay 1937). Much of the controversy today, more than 100 years after the first discussions, follows the same themes.

12.3

What Are Patents?

The history of patent law dates back several centuries, with the first example sometimes considered to be a Venetian statute of 1474. A summary definition states: “A patent gives an inventor a period of exclusive exploitation (up to 20 years in the UK) in return for a disclosure of the invention” (Huskisson 1996). According to the UNCTAD site (http://www.iprsonline.org/guide/index.htm), a patent application must satisfy the patent examiners that the invention is: – Useful (i.e., has industrial application): ideas, theories, and scientific formulas are not sufficiently useful or industrially applicable to be patentable – Novel: the invention should be recent and original, but perhaps most importantly it should not already be known (in the public domain). In most countries (except the USA) the patent is awarded to the first person to apply, regardless of whether this person was the first to invent – Non-obvious or must involve an inventive step: not obvious to a person skilled in the technology and more inventive than mere discovery of what already exists in nature (such as a gene with no known function). The invention must be disclosed to the patent examiners in a detailed way that would enable a skilled technician to make and use it. In the case of an invented process, the patent can cover a nonobvious way of making something already known (i.e., previously invented or discovered). In the case of an invented product, the non-obvious/inventive step requirement does not require it to be made by a novel method. This disclosure of an invention (Fromer 2007) takes the form of a publication from the relevant patent office. In the case of most authorities, the patent application is published 18 months after the date of filing and is then available for inspection. A former exception to this rule is that the United States Patent and Trademark Office (USPTO) (http://www.uspto.gov/), until 15 March 2001, maintained secrecy until the time the patent was granted, a period that can range from an average of 2–3 years upwards to more than 20 years. An extreme example of the length of time

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sometimes involved is the approximate 20 years taken for the resolution of a dispute concerning key patents which cover elements of Agrobacterium-mediated transformation. It was announced on 4 February 2005 that Monsanto, Bayer CropScience, Max Planck Society and Garching Innovation had agreed to cross-license their respective technologies worldwide. Bayer CropScience, Max Planck’s exclusive licensee, and Monsanto agreed to provide each other, in selected areas of the world, nonexclusive licenses related to the development, use and sale of transgenic crops. Monsanto also agreed to provide Max Planck Society with a license in the United States for research purposes. An important difference between the US and the patent systems of other international jurisdictions is that the 17-year duration of a US patent filed prior to 2001 only starts from the time at which it was granted, whereas in Europe (and now in the US) the 20-year period of exclusivity starts from the time of filing the application. Some of the consequences of this change are discussed in more detail later in this review.

12.4

Sources of Patent and Other Related Information

The association between an active IPR system and a strong national or regional economy has been demonstrated in many global studies (Griliches 1990). For example, the Organization for Economic Cooperation and Development (OECD) maintains various patent databases that can be used to assess global and regional trends in all disciplines including biotechnology (Van Beuzekom and Arundel 2006; Anon 2008; Maraut et al. 2008). One of the issues raised in the early discussion of the US Plant Patent system was that access to the patent documents was extremely difficult. In the words of Cook (1933b): “The patent authorities have taken the view that these patents are too valuable to be disposed of casually, and an applicant for a copy must ‘show cause’ why he should be sold one. . . . Until copies of plant patents are available to interested parties this situation can hardly be considered satisfactory from the plant breeder’s point of view.” The situation is now much simpler (Krattiger et al. 2007; Nottenburg 2007). During the preparation of this review extensive use has been made of the freely available patent databases in the US (http://www. uspto.gov/patft/index.html), Europe (http://ep.espacenet.com/), World International Patent Organization (http://pctgazette.wipo.int/) and other international sites (e.g., http://www.surfip.gov.sg/; http://www.google.com/patents; http://www. freepatentsonline.com/; http://www.pat2pdf.org/). The most comprehensive and integrated international site is probably the Patent Lens section of BiOS, Biological Innovation for Open Society, an initiative of CAMBIA (Center for the Application of Molecular Biology to International Agriculture) (http://www.bios.net/daisy/bios/ patentlens.html). A very useful site relating to US ag-biotech patents granted during the period from 1976 to 2000 is provided by the Economic Research Service (ERS)

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of the US Department of Agriculture (USDA) (http://www.ers.usda.gov/Data/ AgBiotechIP/). It should be noted that the most detailed and sophisticated types of patent analysis require commercial subscription. Companies providing this service include Thompson Reuters (http://www.thomsonreuters.com/products_ services/scientific1/Delphion), Micro Patent (http://www.micropat.com/static/index. htm), and patentmaps.com (http://patentmaps.com/shop/v2/shophome.htm).

12.5

Patents and Plant Biotechnology

Apart from the natural genetic protection provided by F1 hybrids (Duvick 1999; Smith et al. 2008), there are a range of legalistic methods that can be used to protect novel types of plants produced by one organization or commercial company from being exploited by competitors, with these methods varying from one country to another (Cahoon 2000; Llewelyn and Adcock 2006; Locke 2007). An introduction to the various approaches, namely plant breeders rights (Kesan and Janis 2002; Helfer 2004; Chen 2006; Ghijsen 2007) and patents, is available from several authors (Brown 2003; Lenssen 2006; Janis and Smith 2007; Kock et al. 2007; Rimmer 2003; Smith 2008), and from the Biological Innovation for Open Society (BiOS) organization (http://www.bios.net/daisy/bios/patentlens/tutorials.html). Information relating to individual countries is available at their respective patent offices. For example, the latest note on patenting of plants in the UK “Examination Guidelines for Patent Applications relating to Biotechnological Inventions in the UK Intellectual Property Office” was published by the Intellectual Property Office in September 2007 (http://www.ipo.gov.uk/biotech.pdf). Similar information is available concerning the patentability of plants in the US (Merrill et al. 2004), Europe (Fleck and Baldock 2003; Schrell et al. 2007), New Zealand (Ministry of Economic Development 2002) and China. In a comparative analysis, the results of a detailed survey of the actual practice of patent examiners in the three key patent offices, US, Europe and Japan, have been published (Howlett and Christie 2003). A complementary study restricted to the present and future position in the US (Merrill et al. 2004) concluded that the continuing high rates of innovation suggest that the patent system there is working well and does not require fundamental changes, although the authors note that both legal and economic changes are putting new strains on the system. There have been several, extensive reviews of the consequences, and implications of applying patent (and other IPR) protection to plants (Farnley et al. 2004; Temmerman 2006; Adcock 2007; Kock 2007) and the reader is referred to these publications, most of which are freely available on the web. In one of the most comprehensive of these reviews (Binenbaum et al. 2003), the important conclusion is reached that as patenting becomes ever more prevalent in biotechnology (Wright 2006; Wright and Pardey 2006a; Chapotin and Wolt 2007) and elsewhere (Straus 2007), the diversity of innovations utilized in the development of modern cultivars

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means that there is an associated increase in the number of separate rights needed to produce each new product (Tokgoz 2003). Where ownership of the relevant IPR rights is particularly dispersed, the problem of multilateral negotiation can become difficult or even impossible to resolve. For example, those who develop new technology by building on existing technologies often know neither the extent to which the latter have been claimed as IP nor the strength of any claims. As a consequence, both the conduct of research and development and any subsequent commercialization entail navigating through a potential minefield of patent applications that have been filed but remain invisible pending publication by the patent office. Fortunately, the uncertainty arising from such so-called “submarine” patents is becoming less important as the US has harmonized with the rest of the world, first by awarding a patent term of 20 years from the date of filing (previously 17 years from the date the patent was awarded), and secondly by publishing (from November 2000) patent applications within 18 months of filing. Despite the increasing complexity of biotechnological IPR (Eisenberg 2006; Kukier 2006), and the difficulties of making accurate investment predictions over extended time scales (Yerokhin and Moschini 2007) it should be noted that a similar position exists in the electronics industry where sophisticated products are assembled from numerous components, sourced internationally, and covered by a multiplicity of patents.

12.6

Patents and Plant Transformation

During the period since the production of the first transgenic plants in the 1980s, a wide diversity of patents have been sought, and granted, on all aspects of the process, ranging from the underlying methods for tissue culture through to the means of introducing the heterologous DNA, and to the composition of the DNA construct so introduced (Dunwell 2005; Pray and Naseem 2007). It would be impossible to summarize all this information in the space available here; the amount of patent information available in the area of plant transformation alone can be judged by the fact that a search of the US application database alone for “transgenic plant” and “method” returned 5,995 records on 1 August 2008. Summaries of relevant recent granted patents and patent applications in the US are given in Tables 12.1 and 12.2. For detailed analyzes of several of the key areas of this subject, the reader is referred to comprehensive summaries published elsewhere, for example in the series of extensive CAMBIA White Papers (Mayer et al. 2004; Roa-Rodrigues 2007; Roa-Rodrigues and Nottenburg 2007a,b); some aspects of these will be considered below. Frequently, the main point of interest in such discussions is the breadth of coverage of the patent(s) in question. There are some well known examples of patents with very broad coverage and this is often a topic of debate and the reason for concerted opposition. For example, European Patent 301749,

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Selection of US patents on transgenic plants published immediately prior to 8 July

Table 12.1 2008 Number 7396979 7396978

Date 8 July 2008 8 July 2008

Named inventors Alexandrov N et al. Miki B et al.

7396977 7393999 7393998 7393997 7393948 7393947 7393946

8 July 2008 1 July 2008 1 July 2008 1 July 2008 1 July 2008 1 July 2008 1 July 2008

Usami S et al. Acevedo PAN et al. Streatfield S et al. Datla R et al. Sekar V et al. Diehn S et al. Memelink J et al.

7393928 7393922 7390937 7390936 7390655 7390643

1 July 2008 1 July 2008 24 June 2008 24 June 2008 24 June 2008 24 June 2008

7388126 7388125 7388091 7385123 7385107 7385106 7385105 7385104 7385048 7385046

17 June 2008 17 June 2008 17 June 2008 10 June 2008 10 June 2008 10 June 2008 10 June 2008 10 June 2008 10 June 2008 10 June 2008

Chang RC et al. Dean DH Good AG et al. Van Rooijen G et al. Hinchey B Croteau RB, Burke CC Duncan DR, Ubach C Ristic Z et al. Erickson L, Zhang J Sauer M et al. Donovan WP et al. Stein H et al. Medrano L et al. Landschutze V Fujii T et al. Alexandrov N et al.

Applicant Ceres Min Agri-Food Canada Japan Tobacco Pioneer ProdiGene – MS technologies Pioneer Rijksuniversiteit Leiden FibriGen Ohio State Univ Univ Alberta SemBioSys Monsanto – Monsanto Pioneer Univ Guelph SunGene Monsanto Tel Aviv Univ Ceres Bayer Kirin Beer Ceres

Subject Increased biomass Seed coat gene PPDK gene Antimicrobials Insulin Floral shape Polyubiquitin promoter Inducible promoter Transcription factor Gelatin Cry4B Bt gene Nitrogen metab Chymosin Promoters Geranyl diphosphate synthase NO modulation Elongation factor Inducible gene Ketolase CryET33 gene Proline metab Root promoters Starch metab Promoters Ethylene response

granted to Agracetus (then a subsidiary of WR Grace & Co) on 2 March 1994, is an exceptionally broad “species patent,” which grants this company rights to all forms of transgenic soybean varieties and seeds – irrespective of the genes used or the transformation technique employed. Agracetus was purchased by Monsanto in April 1996, after which it withdrew its previous opposition to this patent. However, opposition continued from other companies and organizations, and a hearing was finally agreed by the European Patent Office in May 2003, at which the patent was upheld, with the exception of Claim 25 covering plants other than soybean (http:// www.epo.org/about-us/press/releases/archive/2003/06052003.html; http://www. european-patent-office.org/news/pressrel/pdf/bginfo_soya_e.pdf). After a further series of oppositions the patent was finally revoked on 3 May 2007 (http://www. epo.org/about-us/press/releases/archive/2007/070503.html). Interestingly, there are also IPR aspects of marker-assisted breeding (HensonApollonio 2007), a method sometimes considered as an alternative to transgenic technologies.

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Table 12.2 Selection of US patent applications on transgenic plants published immediately prior to 8 July 2008 Number Named inventors Applicant Subject 20080168587 Yao K et al. – High oleic acid 20080168586 Laga B et al. Bayer Mutant fatty acid desaturase 20080168585 da Costa e Silva O et al. BASF Stress-related GTP binding protein 20080168584 Zheng Z et al. NRCC Brassica seed metabolism 20080168578 da Costa e Silva O et al. BASF Stress-related protein kinase 20080166811 Maliga P et al. – Site-specific recombination of plastids 20080166754 Cahoon E et al. – Caffeic acid 3OMT homologs 20080163402 Wilkinson JQ et al. Pioneer Non-plant 30 termination sequences 20080163399 Carozzi N et al. Athenix Delta-endotoxin 20080163398 Kakefuda G et al. BASF AHAS small subunit protein 20080163397 Ratcliffe OJ et al. Mendel Biotech CCAAT-binding transcription factor 20080163396 Allen SM et al. – Gene expression 20080163395 Song H-S et al. BASF Gene expression 20080163394 Frankard V et al. CropDesign Cyclin and yield 20080163393 Spangenberg G et al. Agric Victoria CAD genes Services 20080163392 Zink O et al. Bayer Herbicide resist 20080163391 Sussman SM et al. Penn State Stomatal closure 20080163390 Kachroo P et al. Univ Kentucky Fatty acids and disease 20080161191 Falco SC et al. Du Pont Methionine sulfoxide reductase 20080160162 Davies JP et al. Agrigenetics Improved oil 20080155717 Kaeppler SM et al. Wisconsin Polycomb genes Alumni 20080155715 Komatsu S et al. NIAS Japan Stress response 20080155714 Gontier E et al. Total France Fatty acid synthase 20080155713 Yephremov A et al. Bayer Fatty acids 20080155712 Savidan Y et al. IRD Apomixis

12.6.1

Transformation Methods

There is a great diversity of techniques for the introduction of recombinant DNA vectors containing heterologous genes of interest into plant cells, and the subsequent regeneration of plants from such cells (Moeller and Kan Wang 2008). The two principal methods exploited in commerce are indirect methods based on the use of Agrobacterium tumefaciens (Chung et al. 2006; Lacroix et al. 2008) or the direct introduction of DNA on microparticles of metal, usually gold, a technique known as biolistics (http://www.bioportfolio.com/indepth/Biolistics.html). The most extensive publication in this area is the CAMBIA White Paper (Roa-Rodrigues and Nottenburg 2007a) on Agrobacterium-mediated transformation. This document focuses on the patents directed to methods and materials used for transformation, mainly of plants, and also of other organisms such as fungi. It should be noted that although most of the early development of this technique was performed in universities, most of the patents were consolidated in the hands of a few companies.

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419

DNA Sequences

Almost all the functional components of the various constructs used in plant transformation have been the subject of patent coverage. These include the “effect gene” as well as its associated regulatory sequences (e.g., promoters), the selectable or screenable marker, and additional sequences such as those that might be utilized for the subsequent excision of the transgene (for examples see Dunwell and Ford 2005) or even allow IP protection in their own right (Heider and Barnekow 2007). The present review does not cover details of the gene of interest and the reader is referred to other recent reviews that include summaries of the range of present and future transgenic crops (Dunwell 2002, 2004) (see also Tables 12.1 and 12.2). Much of the debate in this area concerns the ability to apply for patents on DNA sequences of unproven function. There have been several attempts to do so, and the decisions on such applications have not been finalized (Howlett and Christie 2003; Rimmer 2007). However, the important fact remains that patent databases contain a great deal of useful sequence information that is frequently ignored by research scientists in the academic sector. Specifically, it is estimated that some 30–40% of all DNA sequences are only available in patent databases, since there is of course no obligation for commercial (or other) applicants to submit their sequences to public databases. Free access to some patent sequence data is now available via the latest version of the Blast search system at NCBI, and via Patent Lens (http://search. patentlens.net/sequence/blast/blast.html). Access to this information is also available via the GENESEQ system, a commercial service from Thompson Reuters (http://scientific.thomsonreuters.com/bondplus/geneseq/). Regulatory elements are crucial to gene expression in all organisms. The patent landscape of transcriptional regulators that are constitutively active, spatially active (e.g., tissue-specific), or temporally active (e.g., induced or active in response to a certain chemical or physical stimulus) has been well summarized (Roa-Rodrigues 2007). In this review, an assessment is presented of the possibilities for, and limitations on, further development of regulation of gene expression. Although the inventions protected by individual patents cannot be exactly the same, in certain cases there are patents that due to the breadth of their scope may encompass other protected inventions or there may be patents which share common features. Is this case, this review points out the juxtaposition of the different inventions and the possible room left to maneuvre around the different entities in the field. The science behind some of these issues has also been reviewed recently (Century et al. 2008). It also needs to be considered that there are patents that while not totally directed to promoters may have an effect on the control of gene expression. This is the case for the restrictive reproductive technologies, for example, those termed as “Terminator” technologies (Van Acker et al. 2007; Van Dooren 2007), which may have a great impact on the use and development of methods to regulate the expression of genes related to plant reproduction and seed generation (Dunwell and Ford 2005; Hills et al. 2007).

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Selection and Identification of Transformants

The production of transgenic organisms, including plants, involves the delivery of a gene of interest and the use of a selectable marker that enables the selection and recovery of transformed cells. This is necessary because only a minor fraction of the treated cells become transgenic while the majority remain untransformed. It has been estimated recently (Miki and McHugh 2004) that approximately 50 marker genes used for transgenic and transplastomic plant research or crop development have been assessed for efficiency, biosafety, scientific applications and commercialization. These selectable marker genes can be subdivided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or nonconditional on the presence of external substrates. The most common strategy currently used for selection is negative selection, the elimination of nontransformed cells in conditions where the transformed cells are allowed to thrive. Elimination is often effected by the treatment of cells with compounds, (e.g., antibiotics or herbicides) in conjunction with a transgene that confers resistance or tolerance to the chemical through detoxification or modification of the chemical. A summary of the most important scientific aspects of such resistance genes has been published recently, together with an analysis of selected patents that relate to the most widely used ARMs (Roa-Rodrigues and Nottenburg 2007b). Many of these marker genes are covered by patents or patent applications with the most thorough IP analysis available probably being that published on antibiotic markers and Basta resistance by CAMBIA (Mayer et al. 2004).

12.7

Novel Products and the Freedom to Operate

One of the issues of overriding importance to all companies is whether or not they are free to commercialize any particular product (Lence et al. 2002). Such “freedom to operate” is determined by the status of any IPR that might cover the product in question and analysis of such IPR requires continuous (and therefore expensive) surveillance. A well known example that can be used to demonstrate the complexity of this issue is “golden rice,” a transgenic line enhanced for beta-carotene (provitamin A) (Ye et al. 2000; Mayer 2007) and provides hope for alleviating the severe vitamin A deficiency that causes blindness in half a million children every year. It has been suggested that extensive patenting has hampered delivery of this rice to those in need since some 40 organizations hold 72 patents on the technology underlying its production (Kryder et al. 2000). The range of patents covering various components of the pBin 19 hpc plasmid used in the production of this rice include ones on the phytoene trait genes, the promoter sequences, the selectable marker and the transit peptide. This issue has now been overcome by a coordinated international program designed to streamline the production and distribution of this material (http://www.goldenrice.org/) (Potrykus 2007). However, perceived

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problems with access to golden rice (Enserink 2008) and essential medicines have stimulated debate within the US on the obligations of American universities to facilitate the provision of goods for the public benefit (Kowalski and Kryder 2002; Phillips et al. 2004), an issue also considered below.

12.8

Economic Benefits of Transgenic Crops

The exploitation of IPR associated with transgenic crops is central to the generation of economic benefit (Moschini and Yerokhin 2007) for the companies producing such crop and the farmers growing them. The global economic benefits of the first decade of these crops have been summarized recently by Barfoot and Brookes (2008) whereas the specific benefits of individual crops have also been described (Anderson and Valenzuela 2008). One consequence of the IPR system is that companies place considerable emphasis on legal protection of their investment and this includes the prosecution of those deemed to be infringing any proprietary IPR (Kesan and Janis 2002, 2003; Ziff 2005; Munzer 2006; Pila 2008).

12.9

Patents and Commercial Consolidation

Several summaries of this subject has been provided in the last few years (Brennan et al. 2005; Bulut and Moschini 2005, 2006; Chan 2006; Schimmelpfennig and King 2006; Karapinar and Temmerman 2008; Marco and Rausser 2008). The latter authors conducted an analysis of agbiotech patents issued between 1976 and 2000, classified by their original patent holders and their 2002 owners. These data showed how 95% of patents originally held by seed or small agbiotech firms had been acquired by large chemical or multinational corporations. Furthermore, none of the smaller firms acquired patents from the larger ones, and none of the patents changed hands among the different types of large firms. Specifically, chemical companies retained all 651 patents that they originally owned, but they also acquired 219 patents from agbiotech firms and 451 patents from seed companies. A similar, related study of the role of patents in the pharmaceutical industry has also been published (Brusoni et al. 2005), together with a summary of the consequences of having a large portfolio of patents (Chan 2008), and a legal discussion of using IPR as collateral in trade (Dequiedt et al. 2007; Dunn and Seiler 2007).

12.10

Public and Private Sector Issues

The most detailed review of this aspect of agbiotech patents is probably that conducted by Graff et al. (2003) who summarized both the ownership of critical patents and compared the relative significance of the private and public sectors in

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each area of research relevant to the commercialization of transgenic plants. The main findings of this review and others (Heisey et al. 2005; Schimmelpfennig and King 2006; Eaton 2007) are as follows. Six companies hold 75% of all agricultural patents and it has been suggested that such concentration exacerbates the challenge of delivering agricultural inventions to the neediest segments of the world’s population. One solution could be the compulsory licensing of patented inventions that have failed to reach the neediest markets (see below for further detail). An alternative would be based on the fact that while the public sector holds less than 3% of all patents, it does have 24% of agricultural biotechnology patents, many covering genes of great potential interest. By exploiting these resources, universities and other public organizations, therefore, do have opportunities to deliver affordable biotechnological innovations (Morris et al. 2006). Concern has also been expressed about the potential dangers (financial or otherwise) associated with the use of patented technologies by academic establishments (Kesselheim and Avorn 2005). This is a complicated issue involving “experimental use exception” (Faye 2005a,b), the policy that allows others to examine and test a patented discovery, but not to use it routinely.

12.11

Patents, Ethics and International Development

The moral and ethical aspects of transgenic plants have recently been considered (Myskja 2006; Cooley 2007; DeBeer 2007; Wilson 2007), and in a broader context, some authors consider that the commercialization of biotechnology, especially research and development by transnational pharmaceutical and ag-biotech companies, is already excessive and is increasingly dangerous to distributive justice, human rights, and access of marginal populations to basic goods required for human prosperity (Shrader-Frechette 2005). The various trends associated with these socio-economic aspects of ag-biotech development have also been reviewed (Parayil 2003; Lipton 2007; Lea 2008). Amongst the agencies involved, the various Consultative Group on International Agricultural Research (CGIAR) centers add value through selective breeding, and the superior varieties they generate are widely distributed without charge, thereby benefiting both developing and developed countries (Anon 2001). During the Gene Revolution, the situation changed, and much has been written over the last few years on the potentially deleterious effects of plant IPR on the freedom and commercial opportunities of farmers in developing countries (Bastuck 2006; Chiarolla 2006; Fukuda-Parr 2006; Garrison 2006; Hamilton 2006; World Bank 2006; Wright and Pardey 2006b). One of the major reasons that IPR have become an important factor in plant breeding (Lence et al. 2002; Louwaars et al. 2006; Kingston 2007) is through the greater use of utility patents (Summers 2003; Kevles 2007). Such patents have stimulated greater investment in crop improvement research in industrialized countries, but they are also creating major problems and potentially significant additional expense for the already financially constrained

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public-sector breeding programs that produce seeds for poor farmers (Spielman 2007; Spielman et al. 2007). For example, it has been calculated (Phillips et al. 2004) that developed countries spend about $5 in research and development for every $100 in agricultural output whereas developing countries spend only 66 cents. Patents on biotechnology methods and materials, and even on plant varieties (Tripp et al. 2007), are thus potentially complicating and undermining the collaborative relationships between international institutions. There is some concern that public-sector research institutions in industrialized countries no longer fully share new information and technology. Instead, they are inclined to patent and license and have special administrative offices charged with maximizing their financial return from licensing (Brazell 2000). Commercial production of any GM crop variety requires dozens of patents and licenses (see above), and it is only the large companies that can afford to assemble the IPR portfolios necessary to give them the freedom to operate. Additionally, now, under the TRIPS agreement of the World Trade Organization (http://www.iprsonline.org/unctadictsd/docs/ RB_Part2_2.5_nov02_fullpatents-updated.pdf), most developing countries are required to put in place their own IPR systems, including IPR for plants (Giannakas 2001; Gaisford et al. 2007; Watal and Kampf 2007). Several proposals have been made on how the international community should deal with these present IPR realities affecting agriculture and horticulture (Delmer et al. 2003; Delmer 2003; Ramanna 2005; Brewster et al. 2007; Lence et al. 2003). With little competitive loss, seed companies could agree to use the Plant Variety Protection (PVP) system (including provisions allowing seed saving and sharing by farmers) in developing countries in cooperation with public plant-breeding agencies, rather than using patents to protect their varieties (Singh 2004). To speed the development of biotechnology capacity in developing countries (Louwaars et al. 2005; Salazar et al. 2006; Le´ger 2007), companies that have IPR claims over certain key techniques or materials might agree to license these for use in developing countries at no cost (Nottenburg et al. 2002). These authors also propose an agreement to share the financial rewards from IPR claims on crop varieties or crop traits of distinct national origin (Wu and Lu 2005; Mgbeoji 2006; Kartal 2007; Mahop 2007; McManis 2007), such as Thailand’s Jasmine rice or South Asian Basmati rice. There have been several reviews of this topic each with a particular regional emphasis. For example, biotechnology and GM crops has been discussed specifically in relation to development in Africa (Eicher et al. 2006; Juma and Seregeldin 2007; Virgin et al. 2007; Asante 2008), India (Demangue 2005; Sreedharan 2007), China (Stone 2008), the Philippines (Cabanilla 2007) and South America (Orozco et al. 2007; Scoones 2008). There is also a series of published case studies in which the IPR aspects of specific transgenic crops have been explained. Such examples include those for corn (Gracen 2007), papaya (Goldman 2007; Gonsalves et al. 2007), herbicide tolerant rice (Espinoza-Esquivel and Arrieta-Espinoza 2007) and canola (Smyth 2006), insect resistant eggplant (Medakker and Vijayaraghavan 2007) and cotton (Rao and Antharvedi 2007), and biopharming (Durell 2006; Krattiger and Mahoney 2007).

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Many of the concerns about the scope and application of IPR in the area of agriculture (Tencalla 2006; Laxmi et al. 2007) also extend to the more general issue of food from the perspective of safety (Klaasen 2007; Key et al. 2008) as well as long term security (Malik and Zafar 2005; Tansey 2006; Hossain 2007). There are also discussions about the need to regulate the whole process (Rimmer 2006; Rhodes 2007; Roff 2008). For all the reasons discussed above, new organizations such as Public Intellectual Property Resource for Agriculture (http://www.pipra.org/) and the African Agricultural Technology Foundation (http://www.aftechfound.org/) have been established as a means of rationalizing the huge proliferation of patents, especially in plant biotechnology. It is the intention of these organizations to develop a freedom-to-operate information database, and to help public sector agricultural research institutions achieve their public missions (Cantley 2004) by ensuring access to intellectual property required to develop and distribute improved staple crops and specialty crops (Anon 2006; Dodds et al. 2007).

12.12

Conclusion

Although the 1906 “Genetics” conference (Anon 1907) did receive commercial support and there were 20 pages of advertisements in the proceedings, this funding was restricted to horticulture and associated gardening items. Bateson, in his after dinner speech to foreign guests, concluded: “I expect a century must elapse before the . . . complete union of Science and Practice will be achieved.” Some 25 years after Bateson, the following comment was being made: “It will be extremely interesting to follow the new developments in plant breeding in order to determine the influence of the new patent protection on agriculture. In years to come, much of the food consumed, many of the clothes worn and even the houses occupied by man may be radically changed by the mass attack of plant breeders so that the future generations may speak of a horticultural revolution rivaling, if not surpassing the great industrial revolution” (Rossman 1931). A century has now elapsed since the first of these comments and indeed the value of genetics in agriculture and horticulture has been proven. Moreover, the role played by patent protection in this period has been critical to the commercial exploitation of genetics during this period.

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Chapter 13

Transgenic Crop Plants: Contributions, Concerns, and Compulsions Brian R. Shmaefsky

13.1

Introduction

No one discovery, event, person, or product alone defines or typifies plant biotechnology. Biotechnology plants, known scientifically as transgenic plants or genetically modified plants (GMPs), are derived from a blend of ancient agricultural practices and modern genetics-based technologies. Traditionally, plants served societies primarily for basic needs, such as food and shelter from the environment. Some early cultures made use of whole plants and plant compounds for medical and religious purposes. Plants took esthetic roles as civilizations grew. Many plants in early civilizations were selected for their beauty and fragrance to grow in gardens and in homes. Biotechnology significantly improved the traditional use of plants by improving the way plants are grown and the quality of the plant products. It has also greatly expanded the roles of plants within the past 20 years. Plants are now used for biomanufacturing a variety of commercial and industrial products. They are also put to work for a host of bioremediation purposes (Shmaefsky 2007). The principal people of biotechnology plant development are from a variety of scientific disciplines. Many of the contributors to plant biotechnology are biologists. However, the field also uses the research efforts of chemists, computer information scientists, engineers, medical doctors, mathematicians, and physicists to provide the knowledge base and application potentials for biotechnology plant development. Plant biotechnology innovations and research are not restricted to the wealthiest developed nations. Many new developments are coming out of China, India, Korea, and Mexico. Plant biotechnology is now a global effort, with each country using the technology to meet local and universal needs (Ernst and Young 2007). Venture capital and technology transfer agreements are providing many

B.R. Shmaefsky Lone Star College – Kingwood, HSB 202V, 20,000 Kingwood Drive, Kingwood, TX77339-3801, USA e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_13, # Springer-Verlag Berlin Heidelberg 2010

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incentives for unprecedented growth of the field in both the basic and applied scientific aspects. Unlike many sciences, marketplace demand is fueling new directions in plant biotechnology. Plant biotechnology developments have made many valuable contributions to agriculture, environmental remediation, horticulture, and medicine. Many of these advances go unnoticed. Few people are likely to be aware of the scope of biotechnology during a typical purchase of foods and products at a department store or a supermarket (Fritz et al. 2003). In spite of the benefits, many advances in plant biotechnology are not applauded by governmental officials, the public, and scientists. In contrast, these developments are criticized for a wide variety of reasons. Certain trepidations are founded on legitimate issues related to environmental quality, food safety, public health, and sustainable economic development. Other criticisms of plant biotechnology are provoked by compulsions rooted in culture, morality and public perception (Morris 2007). Both categories of criticism have hindered the progress of plant biotechnology. However, they have also contributed to refinements in rationale for developing biotechnology plants. In addition, they have fostered new ways of improving the safety of this upcoming technology.

13.2 13.2.1

Contributions of Plant Biotechnology Sciences Contributions to Biotechnology

Plant biotechnology made use of ancient and modern discoveries and technologies to produce new types of plants and to find new uses for plants. Researchers not only improved the plants with innovative ideas, but they also advanced the field by applying animal, bacterial, and fungal research to plant models. In some situations, plants even became more viable substitutes for the original research model associated with the discovery or new technology (Shmaefsky 2007). For example, the production of human therapeutics produced in genetically modified (GM) animal cell lines were proven to be safer if biomanufactured in plant cell cultures. Plants do not carry the hidden cytoplasmic and genomic parasites and pathogens transmitted through animal cell lines (Shmaefsky 2005). For many centuries, people have been choosing and modifying the characteristics of native and imported plants for agricultural and medical purposes. The intentional cultivation of plants began in the Middle East about approximately 15,000 years ago as societies moved away from hunting-and-gathering lifestyles (Mannion 1999). Small farms were set up to grow native plants used for human and animal feed. Unique characteristics of the plants were preserved by collecting and sowing only the seeds from plants with sought-after traits (Kaniewski et al. 2007). This cultivation of plants gave communities a consistent supply of food from one season to another. However, cultivation eventually led to the domestication of plants having a combination of desirable qualities not available by standard

13

Transgenic Crop Plants: Contributions, Concerns, and Compulsions

Table 13.1 Major events leading to plant biotechnology Year Event Pre-12000 BCE Origins of crop cultivation Circa 8000 BCE Selective domestication of crops Circa 8000 BCE Domestication of ornamental plants Circa 7000 BCE Corn domesticated Circa 4000 BCE Most modern-day crops domesticated Circa 2000 BCE 700 BCE 300 BCE 100 BCE 1663 1860s 1890s 1890s 1900s 1939 1944 1952 1953

Globalization of domesticated crops Crops irrigation and pest control First books on plant domestication Sowing and harvesting technology First description of cells Principles of trait inheritance Inheritance applied to crop production Natural selection applied to agriculture Gene locus mapping First successful plant tissue culture First transformation experiment Transposable elements discovered Role and structure of DNA confirmed

1972 1973 1977 1978 1983 1983 1987 1994

DNA splicing with restriction enzymes First genetically modified organism DNA sequencing Discovery of RFLP and gene markers Polymerase chain reaction discovered First GMO plant – tobacco Bioinformatics developed First commercial GMO Tomato

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People/place Middle and Near East Middle and Near East Asia, Europe, and Middle East South America Africa, Northern Europe and Far East Worldwide Europe and Near East Theophrastus China R. Hooke G. Mendel H. DeVries C. Darwin T.H. Morgan P. Nobe´court O. Avery B. McClintock E. Chargaff , F. Crick, L. Pauling, J. Watson P. Berg H. Boyer, S. Cohen F. Sanger D. Botstein K. Mullis D.T. Gibson E. Lander Calgene Inc, Davis, CA

cultivation (Betz 1998). This desire to cultivate crops contributed to the cultivation of food animals and thereby improved the likelihood of gathering a balanced diet (Diamond 1999). Cattle and deer were attracted to cultivated fields and were then corralled for easy gathering when preparing to harvest the animals (Mannion 1999). The major events leading to plant biotechnology are furnished in Table 13.1. Early attempts at selective breeding were used 10,000 years ago for domestication to produce a monoculture of plants that can be grown for consistent features from one generation to the next. The early domesticated plants, known as Neolithic founder crops, included purebred and hybridized cereals, fiber plants, and pulses (Zohary and Hopf 2000). The production of custom-designed plants was generally limited to food and medical plants (Woods and Woods 2000). However, ornamental plants were developed in various countries including Asia and parts of Europe and the Middle East (Harlan 1980). Greek, Persian, and Roman agriculture from 700 BC experimented with irrigation, pesticides, and weed control to improve crop yield (Isager and Skydsgaard 1992). The domestication of plants gave people the principles to selectively breed animals with characteristics favorable for

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agricultural purposes (Mannion 1999). Iron Age people in northern Europe drank the milk from domesticated cows over 3,000 years ago (Shmaefsky 2005, 2007). After 1866, selective breeding of commercially important plants led to a finer degree of precision upon the publication of G.J. Mendel’s lectures in the Proceedings of the Natural History Society (Mendel 1866). In the 1900, H. de Vries, C. Correns, and E. von Tschermack rediscovered Mendel’s cross ratios in plants (Goldschmidt 1950). With this information they were able to breed field crops with predictable characteristics. At the same time, this information was melded with C. Darwin’s ideas of species selection in his work On the Origin of Species by Means of Natural Selection, or The Preservation of Favoured Races in the Struggle for Life, published in 1899 (Bowler 1989). Plants were being produced for what was identified as superior qualities usually related to ease of cultivation and consistency of quality (Mannion 1999). Later, the need for resistance to pathology and predation was recognized as essential elements of plant worthiness. The evolutionary principles of plant selective breeding were then applied to agricultural animals and microorganisms involved in the fermentation of beverages and foods (Shmaefsky 2005). Trait variation and distribution in crop selective breeding were better understood after T.H. Morgan’s work on mutations and gene locus mapping in the early 1900s (Morgan et al. 1915). The knowledge of gene loci distances facilitated the predictability of producing purebred crops with several desirable characteristics in a particular variety of crop. In 1977, F. Sanger opened the door for analyzing crop genes with the development of the chain termination method for DNA sequencing (Sanger 2001). The identification and isolation of potentially valuable crop genes was facilitated by D. Botstein’s 1978 work on restriction fragment length polymorphisms (RFLP) and gene markers (Tanksley et al. 1989; Heesacker et al. 2008). Contributions of modern plant gene loci engineering are serving as models for other eukaryotic systems (Roden et al. 2005). Work on plant genome sequences are providing generalized data for molecular genetic studies at the gene locus and chromosome levels. Many of the plant studies can directly translate to animal and yeast genomic research. Unfortunately, in the middle twentieth century Soviet Union, T.D. Lysenko abused the concept of crop superiority by breeding for arbitrary crop characteristics related to the political ideology of Russian botanist I. Vladimirovich Michurin (Graham 1998). This work was an extension of the eugenics doctrine favored by F. Galton who in 1883 misinterpreted C. Darwin’s and A.R. Wallace’s views of selective fitness (Bulmer 2003). It forced the scientific community to establish a realistic interpretation of crop fitness that is reflected today in contemporary agronomic principles (Williams et al. 1998). Most conventional crop researchers did not follow the premises and protocols of Lysenko contributing to the integrity of evolutionary biology and plant genetics applications. The technology of plant cultivation remained unchanged from classical Greek, Persian, and Roman times until the advent of plant tissue culture. Initial advances in animal cell culture in the 1920s conducted by Alexis Carrel paved the way for plant tissue culture. In 1934, R.J. Gautheret performed preliminary studies on plant tissue

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with limited success. He based his research on the animal cell culture work of A. Carrel. Plant tissue culture became more feasible in 1939 following the work of P. Nobe´court. Further refinements in growing conditions and media made it possible to grow a wide variety, based on the findings of L. Knudson, F. Skoog, K.V. Thimann, F. Went, and P. White. They developed precision growing conditions by identifying the roles of hormones on plant cell differentiation, growth, and organogenesis (Gautheret 1983). Advances in plant hormone and growth factor research provided the foundations for understanding similar cell-to-cell channelmediated communication systems in other organisms (Fleming 2005). These channels, which resemble plasmodesmata, convey critical developmental signals in mammalian embryos (Hertzberg and Skibbens 1984). Plant tissue culture permitted the rapid clonal propagation of plants in a diseasefree environment. Cloning eliminated the need for continually bred hybrid plants each generation and it also facilitated the propagation of seedless varieties. Culturing plant tissues free from bacteria and viruses aided in the removal of untreatable diseases during clonal propagation (Bhojwani and Soh 2003). Plant propagation entered a new era with the first successful genetic engineering conducted by P. Berg in 1972 and the first genetically modified organism produced by H. Boyer and S. Cohen in 1973. Genetic engineering from bacteria and yeast models was applied to plants in the middle 1980s with the development of genetically engineered crops tobacco and tomato produced for field use (Christipeels and Sadava 2003). In turn, it became simple to grow experimental plants in vitro and as field models for other transgenic eukaryotic systems (Piruzian et al. 2006; Bassett 2007). The “biotechnology era” was fully established in agriculture in the 1980s with the development of polymerase chain reaction (PCR) by K. Mullis’s team at Cetus Corporation (Mullis et al. 1994). It made possible the amplification desirable genes from minute samples collected from archived and fresh specimens. PCR also made it possible to store isolated genes as well as whole genomes in germplasm banks (Committee on Managing Global Genetic Resources 1993). Advances in transfection vectors with reporter genes for eukaryotic systems in the late 1980s increased the feasibility of producing genetically modified crops (Marillonnet et al. 2005). Another boon was the creation of reverse transcription PCR (RT-PCR) in the late 1980s (Stoflet et al. 1988). It permitted the amplification of genes from rare transcripts extracted from a cell or synthesized from protein sequences. PCR studies in plant pathology contributed to disease investigation in other agriculture and food production fields (Kageyama et al. 2003). Soil PCR studies, in particular, have led to advances in understanding the spread of animal and human pathogens on various environmental surfaces. Biotechnology plants have been developed for a variety of marketable applications since the first GMP was commercialized in 1994 (Piruzian et al. 2006). The Flavr Savr tomato, produced by Calgene Incorporated of Davis, California, showed that GM plants were safe for general human consumption, which is one of most strictly regulated applications of novel plants (Martineau 2001). GM plants could be developed rapidly with precise characteristics almost unattainable by breeding crosses. New traits could be introduced without sacrificing other characteristics

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unintentionally lost in the production of monoculture plants. However, this initial success did not show that GM plants were economically viable alternatives to plants produced by traditional selective breeding (Piruzian et al. 2006). The growing field of extrachromosomal genetic engineering was established in plants during the 1990s. The genetic modification of chloroplasts contributed to metabolic engineering trial in algae and the mitochondria in animal cells (Shmaefsky 2007). Cultivation of GM plants in different countries in 2007 is shown in Fig. 13.1. The trend in increase in cultivation of transgenic crop plants is depicted in Fig. 13.2. The late 1990s proved different from early periods of plant development with the creation of drought, herbicide, and insect-resistant crop plants (Table 13.2). Plants were now being developed for dealing with environmental stress as well as market quality and consistency. They were practicable to grow for field production and inspired the other commercially feasible plants. Plant biotechnology in twenty-first century moved out of the field and into the industrial manufacturing setting. GM plants were diversified for uses in biopharming and chemical production (Berger 2007). Forestry also benefited with GM trees capable of doubling the output of wood fiber per tree (Shmaefsky 2000). A new generation of GM field plants was being developed and exploited during that period. Field trials of GM plants contribute greatly to the field of bioremediation. Plants capable of bioremediation in the field were developed to degrade and uptake a variety of environmental pollutants (Mulligan 2002). This led to a new technique called phytoremediation that augmented the roles of bacteria and fungi in bioremediation (Willey 2007). Philippines

0.3

Uruguay

0.5

South Africa

1.8

Paraguay

2.6

China

3.8

India

6.2

Canada

7

Brazil

15

Argentina

19.1

United States

57.7 0

10

20

30

40

50

60

millions of hectares

Fig. 13.1 Cultivation of biotechnology plants in 2007 (From: EarthTrends, June 2008 Monthly Update: Genetically Modified Crops and the Future of World Agriculture (http://earthtrends.wri. org/updates/node/313))

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100,000 90,000 World 80,000

Thousand hectares

70,000 Developed Countries

60,000 50,000 40,000

Developing Countries

30,000 20,000 10,000 0 1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

Fig. 13.2 Biotechnology plant cultivation from 1996 to 2005 (From: EarthTrends, June 2008 Monthly Update: Genetically Modified Crops and the Future of World Agriculture (http://earthtrends.wri.org/updates/node/313))

Table 13.2 Global area of genetically engineered crops, 1996 to 2006: by trait (million hectares) Trait HT IR (Bt) IR/HT VR/others Total 1996 0.6 1.1 – <0.1 1.7 1997 6.9 0.4 <0.1 <0.1 11 1998 19.8 7.7 0.3 <0.1 27.8 1999 28.1 8.9 2.9 <0.1 39.9 2000 32.7 8.3 3.2 <0.1 44.2 2001 40.6 7.8 4.2 <0.1 52.6 2002 44.2 10.1 4.4 <0.1 58.7 2003 49.7 12.2 5.8 <0.1 67.7 2004 58.6 15.6 6.8 <0.1 81 2005 63.7 16.2 10 <0.1 90 2006 69.9 19 13.1 <0.1 102 HT Herbicide tolerance; IR Insect resistance (mostly Bt); VR Resistance to virus diseases Source: ISAAA, Clive James, 2006 (From: Global Status of Commercialized Biotech/GM Crops: 2006 (http://www.isaaa.org/ RESOURCES/PUBLICATIONS/BRIEFS/35/EXECUTIVESUMMARY/default.html)) Permission to publish table granted by the International Service for the Acquisition of Agri-Biotech Applications

Bioinformatics heralded in another period of plant biotechnology advances. It provided the computational power needed to interpret complex genomic expression information (Lander et al. 1987). This branch of computational science integrates information on plant gene expression, genotype, phenotype, and taxonomic

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relationship with analytical tools for interpreting multivariate data. It is currently providing unprecedented information for building models that give a better understanding about how biotechnology plants interact with abiotic and biotic environmental factors. Bioinformatics is also paving the way for new types of plant derived products from novel biotechnology plants (Shmaefsky 2007). Advances in plant bioinformatics are contributing to other organismic models particularly related to long-term environmental factors that regulate gene expression (Edwards 2007). The complexity of data interpretation provided by bioinformatics has led to the compartmentalization of plant biotechnology research into genomics, proteomics, cellomics, metabolomics, physiomics, and enviromics (Edwards 2007; Shmaefsky 2007). Genomics is the oldest of the endeavors and is rooted in traditional genetics that is continually updated by contemporary molecular biology investigations. Genomics investigates the DNA level of plant function and is tentatively categorized into subspecialties such as chromatinomics, chromonomics, and epigenomics. Chromatinomics is defined as “the total differentiation capacity, gene expression as well as other stem cell functional characteristics that vary throughout a cell cycle transit” (Cerny and Quesenberry 2004). Although chromatinomics started out as an animal cell field, recent advances in botany showed that plants have stem cell with characteristics similar to animals. This adds to the validity that plants can serve as accurate genomic models for animals for understanding organismic development (Dinneny and Benfey 2008). Chromonomics, coined by H. Willard of Case Western Reserve University, is similar in meaning to chromatinomics and deals with how genetic sequences relate to the overall function of an organism. Epigenomics is a holistic approach to understanding an organism’s genome. It is described as an approach that views imprinting, metabolic networks, genetic hierarchies in embryonic development, and epigenetic mechanisms of gene activation and other complex phenotypes from the genomic level down, rather than from the genetic level up (Beck et al. 1999). Crop plant research in epigenomics is contributing to a vast database of knowledge applicable to most multicellular organisms. Plant epigenomics is also contributing in plant usage to a human genomics field called ethnogenomics. Ethnogenomics was defined by E. K. Khusnutdinova as “The main task of ethnogenomics is to study the characteristics of genomic polymorphism and genomic diversity of various groups of population: separate communities, ethnoses, and ethnoterritorial communities” (Khusnutdinova 2003). Genomic properties of early cultivated and domesticated plants correlate with population-specific single nucleotide polymorphisms that affect diet-related health (Box 1981; Limborska 2004). Proteomics investigates the protein makeup of an organism in contrast to the DNA information. According to Stephen M. Beverley of the Mitsubishi Kagaku Institute of Life Sciences (MITILS) of Japan, proteomics is study of the structure and function of proteins, including the way they function and interact with each other inside cells. It is related to the term proteogenomics and transcriptomics, which also investigate the roles of transcribed RNA (Anon 1999). Plants serve as excellent proteomic model organism and GM plants are being exploited for unique and practicable transgenic proteins having a variety of agricultural, alternative

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energy, commercial, and medical uses (Gibson 2008). It is hoped that GM plants may serve as a sustainable and “green” source of biomanufacturing many produces made from fossil fuels (Shmaefsky 2007). Cellomics, metabolomics, and physiomics all investigate different levels of biochemical functions within an organism. The term cellomics was used to describe cell function particularly related to drug impacts at the cellular level (Russo 2000). Transgenic plants that modify cellomic processes are under development for precision disease treatment (Shmaefsky 2005). Metabolic and physiomics research is conducted on the organismic levels. These fields have expanded beyond the study of human pathology to an understanding of processes in any type of multicellular organism and are now covered under the category of functional genomics (Fiehn 2001). Biotechnology plant metabolic engineering trials are serving as research models for other colonial and multicellular eukaryotic organisms. Enviromics is the most global study of genomic function. It was coined by J. C. Anthony of Michigan State University School of Medicine to mean “The envirome is the total complement of environmental characteristics, conditions, and processes required for life form viability and successful adaptation. The genome dwells within the environment, and genomic expression shapes and is shaped by environment.” Enviromics was originally used in the human context for studying drug interactions. It is now being generalized for any organism. New developments in GM medicinal plants are contributing to two fields of human enviromics called pharmacomics and plant nutriomics (Yan et al. 2006). It is hoped to develop medical plant products tailored to pharmacogenetic differences in animals and humans. Transgenic plants are valuable contributors to modern biotechnology as well being benefactors of genetic research on other organisms. Many of the investigations on transgenic plants are leading to innovative technologies with far-reaching economic potential. The biotechnology platform currently includes several specialty areas and research competencies centered on biotechnology plants. Included in the transgenic plant platforms are efforts in biochemical and genetic diversity, large scale biochemical and organic molecule production, metabolic pathway engineering, phenotype development and improvement, and protein design (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007).

13.2.2

Plant Platform Contributions

The development and uses of transgenic plants are still in the infancy stage. However, transgenic plants are forming the foundation of biotechnology research and development (R&D) platforms. The European Community developed an internationally acknowledged classification of biotechnology platforms according to a particular industrial strategy unique to that type of biotechnology. These platforms are the basis of R&D efforts that have agricultural, commercial, or medical applications (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007).

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The Plant Industry Platform is primarily involved in investigating and producing genetically exceptional plants used in agriculture, forestry, and horticulture. It also provides a source of genes used in genetic engineering of microorganisms and plants for commercial applications. This platform is investigating and developing applications for plants that produce commercial proteins, dietary supplements, herbal therapeutics hormones, medical diagnostics compounds, pharmaceutical compounds, research chemicals, and vaccines. Another aspect of this platform is phytoremediation or the use of plants to clean up contamination of air, soil, and water with pollutants (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shimoda 1998; Shmaefsky 2007). Lower plants are also serving as a valuable source of genes and are currently being used in environmental quality biomonitoring systems (Martin 1998). Transgenic plants are making significant contributions to the Plant Structural Biology Industrial Platform. This platform focuses on the functional chemistry of organisms. It includes investigations into the structural analysis of biological molecules at every level of organization. This platform is studied using all methods that lead to an understanding of biological function in terms of molecular and supermolecular structure. Supermolecular structure refers to the forces that cause molecules to interact with other molecules and carry out various tasks. The Structural Biology Industrial Platform looks at the technology transfer potential of carbohydrates, lipid, nucleic acids, and proteins applications (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). Current products of this platform include commercial cements, industrial enzymes, medical adhesives, nanotechnology devices, preservatives, and synthetic plastics all of which can be derived from transgenic plants. For example, lignin pathways are modifiable to produce a wide array of organic polymers ranging from synthetic fuels to plastics (Hans-Joachim and Weiting 1998). The Biotechnology for Biodiversity Platform is a basic research platform that uses information about biodiversity for technology transfer into industrial applications. Biodiversity is generally defined within this platform as the number and variety of living organisms. It takes into account the genetic diversity, species diversity, and ecological diversity of all organisms on the Earth and even on other planets. The biodiversity platform primarily investigates the potential commercial applications of particular genes identified through biodiversity investigations. The botanical aspect of this platform identifies genes from wild plants ancestors of crops. These genes can be used to help crops resist diseases, drought, insects, herbicides, and poor soil quality (Eizenga et al. 2006). A significant proportion of the research in this platform involves the establishment of gene banks (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). The DNA information collected within this platform’s gene bank is stored as a bioinformatics catalog of the DNA sequence and the various traits imparted by a particular sequence of DNA. One of the newest areas is the Environmental Biotechnology Industrial Platform. It is engaged in the pursuit of novel environmental biotechnologies. Environmental biotechnology is a broad field that includes a wide variety of agricultural and

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industrial applications. The Environmental Biotechnology Industrial Platform uses biological systems to remediate anthropogenic and natural chemical degradation of the atmosphere, land, and water. Some current applications include soil and sediment remediation, water purification, the removal of organic and inorganic pollutants, the breakdown or biodegradation of organic pollutants, introduction of natural or genetically modified organisms to treat solid wastes, water treatment, marine clean-up, and the conversion of wastes into other materials and energy (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). This platform overlaps with the Plant Industry Platform. The Biotechnology for Biodiversity Platform also contributes to this platform by identifying genes relevant to environmental protection and remediation. Transgenic plants are also making significant contributions to the In Vitro Testing Industrial Platform. This platform was formed from economic, ethical, political, moral, and scientific arguments in favor of reducing or replacing the need for animal tests commonly used in medicine and research. The platform investigates technologies that comply with the same governmental regulations that set the guidelines for animal testing. It involves the development of in vitro tests that model the chemistry and functions of animals, microorganisms, and plants. The technologies used in this platform currently involve the use of animal cell cultures to replace the role of whole live animals for testing the effectiveness and safety of many consumer products (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). Model plant systems using hairy root cell cultures are being used as a GM plant in vitro model within this platform (Hu and Du 2006). However, plant models are also being investigated as adjuncts to animal and microbial models. For example, it is known that cytoskeleton components of plants can be generalized to other eukaryotic organisms (Grabski et al. 1998). In vitro models are used for safety testing on chemicals such as cleaning agents, cosmetics, dietary supplements, dyes, food ingredients, fragrances, inks, preservatives, and soaps (Shmaefsky 2007). The plant models in this platform must be based on sound scientific principles and have ample evidence to show that they provide equivalent data to animal studies. Current European Community Platforms directly and indirectly related to plant biotechnology are listed in Table 13.3.

13.3 13.3.1

Concerns Assessing the Risks of Transgenic Plants

As with almost any scientific application or technology, biotechnology poses potential risks associated with the benefits it provides. Most technologies are used in spite of the risks they create. For example, the Internet has benefited many people throughout the world with the conveniences of electronic commerce. However, the risk of identity theft and credit fraud comes along with the advantages of shopping

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Table 13.3 Current European community biotechnology platforms ACTIPa Animal Cell Technology Industrial Platform: The Animal Cell Technology Industrial Platform includes animal cell technologies involved in a variety of industrial and medical applications. BACIP Bacillus Subtilis Genome Industrial Platform: The main goal of this platform is bring together information and technological applications related to the genetics the Bacillus bacterial. Bacillus bacteria carry out a variety of metabolic activities that have important commercial value. Biotechnology for Biodiversity Platform: This is a basic research platform that uses BBPb information about biodiversity for technology transfer into industrial applications. EBIPb Environmental Biotechnology Industrial Platform: This is one of the newer platforms and is engaged in the field of environmental biotechnology. ENIPa European Neuroscience Industrial Platform: This platform focuses on medical and pharmaceutical applications related to information about the nervous system. FAIPa Farm Animal Industrial Platform: This platform is composed of small and large agricultural operations involved in farm animal reproduction and selection. Fungal Industry Platform: The Fungal Industry Platform represents research and FIPa technology transfer efforts interested in biotechnology applications of filamentous fungi. HAE Healthy Aging Europe Industrial Platform: This platform combines research on human aging with biotechnology innovations that may reduce ailments and diseases attributed to age. Industry Platform for Microbiology: This is a basic science platform that provides IPMb information on microbial physiology, microbial ecology, microbial taxonomy, and microbial biodiversity. IVTIPb In Vitro Testing Industrial Platform: This platform was formed from economic, ethical, political, moral, and scientific arguments in favor of reducing or replacing the need for animal tests commonly used in medicine and research. LABIP Lactic Acid Bacteria Industrial Platform: The main goal of this platform is coordinate information and technological applications related to the genetics the lactic acid producing bacteria. Plant Industry Platform: The Plant Industry Platform is primarily involved in genetically PIPb unique plants used in agriculture, forestry, and horticulture. SBIPb Structural Biology Industrial Platform: This platform focuses more on the chemistry of organisms. It includes any investigations into the structural analysis of biological molecules at every level of organization. TSE IP TSE Industrial Platform: This platform deals with research related to transmissible spongiform encephalopathies. Yeast Industry Platform: This platform is founded on any applications of yeast related YIPa biotechnology. a Indirectly supplemented by plant biotechnology b Directly related to plant biotechnology

online. Over time, societies provide protective measures to reduce risk, and secure sites that encrypt Internet data in order to reduce information theft. A risk is traditionally defined as the probability of harmful consequences associated with an entity or an action. However, it is important that the risks associated with scientific applications and technologies are evaluated in a detectable or quantitative context (Hansson 2004). Risks of transgenic plants, as is done for other scientific and technological developments, are determined using a risk assessment (Apostolakis 2004). A risk

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assessment is the determination of qualitative and quantitative values of risk related to an entity or situation and recognized as a measurable threat (Wilson and Shlyakhter 1997). Transgenic plants are subject to different types of risk assessment models based on the risks related to their applications, development, or platform categories (Wilson and Shlyakhter 1997; Apostolakis 2004; Shmaefsky 2007). A risk is first determined by identifying any hazards related to the entity of action. The hazard identification determines the nature of the potential adverse consequences of the entity or activity and the strength of the evidence supporting that it can have that type of hazardous effect. Evidence of a hazard must be determined by empirical studies that should have a cause and effect relationship or mechanism for the type of harm being induced. It is also necessary to identify the recipient of the hazard such as agricultural animals, an ecosystem, or humans (Wilson and Shlyakhter 1997; Apostolakis 2004). It is generally accepted by governmental regulatory agencies that it is impracticable and likely impossible to adopt a zero-risk policy (Wilson and Shlyakhter 1997). So, once a risk identification study is completed, an acceptable risk determination is calculated. There is no universally acceptable model for computing how much harm is tolerable for a particular risk. However, the calculation must be represented as a numerical value of what is determined to be a negligible increase in risk or harm from the technology. The negative control for determining negligible is a comparison to not having the technology (Apostolakis 2004). It is in many cases unclear how the consensus on a particular figure is established. This apparent arbitrariness in quantifying acceptable risk leaves the determination open to controversy and debate amongst the public, regulators, and scientists (Flyvbjerg 2006). The risk assessment must also take into account acute versus long-term or lifelong risks (Wilson and Shlyakhter 1997; Apostolakis 2004). A benefits assessment is essential to weight the importance of accepting a risk by using a technological development (Wilson and Shlyakhter 1997; Flyvbjerg 2006). The benefits are characterized in a variety of ways based on the intended application of the technology. For almost all applications of transgenic plants, the benefits need to be accurate, quantitative, and realistic. In addition, the benefits must be based on empirical evidence supported by the mode of action of the technology (Flyvbjerg 2006; Shmaefsky 2007). Upon evaluating the benefits, risk–benefit determination is then calculated for the technology (Fig. 13.3; Virine and Trumper 2008). The assessment that it assumes exposure to personal risk is a normal aspect of everyday life and that a risk should not be used to completely negate the value of the benefits. Several types of risk–benefits analyses can be performed on transgenic plant applications. A statistical risk–benefits analysis is determined using currently available data on the related past benefits and risks. Novel products and technologies require at projected risk–benefits analysis. This analysis uses theoretical models structured from historical on similar, but not identical, benefits and risks. Some types of innovations are subject to perceived risk–benefit analyses. This assessment is based on intuition and weighs emotional factors that can cause people to overestimate the risks to the degree of diminishing the value of the benefits (Virine and Trumper 2008). Many types of transgenic plants applications

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Risk Assessment

Risk

Experimentation

Entry of Action

Hazard

No

Yes

Risk Determination

No Action Exposure Estimation

Consequence Assessment

Yes

Risk Management Program

Fig. 13.3 Risk assessment flowchart

Benefits

Risk Estimation Deterimnation

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unfortunately stir fears that compel regulators to carry out a perceived risk–benefit study (Shmaefsky 2005; Morris 2007). Positive outcomes of perceived risk are critically important in ensuring the marketability of biotechnology plants (Spehar 2000). Transgenic plant risk assessment and regulations are carried out in the US by three governmental agencies: the Department of Agriculture (USDA), the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA). Each agency is individually responsible for oversight of genetically engineered plants and products developed in the US and imported from other countries (Farm Foundation 2005). Their responsibilities are determined by the nature of the biotechnology plant application. For example, the USDA oversees applications related to animal feed and human food. A division of the USDA called the Animal and Plant Health Inspection Service (APHIS) has authority over the field cultivation of transgenic plants. It also oversees veterinary biological substances, including animal vaccines that are products of biotechnology under the Virus, Serum, Toxin Act. The EPA has jurisdiction over the testing of transgenic plant products containing pesticidal compounds. This covered primarily under the Toxic Substance Control Act Biotechnology Program of the Office of Prevention and Toxic Substances. They also develop regulations on plants grown in the field and used for phytoremediation. The FDA is responsible for overseeing biotechnology plant products used for manufacturing nutritional supplements and pharmaceutical agents (Farm Foundation 2005; Shmaefsky 2007). The FDA usually regulates biotechnology plants under the Federal Food, Drug, and Cosmetic Act. In many situations, the supervisory authorities of these agencies have common characteristics that overlap, sometimes causing jurisdictional confusion. Current United States regulations over biotechnology plants can be found on the USDA (USDA Animal and Plant Health Inspection Service 2007), EPA (EPA Biotechnology 2008; EPA Pesticides 2008), and FDA websites (FDA Biotechnology 2008). As in the United States, transgenic plants are regulated to differing degrees in other nations. Transgenic plants in Canada are regulated by the Canadian Food Inspection Agency (Canadian Food Inspection Agency 2008). They are charged with safeguarding Canada’s food supply and the plants and animals to ensure consistent standards for safe and high quality foods and agriculture-related products. They share this responsibility with another government agency called Health Canada (Health Canada/Sante 2008). Health Canada evaluates the human health safety of products developed from transgenic plants including cosmetics, foods, pharmaceuticals, medical devices, pesticides. Environmental health concerns of biotechnology plants are also assessed by Health Canada. Canada also has the Canadian Biotechnology Advisory Committee, which advises on biotechnology plant risk policy (Canadian Biotechnology Advisory Committee 2008). European Union (EU) nations regulate biotechnology risks under the European Commission on Biotechnology (European Commission on Biotechnology 2008). This role is carried out by the Secretariat-General who supervises DirectorateGeneral that oversee specialized areas of technology. The Directorate-General

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over Life Sciences and Biotechnology works with transgenic plants. It develops new policies and evaluates how the policies fit into other EU institutions. Risk issues are covered under guidelines related to the Confidence in Science-Based Regulatory Oversight recommendations (Commission of the European Communities 2002). The European Parliament deals with legal aspects of biotechnology plant risks (European Parliament 2006). They have jurisdiction over biotechnology risks categorized as industrial biotechnology and bioremediation, nonfood agricultural and silvicultural biotechnology, and pharmaceuticals. Many Asian nations are collectively investigating harmonization agreements that are in agreement with America, Canadian and European risk assessments. The Food and Agriculture Organization of the United Nations is serving as a neutral forum for assessing biotechnology plant risks in Asia (Food and Agriculture Organization of the United Nations 2008). The cooperating nations include Bangladesh, China, India, Indonesia, Malaysia, Pakistan, Philippines, Sri Lanka, Thailand, and Viet Nam. The Organization for Economic Co-operation and Development is another harmonization agency with a more global charge of making biotechnology plant risk evaluation studies for countries worldwide. They state their mission as, “The main focus of the work is on international harmonization of regulatory oversight in modern biotechnology, which will ensure that environmental health and safety aspects are properly evaluated, while avoiding nontariff trade barriers to products of the technology” (Organization for Economic Co-operation and Development 2008). Biotechnology applications are likely the most highly scrutinized and regulated contemporary technologies. Much of this is due to the newness of the applications meaning that there is a large lack of past data about probable risks. It is also possible that the strict regularly environment of biotechnology plants is to ensure that almost no harm is produced to appease public misunderstanding and unwarranted pubic fear of biotechnology (Fritz et al. 2003; Shmaefsky 2005, 2007). Tight regulation has not stifled the advancement of transgenic plant development. However, it has slowed the pace of getting products to market and produced a technology transfer environment requiring high costs to accommodate for comprehensive risk assessments (Caruso 2006).

13.3.2

Case Studies in Biotechnology Plant Risks

The regulations placed on biotechnology plant applications are meant to reduce the chance of significant concerns of risks to the end user and the environment. However, this does not always allay concerns about transgenic plants and their products. Scientifically valid problems have arisen from transgenic plants causing regulators to reconsider risk assessments. Four representative risk concerns came about with the earliest successful transgenic plants released on the market: horizontal gene transfer, genetic pollution and super weeds, biopesticide safety, longterm toxicological effects, and biodiversity loss (Shmaefsky 2005).

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Horizontal Gene Transfer

Horizontal gene transfer, which is also known as lateral gene transfer, is described as the natural genetic transformation of bacteria believed to be the essential mechanism for genetic plasticity within and between species (Davison 1999). This inherent tendency of bacteria to exchange DNA in the environment raised safety concerns about the production of transgenic bacteria. It was believed that novel genes introduced into bacteria could spread to wild type bacteria thereby producing potentially harmful wild bacteria. This genuine concern compelled the biotechnology community to hold the Asilomar Conference on Recombinant DNA in 1974 (Berg and Singer 1995). As a result of the conference, guidelines were developed to reduce the risk of horizontal gene transfer from genetically modified microorganisms. The widespread field cultivation of transgenic plants in the late 1990s raised concerns if horizontal gene transfer was possible in plants. Michael Syvanen in 1994 reported that studies on plant phylogeny showed horizontal gene transfer as a natural phenomenon involved in evolutionary change (Syvanen 1994). Plants contained genes from the genomes of other plants and of soil microorganisms. His other supporting work consisted of various genetic studies dating back to the early 1980s, including recent genomic investigations on Archaea. Other researchers have reported evidence that functional chunks of DNA persisted in the environment. Plus they found that significant quantities of gene vector-like plasmid DNA survived passage through a mouse digestive system and even stayed intact after macrophage ingestion. At the time of his initial research, Syvanen had no confirmed studies of this type of gene transmission occurring in nature. However, the compelling laboratory studies were evidence enough to warrant Syvanen’s concern of horizontal gene transfer in the environment by plants. Much of the scientific community was not persuaded by Syvanen’s extrapolations about horizontal gene transfer in plants. They felt the risk was unfounded because of the lack of direct experimental evidence in the field (Ho 2002). This skepticism was undermined in 2001 when a plausible mechanism for horizontal gene transfer in plants was proposed. A study reported the transmission of complete gene cassettes by rhizosphere Pseudomonas (Sengelov et al. 2001). A host of phylogenetic, laboratory, and greenhouse studies further supported the risk of horizontal gene transfer by a variety of rhizosphere and pathogenic bacteria including Agrobacteria (Broothaerts et al. 2005). Concerns were now being raised about wild plants picking up novel genes and soil bacteria obtaining kanamycin resistance reporter genes from transgenic plants. Unfortunately, there is not enough research about the frequency of horizontal gene transfer in the field. So, it is difficult to make any policies or regulations that reduce the incidence of horizontal gene transfer to other plants or to soil microorganisms. Estimates of horizontal gene transfer from transgenic plants to microorganisms in the environment is likely at a frequency one trillion times lower than the current risk assessment from laboratory data (Heinemann and Traavik 2004). Sensitive methods for finding reporter genes or other components of transgenic

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gene vectors will have to be developed to come up with acceptable limits of transfer. Metabolic engineering of transgenic plants creates other concerns of horizontal gene transfer. There is now experimental evidence that the plant organelle genes can by transferred in the field (Richardson and Palmer 2007). The studies showed that parasitic plants were donated and received mitochondrial genes when attached to host plants. Data indicated that most of the transfer involved a single gene. However, horizontal gene transfer of large sets of mitochondrial genes was also demonstrated. The studies showed the transfer in bryophytes and eudicots. It is shown that the same mechanism can transfer chloroplast genes. Parasitic plants are common invaders of agricultural lands adjacent to natural areas. They are particularly invasive in the ecotomes formed by plant cultivation (Pennisi 2006). Frequency studies capable of doing a risk assessment are not available. The apparent risk of horizontal gene transfer with transgenic plants is a valid scientific claim. Laboratory evidence supports that intact functional genes can be transmitted from plants somewhat readily to other plants and biosphere microorganisms. The precedence for transferring deleterious traits from one organism to another is well supported in the microbiological literature. Phylogenetic studies on plants confirm genomic and epigenomic sharing through the rhizosphere microorganisms and parasitic plants. In spite of these findings, there is no compelling evidence that the amount of horizontal gene transfer from transgenic plants poses harm to people or the environment. As follows, it is nearly impossible to come up with rational risk assessments. In addition, there are no definitive strategies for reducing horizontal gene transfer from transgenic plants.

13.3.2.2

Genetic Pollution and Super weeds

Natural plant breeding mechanisms raised another set of legitimate risk concerns for transgenic plants grown in the field. A growing number of studies confirmed the gene flow between closely related plants during normal pollination mechanisms (Arias and Rieseberg 1994). The consequence of gene flow is the transmission of traits between crop plants and wild type ancestors living within pollination distance. Earlier papers used the term genetic pollution to describe this contamination of the environment with domesticated plant genes (Dubois and M’orere 1980). Many proponents of transgenic plants see this as a mechanism for producing uncontrollable wild plants called super weeds (Ellstrand and Schierenbeck 2000). The term super weed was coined in 1949 by E. Anderson to explain newly invasive plant lines that resulted from hybridization between crops and related wild plants. N. C. Ellstrand reintroduced the term to mean wild plants that picked transgenes from transgenic plant relatives. He mainly was concerned about weeds picking up characteristics that make them more invasive and difficult to control. Genetic pollution at first was held with the same skepticism as horizontal gene transfer by the scientific community. Initially, there was not a body of evidence supporting the creation of super weeds through gene flow. However, in 2000,

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N.C. Ellstrand used DNA analyses to document 28 examples of hybridizations that increased the aggressiveness of plants (Ellstrand and Schierenbeck 2000). This increase in vigor promoted invasive traits in the recipients of the gene flow. He showed pollen transfer and compatibility as the mechanism of gene flow. Compelling data was presented for the following gene flow events: Male sterile canola (Brassica napus) picked up fertility genes from wild radish (Raphanus raphanistrum) and birdsrape mustard (B. rapa L.), oilseed rape (canola) passed transgenes for herbicide resistance to turnip rape (B. rapa syn. B. campestris), and sorghum (Sorghum bicolor) exchanged flowering time genes with Johnsongrass (S. halepense). Transgenes also make their way from genetically modified alfalfa, berries, cotton, cucumbers, potatoes, rice, strawberries, and sunflowers. The probability of the transgenes appearing randomly through mutation in the wild population was highly unlikely. The possible deleterious effects of herbicide resistance genes were particularly being criticized by opponents to transgenic plants. A concern about gene flow patterns that produce herbicide-resistant weeds was fueled by the abundance of studies on the transmission of other types of transgenes (Cavan et al. 2008). Gene flow of herbicide resistance genes would in effect render weed plants resistant to the popularly used broad spectrum agricultural herbicides. This valid concern compelled government investigators to seek proactive strategies for controlling herbicide-resistant super weeds. The early research did not evaluate transgene transfer frequency and did not establish an accurate determination of the environmental proximity needed for successful transgene transmission in the field. However, the studies did establish the fact that traits that are most likely to be retained in hybrids are those, which improve fitness by encouraging invasiveness, drought tolerance, disease resistance, herbicide resistance, and winter hardiness. The research also recognized that the repeated introduction of transgenes produces an effect called swamping, which ensures establishment of the new genes in the population (Rissler and Mellon 1996). Evidence of super weeds being created in the field was confirmed in a 2004 study conducted by the Department for Environment, Food and Rural Affairs in the UK. The study showed gene flow of herbicide resistance genes from GM oilseed rape to wild rape (Daniels et al. 2004). Initially, transgenic plants grown in the field were designed with excision genes and genes that cause male infertility (Daniell 2002). Excision genes remove the transgene when not expressed. Infertility genes prevented pollen formation. These technologies were used to prevent the spread of transgenic plants into the wild. Concerns were raised that these strategies were not effective at preventing gene flow and even horizontal gene transfer (Jackson et al. 2002). For example, the excision genes could be separated from the desirable genes on another expression vector. In 1999, J. Gressel of the Weizman Institute of Sciences proposed another approach to reducing the risk of gene flow between GM crops and wild plants. He suggested using tandem construct vectors to reduce the chance of successful gene transfer through pollination (Gressel 1999). Tandem repeat technology

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piggybacks two genes that restrict gene flow. For example, genes affecting germination by altering seed dormancy, ripening, and dissemination closely linked to genes for the desired trait in the construct. Other strategies include adding traits that cause dwarfing, inhibit flower production, and prevent maturation. The basis of the constructs were to use tightly linked genes that do not segregate separately, use traits that are harmless to crops but deleterious to typical weeds, and use genes that are disadvantageous to the successful reproduction of weeds within a population lacking the construct trait. The risk analysis of gene flow in transgenic plants requires an understanding of several factors that affect the successful expression of transgenes in nontarget plants. First, the probability of gene flow must be determined. This involves looking at the potential gene flow candidate plants growing within pollination distance to the transgenic plants. Factors designed to limit gene flow are taken into account when determining the probability. Another factor that must be assessed is the effect of gene flow on improving the fitness of the wild plant receiving the transgenes. Another consideration in a risk analysis is the ability for gene flow that disables factors such as suicide genes in the transgenic crop plants. Gene flow is a valid scientific risk that is already being addressed in the design of transgenic plants. It is more likely a greater risk than horizontal gene transfer. There is ample evidence that the gene flow has occurred. Improved strategies for reducing the back and forth gene exchange between transgenic plants and wild relatives will be needed for the development of each novel genetically modified crop grown in greenhouses and in the field. Continuous monitoring of super weeds and transgenic plants disabled by gene flow is required to develop future strategies to reduce gene flow. The scientific community needs to compromise on accepting worst-case scenario analysis models until more information is collected to fully evaluate any risks of deleterious gene flow (Thompson et al. 2003).

13.3.2.3

Biopesticide Safety

Some of the earlier biotechnology plant applications were intended at reducing pesticide use in agriculture while seeking less crop damage by herbivorous pests. This was driven by a growing number of environmental and public health regulations mandating reductions in agricultural chemical use (Shmaefsky 2005; Piruzian et al. 2006). In the late 1980s in the United States, transgenic plant development was influenced by a revitalization of Delaney Amendment-type food safety concerns. In 1958, Vermont Senator Charles L. Delaney added a provision to the US Food, Drug, and Cosmetic Act, which stated that no food additive shall be deemed safe after it is found to induce cancer when ingested by human beings or animals, at any dose level. Such an additive therefore must not be used (Benders 1995). This zero tolerance thinking became the acceptable risk held by the public. It was spurred by media reports about the possible harmful effects of agricultural chemicals, food additives, food irradiation, food-borne microorganisms, and drugs used in agricultural animals (Jones 1992). As a result, crop growers were looking

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for ways of reducing pesticide use. Pesticides in particular were targeted for the harmful effects as possible carcinogens and endocrine disrupters (Colborn et al. 1996). Harmless natural pesticides such as pyrethrums were not efficacious for large scale field crop production (Shmaefsky 2005). So, other options to safely control crop pests had to be evaluated resulting in the development of biopesticides. The EPA defines biopesticides as “Certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. For example, canola oil and baking soda have pesticidal applications and are considered biopesticides” (Environmental Protection Agency 2008). There are approximately 200 registered biopesticides found in a variety of products. Developers of transgenic plants took advantage of microbial pesticide genes with a cytotoxic specificity towards insect digestive systems. The most effective of these was Bacillus thuringiensis toxins, or Bt toxins, derived from a Gram-positive soil bacterium that is also found in the guts of insects. Bt toxins are delta-endotoxin that lyse cells lining the digestive system of insects and nematodes susceptible to a specific type of Bt toxin. The toxin binds cadherin-like proteins and forms ion channels that cause an efflux of potassium ions from the cells. Bt toxins are active within hours and ultimately result in starvation within a few days (Harper 1974). There are many Bt toxin genes. Common ones used in biotechnology plant insect control applications are the Cry1Ab, Cry1Ac, Cry1F, Cry3Bb, Cry34Ab1, and Cry35Ab1. The genes are modified in various ways to increase transcriptional functionality in plant cells and are inserted into cauliflower mosaic virus (CaMV) 35S promoter expression vector (Lewin et al. 1998). Bt gene products proved safe for agricultural use by the EPA and USDA. This led to the release of Bt corn, cotton, and potatoes as field crops in the 1990s (Piruzian et al. 2006). The field cultivation of these crops was uneventful until concerns arose about the potentially harmful effects of Bt on nontarget insects, pest insect resistance to Bt, and vector–virus recombination. Bt corn carrying the Cry1ab gene used against corn borer became the focus of criticism about its effects on nontarget insects. Two factors precipitated this concern. Corn is wind-pollinated and the corn borer is a lepidopteran. The wind pollination factor raised legitimate concerns about the spread of the Bt toxin into nontargeted areas. The Bt toxin was meant to be effective as the corn borer larvae were feeding on plant tissues expressing the gene. Several studies cautioned that it was possible that Bt corn pollen could be toxic to beneficial Lepidoptera in areas downwind from the fields (Sears et al. 2001). The study focused on monarch butterflies and correlated with an apparent decline in monarch population migrating between Eastern North American Bt fields and Mexico. It also contained a risk assessment model that supported the potential hazardous effects of Bt toxin corn pollen on wild insect populations outside of the field. Studies by the EPA confirmed that harmful levels of Bt toxin protein were present in the corn pollen and that monarch larvae feeding on the pollen could be harmed (US Environmental Protection Agency 1995). However, there was no study confirming a direct link between dwindling monarch populations related to the distribution of Bt corn fields.

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Researchers at the University of Illinois conducted a study in 2001 to look into the corn pollen density of the monarch feeding areas adjacent to corn fields. They discovered that within a corn field the pollen densities are below 600 grains cm–2 of leaf for 95% of the plants. The highest pollen density was 1,400 grains cm–2 under rainless conditions just after anthesis (Pleasants et al. 2001). The USDA discovered in 2002 that “eating leaves with pollen coating densities below 1,000 grains cm–2 had no effect on caterpillars’ weight or survival rate. Above 1,000 grains cm–2, caterpillars were smaller than those from the control treatments, but their survival rate was no different from that of controls” (Kaplan 2002). So, the USDA report invalided the claims that Bt corn was responsible for monarch population loss. It is now believed that the monarch population incongruity way have been due to errors in reporting the population or due to a variety of density-independent and densityindependent environmental factors (Borland et al. 2004). Pesticide resistance is a realistic and scientifically valid risk of using any type of pesticide. Biopesticides are equally likely candidates for being counteracted by evolutionary mechanisms of target organisms. There are three major mechanisms for pesticide resistance at the biochemical level against biopesticidal compounds: alterations of pesticide toxicity target proteins, the development of enzymemediated resistance, and thermal stress response (Patil et al. 1996). Biopesticides that bind to surface proteins, such as Bt toxin, malathion, and pyrethroids, require a specific ligand binding conformation site on the particular target protein. Mutations that affect the amino acid composition of the binding site can render the biopesticide less effective by reducing binding tenacity (Williamson et al. 1996). For Bt toxin, this lack of binding specificity to the target receptor means reduced capability to lyse intestinal cells of the insect pest. Biopesticides are treated as xenobiotics by target organisms and are thereby subject to degradation by detoxification metabolic pathways. Typical detoxification enzymes found in many organisms include esterases, glutathione S-transferases (GST), and oxidases (Cygler et al. 1993). Most types of Bt toxin crystal proteins were initially thought to be resistant to enzymatic decay. Studies confirm that multiple types of toxins derived from B. T. israelensis are subject to resistance by enzymatic degradation in certain lepidopterans (Keller et al. 1996). Heat or thermal shock proteins likely play a role in Bt toxin resistance (Patil et al. 1996). They have a homeosis in which exposure to high levels of a toxin inhibits the defenses while low or subtoxic exposure stimulates protective mechanisms against the toxin (Cohen 2006). As with other biopesticides, Bt toxin is likely to induce thermal shock because they induce lysis by apoptosis. Apoptosis induction is one factor having a homeosis effect (Samali and Cotter 1996). Methods have been developed in which the Cry2A class of Bt toxin genes can be modified to reduce thermal shock induced resistance (Mandal et al. 2007). Transgenic plants using the CaMV expression vector, and related vector types, have been criticized by M.-W. Ho of the Open University in the UK as being a “promiscuous” sequence of genetic material. She used this term to describe the vector’s ability to recombine readily in unpredictable ways with genomic DNA, extrachromosomal DNA, and intracellular organisms such as viruses (Ho et al.

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2000). The ability for Bt CaMV expression vectors to combine with plant viruses during field infections is supported in the literature (Kohli et al. 1999). There is concern that this ability to recombine with wild type viruses can produce new types of viruses with harmful characteristics produced by vector–virus recombination (De Vries and Wackernagel 1998). Ho provides compelling theoretical evidence that the CaMV vector can transfer Bt cassettes to viruses that then transfer the functional gene to wild plants growing in the vicinity of transgenic plants. She believes that this hazard can spread far from the fields of transgenic plants (Ho and Steinbrecher 1998). Laboratory studies and knowledge of the behavior of transgenic plant expression vectors support the idea of possible hazards associated with biopesticides expressing plants. Evidence is available to assess nontarget insect toxicity, biopesticide resistance, and vector–virus recombination as measurable risks associated with transgenic plants that express biopesticides. However, as with other concerns about transgenic plants, there is little field evidence about the degree of risk posed by the technology. So, it is difficult to determine the severity of the risk. Consequently, it is not possible to calculate if the risk outweighs the benefits of the biotechnology plants. Older regulations were apparently not adequate for preventing these risks based on the current findings (Rechcigl 1998).

13.3.2.4

Long-Term Toxicological Effects

A growing number of biotechnology plants are being genetically modified for the food supplement and nutraceutical (or nutriceutical) markets (Hugenholtz and Smid 2002). This usually involved enhancing plant metabolite expression or introducing transgenes for a nutritional supplement such as an amino acid or a vitamin. The most common types of nutraceutical biotechnology plants are engineered to produce antioxidants, phytoestrogens, prebiotic compounds, and vitamins (Ajjawi and Shintani 2004). These plants were developed partly based on global consumer demand for nutritional supplements and to a certain extent to provide higher nutrition plants for developing nations (Piruzian 2005; Shmaefsky 2005). Many of the ingredients of transgenic plants go into functional foods. Functional foods are foods formulated to provide a certain wellness benefit and may contain plants materials designed to overexpress omega-3 fatty acids that are believed to reduce heart disease (International Life Sciences Institute 2002). For the good they were intended to provide, certain biotechnology-based food supplement and nutraceutical plants were received with much criticism from the scientific community (Shmaefsky 2005). Two products of particular concern were golden rice and high-isoflavone soybeans. There were fears that improper control over the consumption of these products can lead to toxicological dosing (Millstone et al. 1999; Domingo 2000). Golden rice was developed by P. Beyer and I. Potrykus in 1999 to investigate the possibility of b-carotene production in grains (Ye et al. 2000). It used the phytoene synthase (psy) gene from daffodil and the carotene desaturase (crtI) gene from the

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bacterium Erwinia uredovora to produce b-carotene, which in turn is converted to vitamin A in the human digestive system. The ultimate goal of overexpressing vitamin A was to reduce malnutrition-induced blindness primarily in the developing nations. Rice is normally low in vitamin A. Yet, rice makes up a bulk of the caloric intake of people in the developing nations. This first generation of golden rice did not produce sufficient enough quantities of b-carotene to meet the 300 mg per day vitamin A requirement for young children. So, a new generation of transgenic rice called golden rice 2 was produced by modifying the expression vector. It synthesized 20-times the amount of b-carotene as the original golden rice (Paine et al. 2005). Nutritional concerns about golden rice ranged from furthering malnutrition to inducing vitamin A toxicity. Research shows that improper or prolonged storage of golden rice significantly reduces the b-carotene content (Zimmermann and Qaim 2004). People not educated about storing the rice may end up not being benefited by the golden rice. It is feared that they may even avoid other forms of vitamin A supplementation provided by public aid organizations. Vitamin A toxicity is particularly a concern with golden rice 2 because of its high b-carotene content (Anderson et al. 2004). Pregnant women were a special precautionary group because of the known teratogenic effects of vitamin A in large doses. Overdosing concerns were a valid argument based on experience with foods fortified with iron (Hurrell 1997; Van den Berg 1999). This concern haunts the use of transgenic rice developed by the International Rice Research Institute to sequester iron equivalent to food fortification levels (Florentino 2001). These concerns are being addressed and remedied by risk–benefit studies related to food distribution and public education programs (Al-Babili and Beyer 2005). Tomatoes and other crops developed for high flavonoid content created scientifically valid concerns about phytoestrogens in the diet. These crops were primarily grown for the purported health benefits of the antioxidant properties of flavonoids (Reddy et al. 2007). Ample in vitro research shows that dietary antioxidants can reduce the incidence of cancer and cardiac diseases (Goodman et al. 2004). Other studies show that antioxidants reduce inflammation induced disease by suppressing serum C-reactive protein (Chun et al. 2008). In addition, some plants were developed for the phytoestrogen effect of flavonoids as a hormone supplement therapy (Ji and Peterson 2004). The plants are developed by inserting or enhancing flavonoid biosynthetic pathways (Jung et al. 2000). Studies on increased phytoestrogens in diet showed risks that in some people may outweigh the benefits. A high to moderate intake of dietary phytoestrogens can promote breast cancer in women with breast cancer susceptibility genes (Bernstein 2002). Other studies show that increased levels of dietary phytoestrogens can impair estrogen function in any individual (Kuiper et al. 1998). Phytoestrogens may inhibit testosterone function during human male embryogenesis in mothers who consume large levels of estrogen-like compounds (Hoei-Hansen et al. 2004). The US FDA currently places precautions on antioxidant and phytoestrogen nutraceuticals, under Title 21 – Part 101, because of the lack of adequate long-term human health risk–benefit studies (Code of Federal Regulations 2008). Other

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precautions and guideline about functional food safety and utility are provided by the Institute of Food Technologists (Institute of Food Technologists 2005).

13.3.2.5

Biodiversity Loss

The danger of GMOs to biodiversity has been a concern to many scientists since the 1986 field trials of ice-minus Pseudomonas syringae that were genetically engineered to protect plants from frost damage (Amarger 2003). It was released with great protest from various environmental protection groups by Advanced Genetic Sciences of Oakland, California. Greenpeace International was one of the earlier groups to mount an anti-GMO campaign (Greenpeace International 2008). They support their opposition to field GMO crops by stating, “GMOs should not be released into the environment as there is not adequate scientific understanding of their impact on the environment and human health.” Greenpeace believes that there is a long-term risk of biodiversity loss created by field grown biotechnology plants. They rightfully defend their view with the fact that there are no adequate studies showing that biodiversity would not be affected. However, there is little direct scientific evidence that biodiversity would be significantly and irreversibly be harmed. Their outlook on biodiversity impairment by biotechnology is supported by other conservation organizations such as World Wildlife Fund (World Wildlife Fund 2008). A science based nonprofit organization called the Union of Concerned Scientists (UCS) uses extrapolative field studies and laboratory research to support their opposition to GMOs. In particular, they are opposed to field grown transgenic plants (Union of Concerned Scientists 2008). They state, “UCS does not support or oppose genetic engineering per se. With respect to some applications, such as the production of pharmaceuticals by genetically engineered bacteria, the benefits are clear and compelling. In the food system, however, we find the risk–benefit calculus more difficult.” As consistent with Greenpeace, the UCS feels the risks of GMO field crops have not been fully explored and that these crops have the potential to disrupt biodiversity. A variety of research studies support the biodiversity hazards of field grown transgenic plants. Some studies show that GMO crops could affect fauna and flora through intensification of agricultural activities into wilderness fueled by easier cultivation in previously non-arable areas and by greater profits from transgenic plants (Manteo 1998; Johnson 2000). Nontarget wild insect deaths have also been confirmed with filed use of biopesticides (Birch et al. 1997; Jensen 2000). The impacts of horizontal gene transfer and gene flow on plant biodiversity near agricultural lands has also been studied and assessed for potential biodiversity impairment risks (Ammann et al. 1999; Hodgson 2000; Crawley et al. 2001). Aquatic biodiversity is also been studied because of the possible deleterious effects caused by phytoremediation and biopesticides biotechnology plants. A variety of studies also investigated the impacts of biotechnology field crops on different

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aspects of soil biodiversity (Donegan et al. 1997; Crecchio and Stotzky 1998; RosiMarshall et al. 2007). Accurate and research based risk assessments are currently being determined for the impacts of transgenic plants on local and global biodiversity. The risks associated with agricultural development have to be assessed separately from the direct impacts of the plants on the environment. Earlier risk assessments are being combined with contemporary findings to develop rational risk–benefit models for field grown transgenic plants (Regal 1989; Williamson 1993; Ammann et al. 1996; Clark and Lehman 2004; Garcia and Altieri 2005; Raybould 2006; Raybould 2007). So far, there is currently no measurable biodiversity loss directly associated with the unique attributes of transgenic plants compared to traditional field grown plants.

13.4

Compulsions

There are many public health fears of transgenic plants in spite of the best regulations that assure low risks of harm to humans and the environment (Fig. 13.4). Many of these fears in the early days of biotechnology were not founded on scientific evidence (Lewis 1998). These fears are usually fueled by pseudoscientific claims from anti-biotechnology groups or are founded on anecdotal evidence. Unfortunately, these compulsions are reinforced by negative or inaccurate media coverage and are promulgated by certain opponents of biotechnology (Baker 2005). Today, many of the compulsions are due to a lack of public understanding of the science behind transgenic plants and the regulations that reduce risks to environmental and public health (Gaskell and Bauer 2001). The emotions behind compulsions are not something to be ignored or taken nonchalantly. They become myths that are difficult to erase from public memory. Many of the myths remain on anti-biotechnology websites that are regularly cited as factual information by consumer and public interest groups. Public

Fig. 13.4 Perceived risk

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misunderstanding of transgenic plants in the past has led to anti-biotechnology legislative policies, decreasing capital investments in biotechnology companies, governmental restrictions on biotechnology funding, product prohibitions, protests by public interest groups, and terrorist attacks against biotechnology facilities (Querling 2000). This negative publicity is compelling some companies such as Unilever in Europe to avoid using plant materials made with transgenic plants (Informa Economics, Inc. 1999). Typical new coverage of transgenic plant protests and moratoriums include: 1. 60 Arrested In Sacramento Bio-Tech Protests Demonstrators Dress As Corn, Butterflies And Tomatoes. Oakland Tribune, June 24, 2003: More than 1,500 protesters rallied at the state Capital then marched through downtown as the Ministerial Conference and Expo on Agricultural Science and Technology began and discussed for three days on genetically engineered crops (BNET Business Network 2008) 2. Eight arrests at GM crop protest. BBC News, July 14, 2001: Approximately 40 protesters broke into the field and began ripping up the crop of GM fodder maize (BBC News On-line 2008) 3. French Farmers Protest Against GMO Maize Destruction. Reuters News Service, August 2, 2006: Around 300 maize growers protested on Tuesday in southwest France in defense of a local farmer whose field of genetically modified (GMO) maize was destroyed at the weekend by activists linked to Jose Bove (Planet Ark 2008) 4. GM crops banned in Switzerland until 2012. Animal Feed & Animal Nutrition News, May 29, 2008: The Swiss Federal Council (government) has voted to extend the country’s moratorium on genetically modified (GM) plants for a further 3 years beyond the current expiry date of November 2010, Dow Jones reports (Reed Business 2008) 5. Mass Protests against GM Crops in India. ISIS Press Release, March 30, 2008. As India edges closer to what is probably the last year of field trials for Bt Brinjal (eggplant, aubergine) before commercial approval may be granted, large scale resistance has been building up all over the country. Bt Brinjal, if allowed in India, would be the first food crop in the world with the Bt gene inserted into it that is to be directly consumed by human beings. Indians feel that they are about to be made guinea pigs by USAID, and by Monsanto and Cornell University that have developed this crop (Institute of Science in Society 2008) 6. Kenya’s Biosafety Bill faces opposition. African Science News Service, August 21, 2007: The debate on agricultural biotechnology in Kenya boiled over again last week when peasant farmers and GMO critics staged a demo against GMO proponents who see it as a panacea to low yields against . . . (African Science News Service 2008) 7. Landless peasants occupy Syngenta plants (Justic¸a manteˆm multa de 1 milha˜o para Syngenta). Reuters News Service, December 15, 2007: Hundreds of activists broke into a Swiss-owned Syngenta agrochemical plant in the state of Sao Paulo, expelling 50 employees and shutting down production, a company

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spokeswoman told Reuters. Members of the Landless Rural Workers’ Movement, or MST, and the allied group Via Campesina also destroyed genetically modified corn and soy seedlings at a Syngenta farm in the northeastern state of Ceara, the groups said. The groups demand Syngenta leave Brazil, accusing the company of attacking landless workers and violating environmental laws (Terra de Direitos 2008) 8. GM Crops in Australia – will the moratoria end? GMO Compass, August 31, 2007. The cultivation of GM crops is banned in all Australian states except Queensland. However, moratoria will expire in New South Wales and Victoria next year. A report written on behalf of the Australian government now supports the commercial use of GM plants to promote competitive agricultural production. This has raised the debate on the future role of GM plants in Australia (GMO Compass 2007) It is difficult to predict the full extent of human behavior when compulsions are based on misunderstanding and fear of a technology. Some people react by avoiding the technology and others react by violently opposing it. Given normal perceptions of a technology, risk is assessed in the mind as the threat impact multiplied by the probability of impact. Fear and misunderstanding can elevate either or both the threat impact and probability values. People generally prioritize the dangers of a technology by multiplying the risk by their ability to control that risk (Gardner 2008). Misunderstanding and fear exaggerate a person’s inability to control thereby greatly decreasing their acceptance of the technology. Major compulsions about biotechnology fall into the categories of “Frankenfoods,” food allergies and vaccine resistance, and erosion of the global economy.

13.4.1

Frankenfoods

The pejorative term “Frankenfood” was coined in 1992 by Boston University English Professor Paul Lewis in a letter about biotechnology foods to the New York Times (Katz 2005). This term became a symbol of the perceived dangers of GMO foods. It is commonly used in publications and dialogs by opponents of transgenic plants for animal and human consumption. The Frankenfood term has effectively associated transgenic plants with the unethical and unnatural methodologies used to produce the fictitious monster in Mary Shelley’s book Frankenstein or The Modern Prometheus. Unfortunately, the use of the term has been very successful in producing public compulsions about transgenic plants (Rowan 1994). A news story entitled Frankenfood concerns are valid, published in 2000 in the Albuquerque (New Mexico) Journal is a moderate view of the public sentiment about Frankenfoods. It stated: The United States moved away from its near-isolationist opposition to the labeling of genetically modified foods last weekend when it accepted a new international agreement that will speed the labeling of such foods on the world market.

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But it has yet to shed fully its unconditional support of the biotech industry, as evidenced by the compromise it exacted in the UN Biosafety Protocol: For 2 years after the protocol takes effect, labels need merely say a product has been engineered, without specifics. During those two years, negotiators will work out more specific labels. The United States had only a handful of allies in opposing labeling; 125 other nations supported the move, including members of the European Union. EU resistance to so-called “Frankenfoods” is strong in light of member nations’ experiences with food scares, from Mad Cow fatalities in the ’90s to historical episodes of famine. While there is no evidence genetically modified foods are unsafe to eat, consumers do have legitimate concerns that make detailed labeling imperative. (Albuquerque Journal Online 2000)

More extreme news coverage sternly denigrates any benefits of transgenic plants while stressing unsubstantiated risks to human health. Greenpeace International promoted the following news story in Europe entitled Monsanto maize approved for human consumption potentially toxic warns new study. It was released by Media-Newswire in 2007: The study, carried out by French scientific research institute CRIIGEN on the results of rat feeding trials using a GE maize made by biotech firm Monsanto, highlights 60 significant differences between the rats that were fed the GE maize and those fed normal maize (all for 90 days). The first group showed differences in their kidney, brain, heart and liver measurements, as well as significant weight differences. These could be warning signs of toxicity, but have not been further investigated. (Media Newswire 2007)

This story had a high impact on public sentiment in Europe even though the study finding were not substantiated by follow-up research that indicated any causality or mechanisms of possible toxicity from the transgenic plants (Miller and Conko 2004). The health fears of Frankenfoods fall into four commonly publicized categories: there is little scientific study about their health risks, safety testing technology is inadequate to assess potential harm, they can carry unpredictable toxins, and they may increase the risk of allergenic reactions (Ewen and Pusztai 1999; Pusztai 2000). Unfortunately, it is nearly impossible and unrealistic to do a complete risk assessment about the potential hazards of any foods (Kuiper et al. 1999). In spite of this lack of information, most food causes few unpredictable health problems if consumed in normal quantities. In addition, foods derived from transgenic plants should not be any more at risk for toxicological dangers than traditionally cultivated plants (Momma et al. 1999). Their metabolic processes are known just as thoroughly as other plants. This does not quell the fears that the risks of genetically modified foods are not fully known. The epidemiological evidence of no ill harm to human populations consuming genetically modified foods is not compelling enough to quell concerns. Therefore, the compulsion over Frankenfoods will not likely be completely eliminated from public sentiment (Domingo 2000). Evidently, the bad press perpetuating the unsubstantiated health risks of transgenic plants is effective and contributing to consumer buying trends and legislative decisions (Fox 1999). An informal 2008 poll of attitudes about biotechnology foods conducted by CBS (Columbia Broadcasting System) found that “87% of (American) consumers would like GMO ingredients to be labeled, just as they are in

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Europe, Japan and Australia.” In the same poll they discovered that “53 percent of Americans say they won’t buy food that has been genetically modified (CBS Evening News 2008).” A 2008 commentary in “The Economist entitled Son of Frankenfood?” reported that biotechnology companies, food manufactures, and food retailers will have a difficult time convincing consumers to purchase biotechnology food products in spite of regulatory approvals that ensure safety (The Economist 2008). Books dispelling the Frankenfood myths have done little to improve consumer confidence in transgenic plants (Schacter 1999; Fedoroff and Brown 2004).

13.4.2

Food Allergies and Vaccine Resistance

Food allergies and other immunological problems are probably one of the most common compulsions associated with transgenic plants used for consumer chemicals, foods, and pharmaceuticals (CNN.com 2000; Shmaefsky 2007) There is a general belief that some unknown factor in transgenic plants can induce allergies. Others biotechnology opponents argue that certain known components, such as specific fragments of bacterial and viral nucleic acids, can induce immune hypersensitivies and autoimmune disease. Another criticism of edible vaccine transgenic plants claims that they contribute to the growing number of disease organisms resistant to vaccines. These compulsions are seemingly supported by compelling research studies and epidemiological data (US. EPA. FIFRA Scientific Advisory Panel 2001; Genetically Engineered Organisms 2008) A typical example of public hysteria caused by a fear of transgenic plants involved the Taco Bell brand packaged taco shell scare in 2000. Kraft Corporation of the US voluntarily recalled approximately three million boxes of taco shells unintentionally contaminated with StarLink corn intended for animal feed (CNN. com 2000). StarLink was genetically modified to express Bt toxin using the Cry1A for the cry9C protein and was not approved for human use (Halford 2005). Most of the news coverage included statements such as the Taco Bell shells “contained at toxin” or “the new protein could be an allergen to humans” (Genetically Engineered Organisms 2008). These news stories led to 210 people complaining about allergic reactions after purportedly eating the contaminated taco shells (US, EPA, FIFRA Scientific Advisory Panel 2001). Seventy-four of the people went to a physician with various medical complaints and another 20 admitted themselves to an emergency room seeking treatment for self-reported allergic reaction symptoms. This is almost five times the number of people that normally report about illnesses from eating related corn products. Ultimately, the Centers for Disease Control (CDC) formed a study to investigate the allergy claims to the relationship of the illnesses to a GMP (Centers for Disease Control 2001). The CDC established that 28 of the people who were confirmed to have eaten corn products containing the Cry9C protein had experienced a true allergic reaction

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unrelated to any other medical condition. However, none of the subjects possessed immunoglobin E (Ig E) antibodies that responded to Cry9C protein. Ig E is the definitive indicator of an allergic response in humans. So, the allergic response was determined by the CDC to be due to another factor. Plus, purposed illnesses in the subjects not investigated by the CDC were unrelated to food allergies. Critics debate the validity of the CDC study and argue that allergic reactions to the Bt protein or other transgenic plant proteins have not been ruled out (Organic Consumers Association 2008). Another compulsion about transgenic plants is illness associated with the consumption of bacterial and viral DNA and RNA used in the transgene vectors (Ho and Ching 2004). M.-W. Ho and A. Pusztai are the primary supporters of this view and foresee the possibility of autoimmune diseases developing from the consumption of these nucleic acids (Reisner 2001). Their arguments are very convincing and they cite laboratory animal studies that allude to the possibility of antibody production in response to high levels of transgene vectors in the diet (Violand et al. 1994; Schubbert et al. 1997). Much of this is based on the claims that the CaMV virus found in normal foods is not highly infectious and cannot be absorbed by mammals. It is believed that instability of the CaMV vector can possibly recombine in ways that initiate autoimmunity in consumers (Green and Allison 1994). However, no distinct mechanism explaining how the vector causes autoimmunity has been confirmed (Kuiper et al. 2001). Edible vaccines have created a different set of compulsions that are hampering their development for human use (Bonetta 2002). The primary concern, which is scientifically valid if proper quality control is not followed, is their effectiveness at inducing adequate immunity (Toonen 1996). However, this may be true with any vaccination program. The main compulsion is the fear that edible vaccines are highly likely to induce resistance in the organisms targeted by the vaccination (Sharma et al. 1999). This in turn will make the edible vaccine plants and the tradition vaccines ineffective. However, there is no evidence that edible vaccines are more likely to do this than traditional vaccines (Daniell et al. 2001). Overall, a consensus of the scientific community is that edible vaccines are effective and likely safer than traditionally vaccine administration strategies (Washam 1997).

13.4.3

Erosion of the Global Economy

There are many types of criticisms about the professed economic benefits provided by transgenic plants. Much of this negative sentiment is targeted at agricultural field applications (Piruzian et al. 2006). A global disapproval of agricultural biotechnology plants is their alleged interference with sustainable development programs in developing nations (World Summit on Sustainable Development 2002). The sentiment of the 2002 World Summit on Sustainable Development was reflected in the following statement: “It was impossible to determine whether GMPs could contribute to sustainable agriculture in the absence of a high level of scientific knowledge

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and technology to carry out credible risk assessments.” It was debated that the costs of remediating any risks may outweigh long-term economic benefits of GM crops. In addition, the conference sentiment believed the R&D and production costs of GM crops was contrary to economic sustainability in developing nations and in small farms in developed nations. Apparently, the general model of sustainable development rejects the current contributions of transgenic plants (Arrow et al. 2003). Sustainability is best defined as, “a pattern of resource use that aims to meet human needs while preserving the environment so that these needs can be met not only in the present, but in the indefinite future” (United Nations 1987). The United Nations has several economic criteria of sustainability that are not currently met by transgenic plant development. Major economic determinants of sustainability include the use of technologies that encourage the ratio of share in national income of highest to lowest quintile, increase the gross domestic product (GDP) per capita, raise the investment share in GDP, and develop material intensity of the economy (The United Nations Division for Sustainable Development 2008). The World Health Organization believes that transgenic plants currently used in developed and developing nations do not overall contribute to the equal distribution of wealth and resources. In developing nations the plants are perceived as not contributing much to the longterm sharing of a nation’s wealth. They also do not improve the country’s share in GDP particularly if the plants are owned or managed by foreign entities (U.S. Department of Agriculture 1999a, U.S. Department of Agriculture 1999b). Another purported limitation of GM crops is that the improvements they provide do not necessarily provide economic benefits to the availability and affordability of human foods. Due to safety regulations, most of the food-related plant products are used as animal feed (U.S. Department of Agriculture 1999b). A criticism presented to the US Department of State by Jennifer Kuzma, Director of the Center for Science, Technology, and Public Policy at the University of Minnesota, reflects a common view of transgenic crop plants. She stated, “But these and other applications carry risks that need to be addressed through regulatory and safety regimes. Governments and other organizations also need to step in and invest in biotechnology research and development tailored toward products that can help developing countries and assist these nations in building the capacity to benefit from bioinnovation” (Kuzma 2005). Little has been done to remediate the economic investment situation since her comments were made in 2005. Apparently, transgenic plants still require serious economic commitments and a research infrastructure available primarily in wealthier nations. The efforts of wealthier nations do not necessarily contribute to sustainable economic growth of the transgenic plant sector for developing nations (Suter and Oegerli 2002). There is data disputing the economic value of transgenic crop plants. A 2006 assessment evaluated the economic gains of using transgenic crop plants by the developing and developed nations. A conclusion was made that, “In terms of the division of the economic benefits obtained by farmers in developing countries relative to farmers in developed countries, data shows that in 2006, just over half of the farm income benefits (53%) have been earned by developing country

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farmers. The vast majority of these income gains for developing country farmers have been from GM IR cotton and GM HT soybeans. Over the 11 years, 1996– 2006, the cumulative farm income gain derived by developing country farmers was $16.4 billion (48.5% of the total)” (Brookes and Barfoot 2007). The authors’ conclusions show that substantial gains in net economic benefits were made at the farm level amounting to $6.94 billion in 2006 and $33.8 billion for the period 1996– 2006. Some critics debate the sustainability and equitable distribution of this increased wealth. They believe that most of the profits go consistently to the developers of the plants and not necessarily the growers (International Service for the Acquisition of Agri-Biotech Applications 2004). Many Asian countries are finding economically sustainable to invest in local niche market and global specialty market transgenic plants. They are already seeing a demand for plant technologies that would provide equitable economic development for their nations (International Food Policy Research Institute 2007). Countries, such as India, see a great need for developing food and pharmaceutical GM plants that meet the needs of Asian consumers. In addition, they are hoping that they produce a large export market of functional food GM plants. African nations are convinced that investing in the potential for economic benefits is not worth the risk. They have a greater infrastructure to build for achieving a sustainable transgenic plant market (Eicher et al. 2005). Overall, the information about the benefits of transgenic plants to the global economy is filled with emotionally fueled concerns. Many of these concerns are based on the perceived economic gain compared to the overall picture of the risks.

13.5

Conclusion

Transgenic plants have their obvious merits to science and society. Plus, the science behind transgenic plants have contributed to the overall growth of biotechnology and supplemented developments in botanical knowledge. However, there are also real and perceived society benefits and risks that must be addressed for the full utility of these plants to be applied globally (Doyle 1995; EPA Biotechnology Program 2008; Mae-Won 2000). There are measurable attributes to the benefits and real risks have data that can be used to make rather reliable prognostications about the economic value and safety of transgenic plants. Unfortunately, the compulsions or perceived risks are not fully measurable. However, they have significant impacts on the future of transgenic plants. Efforts are being made to help reconcile perceived risks of transgenic plants. A report by the Riso National Laboratory in Roskilde, Denmark recognized that, “Rapid developments in, and the controversial nature of, biotechnology call for communication, networks, partnerships, and collaboration in research, not just among researchers but also between researchers and research ‘users’ in industry, government, and elsewhere. Technological

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foresight appears to offer a coordinating method for developing and strengthening those linkages” (Borch and Rasmussen 2003). The study in the Riso National Laboratory report concluded: However, the current debate characteristically involves sharply opposed fronts. In it, stakeholders and experts on both side of the conflict advocate widely differing opinions. Without a proper, generally intelligible dialog, the broader public audience finds it hard to comprehend this type of debate. The study pursues the notion that public dialog can act as a driver of future applications in the technological domain, specifically GM crops. The study concluded with a stakeholder workshop that revealed three key issues that might provide helpful starting points for a more free-flowing and open-minded debate about the future of GM crops. The issues were those arising from the following statements: a broad perspective on risk is crucial; international regulation must make allowance for developing countries; a better configuration of the risk debate is needed. These issues are discussed in more detail in the report, along with the foresight method we used to reveal these issues.

Currently, there are few systemic global initiatives that follow the recommendations of this report.

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Index

A Abiotic stress, 68 Abscission, 212, 222 Absicisic acid, 73 Acclimation, 100 Acidothermus cellulolyticus, 258 Acquired resistance, 38 Activation tagging, 379 Activator, 373 Adaptation, 116 Agbiotech firm, 421 Agglutinins, 347 Agrobacterium, 26, 272, 418 A. tumefaciens, 272, 287 Algae, 343 Allergenic, 142, 402 Allergenicity, 142, 188 Allergic reaction, 465 response, 465 Allergies, 464 Amylase, 190 Annotation, 351 Anti-apoptotic, 231 Antibacterial, 348 Antibodies, 285 Anticancer, 349 Antigen, 283 Anti-HIV, 349 Antimicrobial, 41 Antioxidant, 39, 70, 89, 232 Antirrhinum, 175

Antisense, 96, 218 suppression, 225 Antiviral, 348 ANVISA, 408 Aphid, 4 APHIS, 393, 449 Apoplast, 43 Aquaporin, 86 Arabidopsis, 14, 44, 73, 170, 209 A. thaliana, 277, 290 Arabinose, 258 Armyworm, 15 Arsenic, 319 Arthropod, 2 Astaxanthin, 346 Atropa belladonna, 322 Autoimmune disease, 465 Autoimmunity, 465 Auxin, 200, 226 Avicennia marina, 93

B Bacillus thuringiensis (Bt), 6 Bt corn, 455 Bt cotton, 9 Bt gene, 455 Bt maize, 7 Bt pollen, 20 Bt potato, 9 Bt resistance, 10 Bt toxin, 7, 134, 455

479

480

Bamboo, 177 barnase gene, 206 barstar gene, 206 Basmati rice, 423 b-carotene, 457 Beta vulgaris, 329 Billion dollar bug, 4 Bioaccumulation, 309 Bioalcohol, 251 Biobutanol, 251 Biodegradability, 252 Biodegradation, 309, 324 Biodiesel, 186, 252 Biodiversity, 5, 444 loss, 459 Biodiversity Platform, 444 Bioengineering, 258 Bioethanol, 251 Biofuel, 249 Biogas, 252 Bioinformatics, 442 Biomagnification, 315 Biomass, 91, 258 Biomolecule, 346 Biomonitoring, 302 Biopesticide, 11, 450 safety, 454 Biopharmaceutical, 269 Bioplastic, 328 Biopolymer, 328 Bioreactor, 277, 352 BiOS, 415 Biosafety, 281 Biosimilar, 270 Biosorption, 309 Biosynthesis, 259 Biotechnology, 436 Biotic stress, 1 Biotransformation, 309 Biotype, 25 Bradyrhizobium, 138 Brassica, 83 B. carinata, 320 B. juncea, 318 B. napus, 94 B. rapa, 150, 453 Bromoxynil, 134 Brown planthopper, 12

Index

C Calmodulin, 81 Canola, 26 Carcinogen, 455 Carotenoid, 346 Carrageenan, 347 cDNA, 370 Cell line, 269 Cellomics, 443 Cellulose, 257 Cellulosic, 257 Centers for Disease Control, 464 CEPA, 402 CFIA, 399 CGIAR, 422 Chaperone, 81 Chaperoning, 81, 102 Chenopodium album, 22 Chickweed, 22 Chilling, 98 resistance, 101 resistant, 101 sensitive, 101 stress, 98, 101 Chill injury, 98 Chimeric gene, 205 Chitinase, 41 Chlamydomonas, 352 C. reinhardtii, 272, 287, 352, 353 Chloroplast, 258 Chromatinomics, 442 Circadian clock, 208 Citrullus lanatus, 92 Climate change, 67 Cloning, 257, 439 Cold acclimation, 98, 99 chain, 271 responsive, 103 stress, 97 tolerance, 97, 99 Coleoptera, 7 Colorado potato beetle, 4 Compatible solute, 84 CONABIA, 407 Contamination, 302 Copyright, 412 Corn earworm, 7

Index

Cotton, 26 Cry protein, 7 toxin, 10 cry gene, 7 CTNBio, 408 Cuticle, 219 Cyanidioschyzon, 351 Cyanobacteria, 259, 350 Cybrid, 207 Cytokinin, 177, 287 Cytoplasmic male sterility, 203 Cytotoxicity, 88

D Daktulosphaira vitifoliae, 3 Decontamination, 309 Defensin, 42 DEFRA, 404 Dehiscence, 222 Dehydration, 220 Dehydrin, 102 Deletion, 381 Dendroclamus strictus, 177 Desaturation, 101 Desiccation, 90, 102, 220 Detoxification, 5, 81, 93, 307 Diacylglycerol, 80 Dietary supplement, 346 Digitalis, 289 Diptera, 7 Disease resistance, 40, 43 Dissociation, 373 DNA footprint, 377 sequence, 419 sequencing, 438 DNMA, 407 Domestication, 213, 436 Domestic Substances List, 402 Drought avoidance, 72 resistance, 70 stress, 69 tolerance, 69

E Economic Research Service, 414 Ecosystems, 302

481

Ectopic, 231 EFSA, 405 Electrolyte leakage, 222 Electron transport chain, 100 Eleucine, 139 Elicitor, 44 Embryogenesis, 458 Endosymbiotic, 273 Enhancer, 373 Enterobacter cloacae, 320 Enterotoxin, 283 Enviromics, 443 Environment, 307 Environment Canada, 402 Environmental biotechnology, 444 Environmental Impact Statement, 396 Enzyme inhibitor, 13 Epigenomics, 442 Erwinia uredovora, 458 Escherichia, 318 E. Coli, 381 Estrogen, 458 Ethanol, 257 Ethnogenomics, 442 Ethylene, 38, 211 receptor, 217 European corn borer, 7 European Union, 403, 449 Eutrophication, 344 Explosive, 323 Expressed sequence tag (EST), 351, 370 Expression knock-outs, 383

F Fat hen, 22 Fermentation, 253 Fertility restorer (Fr/Rf ), 203 Flagellin, 44 Flanking sequence tag, 373 Flavonoid, 203, 458 Floral transition, 168 Florigen, 169 Flowering time, 169 FONSI, 395 Food and Agriculture Organization, 450 Food and Drug Administration (FDA), 396, 449

482

Forward genetics, 373 Fossil fuel, 249 Frankenfood, 462 Fucus vesiculosus, 349 Functional food, 457 Functional genomics, 87 Fusarium, 10 Fusion protein, 15

G Gain-of-function, 379 Gelatinization, 253 Gene discovery, 68 disruption, 373 expression, 44, 371 flow, 32, 148, 452 knock-down, 383 prediction, 370 silencing, 316, 383 stacking, 15 targeting, 381 transfer, 451 trapping, 378 Generics, 270 Genetic alternation, 206 diversity, 310 pollution, 452 resource, 50 Genetically modified (GM), 6, 368, 391 GM cotton, 9 GM crop, 396 GM food, 406 Genetically modified organisms, 392 Genetically modified plants, 435 Genetic engineering, 393 Genic male sterility, 203 Genome genome-wide expression, 371 organization, 204 replication, 204 Genomics, 442 Gibberellic acid, 180 Gibberellin, 170 Ginseng, 181 Glufosinate, 134 Glutathione, 94

Index

Glutathione reductase, 93 Glycine betaine, 84 Glycophyte, 88 Glycosylation, 270, 276, 277 Glyphosate resistance, 25 Glyphosate resistant (GR), 25, 133 alfalfa, 149 canola, 134 cotton, 134 crop, 136, 137 gene, 150 maize, 134 soybean, 134 transgene, 150 varieties, 134 GMPO, 407 Golden rice, 457 Good manufacturing practice, 270 Gossypium hirsutum, 9 Green leafhopper, 12, 13 Greenpeace International, 459 Green peach aphid, 18 Gross domestic product, 466 GUS expression, 45

H Halophytic, 93 Health Canada, 401, 449 Heavy metal, 108 Helicoverpa zea, 7 Helminthosporium oryzae, 37 Hemicellulose, 258 Hepatitis B, 286 vaccine, 271 Herbal therapeutic, 444 Herbicide (HT), 24 resistance, 25 resistant crop (HRC), 133 tolerant, 27 canola, 28 cotton, 29–30 maize, 29 soybean, 30 Heterosis, 202 Hexapeptide, 289 Homeostasis, 186, 217

Index

Homoptera, 7 Honeybee, 30 Hordeum, 83 Human interleukin, 288 Hybrid, 207 Hybridoma, 286 Hydrogen peroxide, 80 Hydrolysis, 253 Hydrophilicity, 83 Hymenoptera, 7 Hyperaccumulation, 311 Hypersensitive response, 38 Hypersensitivity, 220 Hypertensive, 289

I Imidazolinone, 150 Immunity, 465 Immunization, 282 Immunogenicity, 280 Immunoglobin, 465 Inbred line, 187 Insecticide, 4 Insertion, 381 library, 374 line, 261 Insertional mutagenesis, 377 Insulin, 269 Integrated pest management, 6 Intellectual Property Rights, 411 Interleukin, 288 Invasiveness, 33 Iron, 108 Isogenic line, 143 Isopentyl transferase, 72

J Jasmonic acid, 16, 38 Jatropha, 250 Jumping gene, 373

K Kanamycin resistance, 451 Klebsiella, 138 K. pneumoniae, 27 Knock-out, 373

483

L Lacanobia oleracea, 16 Laminaria, 344, 349 L. japonica, 353 Late embryogenesis abundant, 81 LD50, 146 Lectin, 12, 347 Lepidoptera, 7 Leucaena leucocephala, 311 Leucine-rich repeats, 41, 44 Leymus, 83 Lignin, 258 Lipoprotein, 273 Lipoxygenase, 189 Liriodendron tulipifera, 315 Locust, 4 Lolium, 139

M Maintainer line, 207 Maize, 26 Male sterility, 203 Mammalian cell culture, 270 Mannitol, 84, 90 Marker -assisted breeding, 417 -assisted selection, 87 gene, 420 Medicago, 221 M. sative, 80 Mercury, 311 Metabolic engineering, 452 Metabolism, 260 Metabolite, 18, 372 Metabolomics, 372, 443 Metallothionein, 112 gene (MT), 318 Methylation, 46 Methylmercury, 311 Microalgae, 272, 348 Microarray, 16, 95, 220 Microcystis, 348 Microfissure, 220 MicroRNA, 46, 47 Mirabilis, 226 MITILS, 442 Mitochondrial DNA (mtDNA), 204 Model plant, 445

484

Molecular breeding, 213 Molecular pharming, 272 Monarch butterfly, 20 Monoclonal antibodies (MAbs), 269, 285 Mouse, 288 mRNA, 187 Mutagenesis, 375 Mutagenic, 375 Mutation, 139, 174, 215 spontaneous mutation, 225 Mutator, 373

N NCBI, 419 Near-isogenic lines (NILs), 290 Necrosis, 38 Nematode cyst nematodes, 13 foliar nematodes, 13 rootknot nematodes, 13 NEPA, 395 Neutropenia, 288 Nicotiana N. glutinosa, 318 N. tabacum, 260, 286 Nitrogen, 105 assimilation, 105 Notice of Intent (NOI), 396 Novel trait, 401 Nucleotide binding site (NBS), 44 Nutraceutical, 457 Nutriomics, 443

O OECD, 414 Oleosin, 288 Oomycete, 36 Organelle biogenesis, 205 transformation, 207 Organic agriculture, 6 pollutant, 321 Orobanche, 31 Oryza, 288

Index

O. sativa, 289, 316 Osmoprotectants, 88 Osmoprotection, 84 Osmosensors, 79 Osmotic, 219 Ostrinia nubilalis, 7 Overexpression, 45, 209 Oxidative burst, 231

P Panax gensing, 181 Papaya ringspot virus, 47 Patent, 413 Patent discovery, 422 pat gene, 29 Pathogen mimicry, 46 related (PR), 39 Pathogenesis-related (PR), 231 Pathogen-related (PR) PR gene, 46 PR transcript, 46 Pearl millet, 177 Peroxidaton, 85 Peroxiredoxin, 93 Pesticide, 6 Petunia, 213 Phaeodactylum, 352 Phanerochaete chrysosporium, 259 Pharmaceutical, 444 Pharmacomics, 443 Phosphatidic acid, 79 Phospholipid, 100 Phospholipid-cleaving enzymes (PLEs), 79 Phosphorus, 107 Photoperiod, 170, 208 Photosynthate, 259 Phragmites, 317 Phycocolloids, 347 Phylloxera plaque, 3 Physiomics, 443 Phytoaccumulation, 317 Phytoalexin, 43 Phytochelatins (PCs), 318 Phytoestrogen, 457 Phytoextraction, 112

Index

Phytohormone, 181 Phytoimmuno-remediation, 324 Phytomonitor, 328 Phytophthora, 221 P. infestans, 36, 43 Phytoremediation, 110, 302, 303, 444 Phytoremediator, 310 Phytorestoration, 326 Phytosensing, 45 Phytotoxin, 136 Phytovolatilization, 110, 315 Pink bollworm, 9 Pisum sativum, 181 Plant Industry Platform, 444 Plants with novel trait (PNT), 399 regulation, 399 Plasmid, 272 Plasmodium, 284 Plastid transformation, 207 Pleiotropic, 72 Pollutant, 302 Pollution, 302 Polyamine, 89 Polymerase chain reaction (PCR), 383 Pongamia, 250 Poplar, 316 Populus, 111, 209, 316 P. tremuloides, 260 Powdery mildew, 42 Programmed cell death (PCD), 227 Prolamin, 289 Proline, 84 Protease inhibitor, 11 Protein bodies, 271 denaturation, 81 kinase, 80 trafficking, 273 Proteinase inhibitor, 16 Proteomics, 371 Protoplast, 381 Provitamin A, 346 Pseudomonas, 459 Pyrus communis, 89

Q Quantitative trait loci (QTL), 44, 87, 174, 377

485

R Ralstonia, 155 R. eutropha, 323 Raphanus, 453 Reactive oxygen species (ROS), 79 Recombinant DNA (rDNA), 393, 451 engineering, 351 protein technology, 271 Recombinant inbred lines (RILs), 290 Reporter gene, 377 Resistance (R) durable resistance, 44 major-gene resistance, 44 polygenic resistance, 44 R-gene, 38 single-gene resistance, 44 Restorer gene, 204 Retrotransposon, 373 Reverse genetics, 382 Rhizobacteria, 38 Rhizosecretion, 325 Ripening, 216 Risk assessment, 446 RNA datasets, 371 expression, 371 interference (RNAi), 261 polymerase, 385 silencing, 47, 373, 383 RNA-interference (RNAi), 19 Roses, 181 Roundup ready, 29 Royal Horticultural Society (RHS), 411 Ryegrass, 25

S Saccharomyces, 82, 254 SAGPyA, 407 Salicylic acid, 38 Salinity stress, 88 tolerance, 87 Salt sensitivity, 92 tolerance, 87 Schizosaccharomyces, 92 Sclerotinia, 42

486

Selenium, 320 SENASA, 407 Senescence, 211 Sequencing, 351 Sequestration, 5, 114, 309 Shoot apical meristem (SAM), 168 Short interfering RNA (siRNA), 46 Signal perception, 78 sensing, 78 transduction, 78, 96 Signaling, 169, 226, 231 cascade, 18, 231 Signal transduction, 45, 72 Single-stranded RNA (ssRNA), 385 Snowdrop lectin, 15 Solanum, 104 S. commersonii, 104 S. lycopersicum, 38 S. tuberosum, 9, 104 Somaclonal mutation, 374 Sorghum S. bicolor, 453 S. halepense, 453 Soybean, 26, 181 Spartina, 317 Specialty crops, 424 Spider mite, 16 Spodoptera frugiperda, 7 Stellaria media, 22 Sterols, 346 Striga, 22 Structural genomics, 369 Structural protein, 218 Substantially equivalent (SE), 397 Sulfonylurea, 27 Superoxide, 42 Superweed, 32, 453 Suppressor, 373 Synechocystis, 352

T Tamarix, 84 T-DNA, 261, 374 Technology transfer, 444 Teosinte, 200 Teratogenic effects, 458

Index

Terminator, 419 Thalassiosira pseudonana, 352 Therapeutic proteins, 287 Therapeutics, 277 Thlaspi T. caerlescens, 114 T. caerulescens, 319 Tillering, 200 Tobacco budworm, 9 Tomato moth, 16 Tonoplast, 85 Trademark, 413 Transactivation, 379 Transcription, 95 factor (TF), 46, 76, 95 Transcriptome, 371 Transcript tryncation, 205 Transgene, 21, 148, 372 docking, 382 mitigation, 32 upgrades, 382 Transgenic goats, 270 Translation, 206 Transporter, 91 Transposase, 382 Transposon, 373 tagging, 322, 375 Trehalose, 84, 90 Trichederma, 82 Trichloroethylene (TCE), 321 Triticum aestivum, 286 Trypsin inhibitor, 9 Typha, 317

U Ubiquitin, 86 Umbelopsis ramanniana, 261 Undaria, 344 Union of Concerned Scientists (UCS), 459 USDA, 393, 415, 449 USPTO, 413

V Vaccine, 279 Variety Registration Office (VRO), 399 Vector, 272

Index

Vernalization, 170, 208 Vitamin A, 458 Vitis V. kabrusca, 3 V. vinifera, 3

487

X Xanthomonas, 41, 221 Xanthotoxin, 19 Xenopus, 96

W

Y

Water use efficiency (WUE), 73 Western corn rootworm, 4 Wild-type (WT), 73 Wilting, 94 Witchweed, 22 Wounding, 231

Yeast, 289

Z Zea, 171 Zea mays, 171