Transgenic Horticultural Crops: Challenges and Opportunities

Transgenic HorTiculTural crops Challenges and Opportunities

EditEd by

Beiquan Mou and ralph scorza

Transgenic HorTiculTural crops

Transgenic HorTiculTural crops Challenges and Opportunities

EditEd by

Beiquan Mou and ralph scorza

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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Contents Preface......................................................................................................................vii Editors........................................................................................................................ix Contributors...............................................................................................................xi Chapter 1 Transgenic Fruit and Nut Tree Crops Review.......................................1 Ana M. Ibáñez, Cecilia B. Agüero, Mathew A. Escobar, and Abhaya M. Dandekar Chapter 2 Transgenic Vegetables......................................................................... 31 Owen Wally, J. Jayaraj, and Zamir K. Punja Chapter 3 Transgenic Ornamental Crops............................................................ 55 Beverly A. Underwood and David G. Clark Chapter 4 Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants.................................. 83 Qiang Chen Chapter 5 Transgenic Fruit Crops in Europe..................................................... 125 Henryk Flachowsky and Magda-Viola Hanke Chapter 6 Transgenic Horticultural Crops on the African Continent................ 147 Idah Sithole-Niang Chapter 7 Transgenic Horticultural Crops in Asia............................................ 155 Desiree M. Hautea, Von Mark Cruz, Randy A. Hautea, and Vijay Vijayaraghavan Chapter 8 The Economic and Marketing Challenges of Horticultural Biotechnology.................................................................................... 175 Steven Sexton and David Zilberman

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Chapter 9 Consumer Acceptance of Genetically Modified Foods: Traits, Labels, and Diverse Information............................................ 193 Wallace E. Huffman Chapter 10 Intellectual Property and Development of Transgenic Horticultural Crops........................................................................... 219 Cecilia L. Chi-Ham and Alan B. Bennett Chapter 11 Structuring University–Private Partnerships for Developing and Commercializing Transgenic Horticultural Crops..................... 233 Gordon Rausser and Reid Stevens Chapter 12 Why Are Regulatory Requirements a Major Impediment to Genetic Engineering of Horticultural Crops?............................... 249 Steven H. Strauss Chapter 13 Virus-Resistant Transgenic Horticultural Crops: Safety Issues and Lessons from Risk Assessment Studies..................................... 263 Jonathan E. Oliver, Paula F. Tennant, and Marc Fuchs Chapter 14 Molecular Approaches for Transgene Containment and Their Potential Applications in Horticultural Crops................................... 289 Yi Li and Hui Duan Chapter 15 Prospects for the Commercialization of Transgenic Ornamentals...... 305 Michael S. Dobres Chapter 16 Genetic Engineering of Grapevine and Progress toward Commercial Deployment.................................................................. 317 Dennis J. Gray, Sadanand A. Dhekney, Zhijian T. Li, and John M. Cordts

Preface At the dawn of the twenty-first century, food insecurity and malnutrition continue to plague humankind, especially in developing countries. It has been estimated that world food supplies must increase by up to 50% over the next 20 years due to population growth, even while farming land is being rapidly lost to housing, transportation, and industrial uses. Global warming and climate change also pose serious threats to agricultural production and place unprecedented pressures on the sustainability of food supplies. Transgenic field crop production is a major component of modern agriculture and promises to play an important role in meeting the food supply challenges that we face today and in the future. The global planted area of genetically engineered field crops soared to 330 million acres in 25 countries in 2009, of which 158 million acres (48%) were in the United States. Today, more than three quarters of the soybean, nearly half of the cotton, and more than a quarter of the global maize production are from biotech varieties, primarily with herbicide tolerance, insect resistance, or stacked genes for the two traits. As the world debates the risks and benefits of plant biotechnology, the proportion of the global area of transgenic field crops has increased every year, and the safety and benefits continue to be demonstrated. Yet, despite the success of transgenic field crops, the commercialization of transgenic horticultural crops (vegetables, fruits, nuts, and ornamentals) has lagged far behind. Transgenic Horticultural Crops: Challenges and Opportunities examines the challenges, advances, and opportunities for the creation and commercialization of transgenic horticultural crops. The consumption and production of horticultural products continue to increase, and now horticultural crops account for 50% of the value for all agricultural crops in the United States. With the rising demand for fruits and vegetables by healthconscious consumers, there are ever-increasing interests in horticultural crops. Although horticultural crops were the first biotech crops commercialized in the United States, beginning with the Flavr Savr tomato in 1994, they have not made an impact on production due to factors that include consumer concern over genetically modified (GM) food, which results in the reluctance of processors and marketers to accept the biotech products already developed; complex and costly regulatory processes; the limited acreage of most horticultural crops, which makes it difficult to recover the costs of research and development; and costly segregation of GM and non-GM commodities. Current practices in patenting and intellectual property protection have added barriers to the use of biotechnology for the creation and commercialization of new horticultural crop varieties. Additional challenges to the development and commercialization of GM horticultural crops include technical difficulties in the transformation of certain horticultural crops, barriers to regulatory approval in many countries, and the uncertainties of post-commercialization stewardship. Although there are many volumes dealing with plant biotechnology and transgenic plants, those focusing on horticultural crops are rare. In this book, internationally acclaimed experts from different disciplines assess the current status of vii

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Preface

transgenic horticultural crops, examine the challenges for the creation and commercialization of horticultural biotechnology, and identify opportunities, strategies, and priorities for future progress in this important field. The authors of the chapters are leading experts who were asked not to conform to a set outline but to approach their topics in their own unique ways. Chapters 1 through 4 describe the past achievements, newest developments, and current challenges in transgenic fruit, nut, vegetable, ornamental, and pharmaceutical crops. Chapters 5 through 7 provide reviews of transgenic horticultural crops in other parts of the world such as Europe, Africa, and Asia. Chapters 8 through 12 discuss in detail hurdles to the commercialization of transgenic technology in economics and the marketplace, consumer acceptance, intellectual property rights protection, public–private partnership, and regulation. Chapters 13 and 14 describe the risk assessment of transgenic crops and transgene containment. Finally, Chapters 15 and 16 present some case studies and an industry perspective on transgenic horticultural crops. The production and commercialization of transgenic horticultural crops are enormous tasks—their progress and realization need the efforts of the research community, horticultural industry, government, and consumers. Although this book covers many topics, it by no means has exhausted all issues related to transgenic technology in horticulture. We have sought to provide facts, analyses, and insights by leading experts in this field that can inform a wide audience, including graduate and advanced undergraduate students of agriculture—and horticulture in particular, educators at all levels, breeders of horticultural crops, plant geneticists, biotechnologists, biologists, molecular biologists, cell/tissue culture specialists, horticulturists, agronomists, entomologists, plant pathologists, physiologists, nutrition and food technologists, food safety specialists, economists, environmentalists, agricultural extension personnel, growers, farm managers, pest control agents, government regulators, and the interested public. We hope that this book will stimulate more interest and discussion on the subject and promote the advancement of research on transgenic horticultural crops and the development of safe, sustainable GM horticultural crop varieties.

Editors Dr. Beiquan Mou is currently a research plant geneticist with the Agricultural Research Service, U.S. Department of Agriculture, Salinas, California. He obtained his PhD degree in plant breeding and genetics from Oregon State University in 1993. He then worked on the mechanism and inheritance of self- and interspecific incompatibility in Nicotiana at the University of Missouri–Columbia and carried out postdoctoral research on the transgenic modification of cornstarch structure and functionality at Iowa State University. Since 2001, Dr. Mou has been conducting research on the genetics and breeding of lettuce and spinach for disease and insect resistance, nutritional improvement, and horticultural traits. He has released 15 lettuce and spinach varieties possessing unique traits, improved quality, new genes, and/or disease or insect resistance. He currently serves as chair of the USDA Leafy Vegetable Crop Germplasm Committee and the Vegetable Breeding Working Group of the American Society for Horticultural Science. He is sought out for consultation nationally and internationally by other researchers, government agencies, industry, and the media. Dr. Ralph Scorza is a research horticulturist and lead scientist for the Genetic Improvement of Fruit Crops Research Unit at the USDA-ARS Appalachian Fruit Research Station, Kearneysville, West Virginia. He received his BS in agronomy and MS in fruit crops, both from the University of Florida, and his PhD in genetics and plant breeding from Purdue University in 1979. The broad objectives of his research program at the USDA are to develop stone fruit (Prunus) germplasm with improved fruit quality, resistance to biotic and abiotic stress, and improved tree growth habits for high-yielding, mechanically integrated orchard systems. His breeding program combines classical and molecular approaches. Dr. Scorza has released nine stone fruit varieties developed through conventional breeding. His genetic engineering work has included the successful development of disease-resistant grapes, pears, and plums. The plum pox virus–resistant plum cultivar ‘HoneySweet’ developed by Dr. Scorza and his colleagues is the first genetically engineered temperate fruit crop to be deregulated and approved by the APHIS, FDA, and EPA in the United States. Dr. Scorza is a recipient of the Arthur S. Flemming Award and was selected as an ARS-NAA Senior Research Scientist of the Year. He is also a corecipient of three Secretary of Agriculture Honor Awards. He has authored over 190 research publications and is a fellow of the American Society for Horticultural Science.

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Contributors Cecilia B. Agüero Viticulture and Enology Department University of California, Davis Davis, California Alan B. Bennett Department of Plant Sciences University of California, Davis Davis, California Qiang Chen Laboratory of Plant Pharmaceutical Research Biodesign Institute and College of Technology and Innovation Arizona State University Tempe, Arizona Cecilia L. Chi-Ham Department of Plant Sciences University of California, Davis Davis, California David G. Clark Department of Environmental Horticulture University of Florida Gainesville, Florida John M. Cordts Biotechnology Regulatory Services Animal and Plant Health Inspection Service United States Department of Agriculture Riverdale, Maryland Von Mark Cruz Agricultural Research Service United States Department of Agriculture National Center for Genetic Resources Preservation Fort Collins, Colorado

Abhaya M. Dandekar Plant Sciences Department University of California, Davis Davis, California Sadanand A. Dhekney Mid-Florida Research and Education Center Institute of Food and Agricultural Sciences University of Florida Apopka, Florida Michael S. Dobres NovaFlora Inc. West Grove, Pennsylvania Hui Duan J. R. Simplot Company Boise, Idaho Mathew A. Escobar Department of Biological Sciences California State University, San Marcos San Marcos, California Henryk Flachowsky Institute for Breeding Research on Horticultural and Fruit Crops Julius Kühn-Institute Dresden, Germany Marc Fuchs Department of Plant Pathology and Plant-Microbe Biology Cornell University Geneva, New York xi

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Dennis J. Gray Mid-Florida Research and Education Center Institute of Food and Agricultural Sciences University of Florida Apopka, Florida Magda-Viola Hanke Institute for Breeding Research on Horticultural and Fruit Crops Julius Kühn-Institute Dresden, Germany Desiree M. Hautea Institute of Plant Breeding University of the Philippines Los Baños, Philippines Randy A. Hautea International Service for the Acquisition of Agri-Biotech Applications SEAsia Center Manila, Philippines Wallace E. Huffman Department of Economics Iowa State University Ames, Iowa Ana M. Ibáñez Plant Sciences Department University of California, Davis Davis, California J. Jayaraj Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada Yi Li Department of Plant Science and Landscape Architecture University of Connecticut Storrs, Connecticut

Contributors

Zhijian T. Li Mid-Florida Research and Education Center Institute of Food and Agricultural Sciences University of Florida Apopka, Florida Jonathan E. Oliver Department of Plant Pathology and Plant-Microbe Biology Cornell University Geneva, New York Zamir K. Punja Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada Gordon Rausser Department of Agricultural and Resource Economics University of California, Berkeley Berkeley, California Steven Sexton Department of Agricultural and Resource Economics University of California, Berkeley Berkeley, California Idah Sithole-Niang Department of Biochemistry University of Zimbabwe Harare, Zimbabwe Reid Stevens Department of Agricultural and Resource Economics University of California, Berkeley Berkeley, California

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Contributors

Steven H. Strauss Department of Forest Ecosystems and Society Oregon State University Corvallis, Oregon Paula F. Tennant Department of Life Sciences The University of the West Indies Kingston, Jamaica Beverly A. Underwood Department of Environmental Horticulture University of Florida Gainesville, Florida

Vijay Vijayaraghavan Sathguru Management Consultants Pvt. Ltd. Hyderabad, India Owen Wally Department of Biological Sciences Simon Fraser University Burnaby, British Columbia, Canada David Zilberman Department of Agricultural and Resource Economics University of California, Berkeley Berkeley, California

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Transgenic Fruit and Nut Tree Crops Review Ana M. Ibáñez, Cecilia B. Agüero, Mathew A. Escobar, and Abhaya M. Dandekar

CONTENTS Introduction.................................................................................................................2 Genetic Transformation of Fruit and Nut Tree Crops.................................................3 Importance of Genetic Engineering....................................................................... 3 Current Status of Genetic Transformation of Perennial Crops..............................3 Genetic Transformation via Organogenesis......................................................4 Genetic Transformation via Somatic Embryogenesis.......................................4 Methods of Transformation.................................................................................... 5 Agrobacterium-Mediated Transformation.........................................................5 Biolistic Bombardment..................................................................................... 6 Transient Expression..............................................................................................6 Rootstock Transformation...................................................................................... 6 Input Traits.................................................................................................................. 6 Pathogen Resistance............................................................................................... 7 Viral Resistance................................................................................................. 7 Bacterial Resistance..........................................................................................9 Fungal Resistance............................................................................................ 12 Insect Resistance.................................................................................................. 13 Cydia pomonella............................................................................................. 13 Epiphyas postvittana (Light Brown Apple Moth)........................................... 14 Output Traits............................................................................................................. 14 Delayed Fruit Ripening........................................................................................ 16 Improving Fruit Quality and Nutritive Value....................................................... 16 Removal of Undesirable Phytochemicals............................................................ 18 Antigen Production.............................................................................................. 18 Conclusions............................................................................................................... 19 References................................................................................................................. 19

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Transgenic Horticultural Crops: Challenges and Opportunities

INTRODUCTION Fruit crop plants constitute an important group among agricultural crops, Â�adding tens of billions of dollars per year to the global economy. Worldwide, over 45 million hectares of land have been devoted to their production, and millions of growers depend on continued global trade.1 Among fruit crops, citrus production is most significant, with more than 134 million tons produced from an area of 8.7 million hectares in 2008. This is followed by bananas with worldwide production at just under 100 million tons, apples at 76 million tons, and grapes at 74 million tons.2 In California, the production of fruit and nut crops contributes to more than one-third of the total farm gate value of the state’s agricultural commodities. The fruit and nut crops are cultivated using a highly sophisticated production system that uses vegetatively propagated clonal cultivar materials grafted to clonal or seedling rootstocks. The plantings are perennial and orchard systems represent several decades of investment to a grower. These crop plants have a juvenile phase, which can last from 2 to 8 years, during which they grow vegetatively without any fruit production. Thus, traditional fruit tree breeding takes a very long time—20 to 40 years to develop new varieties. This is primarily due to the time it takes to backcross promising selections with current commercial cultivars, which is required to create a new commercial cultivar with optimal fruit characteristics. Transgenic plants offer a more direct and quicker strategy to provide genetic solutions. Fruit and nut crops face many challenges, including the need to be grown on less land with less resources and the need to yield consistently high quality fruit that can sustain the economics of production. Disease and pests pose the greatest challenge to fruit and nut crop production, and we discuss below the different diseases and pests that are important to these crops and the specific solutions that can be provided using transgenic technologies. Many of these are productioneliminating diseases, so finding genetic solutions is critical. In some cases, the disease resistance traits are present in wild germplasm, so introgression of disease resistance by traditional breeding takes place at the cost of fruit quality, which means that many backcrosses have to be conducted to restore fruit quality. In some cases, there is no resistance even in the wild germplasm, so novel sources of resistance have to be developed (e.g., pathogen-derived resistance). For these strategies, transgenic technologies need to be employed. These technologies have been developed in many of the tree crops (as outlined in next section); however, a major challenge is the recalcitrance that these plants display in tissue culture. The percentage of tissue-cultured explants that give rise to a transgenic plant is low and thus it is difficult to transform these plants using Agrobacterium tumefaciens. Successful plant transformation requires high frequencies of both transformation and regeneration, such that a successful overlap of such events can take place at a tissue level to obtain transgenic plants. While there are many examples of success, there are also many fruit tree species that have proved very difficult to transform due to this problem. Two general classes of traits that are of commercial significance are discussed in this review: input traits and output traits. Input traits are specifically important

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for growers, producers, and handlers, because they are focused on sustaining yields and productivity. Disease and pest resistance are examples of important input traits. Output traits, which preserve the quality and nutritional value of the fruit, are of primary importance to the consumer, though they ultimately benefit the grower as well. Increasingly, fruit consumption has been linked with improved nutrition and disease prevention, and thus USDA guidelines suggest that fruit and nuts are an important component of the daily diet. Many fruit quality traits, like flavor, are important for consumption, as a good flavor makes the fruit more desirable. Fruit quality is also important for downstream products like fruit juice and wine. In this chapter, we outline the progress that has been made in different fruit crops to manipulate both input and output traits.

GENETIC TRANSFORMATION OF FRUIT AND NUT TREE CROPS Importance of Genetic Engineering A major breeding objective is to combine fruit quality with other horticulturally important traits such as plant architecture, adaptation to extreme environmental conditions, and decreasing the amount of chemical input required to control pests and diseases. The production of new varieties by conventional breeding is a complex and time-consuming process, especially for fruit trees, due to the high level of heterozygosity, pronounced inbreeding depression, long juvenile phase, and complex reproductive biology. The plant breeder must wait many years to be able to evaluate fruit quality, and, because fruit trees are large perennial plants, a relatively small number of seedling progeny can be evaluated.3,4 In addition, the characters that constitute a good cultivar in most instances are polygenic in their inheritance; thus, the probability of recombining sets of genes that determine the essential properties of a given cultivar is low.5 In wine grape breeding, legal and economic hurdles can be even larger than the biological obstacles described above. New wine grape varieties are not welcome because the wine industry relies predominantly on a few select and ancient cultivars; also, vintners are constrained by tradition, regulation, and economics, so they use classical varieties, such as, ‘Cabernet Sauvignon’, ‘Merlot’, and ‘Chardonnay’.6 Direct genetic modification by the introduction of single genes offers the opportunity to direct very specific changes in existing cultivars, to increase resistance to abiotic stress, diseases, and pests and to modify fruit composition in very precise ways. Vegetative propagation means that modifications to traditional cultivars by genetic transformation leave the essential characters and identity of the cultivars unaltered, a result that would be very difficult to achieve by conventional breeding where two genomes are intermingled.7 Last but not least, genetic transformation also offers the opportunity to study how genes control the growth and development of plants.

Current Status of Genetic Transformation of Perennial Crops A wide variety of transformation and regeneration protocols have been used to produce transgenic fruit trees. However, in most species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few

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Transgenic Horticultural Crops: Challenges and Opportunities

genotypes. Most of the basic protocols were developed several years ago and subsequent efforts have been oriented toward optimization or adaptation of the technique to new genotypes. Unfortunately, the time required to produce transgenic fruit trees is too long, especially for fruit character analysis, and there is a need for faster stable transformation systems. Transient expression systems are less time-consuming methods that are receiving more attention as a tool for gene functional analysis. The production of marker-free transgenic plants is another growing area of interest due mainly to consumer concerns. Genetic Transformation via Organogenesis Although a common factor in many transformation protocols is the use of embryogenic cultures as starting material for transformation, some species have been successfully transformed through the use of organogenesis. This system offers an alternative approach for genotypes that are recalcitrant to plant regeneration by somatic embryogenesis, although the probability of obtaining chimeric plants is higher. Current protocols for the production of transgenic citrus involve the transformation of cells in seedling stem pieces and regeneration of shoots from organogenic cultures. The first reliable protocol reported the transformation of the citrus relative Poncirus trifoliata by cocultivation of etiolated epicotyl segments with Agrobacterium tumefaciens.8 A similar transformation procedure with modifications has been used to produce transgenic plants of other citrus genotypes.9 In general, rooting of transgenic shoots in citrus, except for P. trifoliata and grapefruit, is rather inefficient but can be alleviated by the use of shoot tip grafting.9,10 Mature tissues have also been used as explant material in order to maintain genotype identity and overcome the long juvenile growth phase. Buds collected from trees are grafted onto seedlings of a vigorous rootstock grown under glasshouse conditions; new shoots elongated from them are then used as starting material.9 Transformation via organogenesis has also been applied in several other fruit tree species. For example, apple transformation was first reported by James et al., who used Agrobacterium-mediated transformation of leaf disks from the apple cultivar ‘Greensleeves’.11,12 Although somatic embryogenesis is the most prevalent regeneration method used in grape genetic transformation, transgenic plants of table grape cultivars ‘Silcora’ and ‘Thompson Seedless’ have also been produced via organogenesis. The method is based on the formation of meristematic bulk tissue with a high regenerative capacity, using adventitious shoots as a starting material. Shoot regeneration is obtained 30 days after meristematic bulk slices are inoculated with Agrobacterium.13 Alternatively, shoot tips subjected to wounding and then cocultivated with Agrobacterium have been shown to rebuild complete meristems and produce non-chimeric transgenic ‘Thompson Seedless’ plants.14 Genetic Transformation via Somatic Embryogenesis Embryogenic cultures are the preferred starting materials for transformation with either Agrobacterium inoculation or microprojectile bombardment because they are the most responsive tissues, with competence for in vitro regeneration and genetic transformation.15 The entire process involves induction of somatic embryogenesis,

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maintenance of the embryogenic callus, and transformation, selection, and regeneration of plants. The large number of cultivars and rootstocks increases the complexity due to genotype differences observed at all tissue culture steps. In walnut, somatic embryogenesis techniques were developed for Juglans regia but have been applied to other Juglans species. In this system, immature walnuts are cultured on a basal medium and secondary embryos that originate from cells in the epidermal layer of the embryo display high susceptibility to infection with Agrobacterium.16 A major challenge today in walnut tissue culture is to obtain embryogenesis from maternal tissue. This is important because embryos from zygotic tissue do not allow the exact genotype to be predicted. Repetitively, embryogenic cultures have been obtained from immature anther tissue, but only from the “Chandler” cultivar. Recently, a modified protocol allowed a somatic embryo line to be generated from immature anthers of a “Paradox” hybrid that may provide a source of elite rootstock tissue for genetic transformation.17 In grapevine, anthers collected during first pollen mitosis are the most widely used explant for culture initiation, with the embryogenic callus arising from diploid tissue.18 Embryogenic callus maintenance has been accomplished in liquid, semisolid or solid media supplemented with a variety of growth regulator combinations.19–23 The variety of protocols developed for maintenance of embryogenic cultures is due to the fact that the production of suitable embryogenic cultures for transformation with Agrobacterium has represented a greater challenge than the initiation step, with higher transformation efficiencies being obtained with embryogenic lines composed of fine cells, arrested in a very early pre-embryogenic state.7

Methods of Transformation Agrobacterium-Mediated Transformation The vast majority of transformation protocols use A. tumefaciens (biovar 1) as a vector. Agrobacterium strains that work efficiently for walnut, citrus, and apple transformation include disarmed derivatives of the tumorigenic strains A281 (e.g., EHA101 and EHA105) and C58 (e.g., C58C1).3,12,16 The disarmed strains possess the Ti plasmid-based vir genes required for plant transformation, but lack the native T-DNA (which is associated with pathogenesis). These strains are transformed with broad host range binary plasmids that contain the desired T-DNA region, carrying the gene of interest and selectable marker genes, mainly conferring resistance to antibiotics, such as kanamycin and hygromycin.24 However, given the public concern with the introduction of antibiotic resistance genes into food, methods to eliminate these selectable marker genes from the transformed plants and strategies that avoid selection with antibiotics are being developed. In citrus, workable transformation efficiencies have been achieved using a multi-auto-transformation (MAT) vector combined with an inducible recombinase/recombination sites (R/RS)–specific recombination system and the phosphomannose isomerase/mannose conditional positive selection system.25 In addition, a cotransformation system using a mixture of two Agrobacterium strains (one harboring the gene of interest and the other containing both positive and negative selectable markers genes) has been used to obtain transgenic grapevines free of selectable marker genes.26

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Transgenic Horticultural Crops: Challenges and Opportunities

Biolistic Bombardment The biolistic process has been successfully applied to grape; however, the equipment and expertise required together with the degree of optimization needed to establish a suitable bombardment protocol for specific cultivars have limited the value and wide scale application of this technology. It was initially tested using embryogenic cell suspensions of ‘Chancellor’, a Vitis complex interspecific hybrid, and then successfully extended to Vitis vinifera cultivars ‘Chardonnay’ and ‘Merlot’.27,28

Transient Expression Agrobacterium-mediated transient assays have become an important tool for gene function analysis. Two high quality draft genome sequences have been reported for grapevine.29,30 They are the first genome sequences produced for a fruit crop and will stimulate the search for rapid functional genomics screening systems. Agroinfiltration has been used to transform leaves of in vitro cultured grapevine plants.31 Conversely, transient transformation in grape berry skin has been achieved by treating half-cut berries with an A. tumefaciens suspension.32 In intact fruit of citrus, efficient transgene expression was accomplished through injection of Agrobacterium into the fruit.33 Viral vectors represent an alternative transient expression system that has been shown to be a useful strategy for overexpression or silencing of plant genes in annuals. RNA virus–based vectors, carrying a green fluorescent protein coding gene, have been developed for citrus trees. The most successful vectors were unusually stable and continued producing fluorescence more than 4 years after inoculation of Citrus macrophylla seedlings.34

Rootstock Transformation Nearly all commercial fruit trees are propagated vegetatively. Generally, cuttings or buds from desired varieties are grafted onto rootstocks selected for adaptation to specific soil conditions and resistance to root-destroying diseases and insects. For this reason, genetic transformation of rootstocks has been oriented to improve these characteristics. However, rootstock genetic engineering can be directed to modify the properties of the scion. Currently, the signal peptide sequences derived from mRNAs found in grape xylem exudates are being evaluated for delivery of therapeutic proteins into the xylem.35 Such transgenic products, if synthesized in a rootstock, could move through the graft union and confer resistance to xylem-specific infections such as Xylella fastidiosa, a Gram-negative bacterium that causes Pierce’s disease (PD) in grapevines.

INPUT TRAITS Genetic transformation is a tool for creating new fruit and nut tree crop varieties with improved input traits, which enhance agronomic characteristics and crop performance, conferring great benefit to growers. Traditional breeding to improve input traits like resistance to pathogens and insects is very inefficient, labor-intensive, and

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time-consuming. However, genetic engineering offers a real possibility to create fruit and nut tree crop varieties with desirable agronomic traits that exhibit increased resistance to pathogens and insects. Here, we discuss recent progress in increasing resistance to viruses, bacteria, fungi, and insects through the genetic transformation of fruit and nut tree crops.

Pathogen Resistance Viral Resistance Viruses are the main cause of economic losses to agricultural crops worldwide. For example, Citrus tristeza virus (CTV) causes huge economic losses for the citrus industry and Plum pox virus (PPV) causes one of the most devastating viral diseases of stone fruit (Prunus spp.). Several transgenic approaches have been successfully applied for virus resistance in fruit crops based on pathogen-derived resistance (PDR) strategies. Virus resistant transgenic crops have been developed during the last two decades through both the expression of transgenic virus RNA in host plants and protein-mediated resistance.36–39 Described as a potent genetic vaccination against viral sequences, RNA-mediated virus resistance (RMVR) is less susceptible to variation in virus sequence and is highly sequence-specific, often providing complete immunity to the inoculated virus or RNA.40,41 Proteinmediated resistance has the potential for broader protection but generally results in mild resistance, which only delays symptoms and decreases viral titers. However, the combination of RNA- and protein-mediated resistance in transgenic plants could prove to be potent, broad, and durable.41 Gene silencing is another powerful biotechnological tool used to gain virus resistance for fruit crops. This method has produced transgenic plants able to switch off endogenous genes and invading nucleic acids.42–47 Papaya Ringspot Virus Papaya ringspot virus (PRSV) causes a destructive disease in papaya (Carica papaya L.), an economically important fruit crop in tropical and subtropical areas, resulting in drastic reduction of fruit quality. PRSV is naturally transmitted by aphids in a nonpersistent manner and induces stunting in growth, symptoms of mosaic and distortion on leaves, and streaks on petiole and stem. Resistance against PRSV in papaya has not been obtained by conventional breeding. In the last two decades, effective strategies based on PDR have been widely used. The coat protein (CP) gene of PRSV, HA 5-1, was transferred into papaya via micro-projectile bombardment.48–50 Transgenic papaya lines highly resistant to the severe Hawaiian PRSV strain PRSPV HA were selected under greenhouse and field conditions.49,50 Rainbow and SunUp cultivars have been successfully commercialized in Hawaii since 1998, representing the first practical application of transgenic fruit crop.51,52 However, the CP-hemizygous line Rainbow is susceptible to non-Hawaiian PRSV isolates and the CP-homozygous line SunUp, while resistant to a wider range of isolates from Jamaica and Brazil, is still susceptible to isolates from Thailand and Taiwan.51,53,54 In Taiwan transgenic papaya lines carrying the CP gene of a Taiwan, severe strain, PRSV YK, have also been successfully generated.55 Several transgenic lines provide

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Transgenic Horticultural Crops: Challenges and Opportunities

broad spectrum resistance against homologous and geographically distinct strains of PRSV under greenhouse conditions56 and a high degree of resistance under field trials.57 During the field trial, some transgenic papaya lines showed susceptibility to the new emerging papaya leaf distortion mosaic virus (PLDMV) which indicates that the virus is a threat for PRSV-resistant transgenic papaya in Taiwan and elsewhere.58 Recently, transgenic papaya lines with double resistance to PRSV and PLDMV were generated using a chimeric construct strategy containing a fused cDNA fragment composed of the truncated PLDMV P-TW-WF CP and PRSV YK CP coding regions. Three of nine resistant transgenic papaya lines carrying the chimeric construct showed high levels of resistance to heterologous PRSV strains from Hawaii, Thailand, and Mexico. Transgenic papaya lines look promising for resistance to PRSV strains and PLDMV in Taiwan and elsewhere.59 Citrus Tristeza Virus Citrus tristeza virus (CTV) (Closterovirus) is considered the most economically important virus affecting citrus production worldwide. CTV is spread by several species of aphids with piercing-sucking mouthparts, Toxoptera citricidus being the most efficient vector. This viral disease is now endemic in several economically important production regions around the world. Different strains of CTV can cause diverse disease syndromes that vary in severity. Symptoms may be as mild as weak and scattered vein clearing, whereas quick decline, a syndrome in which a tree with normal appearance starts showing wilt symptoms and completely collapses in a few weeks, is the most dramatic manifestation of disease. The development and identification of CTV tolerant rootstocks was one of the main strategies for the control of quick-decline disease.60 Recently, biotechnology tools like gene silencing (GS) have been successfully applied. Specifically, targeting CTV using RNA interference (RNAi) strategies is the major strategy in plant transformation for CTV resistance. Citrus lines expressing p23 CTV, a silencing suppressor, were reported as resistant to CTV, and transgenic viral protein accumulation was negatively correlated to resistance. In this study, posttranscriptional gene silencing (PTGS) was proposed as the mechanism for resistance to CTV, since resistant citrus plants had multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of the p23-specific small interfering RNAs (siRNAs).61 RNA-mediated resistance for CTV has also been obtained in transgenic grapefruit.62,63 To successfully obtain CTV resistant plants through gene silencing, it is important to understand all the viral counterdefense strategies, like the virus’s RNAi suppression strategies developed during their coevolution with plants.43,45,64 Protein-mediated resistance strategies have also been described for CTV. CTV-resistant citrus plants that express the CP of the virus have been obtained and in these plants virus resistance was related to the accumulation of the transgenic viral protein in plant tissue, with PDR as the proposed mechanism of resistance.65–69 Protoplast fusion is another promising biotech tool because it can produce somatic hybrids, which would be very valuable for the development of CTV-resistant Â�rootstocks. Citrus somatic hybrids with resistance to CTV infection have been successfully obtained by protoplast fusion.70

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Plum Pox Virus Sharka, or plum pox, caused by the plum pox virus (PPV, genus Potyvirus), is one of the most serious diseases for Prunus stone fruit and nut species. This virus has caused serious economic losses to the stone fruit industry in Europe,71,72 and may eventually spread widely throughout North America and South America.73–75 Prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV) are other viruses widespread in production areas worldwide that are very destructive for peach, plum, cherry, and apricot production. Viruses endemic to North America, including peach mosaic virus (PMV) and American plum line pattern virus (APLPV), are also known to cause important diseases of stone fruit.76,77 PPV mainly infects plum, apricot, and peach trees.72,78,79 Sharka disease symptoms range from weak necrosis on leaf blades and stems, to death of young shoots. PPV is transmitted by aphids in a nonpersistent manner, its main vectors being Myzus persicae and Aphis spiraecola.80–83 Due to the severe economic losses in crop production for the stone fruit industry, efforts have focused on the development of PPV-resistant Prunus cultivars either by conventional breeding or by biotechnology approaches. PPV-resistant plant selection by traditional breeding has not been successful, but the use of biotechnology approaches to obtain resistant plants looks promising. Pathogen-derived resistance to PPV has been achieved by the introduction of either wild type or mutated structural and nonstructural genes into host plants.38 A gene silencing approach46 was used to obtain transgenic European plums carrying the CP gene of PPV. The diversity and dynamics of PPV and aphid population in transgenic European plums has also been evaluated in the field during 7 years under Mediterranean conditions.84 After 7 years of experimental trials, the transgenic line C5 (cv. Honey Sweet) was reported to be free from PPV.84–86 The resistance mechanism of line C5 was confirmed to be based on PTGS.46,87 Because PPV is only one of the multiple viruses affecting Prunus stone fruit production, it is highly desirable to engineer multivirus resistance in plums. Accordingly, PTGS-based approaches have been used to provide multiple resistances to important viruses affecting Prunus stone fruit and nut species. A single chimeric transgene, PTRAP6, was created by the fusion of 400–500â•›bp long fragments from six major Prunus fruit viruses (APLPV, PMV, PPV, PDV, PNRSV, and ToRSV). Nicotiana benthamiana plants transformed with PTRAP6 displayed resistance to PDV, PPV, and ToRSV.88 Bacterial Resistance Erwinia amylovora Fire blight, caused by the Gram-negative bacterium Erwinia amylovora, is an incredibly destructive bacterial disease of pear, apple, and other members of the Rosaceae family, as well as grape. Most commercially available apple scion cultivars and rootstocks are particularly sensitive to E. amylovora. Resistance to fire blight in apple and pear has been engineered using several transgenic strategies. Expression of the lytic peptide attacin E in transgenic apples and pears provided good resistance to the pathogenic bacterium.89–96 Other genes used to improve

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Transgenic Horticultural Crops: Challenges and Opportunities

fire blight resistance are epo, a gene encoding EPS-depolymerase,97,98 SB-37,91,99 T4 lysozyme,91,95,99,100 hrpN (harpin),101 cecropin MB 39 (modified SB-37),102,103 and Shiva-1.91 Transgenic apples resistant to fire blight have also been obtained by partially silencing certain genes encoding pathogen-protein receptors.104 There are also opportunities for breeding fire blight-resistant apple and pear cultivars by exploiting genetic variation in germplasm and by developing quantitative trait loci (QTL) markers.105,106 Agrobacterium tumefaciens Crown gall, caused by the soil bacterium Agrobacterium tumefaciens, greatly diminishes tree productivity and is a serious disease for many fruit, nut, and ornamental crops. Walnuts in particular are very susceptible to this disease. Losses are incurred from both contaminated nursery stock and infected orchard trees. Current prophylactic measures and the mechanical removal of galls have not adequately controlled the problem. Pathogenic A. tumefaciens transforms infected plants with the genes iaaM (tryptophan monooxygenase), iaaH (indole-3-acetamide hydrolase), and ipt (isopentenyl transferase). The iaaM and iaaH gene products convert tryptophan into indole-3-acetic acid, an auxin, while the ipt gene product catalyzes the production of adenosine monophosphate (AMP), which is converted to cytokinins by endogenous plant enzymes. The resulting overproduction of auxin and cytokinins induces proliferation of callus tissue at the wound site, resulting in the development of large galls.17,107 To engineer resistance to this pathogen, a binary vector plasmid containing inverted repeats of portions of the iaaM and ipt genes was constructed and transformed into walnut.108 Constitutive expression of this construct induces RNAi-mediated degradation of the iaaM and ipt transcripts, demonstrating the use of RNAi to generate resistance to a major bacterial disease.109 Because the construct and the oncogenes do not need to have perfect homology for silencing to be effective, the resulting transgenic plants are resistant to a very wide range of A. tumefaciens strains, displaying a broad spectrum durable resistance.108 Transgenic walnut plants containing the iaaM- and ipt-silencing constructs are currently in field trials. RNAi has also been used to generate crown gall resistance for tomato,110 apple,111,112 and grape.113 Xylella fastidiosa Xylella fastidiosa (Xf ) is a xylem-limited, Gram-negative bacterium,114 Â�t ransmitted by insect vectors (especially the glassy winged sharpshooter, Homalodisca coagulate). Different Xf strains cause devastating diseases in economically important plants, for example PD in grape, citrus variegated chlorosis (CVC) in citrus,115 and almond leaf scorch disease (ALSD) in almond. Grape PD symptoms include yellowing and gradual necrosis of the petiole attached to the cane after leaf fall. The disease progresses rapidly, resulting in occlusion of xylem vessels and consequent water stress. Vine death often occurs within 2 years.116 Citrus CVC symptoms include leaf variegated chlorosis, defoliation, twig dieback, size reduction, and hardening of fruits.115 The disease management strategy for PD in grape focuses on containing vector transmission using insecticides, with limited success.

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Recently, transgenic grapevines containing xylem-targeted effector proteins like polygalacturonase inhibiting protein (PGIP) and/or chimeric antimicrobial proteins have been obtained.117–121 The use of PGIP to transform grapevines was based on the knowledge that Xf has genes that encode plant cell wall–degrading enzymes, including polygalacturonases.117 Five PGIP expression constructs were designed, each containing different signal peptide sequences in order to identify which most efficiently localizes PGIP to xylem tissues, as well as which provides the best distribution of PGIP through the graft union into untransformed scion tissues.120 Chimeric antimicrobial protein strategy provides a protein-based Â�therapeutic that targets the causative agent specifically resulting in its clearance, which results in resistance to the causative agent. Chimeric proteins have two domains, a surface binding domain and a clearance domain linked by a flexible linker such that both components can act independently.120,121 A chimeric antimicrobial protein was designed to obtain resistance to PD that contained a surface recognition domain (SRD) and a clearance domain (CD). The SRD targeted the Xf surface protein mopB, highly conserved among all Xf strains and believed to be unique to Xf 118 linked to the CD, a lytic peptide with antimicrobial activity against Gram-negative bacteria. Transgenic grapevines expressing PGIP or the chimeric antimicrobial protein have been greenhouse propagated and mechanically inoculated with Xf to validate their efficiency against PD; the results obtained look promising.120 Transgenic grapevines field trials in multiple locations started in 2010. Xanthomonas Citrus canker (CC) is a serious endemic disease caused by Xanthomonas citri (syn.  Xanthomonas campestris pv. citri or related Xanthomonas campestris pv.  aurantifolii). X. citri affects various citrus species and is dispersed by windblown rain, contaminated equipment, and human activity. Canker threatens the Â�existence of citrus industry, affecting leading citrus producers from Brazil and Florida. Canker symptoms are characterized by pustule-like lesions that can cover the surfaces of leaves, stems, and fruits. Such lesions later become corky and surrounded by water-soaked margins with a yellow halo.122 It has been observed that soon after Â�infection, X. citri simultaneously suppresses the host basal defense and induces remarkable changes in the transcriptional profiles of genes associated with cell wall remodeling, cell division and expansion, vesicle trafficking and response to the hormones auxin and gibberellin.123–125 Genetic transformation has been used in citrus cultivars to improve CC disease resistance by the expression of the Sarcophaga peregrine sarcotoxin,126 the attacin E antimicrobial peptide from Tricloplusia ni,127,128 the Xa21 gene from Oryza sativa,129 and the harpin N gene (hrpN) from Erwinia amylovora.130 For example, transgenic Citrus sinensis plants expressing hrpN under transcriptional control of a pathogen inducible promoter (gst1) showed up to 79% reduction in susceptibility to CC compared with non-transgenic plants.130 In addition, researchers at Integrated Plant Genetics have developed the Disease Block™ technology, which consists of the expression of a recombinant antibody fragment directed against PthA, an effector protein associated with division, enlargement, and death of the

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Transgenic Horticultural Crops: Challenges and Opportunities

host cell during the first stages of canker formation.131,132 The cytoplasm-expressed antibody fragment should bind to PthA upon delivery into the host cell and prevent its nuclear localization. This strategy did not produce fully resistant plants but was efficient in slowing the disease process, resulting in a 2000-fold reduction in the number of X. citri cells released after one cycle of infection. Complete immunity is considered possible by improving the stability and expression level of the recombinant antibody.133 Fungal Resistance Venturia inaequalis Apple scab, caused by the fungal pathogen Venturia inaequalis, is a serious disease in almost all apple cultivars grown commercially around the world and causes significant losses worldwide. A scab resistance locus, Vf, has been identified in the crabapple species Malus floribunda.134 The Vf locus confers resistance to five races of V. inaequalis135 but not to races 6 and 7, identified in Europe.105,136,137 A number of other scab resistance genes have been mapped onto the apple genome, but only the Vf locus has been analyzed in detail. Vf is a complex locus containing four paralogs, Vfa1, Vfa2, Vfa3, and Vfa4.138,139 HcrVf2, a homolog to the Cladosporium fulvum resistance gene of tomato, was transformed into the ‘Gala’ cultivar of apple, conferring scab resistance.140 In addition, the Vfa1, Vfa2, and Vfa4 genes were introduced into a plant cloning vector, pCAMBIA2301, and used for Agrobacterium-mediated transformation of ‘Galaxy’ and ‘McIntosh’ apple cultivars. Transformed lines expressing Vfa1 and Vfa2 exhibited partial resistance to apple scab, while transformed lines expressing Vfa4 were found to be susceptible to apple scab.141 The stilbene synthase (Vst)gene from Vitis vinifera L. is also being used to transform apple with apple scab resistance.142 Alternative approaches to engineer resistance include expression of an antimicrobial peptide,143,144 chitinases,96,145,146 exochitinases,147–149 and endochitinases.147,150,151 Transgenic apple plants expressing high levels of endochitinase are resistant to V. inaequalis, but are also stunted.150 One promising strategy for breeding durable scab resistance is to combine several functionally different resistance genes into a single cultivar (pyramiding of resistance genes).105,152,153 Botrytis cinerea Gray mold, caused by Botrytis cinerea, attacks the shoots, leaves, flowers, and fruits of grape, tomato, and strawberry, as well as other crops. It is one of the most destructive fruit diseases, resulting in significant economic losses pre- and postharvest. The most common strategy to control B. cinerea is the regular application of fungicides throughout flowering. However, new strategies utilizing PGIPs, cell wall proteins that can inhibit fungal polygalacturonases, have been shown to be effective in controlling B. cinerea.154 Transgenic expression of pear PGIP in tomato155 and grape117 limits fungal colonization by inhibiting fungal polygalacturonases, reducing susceptibility to B. cinerea. Strategies based upon altering the expression of native plant cell wall–modifying enzymes have also proved effective in controlling gray mold. For example, suppression of the endo-beta-1,4-glucanases Cel1 and Cel2 reduces gray mold in tomato.156 Likewise, simultaneous suppression of genes encoding

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polygalacturonase (LePG) and expansin (LeExp1) in transgenic tomato plants dramatically reduced the susceptibility of ripening fruit to B. cinerea. Pathogen hydrolases targeting the plant cell wall are well-known components of virulence, and it has been experimentally established that wall disassembly by the plant itself also contributes to susceptibility.157 Strawberry has a lack of natural genetic resistance to gray mold, but transgenic expression of genes like ch5B encoding a chitinase158 and thau II encoding thaumatin II159 from other plant species have been used to obtain transgenic strawberry lines with resistance to B. cinerea. Transgenic wild strawberry (Fragaria vesca) fruits overexpressing the Fragaria × ananassa pectin methyl esterase gene (FaPE1) showed increased resistance to B. cinerea. This resistance is related to the increase in pectin-derived oligogalacturonides, essential components for elicitation of defense responses to B. cinerea.160 Phytophthora nicotianae Phytophthora species are considered one of the most important soil borne problems for citrus, leading to considerable losses worldwide.161,162 Gumosis, encompassing both trunk rot and root rot, is the most relevant disease caused by this pathogen and is characterized by necrosis and gum exudation at the ground level. Phytophthora nicotianae has been most frequently associated with the disease. Transgenic orange plants have been engineered using the coding region of the tomato pathogenesis– related protein PR-5, a chitinase with antifungal activity. Transgenic plants as well as their detached bark were challenged with oomycete cultures and P. citrophthora, respectively. A significant reduction in lesion development was observed in one of the transgenic lines when compared with the control plants.163 Somatic hybridization has also been used to produce citrus somatic hybrids tolerant to trunk and root rot caused by Phytophthora nicotianae. In Florida, more than 70 somatic hybrids have already entered into commercial field trials as potential rootstocks.70 Uncinula necator Powdery mildew, caused by Uncinula necator, is one of the most threatening fungal problems for grape. Transgenic plants harboring a constitutively expressing rice class I chitinase gene showed enhanced disease resistance to powdery mildew and anthracnose when compared with the control lines.164 Also, transgenic ‘Chardonnay’ grapevines containing the magainin gene (mag2), a peptide with broad spectrum antimicrobial activity, showed measurable symptom reduction in response to U. necator under greenhouse conditions.165

Insect Resistance Cydia pomonella The larvae of the codling moth (CM) are a severe threat to apple and walnut production worldwide. The CM lays its eggs on fruit or leaf clusters near fruit, and when they hatch, the larvae feed on the fruit, causing considerable economic damage. Application of chemical pesticides, the main method of controlling this insect, has failed to control CM larvae. Biotechnology is an alternative to create resistance

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Transgenic Horticultural Crops: Challenges and Opportunities

to insect pests and to avoid chemical pesticide use. The predominant strategy to engineer CM resistance in tree crops has been through expression of Bacillus thuringiensis (Bt) genes that encode insecticidal crystal proteins (ICP).166–169 In vitro studies have shown that ICPs bind with high specificity and affinity to specific cell receptors on the insect brush border membrane of midgut epithelial cells.170–172 This binding correlates with the formation of pores and membrane lesions in the midgut that lead to swelling, leakage, and lysis of the epithelium, ultimately causing death of the insect through starvation and septicemia.172–174 ICP genes from different Bacillus species are categorized according to the host range of their activity and DNA sequence homology.174,175 The crylA(c) protein was found to be the most toxic to CM larvae,176 but the transformation of apple166 and walnut167 with cry1A(c) produced very low levels of gene expression. This was a result of codon bias in the bacterial gene sequence, which resulted in very low levels of translation.167,169 Codonoptimized synthetic versions of cryIAc have been introduced into apple, where they confer high levels of mortality to CM larvae both under greenhouse and field conditions. Similar experiments have also been done in walnut177 and persimmon,178 where chemically synthesized versions of cryIAc provided excellent protection against target insect larvae. Transgenic tissues expressing cryIAc protein at as low as 0.02% of total cellular protein produced 100% mortality in CM larvae.177 Recent studies have shown evidence that RNA silencing pathways also play a role in plant defense against insects, which can provide an alternative biotechnological strategy to develop pest control in plants.179 Epiphyas postvittana (Light Brown Apple Moth) Epiphyas postvittana, the light brown apple moth, is a native pest of horticultural crops in Australia and New Zealand,180 and is now present in New Caledonia, North-Western Europe, Hawaii, and California. The larvae of the light brown apple moth cause significant damage to apple foliage and fruit. To confer resistance to E.  postvittana, the apple cultivar ‘Royal Gala’ was transformed, incorporating genes encoding the potato (Solanum tuberosum) biotin binding proteins avidin and strepavidin. In the absence of biotin, an essential vitamin for insects, 80%–90% of larvae feeding on the transgenic apple lines died in a period of 3 weeks, compared to 14% on the control plants.181 The recent identification of genes involved in E. postvittana digestion182 has allowed proof-of-concept research focused on the development of an alternative, RNAi-based strategy to combat this pest. In controlled feeding experiments with E. postvittana larvae, oral delivery of dsRNA homologous to the EposCXE1 led to silencing of the native carboxylesterase gene EposCXE1, which encodes a larval midgut enzyme involved in digestion.183

OUTPUT TRAITS The genetic modification of input traits enhances the agricultural properties and performance of a crop, with the grower as the primary beneficiary. In contrast, transgenic crops with altered output traits, such as increased shelf life, enhanced nutritive value, or reduced allergenicity, primarily benefit the consumer. In perennial orchard

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Transgenic Fruit and Nut Tree Crops Review

crops, the modification of output traits necessarily involves characteristics manifested in fruit or seed tissue. Because most fruit and nut tree crops have an extended juvenile period, initial characterization of modified output traits in transgenic trees often cannot occur until 5–10 years after the initial transformation. Thus, with some notable exceptions, most work outlining the genetic modification of output traits in fruit and nut tree crops is preliminary, with many transgenic lines currently described only in USDA field trial listings (Table 1.1). Below, we discuss current progress in the modification of ripening, the improvement of fruit quality and nutritive value, the removal of undesirable phytochemicals, and antigen production in transgenic fruit and nut tree crops.

TABLE 1.1 U.S. Field Trials of Transgenic Fruit and Nut Tree Crops with Enhanced Output Traits Crop

Trait

Apple

Reduced postharvest browning

Polyphenol oxidase (antisense)

Delayed ripening

ACC synthase (antisense); ACC oxidase (antisense) SAM transferase Sorbitol-6-phosphate dehydrogenase (antisense) ACC oxidase (antisense) Xanthosine methyltransferase (antisense) Chalcone synthase, chalcone isomerase, rhamnosyl transferase ACC synthase (antisense) SAM transferase Polygalacturonase inhibitor protein ACC synthase (antisense) Mabinlin ACC oxidase (antisense)

Delayed ripening Altered sugar profile

Coffee

Delayed ripening Reduced caffeine

Grapefruit

Altered carotenoid profile

Papaya

Delayed ripening

Pear Persimmon

Delayed ripening Delayed softening

Pineapple

Delayed ripening

Plum

Increased sweetness Delayed ripening

Gene

Institution(s)

Related Publication

Cornell Univ.; Okanagan Specialty Fruits, Inc. Cornell Univ.; UC Davis; UC Berkeley Excelcis; Agritope UC Davis; Cornell Univ.; Oregon State Univ. Univ. of Hawaii

Murata et al.198,199

Univ. of Hawaii

Ogita et al.201

Univ. of Hawaii

Costa et al.193

Univ. of Hawaii

Dandekar et al.187 — Teo et al.195

—

—

Excelcis; Agritope UC Davis

Gao et al.191 —

Univ. of Hawaii

Botella et al.188 — —

Univ. of Hawaii United States Dept. of Agriculture

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Transgenic Horticultural Crops: Challenges and Opportunities

Delayed Fruit Ripening Many tree fruits are transported long distances from their point of cultivation to their point of sale to the consumer. As demonstrated in several annual plant systems, genetically manipulating the biosynthesis of the hormone ethylene can substantially slow fruit ripening, allowing increased flexibility in transport time and fruit maturity at harvest.184,185 In climacteric fruits, ethylene triggers a rapid increase in respiratory rate and initiates a cascade of biochemical and physiological changes associated with ripening.88 Beginning with the precursor S-adenosyl methionine (SAM), ethylene is synthesized in two enzymatic steps in plants. First, SAM is converted to 1-aminocyclopropane-1-carboxylic acid (ACC), a reaction catalyzed by the ACC synthase enzyme. Second, the ACC oxidase enzyme converts ACC to ethylene.186 The strategies applied to minimize ethylene production in transgenic fruit tree crops, and thereby slow ethylene-associated fruit ripening processes, have primarily focused on posttranscriptional silencing of the genes encoding the ACC synthase and ACC oxidase enzymes. Delayed ripening in transgenic fruit trees has been demonstrated most convincingly in apple and papaya.88,187 Silencing of ACC oxidase in papaya resulted in a 40% reduction in fruit ethylene production, with a corresponding delay in fruit softening and the retention of green peel color. However, the fruit appears to have limited commercial application, as the unripe fruit retains high susceptibility to pathogen infection/ decay, leading to a direct transition from unripe fruit to rotten fruit.88 Dandekar et al. pursued a similar approach in apple, silencing both the ACC synthase and ACC Â�oxidase genes in separate transgenic lines. Apples collected from both ACC synthase- and ACC oxidase-silenced lines displayed >90% reduction in ethylene production. The transgenic fruits were firmer and had a longer shelf life than comparable controls (Figure 1.1), but their sugar/acid balance (a key aspect of fruit flavor) was unaffected. Fruit aroma was likely altered in these lines, however, as total volatile ester production was reduced by 65%–70% compared to controls.187 Several additional studies have described comparable delayed ripening strategies in pineapple,188 mango,189 and avocado,190 though no phenotypic characterization of the transgenic plants has been reported. Similarly, the silencing of ACC oxidase in transgenic pear reduced ethylene production by 85% in in vitro shoots, but effects on pear fruit have not yet been determined.191

Improving Fruit Quality and Nutritive Value Several recently published studies have focused on the generation of transgenic fruit and nut tree crops with enhanced nutritive value or increased levels of specific phytochemicals beneficial to human health. For example, the grape stilbene synthase gene, which is responsible for the synthesis of the phytoalexin resveratrol, has been introduced into apple and kiwifruit.143,192 Resveratrol is an antifungal compound involved in pathogen-induced plant defense responses, but it also has a variety of beneficial effects on human health, including anti-inflammatory and anticarcinogenic properties.143 The accumulation of glycosylated resveratrol derivatives was demonstrated in the leaves of both transgenic kiwifruit and apple plants, though it is currently unclear

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Transgenic Fruit and Nut Tree Crops Review (a)

(b)

(c)

(d)

(e)

FIGURE 1.1â•… (See color insert.) Delayed fruit ripening in transgenic apples. (a) An apple tree expressing an antisense ACC oxidase (ACO) transgene. Ethylene production is reduced >90% in this transgenic line compared to wild-type controls. (b, c) ACO-silenced (b) and wild-type (c) apples stored at room temperature for 1 month. (d, e) ACO-silenced (d) and wild-type (e) apples stored at room temperature for 3 months. (Reprinted with kind permission from Springer Science+Business Media: Transgenic Res., Effect of downregulation of ethylene biosynthesis on fruit flavor complex in apple fruit, 13, 2004, 373, Dandekar, A. M. et al.)

whether the fruit from these plants would accumulate these compounds at levels that could potentially benefit human health.142,192 Preliminary reports have also described attempts to increase vitamin A content in grapefruit (through expression of the Â�carotenoid biosynthetic genes phytoene synthase, Â�phytoene desaturase, and lycopene β-cyclase) and attempts to increase the content of “heart healthy” oleic acid in palm oil (through expression of the fatty acid modifying genes β-ketoacyl-ACP synthase II, Δ9-stearoyl-ACP-desaturase, and palmitoyl-ACP-thioesterase Â�[antisense]).193,194 However, no phenotypic characterization of the transgenic grapefruit and oil palm plants has yet been reported. A unique balance of soluble sugars, starch, acids, and volatiles underlies the flavor and quality of each type of fruit. The recent work of Teo et al. has demonstrated that fruit quality/flavor can be dramatically altered using transgenic approaches.195 Unlike most plant species, apples accumulate and transport photosynthate largely in the form of the sugar alcohol sorbitol, rather than sucrose. Sorbitol is synthesized from glucose-6-phosphate through the action of the enzyme sorbitol-6-phosphate dehydrogenase (S6PDH), and Teo et al. generated transgenic apple plants expressing an antisense copy of the S6PDH gene.195 Leaves from several resultant transgenic lines

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Transgenic Horticultural Crops: Challenges and Opportunities

displayed 80%–90% reductions in S6PDH enzyme activity and 70%–80% reductions in sorbitol levels. Dramatic changes in fruit composition were also observed in the transgenic lines, including significant increases in glucose levels, flesh firmness, and soluble solids content, and corresponding significant decreases in fructose levels, sorbitol levels, starch levels, malic acid levels, and titratable acidity. Overall, these results demonstrate that fruit quality characteristics (and likely flavor) are realistic targets for manipulation/improvement via biotechnology.

Removal of Undesirable Phytochemicals Postharvest enzymatic browning, caused primarily by the enzyme polyphenol oxidase, is a substantial quality problem in a variety of fruit crops, including apple, pear, grape, and pineapple. Polyphenol oxidase comes in contact with its phenolic substrates during fruit ripening or tissue damage, generating reactive quinones that crosslink to form dark-colored phytomelanins.196 Following the lead of studies performed in annual crops,197 several groups have attempted to silence the polyphenol oxidase gene in order to reduce postharvest browning in tree fruits. Most notable is work by Murata et al., in which expression of an antisense polyphenol oxidase gene in transgenic apple lines reduced polyphenol oxidase activity and tissue browning by ~50% compared to wild-type controls.198,199 However, only callus and shoot tissues were examined; it remains to be seen whether reductions in postharvest browning will also be observed in apple fruit.198,199 Similar approaches have been pursued to reduce blackheart in pineapple200 and grape berry darkening,196 though no characterization of the transgenic polyphenol oxidase-silenced plants has yet been reported. Posttranscriptional gene silencing has also been utilized to reduce caffeine content in coffee plants. Caffeine is generated from xanthosine by two sequential methylation reactions catalyzed by the enzymes theobromine synthase and caffeine synthase. Ogita et al. transformed coffee plants with an RNAi vector designed to initiate posttranscriptional gene silencing of the theobromine synthase gene.201 The resultant transgenic lines were morphologically normal, but displayed 50%–70% less caffeine in leaf tissues than controls. Effects on caffeine content in coffee fruit were not described. Although not yet reported in the literature, a similar gene silencing strategy could be used to reduce the levels of allergenic proteins in tree nuts (e.g., walnuts and chestnuts). This approach was recently applied to reduce the levels of the immunodominant Ara h 2 protein in transgenic peanut plants.202

Antigen Production Several studies have shown that transgenic plants that produce viral or bacterial antigen proteins can induce a protective immune response (as measured by the production of antigen-specific serum antibodies) when fed to mice and humans.203,204 These plants, which act as “edible vaccines,” have several significant advantages over traditionally purified and administered vaccines, including ease of human delivery, increased stability, and decreased production and transport costs.205 While most studies on edible vaccines have focused on annual crops, recently the “s” gene of the

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Hepatitis B surface antigen (HBsAg) was expressed in banana plants.206 Hepatitis B is the major cause of persistent viremia in humans, and banana is an ideal host for expression of HBsAg due to its palatability to infants and year-round availability in the tropics. By expressing an HBsAg “s” gene-ER retention sequence fusion under the control of the banana ethylene forming enzyme promoter, Sunil-Kumar et al. successfully produced antibody-reactive antigen in banana leaves and fruits.206 Unfortunately, antigen levels in banana fruit were very low compared to antigen levels achieved in several previously described annual plant systems (1â•›ng/g vs. 16â•›μg/g fresh weight in potato tuber), precluding immunogenicity testing and the use of these bananas for oral vaccination.

CONCLUSIONS The high value of fruit and nut crops and their increasing dietary importance due to their unique nutritional and disease-preventing attributes has focused attention on genetic strategies to solve production limitations. The technology to produce transgenic trees is relatively mature, especially the use of the Agrobacterium-mediated transformation process. However, regeneration of transgenic plants continues to pose the greatest challenge for many of these crops, especially Prunus. In most fruit and nut tree species, only a few of the commercially significant phenotypes have been extensively tested in the field. Viral resistance in plum and papaya has been deregulated, and commercialized in the case of papaya. Many tree crops are promising candidates for the use of transgenic technologies to prevent disease and pest infestation, especially through the use of Bt insecticidal proteins that target lepidopteran insect pests. Resistance to bacterial pathogens poses the greatest challenge, as they present the greatest threat to commercial viability in many perennial crop species. Substantial progress has been made toward gaining resistance to PD in grapevine and crown gall disease in walnut. Many fruit quality traits have also been manipulated, primarily through the RNAi-mediated regulation of ethylene Â�biosynthesis and/or sorbitol biosynthesis, where the phenotypes have been documented in field-grown transgenic fruit. Many interesting transgenic approaches have been developed for other quality traits that are of importance to consumers, including postharvest storage disorders, fruit texture, appearance, sugar and lipid composition, and allergenicity; however, these phenotypes remain to be established in the field. With the exception of papaya, no other transgenic fruit variety has been commercialized and the technology development toward commercialization for scion varieties in particular has been “soft-pedaled” due to the public acceptance issue. Perhaps the greatest immediate promise lies in the potential for commercialization of transgenic rootstocks, as they can provide resistance to disease, pest, and environmental issues while sustaining the production of a wild-type fruit crop from the grafted (non-transgenic) scion.

REFERENCES

1. Rieger, M., Introduction to Fruit Crops, Food Products Press, New York, p. 462, 2006. 2. FAOStat. http://faostat.fao.org/, 2008, Accessed on March 3, 2010. 3. Gomez-Lim, M. A. and Litz, R. E., Genetic transformation of perennial tropical fruits, In Vitro Cell. Dev. Biol. Plant, 40, 442, 2004.

20

Transgenic Horticultural Crops: Challenges and Opportunities

4. Petri, C. and Burgos, L., Transformation of fruit trees: Useful breeding tool or continued future prospect? Transgenic Res., 14, 15, 2005. 5. Perl, A. and Eshdat, Y., Grapes, Springer-Verlag, Berlin, p. 189, 2007. 6. Meredith, C. P. and Reisch, B., Grapevine genetics: New tools for vine improvement, gene mapping, genetic engineering and DNA fingerprinting, Am. J. Enol. Vit., 48, 265, 1997. 7. Thomas, M., Iocco, P., and Franks, T., Transgenic grapevines: Status and future, Acta Hort., 528, 281, 2000. 8. Kaneyoshi, J. et al., A simple and efficient gene-transfer system of trifoliate orange (Poncirus-trifoliata raf ), Plant Cell Rep., 13, 541, 1994. 9. Pena, L. et al., Agrobacterium-mediated transformation of citrus, in Transgenic crops of the world—essential protocols, ed. Curtis, I. S., Kluwer Academic Publishers, Dordrecht, the Netherlands, p. 145, 2004. 10. Pena, L. et al., Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants, Plant Cell Rep., 14, 616, 1995. 11. James, D. J. et al., Genetic-transformation of apple (Malus-pumila mill) using a disarmed Ti-binary vector, Plant Cell Rep., 7, 658, 1989. 12. Dandekar, A. M. et al., Apple (Malus x domestica), in Methods in Molecular Biology, ed. Wang, K., Humana Press Inc., Totowa, NJ, p. 253, 2006. 13. Mezzetti, B. et al., Genetic transformation of Vitis vinifera via organogenesis, BMC Biotechnol., 2, 1, 2002. 14. Dutt, M. et al., Transgenic plants from shoot apical meristems of Vitis vinifera l. “Thompson seedless” via Agrobacterium-mediated transformation, Plant Cell Rep., 26, 2101, 2007. 15. Suprasanna, P., Ganapathi, T. R., and Bapat, V. A., Genetic transformation of woody plants using embryogenic cultures, J. New Seeds, 7, 17, 2005. 16. Leslie, C. A. et al., Walnut (Juglans), in Methods in Molecular Biology, ed. Wang, K., Humana Press Inc., Totowa, NJ, pp. 344, 297, 2006. 17. Britton, M. et al., Juglans—Walnuts v. biotechnology in agriculture and forestry, Transgenic Crops: Biotechnol. Agric. Forest., 5, 349, 2007. 18. Rajasekaran, K. and Mullins, M. G., The origin of embryos and plantlets from cultured anthers of hybrid grapevines Vitis vinifera x Vitis repestris, Am. J. Enol. Vitic., 34, 108, 1983. 19. Mauro, M. C. et al., Analysis of 41 B (Vitis vinifera x Vitis berlandieri) grapevine rootstocks for grapevine fanleaf virus resistance, Acta Hort., 528, 313, 2000. 20. Perl, A., Colova-Tsolova, V., and Eshdat, Y., Agrobacterium-Mediated Transformation of Grape Embryogenic Calli, Academic Publisher, Dordrecht, the Netherlands, p. 229, 2004. 21. Perrin, M., Gertz, C., and Masson, J. E., High efficiency initiation of regenerable embryogenic callus from anther filaments of 19-grapevine genotypes grown worldwide, Plant Sci., 167, 1343, 2004. 22. Aguero, C. B., Meredith, C. P., and Dandekar, A. M., Genetic transformation of Vitis vinifera l. cvs Thompson seedless and Chardonnay with the pear PGIP and GFP encoding genes, Vitis, 45, 1, 2006. 23. Bouquet, A. et al., Grapevine (Vitis vinifera l.), in Methods in Molecular Biology, ed. Wang, K., Humana Press Inc., Totowa, NJ, pp. 344, 273, 2006. 24. Torregrosa, L., Iocco, P., and Thomas, M. R., Influence of agrobacterium strain, culture medium, and cultivar on the transformation efficiency of vitis vinifera l, Am J. Enol. Vitic., 53, 183, 2002. 25. Ballester, A., Cervera, M., and Pena, L., Evaluation of selection strategies alternative to NPTII in genetic transformation of citrus, Plant Cell Rep., 27, 1005, 2008. 26. Dutt, M. et al., A co-transformation system to produce transgenic grapevines free of marker genes, Plant Sci., 175, 423, 2008. 27. Reisch, B. et al., Genetic transformation of Vitis vinifera to improve disease resistance, Acta Hort., 603, 303, 2002.

Transgenic Fruit and Nut Tree Crops Review

21

28. Vidal, J. R. et al., Biolistic transformation of grapevine using minimal gene cassette technology, Plant Cell Rep., 25, 807, 2006. 29. Jaillon, O. et al., The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla, Nature, 449, 463, 2007. 30. Velasco, R. et al., High quality draft consensus sequence of the genome of a heterozygous grapevine variety, PLoS ONE, 2, 1326, 2007. 31. Zottini, M. et al., Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells, Plant Cell Rep., 27, 845, 2008. 32. Azuma, A. et al., Genomic and genetic analysis of MYB-related genes that Â�regulate anthocyanin biosynthesis in grape berry skin, Theoret. Appl. Genet., 117, 1009, 2008. 33. Ahmad, M. and Mirza, B., An efficient protocol for transient transformation of intact fruit and transgene expression in citrus, Plant Mol. Biol. Rep., 23, 419, 2005. 34. Folimonov, A. S. F. et al., A stable RNA virus-based vector for fruit species, Virology, 368, 205, 2007. 35. Aguero, C. B. et al., Xylem sap proteins from Vitis vinifera l. Chardonnay, Am. J. Enol. Vit., 59, 306, 2008. 36. Prins, M. et al., Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants, Biotechnology, 10, 1133, 1992. 37. Lindbo, J. A. and Dougherty, W. G., Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts, Virology, 189, 725, 1992. 38. Liu, Z. et al., Engineering resistance to multiple Prunus fruit viruses through expression of chimeric hairpins, J. Am. Soc. Hort. Sci., 132, 407, 2007. 39. Wilson, T. M. A., Strategies to protect crop plants against viruses: Pathogen-derived resistance blossoms, Proc. Natl. Acad. Sci., 90, 3134, 1993. 40. Baulcombe, D. C., Mechanisms of pathogen-derived resistance to viruses in transgenic plants, Plant Cell, 8, 1833, 1996. 41. Prins, M., Broad virus resistance in transgenic plants, Trends Biotechnol., 21, 373, 2003. 42. Brigneti, G. et al., Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana, EMBO J., 17, 6739, 1998. 43. de Souza, A. J., Mendes, B. M. J., and Filho, F. A. A. M., Gene silencing: Concepts, applications, and perspectives in woody plants, Sci. Agric., 64, 645, 2007. 44. Hannon, G. J., RNA interference, Nature, 418, 244, 2002. 45. Moreno, P. et al., Plant diseases that changed the world—Citrus tristeza virus: A pathogen that changed the course of the citrus industry, Mol. Plant Pathol., 9, 251, 2008. 46. Scorza, R. et al., Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene, Transgenic Res., 10, 201, 2001. 47. Voinnet, O., Pinto, Y. M., and Baulcombe, D. C., Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants, Proc. Natl. Acad. Sci., 96, 14147, 1999. 48. Fitch, M. M. M. et al., Stable transformation of papaya via microprojectile bombardment, Plant Cell Rep., 9, 189, 1990. 49. Fitch, M. M. M. et al., Virus resistance papaya plants derived from tissues bombarded with the coat protein gene of Papaya ringspot virus, Biotechnology, 10, 1466, 1992. 50. Lius, S. et al., Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus, Mol. Breed., 3, 161, 1997. 51. Gonsalves, D., Coat protein transgenic papaya “acquired” immunity for controlling papaya ringspot virus, Curr. Top. Microbiol. Immunol., 266, 73, 2002.

22

Transgenic Horticultural Crops: Challenges and Opportunities

52. Tripathi, S., Suzuki, J., and Gonsalves, D., Development of genetically engineered resistant papaya for papaya ringspot virus in a timely manner: A comprehensive and successful approach, Methods Mol. Biol., 354, 197, 2007. 53. Gonsalves, D., Control of papaya ringspot virus in papaya: A case study, Ann. Rev. Phytopathol., 36, 415, 1998. 54. Tennant, P. et al., Papaya ringspot virus resistance of transgenic rainbow and sunup is affected by gene dosage, plant development, and coat protein homology, Eur. J. Plant Pathol., 107, 645, 2001. 55. Cheng, Y. H., Yang, J. S., and Yeh, S. D., Efficient transformation of papaya by coat protein gene of Papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with carborundum, Plant Cell Rep., 16, 127, 1996. 56. Bau, H. J. et al., Broad-spectrum resistance to different geographic strains of Papaya ringspot virus in coat protein gene transgenic papaya, Phytopathology, 93, 112, 2003. 57. Bau, H. J. et al., Field evaluation of transgenic papaya lines carrying the coat protein gene of Papaya ringspot virus in Taiwan, Plant Dis., 88, 848, 2004. 58. Bau, H. J. et al., Potential threat of a new pathotype of Papaya leaf distortion mosaic virus infecting transgenic papaya resistant to Papaya ringspot virus, Phytopathology, 98, 848, 2008. 59. Kung, Y. J. et al., Generation of transgenic papaya with double resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus, Phytopathology, 99, 1312, 2009. 60. Bar-Joseph, M., Marcus, R., and Lee, R. F., The continuous challenge of Citrus tristeza virus control, Ann. Rev. Phytopathol., 27, 291, 1989. 61. Fagoaga, C. et al., Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime, Plant Mol. Biol., 60, 153, 2006. 62. Rai, M., Refinement of the Citrus tristeza virus resistance gene (CTV) positional map in Poncirus trifoliata and generation of transgenic grapefruit (Citrus paradisi) plant lines with candidate resistance genes in this region, Plant Mol. Biol., 61, 399, 2006. 63. Febres, V. J., Lee, R. F., and Moore, G. A., Transgenic resistance to Citrus tristeza virus in grapefruit, Plant Cell Rep., 27, 93, 2008. 64. Tatineni, S. et al., Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus trees, Virology, 376, 297, 2008. 65. Mauro, M.C. et al., High efficiency regeneration grapevine plants transformed with the GFLV coat protein gene, Plant Sci., 112, 97, 1995. 66. Valat, L., Fuchs, M., and Burrus, M., Transgenic grapevine rootstock clones expressing the coat protein or movement protein genes of grapevine Fanleaf virus: Characterization and reaction to virus infection upon protoplast electroporation, Plant Sci., 170, 739, 2006. 67. Zanek, M. C. et al., Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus, Plant Cell Rep., 27, 57, 2008. 68. Dominguez, A. et al., Efficient production of transgenic citrus plants expressing the coat protein gene of Citrus tristeza virus, Plant Cell Rep., 19, 427, 2000. 69. Dominguez, A. et al., Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene, Molecular Breeding, 10, 1, 2002. 70. Mourao Filho, F. A. A. et al., Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and Citrus tristeza virus, Sci. Hort., 115, 301, 2008. 71. Kegler, H. et al., Some results of 50 years of research on the resistance to plum pox virus, Acta Virol., 42, 200, 1998. 72. Ravelonandro, M. et al., The use of transgenic fruit trees as a resistance strategy for virus epidemics: The plum pox (Sharka) model, Virus Res., 71, 63, 2000.

Transgenic Fruit and Nut Tree Crops Review

23

73. Levy, L., Damsteegt, V., and Welliver, R., First report of plum pox virus (Sharka disease) in Prunus persica in the United States., Plant Dis., 84, 202, 2000. 74. Rosales, M., Hinrichsen, P., and Herrera., G., Molecular characterization of plum pox virus isolated from apricots, plums and peaches in Chile., Acta Hort., 472, 401, 1998. 75. Thompson, D. et al., First report of plum pox potyvirus in Ontario, Canada, Plant Dis., 85, 97, 2001. 76. Gilmer, R. M., Nyland, G., and Moore, J. D., Virus diseases and noninfectious disorders of stone fruits in North America, in U.S. Department of Agriculture Handbook 437, U.S. Government Printing Office, Washington, DC, 1976. 77. American Phytopathology Society, Compendium of Stone Fruit Diseases, eds. Ogawa, J. M., Zehr, E. I., and Bird, G. W., American Phytopathology Society, St. Paul, MN, p. 1, 1995. 78. Garcıa, J. A. and Cambra, M., Plum pox virus and Sharka disease, Plant Viruses, 1, 69, 2007. 79. Moreno, A., Fereres, A., and Cambra, M., Quantitative estimation of plum pox virus targets acquired and transmitted by a single Myzus Persicae, Arch. Virol., 154, 1391, 2009. 80. Glasa, M., Boeglin, M., and Labonne, G., Aphid transmission of natural recombinant plum pox virus isolates to different Prunus ssp.—A contribution for understanding the epidemiology of an atypical PPV, Acta Hort., 657, 217, 2004. 81. Cambra, M. et al., Molecular Methods for Detection and Quantification of Virus in Aphids, Springer, Dordrecht, the Netherlands, p. 81, 2006. 82. Gildow, F. et al., Plum pox in North America: Identification of aphid vectors and a potential role for fruit in virus spread, Phytopathology, 94, 868, 2004. 83. Powell, G., Intracellular salivation is the aphid activity associated with inoculation of non-persistently transmitted viruses, J. Gen. Virol., 86, 469, 2005. 84. Capote, N. et al., Assessment of the diversity and dynamics of plum pox virus and aphid populations in transgenic European plums under Mediterranean conditions, Transgenic Res., 17, 367, 2008. 85. Malinowski, T. et al., Field trials of plum clones transformed with the plum pox virus coat protein (PPV-CP) gene, Plant Dis., 90, 1012, 2006. 86. Scorza, R., The development of ‘Honeysweet’—A transgenic plum pox virus (PPV)resistant plum and the application of intron-hairpin (ihp) RNA technology for PPV resistance in stone fruits, Hortscience, 42, 904, 2007. 87. Hily, J.-M. et al., Accumulation of the long class of siRNA is associated with resistance to plum pox virus in a transgenic woody perennial plum tree, Mol. Plant Microbe Interact., 18, 794, 2005. 88. Lopez-Gomez, R. et al., Ripening in papaya fruit is altered by ACC oxidase co-suppression, Transgenic Res., 18, 89, 2008. 89. Ko, K. et al., Galaxy lines transgenic for attacin E and T4 lysozyme genes have increased resistance to fire blight, Curr. Plant Sci. Biotech. Agric., 36, 507, 1999. 90. Norelli, J. L. et al., Transgenic “Malling 26” apple expressing the attacin E gene has increased resistance to Erwinia amylovora, Euphytica, 77, 123, 1994. 91. Norelli, J. L. et al., Transgenic Gala apple expressing attacin E has increased field resistance to Erwinia amylovora (fire blight), Phytopathology, 89, 56, 1999. 92. Norelli, J. L. et al., Transgenic ‘Royal Gala’ apple expressing attacin E has increased field resistance to Erwinia amylovora (fire blight), Acta Hort., 538, 633, 2000. 93. Aldwinckle, H. S. et al., Development of fire blight resistant apple cultivars by genetic engineering, Acta Hort., 622, 105, 2003. 94. Ko, K. et al., Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple, Biotechnol. Lett., 22, 373, 2000.

24

Transgenic Horticultural Crops: Challenges and Opportunities

95. Ko, K. et al., T4 lysozyme and attacin genes enhance resistance of transgenic ‘galaxy’ apple against Erwinia amylovora, J. Am. Soc. Hort. Sci., 127, 515, 2002. 96. Hanke, V. et al., Tranformation in apple for increased disease resistance, Acta Hort., 538, 611, 2000. 97. Hanke, V., Kim, W.-S., and Geider, K., Plant transformation for induction of the fire blight resistance: Transgenic apples expressing viral EPS-depolymerase, Acta Hort., 590, 393, 2002. 98. Flachowsky, H. et al., Transgenic expression of a viral EPS-depolymerase is potentially useful to induce fire blight resistance in apple, Ann. Appl. Biol., 153, 345, 2008. 99. Aldwinckle, H. S. et al., Genetic engineering of disease resistance in apple fruit cultivars and rootstocks, Curr. Plant Sci. Biotech. Agric., 36, 449, 1999. 100. Hanke, V. et al., Transformation of apple cultivars with T4-lysozyme-gene to increase disease resistance, Acta Hort., 489, 253, 1999. 101. Kader, A. A. et al., Evaluation of the hrpN gene for increasing resistance to fire blight in transgenic apple, Acta Hort., 489, 247, 1999. 102. Liu, Q. et al., Transgenic ‘Royal Gala’ apple plants with cecropin-MB39 gene has increased resistance to Erwinia amylovora, Acta Hort., 560, 95, 1999. 103. Liu, Q. et al., Response of transgenic Royal Gala apple (Malus x domestica Borkh.) shoots carrying a modified cecropin mb39 gene, to Erwinia amylovora, Plant Cell Rep., 20, 306, 2001. 104. Borejsza-Wysocka, E. E. et al., Strategies for obtaining fire blight resistance in apple by rDNA technology, Acta Hort., 738, 283, 2007. 105. Kellerhals, M., Bertschinger, L., and Gessler, C., Use of genetic resources in apple breeding and for sustainable fruit production, J. Fruit Ornament. Plant Res., 12, 53, 2004. 106. Calenge, F. et al., Identification of a major QTL together with several minor additive or epistatic QTLs for resistance to fire blight in apple in two related progenies, Theoret. Appl. Genet, 111, 128, 2005. 107. Britton, M. T., Escobar, M. A., and Dandekar, A. M., The oncogenes of Agrobacterium tumefaciens and Agrobacterium rhizogenes, in Agrobacterium: From Biology to Biotechnology, eds. Tzfira, T. and Citovsky, V., Springer, New York, 2008, Chap. 14. 108. Escobar, M. A. et al., Silencing crown gall disease in walnut (Juglans regia L.), Plant Sci., 163, 591, 2002. 109. Escobar, M. A. et al., RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis, Proc. Natl. Acad. Sci., 98, 13437, 2001. 110. Escobar, M. A. et al., Characterization of oncogene-silenced transgenic plants: Implications for Agrobacterium biology and post-transcriptional gene silencing, Mol. Plant Pathol., 4, 1, 2003. 111. Lee, H. et al., Translation start sequences affect the efficiency of silencing of Agrobacterium tumefaciens t-DNA oncogenes, Plant Physiol., 133, 966, 2003. 112. Viss, W. J. et al., Crown-gall-resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes, Mol. Breed., 12, 283, 2003. 113. Burr, T. J. et al., Breeding for crown gall resistance: Traditional and molecular approaches, Acta Hort., 603, 441, 2003. 114. Lambais, M. R. et al., A genomic approach to the understanding of Xylella fastidiosa pathogenicity, Curr. Opin. Microbiol., 3, 459, 2000. 115. Bove, J. M. and Ayres, A. J., Etiology of three recent diseases of citrus in Sao Paulo state: Sudden death, variegated chlorosis and Huanglongbing, IUBMB Life, 59, 346, 2007. 116. Goodwin, P. H., De Vay, J. E., and Meredith, C. P., Roles of water stress and phytotoxins in the development of Pierce’s disease of the grapevine, Physiol. Mol. Plant Pathol., 32, 1, 1988.

Transgenic Fruit and Nut Tree Crops Review

25

117. Aguero, C. B. et al., Evaluation of tolerance to Pierce’s disease and botrytis in transgenic plants of Vitis vinifera l. Expressing the pear PGIP gene, Mol. Plant Pathol., 6, 43, 2005. 118. Bruening, G. E. et al., Exploiting Xylella fastidiosa proteins for Pierce’s disease control, in Pierce’s Disease Research Symposium, CDFA Pierce’s Disease Board, San Diego, CA, p. 221, 2005. 119. Dandekar, A. M. et al., Design of chimeric antimicrobial proteins for rapid clearance of Xyllela, in Pierce’s Disease Research Symposium, CDFA Pierce’s Disease Board, San Diego, CA, p. 233, 2004. 120. Dandekar, A. M. et al., In planta testing of signal peptides and anti-microbial proteins for rapid clearance of Xylella, in Pierce’s Disease Research Symposium, CDFA Pierce’s Disease Board, Sacramento, CA, p. 117, 2009. 121. Kunkel, M. et al., Rapid clearance of bacteria and their toxins: Development of therapeutic proteins, Crit. Rev. Immunol., 27, 233, 2007. 122. Schubert, T. S. et al., Meeting the challenge of eradicating citrus canker in Florida-again, Plant Dis., 85, 340, 2001. 123. Brunings, A. M. and Gabriel, D. W., Xanthomonas citri: Breaking the surface, Mol. Plant Pathol., 4, 141, 2003. 124. Cernadas, R. A., Camillo, L. R., and Benedetti, C. E., Transcriptional analysis of  the sweet orange interaction with the citrus canker pathogens Xanthomonas axonopodis pv. citri and Xanthomonas axonopodis pv. aurantifolii, Mol. Plant Pathol., 9, 609, 2008. 125. Cernadas, R. A. and Benedetti, C. E., Role of auxin and gibberellin in citrus canker development and in the transcriptional control of cell-wall remodeling genes modulated by Xanthomonas axonopodis pv. Citri, Plant Sci., 177, 190, 2009. 126. Bespalhok Filho, J. C. et al., Transformacao de laranja visando resisteñcia ao cancro cítrico usando genes de peptídeos antibacterianos, Biotec. Cien. Desenv., 23, 62, 2001. 127. Boscariol, R. L. et al., Attacin a gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis ‘Hamlin’, J. Am. Soc. Hort. Sci., 131, 530, 2006. 128. Dutt, M. et al., Resistance to bacterial spot can be correlated with resistance to canker in transgenic citrus, Phytopathology, 99, 30, 2009. 129. Omar, A. A. and Grosser, J. W., Protoplast co-transformation and regeneration of Â�transgenic ‘Hamlin’ sweet orange plants containing a cDNA xa21 Xanthomonas resistance gene and GFP, Acta Hort., 738–235, 2007. 130. Barbosa-Mendes, J. M. et al., Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker, Sci. Hort., 122, 109, 2009. 131. Yang, Y. and Gabriel, D.-W., Xanthomonas avirulence/pathogenicity gene family encodes functional plant nuclear targeting signals, Mol. Plant Microbe Interact., 8, 627, 1995. 132. Duan, Y.-P. et al., Expression of a single, host specific, bacterial pathogenicity gene in plant cells elicits division, enlargement, and cell death, Mol. Plant Microbe Interact., 12, 556, 1999. 133. Powell, W. A. et al., Fungal and bacterial resistance in transgenic trees, in Tree Transgenesis, eds. Fladung, M. and Ewald, D., Springer-Verlag, Berlin, Germany, 2006, 235. 134. Maliepaard, C. et al., Aligning male and female linkage maps of apple (Malus pumila mill.) using multi-allelic markers, Theoret. Appl. Genet., 97, 60, 1998. 135. Hemmat, M. et al., Molecular markers for the scab resistance (Vf) region in apple, J. Am. Soc. Hort. Sci., 123, 992, 1998. 136. Durel, C. E. et al., Genetic dissection of partial resistance to race 6 of Venturia inaequalis in apple, Genome, 46, 224, 2003.

26

Transgenic Horticultural Crops: Challenges and Opportunities

137. Guerin, F. and Le Cam, B., Breakdown of the scab resistance gene Vf in apple leads to a founder effect in populations of the fungal pathogen Venturia inaequalis, Phytopathology, 94, 364, 2004. 138. Vinatzer, B. A. et al., Apple contains receptor-like genes homologous to the Cladosporium fulvum resistance gene family of tomato with a cluster of genes cosegregating with Vf apple scab resistance, Mol. Plant–Microbe Interact., 14, 508, 2001. 139. Xu, M. L. and Korban, S. S., A cluster of four receptor-like genes resides in the Vf locus that confers resistance to apple scab disease, Genetics, 162, 1995, 2002. 140. Belfanti, E. et al., The HcrVf2 gene from a wild apple confers scab resistance to a transgenic cultivated variety, Proc. Natl. Acad. Sci., 101, 886, 2004. 141. Malnoy, M. et al., Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease, Mol. Plant–Microbe Interact., 21, 448, 2008. 142. Szankowski, I. et al., Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa), Plant Cell Rep., 22, 141, 2003. 143. De Cubber, K. et al., Progress in genetic transformation as a tool for increased disease resistance in apple, Acta Hort., 525, 309, 2000. 144. Faize, M. et al., Expression of wheat puroindoline-b reduces scab susceptibility in Â�transgenic apple (Malus x domestica Borkh.), Plant Sci., 167, 347, 2004. 145. Mehlenbacher, S. A., Classical and molecular approaches to breeding fruit and nut crops for disease resistance, HortScience, 30, 466, 1995. 146. Faize, M. et al., Chitinases of Trichoderma atroviride induce scab resistance and some metabolic changes in two cultivars of apple, Phytopathology, 93, 1496, 2003. 147. Bolar, J. P. et al., Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (t. Harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants, Transgenic Res., 10, 533, 2001. 148. Bolar, J. P. et al., Expression of an exochitinase gene from Trichoderma harzianum in transgenic apple lines, Phytopathology, 88, 8, 1998. 149. Bolar, J. P. et al., Expression of fungal chitinolytic enzymes in transgenic apples confers high levels of resistance to scab, Curr. Plant Sci. Biotechnol. Agric., 36, 465, 1999. 150. Bolar, J. P. et al., Endochitinase-transgenic McIntosh apple lines have increased resistance to scab, Phytopathology, 87, 10, 1997. 151. Bolar, J. P. et al., Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor, Phytopathology, 90, 72, 2000. 152. Liebhard, R. et al., Mapping quantitative field resistance against apple scab in a ‘Fiesta’ x ‘Discovery’ progeny, Phytopathology, 93, 493, 2003. 153. Gygax, M. et al., Molecular markers linked to the apple scab resistance gene Vbj derived from Malus baccata jackii, Theoret. Appl. Genet., 109, 1702, 2004. 154. Kars, I. et al., Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris, Plant J., 43, 213, 2005. 155. Powell, A. L. T. et al., Transgenic expression of pear PGIP in tomato limits fungal colonization, Mol. Plant–Microbe Interact., 13, 942, 2000. 156. Flors, V. et al., Absence of the endo-beta-1,4-glucanases cel1 and cel2 reduces susceptibility to Botrytis cinerea in tomato, Plant J., 52, 1027, 2007. 157. Cantu, D. et al., Strangers in the matrix: Plant cell walls and pathogen susceptibility, Trends Plant Sci., 13, 610, 2008. 158. Vellicce, G. R. et al., Enhanced resistance to Botrytis cinerea mediated by the transgenic expression of the chitinase gene ch5B in strawberry, Transgenic Res., 15, 57, 2006. 159. Schestibratov, K. A. and Dolgov, S. V., Transgenic strawberry plants expressing a thaumatin II gene demonstrate enhanced resistance to Botrytis cinerea, Sci. Hort., 106, 177, 2005.

Transgenic Fruit and Nut Tree Crops Review

27

160. Osorio, S. et al., Partial demethylation of oligogalacturonides by pectin methyl Â�esterase 1 is required for eliciting defense responses in wild strawberry (Fragaria vesca), Plant J., 54, 43, 2008. 161. Erwin, D. C. and Ribeiro, O. K., Phytophthora diseases worldwide, J. Agric. Sci., 131, 245, 1998. 162. Graham, J. H. and Menge, J. A., Phytophthora-induced diseases, in Compendium of Citrus Diseases, eds. Timmer, L.W., Garnsey, S.M., and Graham, J.H., APS Press, St. Paul, MN, p. 12, 2000. 163. Fagoaga, C. et al., Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5, Mol. Breed., 7, 175, 2001. 164. Vidal, J. et al., Evaluation of transgenic Chardonnay (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew, Transgenic Res., 15, 69, 2006. 165. Yamamoto, T. et al., Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens, Plant Cell Rep., 19, 639, 2000. 166. Dandekar, A. M. et al., Engineering for apple and walnut resistance to codling moth, in Brighton Crop Protection Conference: Pests and Diseases, Vol. 3, Brighton, England, p. 741, 1992. 167. Dandekar, A. M. et al., Low-levels of expression of wild-type Bacillus-thuringiensis var kurstaki cryIA(c) sequences in transgenic walnut somatic embryos, Plant Sci., 96, 151, 1994. 168. Escobar, M. A. et al., Using GFP as a scorable marker in walnut somatic embryo transformation, Ann. Bot., 85, 831, 2000. 169. Leslie, C. A. et al., Genetic engineering of walnut (Juglans regia L.), Acta Hort., 442, 33, 1997. 170. Hofmann, C. et al., Binding of the delta endotoxin from Bacillus-thuriengiensis to brushborder membrane vesicles of cabbage butterfly (Pieris brassicae), Eur. J. Biochem., 173, 85, 1988. 171. Van Rie, J. et al., Mechanism of insect resistance to the microbial insecticide Bacillusthuringiensis, Science, 247, 72, 1990. 172. Schnepf, E. et al., Bacillus thuringiensis and its pesticidal crystal proteins, Microbiol. Mol. Biol. Rev., 62, 775, 1998. 173. Knowles, B. H. and Ellar, D. J., Colloid-osmotic lysis is a general feature of the mechanism of action of Bacillus-thuringiensis delta endotoxins with different insect specificity, Biochim. Biophys. Acta, 924, 509, 1987. 174. Hofte, H. and Whiteley, H. R., Insecticidal crystal proteins of Bacillus-thuringiensis, Microbiol. Rev., 53, 242, 1989. 175. Crickmore, N. et al., Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins, Microbiol. Mol. Biol. Rev., 62, 807, 1998. 176. Vail, P. V. et al., Response of production and postharvest walnut pests to Bacillus thuringiensis insecticidal crystal protein fragments, Biol. Control, 1, 329, 1991. 177. Dandekar, A. M. et al., High levels of expression of full-length cryIA(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos, Plant Sci., 131, 181, 1998. 178. Tao, R. et al., Engineering genetic resistance against insects in Japanese persimmon using the cryIA(c) gene of Bacillus thuringiensis, J. Am. Soc. Hort. Sci., 122, 764, 1997. 179. Eamens, A. et al., RNA silencing in plants: Yesterday, today, and tomorrow, Plant Physiol., 147, 456, 2008. 180. Wearing, C. H. et al., Tortricid pests of pome and stone fruits, Australian and New Zealand species, in Tortricid Pests, Their Biology, Natural Enemies and Control, Elsevier, Amsterdam, the Netherlands, p. 453, 1991.

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Transgenic Horticultural Crops: Challenges and Opportunities

181. Markwick, N. P. et al., Transgenic tobacco and apple plants expressing biotin-binding proteins are resistant to two cosmopolitan insect pests, potato tuber moth and light brown apple moth, respectively, Transgenic Res., 12, 671, 2003. 182. Simpson, R. M. and Newcomb, R. D., Binding of Bacillus thuringiensis delta-endotoxins cry1AC and cry1BA to a 120-kDa aminopeptidase-n of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells, Insect Biochem. Mol. Biol., 30, 1069, 2000. 183. Turner, C. T. et al., RNA interference in the light brown apple moth, Epiphyas postvittana (walker) induced by double-stranded RNA feeding, Insect Mol. Biol., 15, 383, 2006. 184. Oeller, P. W. et al., Reversible inhibition of tomato fruit senescence by antisense RNA, Science, 254, 437, 1991. 185. Ayub, R. et al., Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits, Nat. Biotechnol., 14, 862, 1996. 186. Adams, D. O. andYang, S. F., Ethylene biosynthesis—Identification of 1-aminocyclopropane1-carboxylic acid as an intermediate in the conversion of methionine to ethylene, Proc. Natl. Acad. Sci., 76, 170, 1979. 187. Dandekar, A. M. et al., Effect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit, Transgenic Res., 13, 373, 2004. 188. Botella, J. R., Cavallaro, A. S., and Cazzonelli, C. I., Towards the production of transgenic pineapple to control flowering and ripening, Acta Hort., 529, 115, 2000. 189. Cruz-Hernandez, A., Gomez-Lim, M. A., and Litz, R. E., Transformation of mango somatic embryos, Acta Hort., 455, 292, 1997. 190. Litz, R. E. et al., Plant recovery following transformation of avocado with anti-fungal protein and SAM hydrolase genes, Acta Hort., 738, 447, 2007. 191. Gao, M. et al., Gene expression and ethylene production in transgenic pear (Pyrus communis cv. ‘La France’) with sense or antisense cDNA encoding ACC oxidase, Plant Sci., 173, 32, 2007. 192. Kobayashi, S. et al., Kiwifruits (Actinidia deliciosa) transformed with a Vitis stilbene synthase gene produce piceid (resveratrol-glucoside), Plant Cell Rep., 19, 904, 2000. 193. Costa, M. G. C., Otoni, W. C., and Moore, G. A., An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes, Plant Cell Rep., 21, 365, 2002. 194. Yunus, A. M. M. and Kadir, A. P. G., Development of transformation vectors for the production of potentially high oleate transgenic oil palm, Electron. J. Biotechnol., 11, 1, 2008. 195. Teo, G. et al., Silencing leaf sorbitol synthesis alters long-distance partitioning and apple fruit quality, Proc. Natl. Acad. Sci., 103, 18842, 2006. 196. Thipyapong, P., Stout, M. J., and Attajarusit, J., Functional analysis of polyphenol oxidases by antisense/sense technology, Molecules, 12, 1569, 2007. 197. Rommens, C. M. et al., Crop improvement through modification of the plant’s own genome, Plant Physiol., 135, 421, 2004. 198. Murata, M. et al., A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential, Biosci. Biotechnol. Biochem., 65, 383, 2001. 199. Murata, M. et al., Transgenic apple (Malus x domestica) shoot showing low browning potential, J. Agric. Food Chem., 48, 5243, 2000. 200. Ko, H. L. et al., The introduction of transgenes to control blackheart in pineapple (Ananas comosus L.) cv. Smooth Cayenne by microprojectile bombardment, Euphytica, 150, 387, 2006. 201. Ogita, S. et al., Producing decaffeinated coffee plants, Nature, 423, 823, 2003.

Transgenic Fruit and Nut Tree Crops Review

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202. Dodo, H. W. et al., Alleviating peanut allergy using genetic engineering: The silencing of the immunodominant allergen Ara H 2 leads to its significant reduction and a decrease in peanut allergenicity, Plant Biotechnol. J., 6, 135, 2008. 203. Thanavala, Y. et al., Immunogenicity of transgenic plant-derived hepatitis-b surfaceantigen, Proc. Natl. Acad. Sci., 92, 3358, 1995. 204. Kong, Q. X. et al., Oral immunization with hepatitis B surface antigen expressed in transgenic plants, Proc. Natl. Acad. Sci., 98, 11539, 2001. 205. Mor, T. S., Gomez-Lim, M. A., and Palmer, K. E., Perspective: Edible vaccines— A concept coming of age, Trends Microbiol., 6, 449, 1998. 206. Sunil-Kumar, G. B. et al., Expression of hepatitis B surface antigen in transgenic banana plants, Planta, 222, 484, 2005.

2

Transgenic Vegetables Owen Wally, J. Jayaraj, and Zamir K. Punja

CONTENTS Introduction............................................................................................................... 31 Insect Resistance....................................................................................................... 39 Nematode Resistance................................................................................................40 Pathogen Resistance..................................................................................................40 Virus Resistance...................................................................................................40 Fungal and Bacterial Resistance.......................................................................... 41 Abiotic Stress Tolerance........................................................................................... 42 Medical Applications................................................................................................ 43 Edible Vaccines.................................................................................................... 43 Therapeutic Products............................................................................................44 Nutritional Modifications..........................................................................................44 Enhanced Nutritional Quality..............................................................................44 Enhanced Taste..................................................................................................... 45 Enhanced Antioxidant Activities..........................................................................46 Fruit Ripening...................................................................................................... 47 Discussion................................................................................................................. 47 References................................................................................................................. 48

INTRODUCTION There are a range of vegetable crop species that are grown worldwide to provide humans with a source of nutrients, vitamins, and fiber. These vegetables are usually consumed fresh, e.g., carrot, cucumber, lettuce, and tomato, while others may be eaten after cooking, e.g., broccoli, cabbage, peas, and squash. Vegetable crops are usually grown under field conditions, and certain high-value crops can be grown in the greenhouse, e.g., tomato and cucumber. These crops have been bred to produce cultivars with high yield, optimal flavor and appearance, and disease and pest resistance. The tools of biotechnology have been applied to vegetable crops to achieve foreign gene insertion and expression of additional and, in some cases, novel traits. The work conducted during the period 1995–2002 on 23 vegetable crop species belonging to 9 botanical families has been summarized by Punja and Feeney.1 In this chapter, recent work over the period 2002–2008 on genetic transformation of vegetable crops for the purpose of introducing potentially useful traits is discussed. The traits that have been engineered are diverse and include insect and nematode resistance, disease resistance (to viruses, fungi, and bacteria), tolerance to abiotic

31

Cabbage, Chinese cabbage and collards

Shallot

Pathogen resistance

Carrot

Three Arabidopsis genes (MAM1, CYP79F1, CYP83A1) involved in generation of aliphatic glucosinolates

Metabolic engineering

Altered glucosinolate composition towards pharmacological important methylsulphinylalkyl glucosinolates

High levels of resistance to Turnip Mosaic Virus Decreased lesions by Xanthomonas campestris

Complete resistance toward armyworm (S. exigua)

Functional measles vaccine production

Measles polypeptides in suspension culture

Bacillus thuringiensis (BT) Cry1Ca or H04 toxin genes Anti-sense Turnip Mosaic Virus Nib gene Aspergillus niger glucose oxidase

[98]

Enhanced production of several keto-carotenoids

[101]

[24] [105]

[74] [8]

[69,70]

[30] [104] [33] [34] [51]

[31]

References

Trichoderma chitinase Pseudomonas fluorescens microbial factor 3 Barley chitinase, wheat lipid transfer protein Rice thaumatin-like protein Carrot betaine aldehyde dehydrogenase, chloroplast expression Haematococcus pluvialis β-carotene ketolase

Result Peroxidase expression enhanced resistance to foliar fungal pathogens Enhanced tolerance to fungal pathogens Enhanced tolerance to foliar fungal pathogens Lower foliar disease due to fungal pathogens Lower foliar disease symptoms to a range of fungal pathogens Increased resistance to salinity up to 400â•›mM NaCl

Wheat chitinase/glucanase, rice peroxidase

Gene Expressed

Insect resistance Pathogen resistance

Abiotic stress resistance Metabolic engineering Medical applications

Traits

Crop

TABLE 2.1 Vegetable Crop Species That Have Been Engineered to Express Specific Transgenes, and their Resulting Effects

32 Transgenic Horticultural Crops: Challenges and Opportunities

Indian mustard

Broccoli/ cauliflower

Insect resistance Nutritional modification

Insect resistance

Nutritional modification

Abiotic stress

Insect resistance

Synthetic BT Cry1Ab BT Cry1AC and Cry1C or Cry1A and Cry1C BT Cry1Ab BT Cry1Ac, Cry1C and co-expressed Cry1Ac and Cry1C Arabidopsis γ-tocopherol methyltransferase

Broccoli ACC synthase 1 and ACC oxidase, antisense expression BT Cry1ab, inducible expression

Broccoli soluble acid invertase, antisense expression

E. coli superoxide dismutase (SOD) and catalase, both constitutive and chloroplastic Brassica napus, LEA protein E. coli trehalose-6-phosphate synthase gene or hot pepper LEA protein gene Broccoli chlorophyllase, antisense expression

BT Cry1AC or Cry1C

Three Arabidopsis genes (CYP79B3, CYP79B2, CYP83B1) involved in indole glucosinolate production BT Cry1ab protein, chloroplast expression

[112]

Shift from γ-tocopherol pools to the more active vitamin E (α-tocopherol)

(continued)

[12] [13,111] [14] [15]

[110]

[108,109]

[107]

[106]

[45] [47]

[59,58]

[10]

[9]

[102]

Enhanced resistance to P. xylostella Reduced spread of P. xylostella Inhibited growth of P. xylostella Resistance to P. xylostella and BT-resistant moth varieties

Collards expressing either gene were highly resistant to P. xylostella Co-expression of both genes enhanced tolerance to SO2 and other reactive oxygen species (ROS) Enhanced resistance to salt and drought stresses Resistance to low levels of NaCl (200â•›mM) and moderate levels of heat stress (45°C) Slowing of chlorophyll breakdown and 1–2 day delay in postharvest yellowing Higher levels of sugars and protein postharvest and slowed floret yellowing Decreased level of postharvest ethylene and delayed chlorophyll loss by 2 days Protection to P. xylostella for up to 8 weeks after induction

High levels of resistance to Plutella xylostella

Altered glucosinolate accumulation, driving accumulation of indole glucosinolates

Transgenic Vegetables 33

Lettuce/ spinach

Cucumber

Pea/bean

Crop

Abiotic stress

Disease resistance

Nutritional modification

Flammulina sp. oxalate decarboxylase gene Arabidopsis ABF3, ABA responsive gene transcription factor B. napus LEA

Coat protein (CP) gene of lettuce big-vein associated virus (LBVaV) in sense or antisense orientation

Thaumatococcus daniellii thaumatin II

[37] [119] [48]

Increased tolerance to salt and drought stress

[20]

[118]

[117]

[115,116]

[114] [29]

[6]

Sweet tasting phenotype, with minor resistance to Pseudoperonospora cubensis Sense CP provided resistance to LBVaV, while anti-sense CP expression gave resistance to LBVaV and mirafiori lettuce virus Reduced Sclerotinia sclerotiorum disease symptoms Higher tolerance to both drought and cold stresses

Enhanced resistance to chilling stress and increased freezing tolerance Higher accumulation of SOD

Wild potato dehydrins

Cassava superoxide dismutase, fruit expression

Increased resistance to Mexican bean weevil High resistance towards B. cinerea

[25]

Increased number of symptomless plants compared to nontransformed Improved resistance to both NaCl and drought stresses Higher levels of free tryptophan accumulation and total tryptophan levels Delayed larval development of the pea weevil

[46] [113]

[26]

References

High resistance towards BGMV

Result

Common bean α-amylase inhibitor 2 Rice class I chitinase

B. napus LEA Modified feedback insensitive rice anthranilate synthase Bean alpha amylase inhibitor 1

Abiotic stress Nutritional modification Insect resistance

Disease resistance Abiotic stress

Bean golden mosaic virus (BGMV) AC1 gene, RNAi expression Mutated rep gene of BGMV

Gene Expressed

Disease resistance

Traits

TABLE 2.1 (continued) Vegetable Crop Species That Have Been Engineered to Express Specific Transgenes, and their Resulting Effects

34 Transgenic Horticultural Crops: Challenges and Opportunities

Sweet potato

Nutritional modifications

Medical applications

Nutritional modification

RNAi of granule-bound starch synthase I

Measles hemagglutinin protein

Rabies virus glycoprotein

Synthetic E. coli enterotoxin B (sLTB), for adjuvant for co-administered antigens Synthetic cholera toxin B subunit (sCTB) HIV-1 Tat protein

Cholera toxin B-proinsulin fusion protein

Chicken α-interferon (ChIFN)

Arabidopsis homogentisate phytyltransferase (hpt) or tocopherol cyclase (TC) Parthenocissus henryana stilbene synthase Taste modifying miraculin from Richadella dulcifica Arabidopsis gamma-tocopherol methyltransferase

E. coli asparagine synthetase A

Arabidopsis R2R3-MYB transcription factor

E. coli asparagine synthetase A

High levels of sCTB, potentially for an edible vaccine Spinach was orally consumed by mice and were primed upon further direct DNA vaccination for high titre production of Tat antibodies Spinach was orally consumed and delivered Rabies virus protection to mice and human volunteers Lettuce orally consumed by mice resulted in high level of measle antibody production Removed over 90% of the amylase in starch

Increased production of resveratrol Enhanced sweetness and taste Altered pool of γ-tocopherol to produce more active α-tocopherols Recombinant ChIFN was correctly folded and active in leaves, with high levels of antiviral activity High levels of proinsulin, and reduced symptoms of pancreatic insulitis in mice Biologically active sLTB were formed in the leaves

Increased asparagine, aspartate and glutamine in addition to enhanced vegetative growth Inhibited production of anthocyanin pigments and less red coloration Decreased sucrose, glucose and fructose and increased inulin levels Elevated vitamin E levels

(continued)

[87]

[75]

[73]

[71] [72]

[82]

[83]

[80]

[122] [89] [93]

[94]

[92]

[121]

[120]

Transgenic Vegetables 35

Tomato

Crop

Abiotic stress

Medical applications Disease resistance

Traits

Resistance to R. solanacearum and E. carotovora High levels of the GLU-AFP resulted in enhanced resistance to Ralstonia solanacearum Enhanced tolerance to Phytophthora capsici Resistance to Fusarium oxysporum f. sp. Lycopersici High level of resistance to TLCV

Sweet pepper ferredoxin I (PFLP) Tomato β-1,3 glucanase (GLU), alfalfa defensin gene (AFP) and bivalent GLU-AFP Pepper basic PR-1 and ascorbate peroxidase-like 1 Tobacco osmotin and bean chitinase Tomato leaf curl virus (TLCV) replicase 1,anti-sense expression RNAi for TLCV rep1 and rep4

Tomato omega-3 fatty acid desaturase Chili pepper ankyrin repeat domain zinc finger

Tomato hydroxycinnamoyl CoA quinate transferase

Mirabilis jalapa antimicrobial peptide and maize glucanase Tomato spotted wilt virus (TSWV) nucleoprotein TLCV coat protein (CP) Arabidopsis thionin (thi2.1), fruit inactive expression

Reduction in foliar lesions by B. cinerea and S. sclerotiorum

Wheat oxalate oxidase

Complete resistance in the field with no outcrossing Symptomless when inoculated with TLCV after 15 days Leaves and roots were significantly more resistant to Fusarium and bacterial wilt Increased phenolic levels and increased tolerance toward UV radiation Resistance to chilling stress at low irradiance Decreased accumulation of free oxygen radicals and enhanced resistance to salinity and oxidative stress

Resistance to TLCV was observed, the addition of the second RNAi prevented evolution of resistance Enhanced resistance to Alternaria solani

High levels of viable potentially therapeutic protein

Result

Human lactoferrin

Gene Expressed

TABLE 2.1 (continued) Vegetable Crop Species That Have Been Engineered to Express Specific Transgenes, and their Resulting Effects

[61] [62]

[60]

[27,28] [19] [125]

[124]

[23]

[35] [32] [22]

[39] [38]

[36]

[123]

References

36 Transgenic Horticultural Crops: Challenges and Opportunities

Nutritional modification

Ocimum basilicum geraniol synthase, ripeningspecific expression Synthetic mammalian GTP cyclohydrolase, fruit-specific expression Bacterial lycopene beta-cyclase (LBc), plastid expression RNAi of endogenous photomorphogenesis regulatory gene, detiolated 1 (DET1), fruit-specific expression Arabidopsis 3-hydroxymethylglutaryl CoA and bacterial 1-deoxy-d-xylulose-5-phosphate synthase RNA silencing of polygalacturonase (PG) and expansin fruit-specific expression T. daniellii thaumatin

Atriplex hortensis betaine aldehyde dehydrogenase Soybean isoflavone synthase Mouse metallothioneins (MT-1) Richadella dulcifica miraculin

E. coli catalase, foliar expression

Poplar boiling stable protein A (bspA) Arthrobacter globiformis choline oxidase

Tomato glycerol-3-phosphate acyltransferase gene

[95] [103] [129] [88]

Significantly firmer fruit throughout ripening and reduced susceptibility to deterioration in long-term storage Fruit noticeably sweeter

(continued)

[96]

Reduction in DET1 activity, with both carotenoid and flavonoid contents increased significantly Elevated total phytosterols and phytoene and β-carotene

Converted high levels of lycopene to β-carotene

[84] [85] [97]

[91]

[49] [127] [128] [90]

[57]

[126] [50]

[63]

Heightened accumulation of pteridine and folates

Increased levels of cis-unsaturated fatty acids in the thylakoid membrane, increasing tolerance toward chilling Increase in water stress tolerance Accumulation of glycine betaine and increase in chilling tolerance Enhanced resistance to the herbicide paraquat and increased tolerance to chilling and drought stresses Enhanced tolerance to moderate levels of NaCl High level of isoflavone accumulation in the fruit skin Elevated levels of Zn and SOD High levels of miraculin accumulation, increasing tomato sweetness Altered terpenoid pool and enhanced flavor

Transgenic Vegetables 37

Insect resistance

Vaccine/ medical

Traits

Hepatitis B virus large surface antigen, fruit-specific expression Human coagulation Factor IX, fruit-specific expression Hepatitis B surface antigen Partial ORF2 of Hepatitis E virus Potato polyphenol oxidase Bacillus thuringiensis Cry6A Poplar chitinase, leaf expression Potato PI-II and carboxypeptidase inhibitors, proteinase inhibitors, leaf-specific expression

HIV-1 Tat protein, fruit-specific expression

Human beta-amyloid

Modified human alpha-1-antitrypsin

Human interleukin-12 (IL12)

Gene Expressed

Note: Gene expression was constitutive unless otherwise stated.

Crop

[81] [67] [68] [2] [16] [3] [5]

High levels of functional protein Immune-primed mice when fruit was orally consumed Produced immunoactive Hepatitis E protein Increased resistance to common cutworms Increased resistance to root knot nematode Delayed development of Colorado potato beetle larvae Increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae

[65]

[131]

[130]

[66]

[79]

References

Reduced bacterial load when fed to mice infected with mycobacteria Accumulation of high levels of biologically active, therapeutic protein Accumulation of high levels of the protein, when fed to mice exhibited an immune response High levels of protein accumulation, that produced an immune response in mice when consumed High levels of protein accumulation in mature fruits

Result

TABLE 2.1 (continued) Vegetable Crop Species That Have Been Engineered to Express Specific Transgenes, and their Resulting Effects

38 Transgenic Horticultural Crops: Challenges and Opportunities

Transgenic Vegetables

39

stresses (salinity and drought), production of edible vaccines and therapeutic products, nutritional enhancement, and metabolic engineering. The published results from these studies show some very interesting trends toward better stress tolerance, pest and disease resistance, and more nutritious vegetable crops (Table 2.1). For the most part, these transgenic vegetable crop species have been evaluated under laboratory and greenhouse conditions, with limited field trials. Several studies, however, have evaluated these materials in trials with animal systems for the assessment of vaccines and therapeutics. Collectively, these studies clearly show the potential of transgenic vegetable crops with beneficial traits, and they are discussed in more detail below.

INSECT RESISTANCE Resistance of plants to insects is a much desired attribute, potentially reducing the number of insecticide sprays required. The following are examples of transgenic strategies used to reduce insect damage on vegetable crops.





1. Manipulation of polyphenol oxidase (PPO) activity could provide resistance simultaneously to both diseases and insect pests.2 Transgenic tomato plants constitutively expressing potato PPO gene were tested for insect resistance. These transgenic plants exhibited up to nearly sixfold higher PPO activity levels compared to controls.2 The PPO transgenic plants clearly showed an increase in resistance; simple growth rates of common cutworms on these plants were up to three times lower than on controls and larvae consumed less foliage. In addition, increased PPO activity led to higher larval mortality.2 These results suggest a critical role for PPO-mediated phenolic oxidation in resistance to this insect.2 2. Insect cytoskeletons and digestive systems contain large amounts of chitin, which can be broken down by certain plant chitinases. Transgenic tomato seedlings expressing a poplar chitinase gene were tested for resistance toward the Colorado potato beetle, which can cause severe losses in tomato and other crops.3 The plants expressing the poplar chitinase gene reduced the larval development allowing approximately 50% to reach the second larval instar, as compared to over 90% in the control plants,3 suggesting the possibility of using a plant chitinase gene for promoting insect resistance.3 3. Plant proteinase inhibitors (PIs) have been well established to play a potent defensive role against predators and pathogens (reviewed by Mosolov and Valueva4). The defensive capacities of plant PIs rely on inhibition of proteases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development. Unlike other insecticidal proteins, for PIs to be active, very high levels of recombinant PI protein are required to achieve pest resistance.4 Tissue specific or inducible expression would yield higher levels at the required site and time. Leaf-specific overexpression of the potato protease inhibitor and carboxypeptidase inhibitors in tomato resulted in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in high-expressing

40



Transgenic Horticultural Crops: Challenges and Opportunities

homozygous transgenic lines compared to controls.5 Seeds of transgenic peas expressing a α-amylase inhibitor reduced pea weevil (Bruchus pisorum L.) survival by nearly 100%, with the larval mortality occurring at an early instar. Conversely, in nontransgenic cultivars, essentially 100% of the pea weevils emerged as adults.6 4. Bacillus thuringiensis (Bt) crystalline toxin genes (cry) have been extensively used to develop transgenic insect resistant crop plants over the past two decades. Several reports are available on different synthetic Bt genes expressed both individually and in a combinatorial way and their efficiency in pest control (reviewed in Christou et al.7). A cry1Ca or a H04 hybrid gene was expressed in leaf tissues of beet and shallot.8 When either of these genes were expressed at high levels in the leaves, it resulted in nearly complete resistance against beet armyworm in shallot or beet.8 Similarly, transgenic garlic plants expressing cry1Ca were completely resistant to beet armyworm in a number of in vitro bioassays.8 There are several other successful examples of increased insect resistance in a variety of transgenic vegetables using Bt cry genes alone or in combination. These include cabbage,9 collards,10 broccoli,11–13 and cauliflower.14 The success of Bt in controlling insects has been very promising, with the only major concern being the development of pest resistance to particular Cry proteins. The use of trap plants and pyramiding different Cry proteins at high levels through chloroplast expression can help alleviate the potential for future pest resistance.13,15

NEMATODE RESISTANCE Some of the Bt toxin genes also have known nematicidal activity. Expression of nematicidal Cry6A in transgenic tomato plants provided protection against plant-parasitic nematodes.16 When Cry6A-expressing roots were challenged with Meloidogyne incognita, the nematode was able to ingest the toxin, which resulted in a significant decrease in gall production by up to fourfold. This report suggests that Bt protein can confer resistance to endoparasitic nematodes, and therefore Bt toxin proteins are potential candidates for developing nematode-resistant transgenic plants.17

PATHOGEN RESISTANCE Virus Resistance The earliest successful development of virus-resistant transgenic vegetable crops utilized the viral coat protein-mediated resistance (CPMR) strategy to engineer pathogen resistance.18 The use of CPMR continues to be one of the most useful mechanisms for viral resistance, having been used for tomato leaf curl virus resistance in tomato19 and big vein-associated virus resistance in lettuce.20 CPMR requires expression of the viral coat (or capsid) protein gene from the virus in transgenic plants. When exposed to the viral inoculum, the transgenic plants displayed reduced number of

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lesions, reduced rate of systemic disease development, and very low levels of virus accumulation compared to control plants. In some instances, the transgenic plant was protected not only against the virus from which the coat protein gene was derived but also against other serologically unrelated viruses (reviewed in Prins et al.18). Despite the widespread use of CPMR, it is believed that multiple molecular mechanisms of resistance exist, which are not fully understood.21 Currently, CPMR is registered for use in transgenic squash and zucchini, with the potential for many new vegetable crops in the future. Expression of viral RNA-dependant RNA-polymerases (replicase) using antisense or sense constructs has also been effective at limiting infection and spread of RNA viruses. Replicase-mediated resistance appears to be strain-specific and functions through lowering the overall level of viral replicases, thus reducing the viral concentration. Replicase-mediated viral resistance has been successfully used in tomato to increase resistance to tomato leaf curl virus.22,23 Plant DNA viruses do not encode polymerases; instead, they require an interaction between viral replication-associated protein (rep) and the host plant’s DNA polymerase. Expression of mutated viral rep gene or rep-gene silencing through antisense technologies can reduce viral titer and has been used to improve viral resistance in cabbage24 and bean.25,26 Other mechanisms of viral resistance include expression of viral nucleoproteins, which serve to disrupt normal movement of viral proteins and nucleic acid packing leading to resistance, reducing viral symptoms. Expression of nucleoproteins has been used in tomato to reduce symptoms in response to tomato-spotted wilt virus.27,28

Fungal and Bacterial Resistance Enhanced resistance to fungi and bacteria has been demonstrated using a number of different strategies for several vegetable crop species (Table 2.1). These include carrot, cucumber, lettuce, and tomato. The genes and corresponding enzymes Â�produced include hydrolytic enzymes (chitinases and glucanases), peroxidase, Â�pathogenesis-related (PR) proteins (thaumatin), and peptides. Expression of chitinases or β-1,3-glucanases, which are PR-proteins that can hydrolyze the major structural polysaccharides in the fungal cell wall (chitin and laminarin), provided varying levels of resistance to Botrytis cinerea in cucumber29 and to a number of fungal pathogens infecting carrot.30,31 Chitinase expression has been achieved in combination with other genes including osmotin in tomato32 to reduce Fusarium wilt and with lipid-transfer protein in carrot,33 which resulted in reduced fungal infection. Expression of another PR-protein, thaumatin, also enhanced fungal disease resistance in carrot.34 Peroxidase expression reduced Botrytis and Sclerotinia infection of carrot leaves31 and, when expressed in combination with PR-1, reduced Phytophthora development in tomato.35 A secondary approach has been to engineer expression of enzymes that degrade pathogen virulence products, e.g., oxalic acid oxidase or decarboxylase to break down oxalic acid. For example, expression of a wheat oxalate oxidase gene in tomato reduced the development of Botrytis and Sclerotinia36 while an oxalate decarboxylase gene from the fungus Flammulina also led to reduced Sclerotinia development

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Transgenic Horticultural Crops: Challenges and Opportunities

in transgenic lettuce.37 Both of these genes exhibited the ability to degrade oxalic acid, an important pathogenicity factor for this fungus.36,37 For bacterial resistance, gene products such as peroxidase, ferredoxin, and defensins reduced disease development on tomato (Table 2.1) caused by several pathogens. In tomato, bacterial wilt development was reduced through combined expression of a β-1,3-glucanase and defensin gene38 while expression of a sweet pepper ferredoxin gene also reduced the development of bacterial blight.39

ABIOTIC STRESS TOLERANCE Abiotic stresses, including salinity, temperature, and water stresses, are among the most limiting stresses, lowering crop yield and quality worldwide. Growing areas that experience extreme salt and water stress typically grow limited amounts of vegetable crops, which can lead to local human nutritional deficiencies. Transgenic vegetable crops capable of growing on saline soil or with minimum irrigation in these areas could help stabilize food and nutritional production in these regions.40 Osmotic adjustment at the cellular level is a vital response in dealing with water, salinity, and freezing stresses. Natural plant defenses to these stresses include the synthesis and accumulation of plant compatible solutes, including glycine betaine, sugars, proline, and compatible proteins (late-embryogenesis abundant protein [LEA], dehydrins, and heat shock proteins) (reviewed in Cheong and Yun41). Compatible osmolytes protect the plant through osmotic adjustment, which helps maintain turgor, scavenging of reactive oxygen species (ROS), and the stabilization of protein structure.40 The ability to regulate osmotic potential using compatible solutes has increased yield under drought conditions in many crop plants.42,43 There are reports of transgenic vegetables with increased production of compatible solutes, resulting in increased resistance to abiotic stresses (Table 2.1). Two of the more commonly used strategies in transgenic vegetables are the overexpression of LEA genes and overproduction of glycine betaine. Normally, LEA genes are expressed during the late stages of seed development; however, many LEA genes are also expressed in vegetative tissue when the plants undergo an abiotic stress.44 Many of the LEA proteins have been shown to confer tolerance to drought, salinity, and freezing stresses.45 Overexpression of heterologous LEA proteins has been successful in increasing abiotic stress resistance in kidney bean,46 Chinese cabbage,45,47 and lettuce.48 Glycine betaine is naturally produced in many plant species, and high accumulation levels are associated with drought and salt tolerance. Tomato plants do not produce endogenous glycine betaine; therefore, plants were transformed to express heterologous betaine aldehyde dehydrogenase (BADH)49 or choline oxidase.50 These tomato plants were able to withstand moderately high levels of salinity and produced viable fruit and seeds. Carrot accumulates low levels of glycine betaine naturally, but chloroplastic overexpression of endogenous BADH resulted in enhanced glycine betaine levels.51 These transgenic carrots were able to grow on soil containing physiologically very high salt levels. Another strategy used by drought and salinity-resistant plants is to compartmentalize the abundant but toxic Na+ ions to utilize them for osmotic adjustment

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by transporting the ions into the vacuole using tonoplastic Na+/H+ antiporters.52 Transgenic tomato plants, overexpressing the Arabidopsis Na+/H+ antiporter (Atnhx) gene, were able to grow and produce fruit at relatively high levels of Na+, without accumulating excess Na+ in the fruit.52 The Atnhx gene has also been overexpressed in sugar beet, which became more resistant to drought stress, in addition to increasing the natural level of sucrose in the taproot.53,54 Abiotic stresses disrupt normal plant metabolism by affecting reaction rate, protein stability, and membrane integrity. This alteration in metabolism leads to an increase in accumulation of ROS to potentially toxic levels (reviewed in Ashraf 55). Detoxification of ROS occurs naturally to some extent in all plant species through the production of detoxifying enzymes and antioxidants. 56 The recently employed strategies for detoxifying ROS in vegetable crops include the overexpression of the detoxifying enzymes superoxide dismutase and catalase in tomato57 and Chinese cabbage, 58,59 resulting in enhanced tolerance to abiotic stresses. Additionally, more complex metabolic engineering to increase the production of antioxidants has been made to enhance resistance toward abiotic stresses in tomato.60–63

MEDICAL APPLICATIONS Edible Vaccines Transgenic vegetables offer an attractive option for vaccine production, since nearly any antigenic protein can be expressed in plants at a large scale, and the crops can be grown locally.64 Vaccines can also be produced in edible parts of plants, which aids in delivery. Edible plant-based vaccines can be made against an entire protein or just the desired antigenic peptides from viruses, parasites, or bacterial pathogens.64 Development of edible vaccines has been primarily directed to pathogens, which infect the host via the mucosal surfaces lining the digestive, respiratory, and urinoreproductive tracts of the body, since these vaccines need to be absorbed in a similar fashion. Much of the research on edible vaccines has focused on tomato fruit, carrot taproots, and leafy greens,65–73 since they can be consumed raw, eliminating the possibility of degradation of the vaccine during the cooking process. Expression of these polypeptides has been mainly with constitutive promoters while some research in tomatoes used fruit-specific E8 promoter.65,66 In recent years, successful Â�vegetable-based edible vaccines have been produced against common viruses such as measles,69,70,74,75 rabies,73 and hepatitis B,66,68 common bacterial diseases such as cholera,71 and HIV,65,72,76 which are extremely difficult to treat. While there are many benefits associated with the production of vegetable-based edible vaccines, there are a number of limitations to this technology. One of the major limitations is obtaining adequate protein concentrations that initiate sufficient immune response to result in complete immunity. Additionally timing of doses and application of multiple doses is crucial and will need to be addressed before these edible vaccines are used for larger scale applications. Despite the promise in alleviating important global diseases, there are currently no commercial transgenic vegetable plants producing edible vaccines.

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Transgenic Horticultural Crops: Challenges and Opportunities

Therapeutic Products A range of other therapeutic agents have been derived from transgenic plants, including potato, rice, wheat, and corn.77 Recently, there has been increasing focus on the use of vegetable crops to produce these therapeutic products, since they can be consumed raw (Table 2.1). The biopharmaceuticals produced to date in vegetables include enzymes, interleukins, and antimicrobial peptides for humans and animals as well as blood-clotting peptides. These therapeutic products were previously or are currently produced in animal, yeast, or bacterial-cell cultures. Plant-based products have the potential for significantly lower costs and reduced potential for contamination.78 Oral consumption of transgenic tomato fruit containing human interleukin-12 resulted in a dramatic reduction in the symptoms of tuberculosis in mice.79 The production of transgenic lettuce expressing chicken interferon has indicated the possibility of using transgenic forage crops for avian feed and potentially for other livestock.80 For the most part, the amount of pharmaceutical proteins produced in the transgenic vegetables would be quite low (less than 1% of total soluble protein). This level of expression is insufficient for compounds that are not highly active, such as blood coagulation factors.81 These proteins would either need to be extracted and further purified, or an alternative expression system would need to be employed. A limitation of using edible biopharmaceuticals is the low absorption of the active agent through the mucosal lining, without excessive degradation. To increase the absorption, the use of nontoxic bacterial adjuvants has been investigated.82 Potentially, very promising results have been seen in lettuce expressing proinsulin fusion protein with a bacterial adjuvant in the chloroplast.83 The fusion allowed for easier uptake of the insulin and alleviated the symptoms of pancreatic insulitis in mice, a condition similar to type I diabetes.

NUTRITIONAL MODIFICATIONS Enhanced Nutritional Quality Vegetable crops are a major nutritional source of many essential human Â�nutrients, including vitamins, carotenoids, and flavonoids, as well as other important Â�nutrients. However, not all vegetables are rich in all these essential nutrients. Folate or Â�vitamin B9 is an essential cofactor for the synthesis of many amino acids and purines. Humans cannot synthesize folates that must be supplemented through the diet, which is mainly from plant sources. Leafy greens contain high natural levels of folate; however, many of the worldwide food staples (grains and tubers) contain inadequate levels of folate, which can lead to widespread deficiencies.84 Folate deficiency can lead to neural tube defects and other human diseases and is a global health problem. Because plants are major folate sources for humans, there is always scope to enhance plant folate levels (biofortification). Plants synthesize folate from pteridine, p-aminobenzoic acid (PABA), and glutamate moieties. A synthetic mammalian GTP cyclohydrolase1 was expressed in tomato in a fruit-specific manner.84 As a result, the fruit pteridine and folate increased relative to controls.84 PABA pools were depleted in engineered fruit that were higher in folate, and supplying such fruit with PABA further increased their

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folate content.84 When transgenic PABA- and pteridine-overproduction traits were combined by crossing, vine-ripened fruit accumulated up to 25-fold more folate than controls.85 Transferring the folate biosynthetic branch developed in tomatoes to staple food plants such as sweet potato, could potentially increase the folate intake in poor countries with limited access to fresh vegetables. Modification of starch composition of tubers enables them to be used for diverse industrial applications. Amylose and amylopectin are the two principal polysaccharide components of most natural starches.86 In sweet potato plants, the range of amylose content (10%–20%) is narrow in comparison with other crops, thereby making them less amenable for industrial uses. Since the ratio of amylase to amylopectin is an important textural property of starch, development of a plant type that contains amylose-free or low amylose would envisage new industrial applications.86,87 Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato granule-bound starch synthase I (GBSSI) gene expression through RNA interference.86,87 GBSSI is one of the key enzymes catalyzing the formation of amylose, a linear alpha (1,4)d-glucan polymer, from ADP-glucose. Due to silencing of GBSSI gene, over 70% of the regenerated transgenic plant roots contained amylopectin but not amylose. These reports suggest that RNA interference is an effective method for manipulating gene expression in the starch metabolic pathway.86,87

Enhanced Taste Fruit taste is a complex genetic trait governed by multiple genes and gene interactions. Although taste improvement in food crops has been classically achieved by careful selection and hybridization for centuries, it is one of the traits that still cannot be directly manipulated by conventional plant-breeding methods but may be possible through genetic engineering. Taste-modifying proteins are alternative sweeteners and flavor enhancers, and manipulation of their expression may enhance the nutritional quality and consumer preference. Thaumatin is a sweet-tasting, flavor-enhancing protein present in the fruits of Thaumatococcus daniellii, an African shrub.88 Transgenic tomato lines expressing recombinant-thaumatin protein in fruits were sweeter than the controls, which was confirmed by organoleptic evaluation.88 Miraculin is a tastemodifying protein present in the red berries of Richadella dulcifica. Miraculin by itself is not sweet; it converts the sour taste of ascorbic acid, acetic acid, and citric acid into a sweet taste after being held in the mouth for some time. This unique property has led to increasing interest in this protein for expression in other plants. Miraculin was constitutively expressed in lettuce, and, as a result, the transgenic leaves accumulated significant levels of miraculin, which eventually attributed sweetness-inducing activity.89,90 Similarly, transgenic tomato plants expressing recombinant miraculin protein accumulated high levels in both leaves and fruits. The purified recombinant miraculin protein from transgenic tomato plants showed strong sweetness-inducing activity, which was similar to that of native miraculin.90 Similar to taste, flavor and aroma are traits that are often rarely available within existing germplasm and hence have to be imported from related or alien gene pool. Ocimum basilicum geraniol synthase gene was expressed in tomatoes in a fruit-� specific manner using the tomato ripening-specific polygalacturonase (PG) promoter.

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Transgenic Horticultural Crops: Challenges and Opportunities

Various monoterpenes that contribute to fruity and floral scents were found to accumulate in the transgenic tomatoes, which eventually enhanced the flavor and aroma of the flesh.91 Inulins are beneficial for human health and are produced naturally in some plants as storage carbohydrates. They serve as replacements for high caloric fats and sugars, since inulins have a sweet taste and are not absorbed through the digestive tract. Transgenic lettuce lines expressing the asparagine synthetase A gene from Escherichia coli accumulated lowered amounts of sucrose, glucose, and fructose, whereas inulin content increased up to 30 times in transgenic leaves.92 These lettuce genotypes with high content of foliar inulin represent useful materials for breeding strategies and a potential source for low-molecular-weight inulin useful for dietary supplements, in addition to the potential for enhanced taste.

Enhanced Antioxidant Activities Enhancement of antioxidant content is an important trait in the light of potential health benefits imparted by these chemicals in plants and animals. α-Tocopherol is the most biologically active form of vitamin E, which is implicated in decreasing the risk of several types of cancers, coronary heart disease, and a number of Â�degenerative human conditions. Manipulating the tocopherol biosynthetic pathway in plants to convert tocopherols into more active α-tocopherol form could have significant health benefits.93 An Arabidopsis γ-tocopherol methyltransferase (γ-TMT) gene was overexpressed in lettuce to improve the tocopherol composition. This resulted in higher TMT activity and the conversion of the γ-tocopherol pool to α-tocopherol in Â�transgenic plants.93 In another attempt, in order to increase tocopherol content by increasing total flux to the tocopherol biosynthetic pathway, two different Arabidopsis genes were constitutively expressed in lettuce. Transgenic plants expressing either of these genes had total tocopherol content increased by more than twofold mainly due to an increase in γ-tocopherol.94 Increased isoprenoid levels, the precursors to many important nutrients, can be achieved in tomato through genetic manipulation of mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), using 3-hydroxymethylglutaryl CoA (hmgr-1) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) genes, respectively. Transgenic tomatoes expressing the Arabidopsis hmgr-1 gene had double the phytosterol levels.95 Additionally, tomatoes expressing a bacterial dxs gene targeted to the plastids resulted in significantly increased carotenoid content, which was attributed to enhanced levels of phytoene and β-carotene.95 In order to increase the carotenoid and flavonoid content in tomato fruits, an endogenous phytomorphogenesis regulatory gene (DET1) was suppressed by RNAi in a fruit-specific manner.96 The resulting transgenic fruits accumulated higher levels of carotenoids and flavonoids compared to the controls.96 Plastid expression of a bacterial lycopene-β-cyclase gene in tomato resulted in the conversion of the main storage carotenoid–lycopene, to β-carotene, leading to a fourfold increase in provitamin A content of the fruits.97 Although β-carotene (provitamin A), α-carotene, and lutein are widely prevalent in many fruits and vegetables, ketocarotenoids, such as canthaxanthin and

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astaxanthin, are rarely present in plants. Ketocarotenoids are strong antioxidants and are chemically synthesized and used as dietary supplements and as pigments in the aquaculture and neutraceutical industries. Ketocarotenoid biosynthetic pathway was engineered in carrot tissues by introducing a β-carotene ketolase gene isolated from the red alga Haematococcus pluvialis.98 In the transgenic carrot taproots, up to 70% of total carotenoids were converted to novel ketocarotenoids, with accumulation up to 2.4â•›mg/g root dry weight. Astaxanthin, adonirubin, and canthaxanthin were most prevalent, followed by echinenone, adonixanthin, and β-cryptoxanthin, and all these ketocarotenoids have neutraceutical importance.98 Glucosinolates are an important group of secondary metabolites found in almost all the members of the family Brassicaceae. Although glucosinolates are well known for their toxic effects in both humans and animals at high doses, at subtoxic doses, their hydrolytic and metabolic products act as chemoprotective agents against chemically induced carcinogens and thereby block initiation of a variety of tumors in addition to having a wide range of bioactivities.99 Sulforaphane, a metabolite of methioninederived 4-methylsulfinylbutyl glucosinolate (glucoraphanin), has attracted attention because of its potential anticarcinogenic activity.100 Glucoraphanin is an aliphatic glucosinolate abundant in broccoli and Arabidopsis ecotype Columbia, but nonexistent in Chinese cabbage. Three Arabidopsis genes, required for aliphatic glucosinolate biosynthesis, were introduced into Chinese cabbage.101 The resulting transgenic lines accumulated significantly higher levels of the aliphatic glucosinolates, gluconapin, and glucobrassicanapin.101,102

Fruit Ripening Although ripening is a process, which renders the fruits to become more edible, the ripening-phenomenon itself can be either beneficial or detrimental to both freshness and processing quality of fruits and vegetables. For instance, during ripening of tomato, cell wall disassembly is closely associated with loss of fresh fruit firmness and subsequently with a loss of viscosity in the processed products. In tomato, PG and expansin (Exp) are among the cell wall proteins that cooperatively participate in ripening-associated cell wall disassembly. Transgenic suppression of either LePG or LeExp1 expression alone resulted in altered softening and shelf-life characteristics of fruits.103 Suppression of LeExp1 or LePG alone did not significantly increase fruit firmness. However, fruits suppressed for both LePG and LeExp1 expression were significantly firmer throughout the ripening phase and were less susceptible to deterioration during long-term storage. The viscosity of juice prepared from the transgenic tomato fruit with reduced LePG and LeExp1 expression was higher than that of controls.103

DISCUSSION Although genetically engineered tomato (Flavr Savr) with enhanced fruit-quality characteristics was the first example of a transgenic vegetable crop to be marketed in the United States and United Kingdom in the 1990s, most transgenic vegetable crop species have not reached the market as rapidly as, for example, transgenic field

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Transgenic Horticultural Crops: Challenges and Opportunities

crops that include canola, corn, potato, and soybean. Transgenic squash and zucchini with virus resistance followed tomato as the next vegetable crop in the U.S. market. This chapter has summarized many other potentially beneficial traits that have been engineered into vegetable crop species. Some of these traits can be of direct benefit to the grower, e.g., insect or herbicide resistance, while others would have benefits to the consumer, e.g., enhanced vitamins, flavor, and quality. While there appears to be continuing interest in the applications of biotechnology to vegetable crop species, several requirements still must be met to ensure success in transformation. A well defined and reproducible tissue-culture system is needed, together with an efficient transformation method as well as an appropriate selection system to distinguish transformed cells from non-transformed cells. While an increasing number of vegetable crop species are becoming amenable to transformation technologies, there are still some limitations. It appears that only a few techniques are available to introduce foreign genes into vegetable crop species, with the most popular being Agrobacterium-mediated transformation. Optimization of gene transfer methods, transformation efficiencies, and tissue-culture procedures still remain a challenge for some vegetable species. The most amenable species appear to be tomato and carrot. There are also other issues that need to be addressed before widespread utility of transgenic vegetable crop species occurs. The traits engineered into the respective crops need to be evaluated under replicated field conditions over several years to ensure the introduced genes confer the desired phenotype. Furthermore, the horticultural attributes in the transgenic lines should be similar to those in the existing non-transgenic cultivar. These lines should demonstrate a clear advantage (to the grower, the consumer) compared to that currently existing in the crop species germplasm. While these criteria have been met with the currently approved transgenic corn, canola, soybean, and potato cultivars, they still require additional effort for the transgenic lines of vegetable crops under study. Consumer acceptance of vegetable crops that contain transgenes will require that the same stringent criteria used to evaluate other transgenic food plants be met. Perhaps more so than transgenic field crops, transgenic vegetable crops that are consumed fresh may face additional consumer concerns that will require that evidence of their benefits be made clear. In the case of vitamin-enhanced tomatoes or high-antioxidant carrots, these could provide a strong example of the potential benefits of the transgenic produce. The other issues regarding use of marker genes, spread of the transgenes through pollen, potential for resistance selection in the case of insect or fungal pests, and allergenicity of the introduced protein are all similar to those required for other transgenic crops and need to be satisfied.

REFERENCES

1. Punja, Z. K. and Feeney, M., Progress in vegetable crop transformation and future prospects and challenges, in Genetically Modified Crops, eds. Liang, G. H. and Skinner, D. Z., Haworth Press, New York, 2004, Chap. 12. 2. Mahanil, S. et al., Overexpression of tomato polyphenol oxidase increases resistance to common cutworm, Plant Sci., 174, 456, 2008. 3. Lawrence, S. D. and Novak, N. G., Expression of poplar chitinase in tomato leads to inhibition of development in Colorado potato beetle, Biotechnol. Lett., 28, 593, 2006.

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4. Mosolov, V. V. and Valueva, T. A., Proteinase inhibitors in plant biotechnology: A review, Appl. Biochem. Microbiol., 44, 233, 2008. 5. Abdeen, A. et al., Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors, Plant Mol. Biol., 57, 189, 2005. 6. Sousa-Majer, M. J. et al., Bean alpha-amylase inhibitors in transgenic peas inhibit development of pea weevil larvae, J. Econ. Entomol., 100, 1416, 2007. 7. Christou, P. et al., Recent developments and future prospects in insect pest control in transgenic crops, Trends Plant Sci., 11, 302, 2006. 8. Zheng, S. J. et al., Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hubner) in transgenic Bt-shallots (Allium cepa L.), Transgenic Res., 14, 261, 2005. 9. Liu, C. W. et al., Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella, Theor. Appl. Genet., 117, 829, 2008. 10. Cao, J., Shelton, A. M., and Earle, E. D., Development of transgenic collards (Brassica oleracea L., var. acephala) expressing a cry1Ac or cry1C Bt gene for control of the diamondback moth, Crop. Prot., 24, 804, 2005. 11. Chen, M. et al., Impact of single-gene and dual-gene Bt broccoli on the herbivore Pieris rapae (Lepidoptera: Pieridae) and its pupal endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae), Transgenic Res., 17, 545, 2008. 12. Viswakarma, N. et al., Insect resistance of transgenic broccoli (‘Pusa Broccoli KTS-1’) expressing a synthetic cryIA(b) gene, J. Hort. Sci. Biotechnol., 79, 182, 2004. 13. Cao, J. et al., Broccoli plants with pyramided cry1Ac and cry1C Bt genes control diamondback moths resistant to Cry1A and Cry1C proteins, Theor. Appl. Genet., 105, 258, 2002. 14. Chakrabarty, R. et al., Agrobacterium-mediated transformation of cauliflower: Optimization of protocol and development of Bt-transgenic cauliflower, J. Biosci., 27, 495, 2002. 15. Cao, J., Shelton, A. M., and Earle, E. D., Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (Brassica juncea L.) and its potential for control of diamondback moth larvae, Plant Cell Rep., 27, 479, 2008. 16. Li, X. Q. et al., Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein, Plant Biotechnol. J., 5, 455, 2007. 17. Li, X. Q. et al., Expression of Cry5B protein from Bacillus thuringiensis in plant roots confers resistance to root-knot nematode, Biol. Control, 47, 97, 2008. 18. Prins, M. et al., Strategies for antiviral resistance in transgenic plants, Mol. Plant Pathol., 9, 73, 2008. 19. Raj, S. K. et al., Agrobacterium-mediated tomato transformation and regeneration of transgenic lines expressing Tomato leaf curl virus coat protein gene for resistance against TLCV infection, Curr. Sci., 88, 1674, 2005. 20. Kawazu, Y. et al., A transgenic lettuce line with resistance to both lettuce big-vein Â�associated virus and mirafiori lettuce virus, J. Am. Soc. Hort. Sci., 131, 760, 2006. 21. Bendahmane, M. et al., Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein, Virology, 366, 107, 2007. 22. Praveen, S. et al., Engineering tomato for resistance to tomato leaf curl disease using viral rep gene sequences, Plant Cell Tissue Organ Cult., 83, 311, 2005. 23. Ramesh, S. V., Mishra, A. K., and Praveen, S., Hairpin RNA-mediated strategies for silencing of Tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants, Oligonucleotides, 17, 251, 2007. 24. Yu, Z. D., Zhao, S. Y., and He, Q. W., High level resistance to Turnip mosaic virus in Chinese cabbage (Brassica campestris ssp pekinensis (Lour) Olsson) transformed with the antisense NIb gene using marker-free Agrobacterium tumefaciens infiltration, Plant Sci., 172, 920, 2007.

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25. Faria, J. C. et al., Partial resistance to Bean golden mosaic virus in a transgenic common bean (Phaseolus vulgar L.) line expressing a mutated rep gene, Plant Sci., 171, 565, 2006. 26. Bonfim, K. et al., RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris), Mol. Plant–Microbe Interact., 20, 717, 2007. 27. Nervo, G. et al., Characterization of two tomato lines highly resistant to tomato spotted wilt virus following transformation with the viral nucleoprotein gene, J. Plant Pathol., 85, 139, 2003. 28. Accotto, G. P. et al., Field evaluation of tomato hybrids engineered with tomato spotted wilt virus sequences for virus resistance, agronomic performance, and pollen-mediated transgene flow, Phytopathology, 95, 800, 2005. 29. Kishimoto, K. et al., Detailed analysis of rice chitinase gene expression in transgenic cucumber plants showing different levels of disease resistance to gray mold (Botrytis cinerea), Plant Sci., 162, 655, 2002. 30. Baranski, R., Klocke, E., and Nothnagel, T., Chitinase CHIT36 from Trichoderma harzianum enhances resistance of transgenic carrot to fungal pathogens, J. Phytopathol., 156, 513, 2008. 31. Wally, O., Jayaraj, J., and Punja, Z. K., Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, B-1,3-glucanase and peroxidase, Eur. J. Plant Pathol., 123, 331, 2009. 32. Ouyang, B. et al., Transformation of tomatoes with osmotin and chitinase genes and their resistance to Fusarium wilt, J. Hort. Sci. Biotechnol., 80, 517, 2005. 33. Jayaraj, J. and Punja, Z. K., Combined expression of chitinase and lipid transfer protein genes in transgenic carrot plants enhances resistance to foliar fungal pathogens, Plant Cell Rep., 26, 1539, 2007. 34. Punja, Z. K., Transgenic carrots expressing a thaumatin-like protein display enhanced resistance to several fungal pathogens, Can. J. Plant Pathol., 27, 291, 2005. 35. Sarowar, S. et al., Constitutive expression of two pathogenesis-related genes in tomato plants enhanced resistance to oomycete pathogen Phytophthora capsici, Plant Cell Tissue Organ Cult., 86, 7, 2006. 36. Walz, A. et al., Expression of an oxalate oxidase gene in tomato and severity of disease caused by Botrytis cinerea and Sclerotinia sclerotiorum, Plant Pathol., 57, 453, 2008. 37. Dias, B. B. A. et al., Expression of an oxalate decarboxylase gene from Flammulina sp. in transgenic lettuce (Lactuca sativa) plants and resistance to Sclerotinia sclerotiorum, Plant Pathol., 55, 187, 2006. 38. Chen, S. C., Liu, A. R., and Zou, Z. R., Overexpression of glucanase gene and defensin gene in transgenic tomato enhances resistance to Ralstonia solanacearum, Russ. J. Plant Physiol., 53, 671, 2006. 39. Huang, H. E. et al., Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein, Phytopathology, 97, 900, 2007. 40. Bohnert, H. J. and Jensen, R. G., Strategies for engineering water-stress tolerance in plants, Trends Biotechnol., 14, 89, 1996. 41. Cheong, M. S. and Yun, D. J., Salt-stress signaling, J. Plant. Biol., 50, 148, 2007. 42. Wang, W. X., Vinocur, B., and Altman, A., Plant responses to drought, salinity and extreme temperatures: Towards genetic engineering for stress tolerance, Planta, 218, 1, 2003. 43. Zhang, J. X., Nguyen, H. T., and Blum, A., Genetic analysis of osmotic adjustment in crop plants, J. Exp. Bot., 50, 291, 1999. 44. Bray, E. A., Bailey-Serres, J., and Weretilnyk, E., Responses to abiotic stresses, in Biochemistry and Molecular Biology of Plants, eds. Buchanan, B., Gruissem, W., and Jones, R., American Society of Plant Physiologists, Rockville, MD, 2000, Chap. 22.

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45. Park, B. J. et al., Genetic improvement of Chinese cabbage for salt and drought tolerance by constitutive expression of a B. napus LEA gene, Plant Sci., 169, 553, 2005. 46. Liu, Z. C. et al., The novel use of a combination of sonication and vacuum infiltration in Agrobacterium-mediated transformation of kidney bean (Phaseolus vulgaris L.) with lea gene, Mol. Breed., 16, 189, 2005. 47. Park, S. H. et al., A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (Brassica campestris) overexpressing CaLEA or otsA, J. Plant. Biol., 46, 277, 2003. 48. Park, B. J. et al., Increased tolerance to salt- and water-deficit stress in transgenic lettuce (Lactuca sativa L.) by constitutive expression of LEA, Plant Growth Regul., 45, 165, 2005. 49. Jia, G. X. et al., Transformation of tomato with the BADH gene from Atriplex improves salt tolerance, Plant Cell Rep., 21, 141, 2002. 50. Park, E. J. et al., Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage, Plant J., 40, 474, 2004. 51. Kumar, S., Dhingra, A., and Daniell, H., Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance, Plant Physiol., 136, 2843, 2004. 52. Zhang, H. X. and Blumwald, E., Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit, Nat. Biotechnol., 19, 765, 2001. 53. Yang, A. F. et al., Efficient transformation of beet (Beta vulgaris) and production of plants with improved salt-tolerance, Plant Cell Tissue Organ Cult., 83, 259, 2005. 54. Liu, H. et al., Transgenic salt-tolerant sugar beet (Beta vulgaris L.) constitutively expressing an Arabidopsis thaliana vacuolar Na+/H+ antiporter gene, AtNHX3, accumulates more soluble sugar but less salt in storage roots, Plant Cell Environ., 31, 1325, 2008. 55. Ashraf, M., Biotechnological approach of improving plant salt tolerance using antioxidants as markers, Biotechnol. Adv., 27, 84, 2009. 56. Zhu, J. K., Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol., 53, 247, 2002. 57. Mohamed, E. A. et al., Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance, Plant Cell Environ., 26, 2037, 2003. 58. Tseng, M. J., Liu, C. W., and Yiu, J. C., Tolerance to sulfur dioxide in transgenic Chinese cabbage transformed with both the superoxide dismutase containing manganese and catalase genes of Escherichia coli, Sci. Hort., 115, 101, 2008. 59. Tseng, M. J., Liu, C. W., and Yiu, J. C., Enhanced tolerance to sulfur dioxide and salt stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts, Plant Physiol. Biochem., 45, 822, 2007. 60. Cle, C. et al., Modulation of chlorogenic acid biosynthesis in consequences for phenolic accumulation and UV-tolerance, Phytochemistry, 69, 2149, 2008. 61. Liu, X. Y. et al., Overexpression of tomato chloroplast omega-3 fatty acid desaturase gene alleviates the photoinhibition of photosystems 2 and 1 under chilling stress, Photosynthetica, 46, 185, 2008. 62. Seong, E. S. et al., Tomato plants overexpressing CaKR1 enhanced tolerance to salt and oxidative stress, Biochem. Biophys. Res. Commun., 363, 983, 2007. 63. Sui, N. et al., Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato, Planta, 226, 1097, 2007. 64. Mason, H. S. et al., Edible plant vaccines: Applications for prophylactic and therapeutic molecular medicine, Trends Mol. Med., 8, 324, 2002. 65. Ramirez, Y. J. P. et al., Fruit-specific expression of the human immunodeficiency virus type 1 Tat gene in tomato plants and its immunogenic potential in mice, Clin. Vac. Immunol., 14, 685, 2007. 66. Lou, X. M. et al., Expression of the human hepatitis B virus large surface antigen gene in transgenic tomato plants, Clin. Vac. Immunol., 14, 464, 2007.

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Transgenic Horticultural Crops: Challenges and Opportunities

67. Gao, Y. et al., Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg, World J. Gastroenterol., 9, 996, 2003. 68. Ma, Y. et al., Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products, World J. Gastroenterol., 9, 2211, 2003. 69. Bouche, F. B. et al., Neutralizing immunogenicity of a polyepitope antigen expressed in a transgenic food plant: A novel antigen to protect against measles, Vaccine, 21, 2065, 2003. 70. Marquet-Blouin, E. et al., Neutralizing immunogenicity of transgenic carrot (Daucus carota L.)-derived measles virus hemagglutinin, Plant Mol. Biol., 51, 459, 2003. 71. Kim, Y. S. et al., Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system, Plant Cell Tissue Organ Cult., 87, 203, 2006. 72. Karasev, A. V. et al., Plant based HIV-1 vaccine candidate: Tat protein produced in spinach, Vaccine, 23, 1875, 2005. 73. Yusibov, V. et al., Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine, Vaccine, 20, 3155, 2002. 74. Bouche, F. B. et al., Induction of broadly neutralizing antibodies against measles virus mutants using a polyepitope vaccine strategy, Vaccine, 23, 2074, 2005. 75. Webster, D. E. et al., Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralizing antibodies in mice following mucosal vaccination, Vaccine, 24, 3538, 2006. 76. Zhou, F. et al., High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes, Plant Biotechnol. J., 6, 897, 2008. 77. Goldstein, D. A. and Thomas, J. A., Biopharmaceuticals derived from genetically modified plants, QJM. Int. J. Med., 97, 705, 2004. 78. Pujol, M. et al., Fighting cancer with plant-expressed pharmaceuticals, Trends Biotechnol., 25, 455, 2007. 79. Elias-Lopez, A. L. et al., Transgenic tomato expressing interleukin-12 has a therapeutic effect in a murine model of progressive pulmonary tuberculosis, Clin. Exp. Immunol., 154, 123, 2008. 80. Song, L. et al., Transient expression of chicken alpha interferon gene in lettuce, J. Zhejiang Univ., 9, 351, 2008. 81. Zhang, H. et al., Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum), Biotechnol. Appl. Biochem., 48, 101, 2007. 82. Kim, T. G. et al., Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa), Protein Expr. Purif., 51, 22, 2007. 83. Ruhlman, T. et al., Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts—Oral administration protects against development of insulitis in non-obese diabetic mice, Plant Biotechnol. J., 5, 495, 2007. 84. de la Garza, R. D. et al., Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis, Proc. Natl. Acad. Sci. USA, 101, 13720, 2004. 85. de la Garza, R. I. D., Gregory, J. F., and Hanson, A. D., Folate biofortification of tomato fruit, Proc. Natl. Acad. Sci. USA, 104, 4218, 2007. 86. Noda, T. et al., Physicochemical properties of amylose-free starch from transgenic sweet potato, Carbohydr. Polym., 49, 253, 2002. 87. Otani, M. et al., Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants, Plant Cell Rep., 26, 1801, 2007. 88. Bartoszewski, G. et al., Modification of tomato taste in transgenic plants carrying a thaumatin gene from Thaumatococcus daniellii Benth, Plant Breed., 122, 347, 2003. 89. Sun, H. J. et al., Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce, FEBS Lett., 580, 620, 2006.

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90. Sun, H. J. et al., Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants, Plant Biotechnol. J., 5, 768, 2007. 91. Davidovich-Rikanati, R. et al., Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway, Nat. Biotechnol., 25, 899, 2007. 92. Sobolev, A. P. et al., Strong increase of foliar inulin occurs in transgenic lettuce plants (Lactuca sativa L.) overexpressing the Asparagine Synthetase A gene from Escherichia coli, J. Agric. Food Chem., 55, 10827, 2007. 93. Cho, E. A. et al., Expression of gamma-tocopherol methyltransferase transgene improves tocopherol composition in lettuce (Lactuca sativa L.), Mol. Cells, 19, 16, 2005. 94. Lee, K. et al., Overexpression of Arabidopsis homogentisate phytyltransferase or tocopherol cyclase elevates vitamin E content by increasing gamma-tocopherol level in lettuce (Lactuca sativa L.), Mol. Cells, 24, 301, 2007. 95. Enfissi, E. M. A. et al., Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato, Plant Biotechnol. J., 3, 17, 2005. 96. Davuluri, G. R. et al., Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes, Nat. Biotechnol., 23, 890, 2005. 97. Wurbs, D., Ruf, S., and Bock, R., Contained metabolic engineering in tomatoes by  expression of carotenoid biosynthesis genes from the plastid genome, Plant J., 49, 276, 2007. 98. Jayaraj, J., Devlin, R., and Punja, Z., Metabolic engineering of novel ketocarotenoid production in carrot plants, Transgenic Res., 17, 489, 2008. 99. Keck, A. S. and Finley, J. W., Cruciferous vegetables: Cancer protective mechanisms of glucosinolate hydrolysis products and selenium, Integr. Cancer Ther., 3, 5, 2004. 100. Fahey, J. W. et al., Sulforaphane inhibits extracellular, intracellular, and antibioticresistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors, Proc. Natl. Acad. Sci. USA, 99, 7610, 2002. 101. Zang, Y. X. et al., Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1, BMB Rep., 41, 472, 2008. 102. Zang, Y. X. et al., Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1, Mol. Cells, 25, 231, 2008. 103. Powell, A. L. T. et al., Simultaneous transgenic suppression of LePG and LeExp1 influences fruit texture and juice viscosity in a fresh market tomato variety, J. Agric. Food Chem., 51, 7450, 2003. 104. Baranski, R., Klocke, E., and Nothnagel, T., Enhancing resistance of transgenic carrot to fungal pathogens by the expression of Pseudomonas fluorescence microbial factor 3 (MF3) gene, Physiol. Mol. Plant Pathol., 71, 88, 2007. 105. Lee, Y. H. et al., Enhanced disease resistance in transgenic cabbage and tobacco expressing a glucose oxidase gene from Aspergillus niger, Plant Cell Rep., 20, 857, 2002. 106. Chen, L. F. O. et al., Transgenic broccoli (Brassica oleracea var. italicia) with antisense chlorophyllase (BoCLH1) delays postharvest yellowing, Plant Sci., 174, 25, 2008. 107. Eason, J. R. et al., Suppressing expression of a soluble acid invertase (BoINV2) in broccoli (Brassica oleracea) delays postharvest floret senescence and downregulates cysteine protease (BoCP5) transcription, Physiol. Plant., 130, 46, 2007. 108. Higgins, J. D. et al., The production of marker-free genetically engineered broccoli with sense and antisense ACC synthase 1 and ACC oxidases 1 and 2 to extend shelf-life, Mol. Breed., 17, 7, 2006. 109. Gapper, N. E. et al., Senescence-associated down-regulation of 1-aminocyclopropane-1carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli, Funct. Plant Biol., 32, 891, 2005.

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Transgenic Horticultural Crops: Challenges and Opportunities

110. Cao, J. et al., Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants, Plant Cell Rep., 25, 554, 2006. 111. Zhao, J. Z. et al., Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution, Nat. Biotechnol., 21, 1493, 2003. 112. Yusuf, M. A. and Sarin, N. B., Antioxidant value addition in human diets: Genetic transformation of Brassica juncea with gamma-TMT gene for increased alpha-tocopherol content, Transgenic Res., 16, 109, 2007. 113. Hanafy, M. S. et al., Accumulation of free tryptophan in azuki bean (Vigna angularis) induced by expression of a gene (OASA1D) for a modified at-subunit of rice anthranilate synthase, Plant Sci., 171, 670, 2006. 114. Nishizawa, K. et al., Assessment of the importance of alpha-amylase inhibitor-2 in bruchid resistance of wild common bean, Theor. Appl. Genet., 114, 755, 2007. 115. Yin, Z. M. et al., Transcriptional expression of a Solanum sogarandinum pGT: Dhn10 gene fusion in cucumber, and its correlation with chilling tolerance in transgenic seedlings, Cell. Mol. Biol. Lett., 9, 891, 2004. 116. Yin, Z. M. et al., Expression of a Solanum sogarandinum SK3-type dehydrin enhances cold tolerance in transgenic cucumber seedlings, Plant Sci., 170, 1164, 2006. 117. Lee, H. S. et al., Transgenic cucumber fruits that produce elevated level of an anti-aging superoxide dismutase, Mol. Breed., 11, 213, 2003. 118. Szwacka, M. et al., Variable properties of transgenic cucumber plants containing the thaumatin II gene from Thaumatococcus daniellii, Acta Physiol. Plant., 24, 173, 2002. 119. Vanjildorj, E. et al., Overexpression of Arabidopsis ABF3 gene enhances tolerance to drought and cold in transgenic lettuce (Lactuca sativa), Plant Cell Tissue Organ Cult., 83, 41, 2005. 120. Giannino, D. et al., The overexpression of asparagine synthetase A from E-coli affects the nitrogen status in leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth, Euphytica, 162, 11, 2008. 121. Park, J. S. et al., Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa), Plant Cell Rep., 27, 985, 2008. 122. Liu, S. J. et al., High content of resveratrol in lettuce transformed with a stilbene synthase gene of Parthenocissus henryana, J. Agric. Food Chem., 54, 8082, 2006. 123. Min, S. R. et al., Production of human lactoferrin in transgenic cell suspension cultures of sweet potato, Biol. Plant., 50, 131, 2006. 124. Schaefer, S. C. et al., Enhanced resistance to early blight in transgenic tomato lines expressing heterologous plant defense genes, Planta, 222, 858, 2005. 125. Chan, Y. L. et al., Transgenic tomato plants expressing an Arabidopsis thionin (Thi2.1) driven by fruit-inactive promoter battle against phytopathogenic attack, Planta, 221, 386, 2005. 126. Roy, R. et al., Transformation of tomato cultivar ‘Pusa Ruby’ with bspA gene from Populus tremula for drought tolerance, Plant Cell Tissue Organ Cult., 84, 55, 2006. 127. Shih, C. H. et al., Accumulation of isoflavone genistin in transgenic tomato plants overexpressing a soybean isoflavone synthase gene, J. Agric. Food Chem., 56, 5655, 2008. 128. Sheng, J. P. et al., Improving zinc content and antioxidant activity in transgenic tomato plants with expression of mouse metallothionein-I by mt-I gene, J. Agric. Food Chem., 55, 9846, 2007. 129. Brummell, D. A. et al., Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansin, Postharvest Biol. Tech., 25, 209, 2002. 130. Agarwal, S. et al., Expression of modified gene encoding functional human alpha-1antitrypsin protein in transgenic tomato plants, Transgenic Res., 17, 881, 2008. 131. Youm, J. W. et al., Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer’s disease, Biotechnol. Lett., 30, 1839, 2008.

3

Transgenic Ornamental Crops Beverly A. Underwood and David G. Clark

CONTENTS Introduction............................................................................................................... 55 Flower Color............................................................................................................. 56 Flavonoids............................................................................................................ 56 Chalcones and Aurones................................................................................... 57 Anthocyanins................................................................................................... 58 Carotenoids..........................................................................................................60 Plant Volatiles............................................................................................................ 62 Floral Fragrance................................................................................................... 62 Fragrance and Color........................................................................................ 65 Volatiles and Insect Plant Defense....................................................................... 65 Flower and Leaf Longevity.......................................................................................66 Floral Longevity...................................................................................................66 Altering Ethylene Responses Genetically by Inhibiting Ethylene Biosynthesis..................................................................................... 67 Inhibiting Ethylene Responses by Disrupting Ethylene Signal Transduction....... 68 Leaf Longevity..................................................................................................... 69 Plant Architecture...................................................................................................... 70 Plant Height.......................................................................................................... 71 Flower Morphology............................................................................................. 71 Fruit and Flower Size........................................................................................... 72 Agrobacterium rhizogenes rol Genes................................................................... 72 Biotic Stress Tolerance.............................................................................................. 73 Current and Future Issues in Ornamental Biotechnology......................................... 74 Acknowledgments..................................................................................................... 75 References................................................................................................................. 75

INTRODUCTION Ornamental crops can be classified as floriculture and nursery plants, shrubs, trees, and foliage plants for outdoor and indoor use. Ornamental crops are produced with the purpose of beautifying, decorating, or enhancing the environment, and exclude plants intended for commercial food production such as vegetables and fruits.1

55

56

Transgenic Horticultural Crops: Challenges and Opportunities

Production of ornamental crops is a global industry, having a multibillion dollar value worldwide driving the development and introduction of novel ornamental plants solely for human use. Novel ornamental plants have been developed by traditional breeding techniques, including hybridization and mutation breeding, and more recently by introducing precise, direct genetic changes through genetic engineering. There are major potential benefits to genetic engineering including the opportunity for the introduction of genes and traits not present in the natural gene pool of the species of interest without altering the genetic background. A variety of consumer and producer traits have been successfully modified in ornamental plants through genetic engineering, including flower color, fragrance, plant architecture, cut flower vase life, disease resistance, abiotic stress, and herbicide tolerance.2,3 This chapter provides an overview of the traits modified by transgenic technology, examines the benefits, and discusses issues that have limited the commercialization of transgenic ornamental crops over the past decade.

FLOWER COLOR Flower color and color pattern are key elements in consumer selection of flowering ornamentals. It has been of considerable interest to develop commercial series of plants with a full spectrum of flower colors. Producing a series of plants having flowers with the full color palette through traditional breeding is challenging for most species since the genetics required for producing all the color compounds is usually limiting. Through identification and introduction of color producing enzymes and pathways from other plants, new colors can be introduced into varieties that would otherwise be difficult to obtain (Table 3.1). Flavonoids, carotenoids, chlorophyll, and betalins are the primary classes of compounds that give flowers their characteristic color. Flavonoids are widely distributed in many plant families, conferring shades of red, orange, yellow, blue, and violet. Carotenoids are derived from isoprenoids and give rise to orange and yellow shades, marigolds being the classic example. Betalins are water-soluble nitrogen-containing compounds derived from tyrosine and are taxonomically restricted to the Caryophyllales.4 Biosynthesis of these compounds has been well characterized in many plant species and readers are referred to published reviews elsewhere for detailed information about their biosynthesis.4,5

Flavonoids Chalcones, aurones, anthocyanins, flavones, and flavonols are the major groups of flavonoids that impart the colorful floral display seen in the angiosperms. Novel and complex coloration patterns have been achieved primarily by manipulation of the flavonoid biosynthetic pathway including preventing or redirecting flavonoid synthesis through gene silencing, introducing biosynthetic enzymes with different substrate preferences or novel activities, and modifying expression of regulatory transcription factors controlling flavonoid biosynthetic genes (Table 3.1).

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Transgenic Ornamental Crops

TABLE 3.1 Genetically Engineered Ornamental Plants with Modified Flower Color Plant Petunia x hybrida

Dianthus caryophyllus Dendrathema grandiflora Eustoma grandiflorum Rosa hybrida

Initial Flower Color

Genetic Modification

Pale pink Purple

35S::ZmDFR Antisense CHS

Purple

Sense CHS

White Purple Pink Red-orange White Pink

35S::MtCHR

Purple Red Pink, mauve Mauve

Gerbera hybrida Lotus japonicus

Red Pale yellow

Torenia hybrida

Purple-blue Purple-blue Purple-blue Purple-blue Purple-blue

Sense CHS Antisense F3H PhF3′5′H and DFR Sense CHS Antisense CHS Antisense CHS Sense CHS 35S::VwF3′5′H RNAi DFR, 35S::IhDFR, 35S::VwF3′5′H Antisense CHS, DFR 35S::crtW Sense CHS Sense DFR Sense F3′5′H Sense F3′5′H 35S:: F3′H Coexpression of Am4′CGT + AmAS1 and RNAi DFR

Transgenic Phenotype

References

Red White, whitepurple patterning White, whitepurple patterning Pale yellow Pale purple Pale pink White Blue

[19] [148]

Pink, white, light pink White, whitepurple patterning Pale pink Bluish flowers Plants with all three genes have bluish flowers Pink Bright yellow, bright orange White White patterning Pink Dark pink

[10]

Yellow flowers

[14]

[6,7] [12] [149] [58] [16]

[8] [149] [150] [150]

[9] [29] [151] [151] [151] [152]

Chalcones and Aurones Chalcone synthesis is the first committed step in flavonoid synthesis. The chalcones serve as the substrates for anthocyanin and, in some plant species, aurone synthesis. The first committed step in flavonoid synthesis is catalyzed by chalcone synthase (CHS), which produces tetrahydroxychalcone (THC) from p-coumaroyl CoA and three molecules of malonyl-CoA (reviewed in Grotewold4). Silencing CHS expression through antisense or cosuppression techniques leads to white or complex coloration patterns in many plant species including petunia,6,7 lisianthus,8 gerbera,9 and chrysanthemums.10 THC has a pale yellow color, but does not accumulate in plants.

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Transgenic Horticultural Crops: Challenges and Opportunities

In nonlegumes, THC is quickly converted to naringenin by chalcone isomerase, which closes the central ring of THC leading to synthesis of flavanones. However, in legumes, such as Medicago truncatula and Glycine max, chalcone reductase (CHR) reduces THC to 6′-deoxychalcone.11 Transgenic petunias expressing Medicago CHR in a white flowering background accumulate novel 6′-deoxychalcones, have lower levels of flavonols, and exhibit a pale yellow corolla color while a pale purple color is observed when the transgene is expressed in a purple petunia background.12 Aurones are a class of flavonoids produced from chalcones that give flowers bright fluorescent yellow colors. They often provide nectar guides for visiting pollinators and have a more limited species distribution in plants.13,14 In Antirrhinum, the chalcone THC undergoes glucosylation and oxidative cyclization to produce aurones.15 Yellow Torenia hybrida flowers were created from a line normally producing blue flowers by silencing native dihydroflavonol 4-reductase (DFR) expression and introducing two Antirrhinum genes for aurone biosynthesis.14 By blocking anthocyanin accumulation and expressing the Antirrhinum aureusidin synthase and chalcone 4′-O-glucosyltransferase genes, aurones accumulated and were not masked by the blue malvidin-type anthocyanins normally accumulating in the Summer Wave Blue Torenia cultivar. Anthocyanins Anthocyanins give flowers red, orange, blue, and violet hues. They are synthesized in the cytosol from flavonones and accumulate in the vacuole where molecular interactions, vacuolar pH, ion cofactors, and cellular shape all contribute to the final visual effect. There are six main groups of anthocyanins: cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin. Their synthesis begins with the production of dihydroflavonols when hydroxyl groups are positioned on the flavonones by flavanone 3-hydroxylase (F3H), flavonone 3′-hydroxylase (F3′H), and flavanone 3′5′-hydroxylase (F3′5′H) (Figure 3.1). Dihydroflavonols are reduced to leucoanthocyanidins by DFR. DFR substrate preference is variable in different plant species and represents a critical entry point in color production. Lack of F3′5′H activity is associated with the absence of blue coloration in many families of flowering plants, including carnation (reviewed by Chandler and Tanaka16). Blue color has been introduced into carnation through introduction of petunia F3′5′H and DFR genes into a white flowered background lacking DFR and F3′H activity.16 Transgenic carnations expressing both of these genes accumulate the delphinidin type anthocyanins.17 These blue-violet transgenic carnations, marketed as the Moon series, were developed by Florigene Pty. Ltd. and Suntory Ltd. and are the first and only transgenic ornamental to date that has been successfully commercialized. While F3′H and F3′5′H activities are important for blue color other factors including co-pigments and coordinating metal ions are also key factors in determining blue color. Cyanidin type anthocyanins are packaged into a supermolecular complex to give blue color to flowers of blue cornflower while the same anthocyanins give red color to rose.18 The brilliant blue color in cornflower is attributed to a tetranuclear complex of cyanidin anthocyanin, a flavone, and chelating Fe3+, Mg2+, and Ca2+ ions. An understanding of the genetics contributing

59

Transgenic Ornamental Crops OH CoAS

+ 3 Malonyl-CoA O

p-Coumaroyl CoA CHS

HO

Chalcones

OH

OH OH O

Tetrahydroxychalcone OH

Flavanones

HO

O

CHI

OH

HO 7 8

OH F3’5’H 6

OH O

O

A C

O OH

F3’5’H

OH O

Dihydromyricetin

Leucoanthocyanidins

O

HO

OH OH

OH OH O

OH

OH

Delphinidin

OH O

OH HO

OH

HO

OH OH

OH

OH HO

OH

Pelargonidin

Luteoforol

LDOX/ANS

OH O

OH

O

OH

Leucocyanidin

LDOX/ANS

HO

OH

O OH OH

Leucopelargonidin

OH

DFR OH

DFR

O

HO

LDOX/ANS

Anthocyanidins

OH

F3’H

OH

OH OH

OH

OH

O

DFR

OH

O

HO

O

OH

Leucodelphinidin

HO

OH

Dihydrokaempferol Dihydroquercetin

OH

OH

F3H OH

OH OH HO

DFR OH

OH O

Eriodictyol

F3H

OH

OH

O

HO

F3’H

5 OH O

F3H

HO

5’

Naringenin

Pentahydroxyflavanone

Dihydroflavonols

OH

3’ 4’ OH

B

O OH OH

Cyanidin

OH

OH HO

OH

O OH

Luteolinidin

FIGURE 3.1â•… Key steps of anthocyanin pigment biosynthesis in plants. CHS, chalcone synthase; CHI, chalcone isomerase; F3′5′H, flavanone 3′5′-hydroxylase; F3′H, flavanone 3′-hydroxylase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX/ ANS, leucoanthocyanidin dioxygenase/anthocyanidin synthase. (Reprinted from Grotewold, E., Annu. Rev. Plant Biol., 57, 761, 2006. With permission. © 2006 by Annual Reviews www. annualreviews.org)

to the formation of this type of complex is of interest for engineering blue color in flowers but has yet to be elucidated. A novel orange color was first introduced into Petunia by incorporating a maize DFR gene into the genome of a variety lacking F3′H and F3′5′H activity.19 The background chosen for introducing the maize DFR was important as petunias lacking F3′H and F3′5′H activity accumulate the dihydroflavonol dihydrokaempferol since the Petunia DFR does not accept dihydrokaempferol as a substrate for leucoanthocyanidin synthesis. The maize DFR, which uses the substrate dihydrokaempferol, opened up a

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Transgenic Horticultural Crops: Challenges and Opportunities

pathway for orange pelargonidin anthocyanin production. However, a commercially acceptable, stable color was not obtained in the initial report.20 Commercially acceptable, stable, improved orange colors were achieved through introgression of the maize DFR into elite breeding lines accumulating cyanidin, peonidin, delphinidin, petunidin, and malvidin anthocyanins and three generations of selfing the F1 crosses.21

Carotenoids Carotenoids are plastid synthesized, lipid soluble, isoprenoid-derived C40 compounds essential to all photosynthetic organisms. Carotenoids are structurally diverse with over 700 structures identified. They function in light harvesting, energy transfer, and photoprotection. They also provide pink, red, orange, and yellow color to flowers and fruits, are precursors of plant hormones and fragrance compounds, and have significant nutritional value in animal diets (reviewed by Lu and Li22). Carotenoid synthesis is initiated by the condensation of two molecules of geranylgeranyl pyrophosphate (GPP) by phytoene synthase (PSY) to form colorless phytoene. Phytoene is converted to all trans lycopene through four desaturation reactions catalyzed by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) and an isomerization reaction catalyzed by carotenoid isomerase (CRTISO). The desaturation reactions introduce conjugated double bonds, leading to a shift in the absorption spectra that causes color to appear. Lycopene represents a branch point in the pathway, where either (1) δ-carotene, α-carotene, and ultimately lutein are produced through the action of lycopene ε-cyclase, lycopene β-cyclase, and carotenoid ring hydroxylases or (2) γ-carotene, β-carotene, and zeaxanthin are produced through the action of lycopene β-cyclase and carotenoid β-ring hydroxylases (Figure 3.2) (reviewed by Hirschberg23). Naturally occurring mutants for many of these carotenoid biosynthetic genes exist in tomato, and fruits from these lines show visible differences in color.24 For example, the tomato R mutant has yellowish-green fruit due to a mutation in the phytoene synthase gene.25 Mutants with ectopic expression of a lycopene β-cyclase have orange fruit, accumulating high levels of β-carotene and less lycopene than control plants.26 Most of the research on engineering carotenoid synthesis in transgenic plants has focused on the alteration of nutritional characteristics in food plants. For example, β-carotene accumulating “golden” rice varieties were developed by introducing daffodil or maize phytoene synthase (Psy) and a bacterial phytoene desaturase (crtl) gene into the rice genome.27,28 Although these plants may be valuable for helping solve worldwide vitamin A deficiency, they also demonstrate the ability to drive heterologous expression of yellow color in plants. By transferring bacterial genes for ketocarotenoid synthesis, deep yellow, orange, and red colors have been introduced into plants normally displaying pale yellow colors (Table 3.1). Bright orange nectaries were created in Nicotiana by transformation with the alga Haematococcus pluvialis β-carotene ketolase gene, causing accumulation of astaxanthin in nectary tissue. This application has more of a neutraceutical value since astaxanthin has many uses related to animal health. While nectaries generally do not contribute much to the overall aesthetic appeal of a plant, it demonstrates that novel carotenoids can be expressed in plants to introduce

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Transgenic Ornamental Crops

O P

GGDP

CrtL-e

HO

Lutein

Zds (crtQ) Lycopene

CrtL-b γ-Carotene

CrtL-b

CrtR-e CrtR-b

GGDP

P O

Pds (crtP)

ζ-Carotene

α-Carotene

P

Psy (crtB) Phytoene

δ-Carotene

P

β-Carotene

OH HO

Zeaxanthin

CrtL-b

CrtR-b

OH

aba2

O HO

O Violaxanthin

?

O HO

OH

OH

OH Neoxanthin

FIGURE 3.2â•… Carotenoid biosynthesis in plants. GGDP, geranylgeranyl diphosphate; Psy, phytoene synthase; Pds, phytoene desaturase; Zds, ζ-carotene desaturase; CrtL-e, lycopene ε-cyclase; CrtL-b, lycopene β-cyclase; aba2, zeaxanthin epoxidase. (From Ronen, G. et al., Plant J., 17, 341, 1999. With permission.)

new  colors. Indeed, bright yellow and orange Lotus flowers were observed in transgenic plants transformed with the Agrobacterium aurantiacum β-carotene ketolase (crtW) gene.29 The CrtW enzyme accepts β-carotene, β-cryptoxanthin, and zeaxanthin substrates for production of ketocarotenoids, which are compounds found in bacteria, fungi, algae, and only a few plant species. Transgenic lotus plants expressing crtW had altered carotenoid profiles with orange lines exhibiting a 1.5fold increase in total carotenoids and ketocarotenoids making up about one-quarter of the total carotenoid content. Flower color range can also be extended through modification of carotenoid breakdown pathways (Table 3.1). Oxidative breakdown of carotenoids is catalyzed by a family of carotenoid cleavage dioxygenases (CCDs) found in plants, animals, and bacteria. The carotenoid breakdown products, or apocarotenoids, are biologically important molecules functioning as hormones, flavor and fragrance compounds, and defense molecules (reviewed by Auldridge et al.30). There are multiple lines of evidence that support a role for CCDs in carotenoid turnover. Arabidopsis ccd1-1 loss of Â�function mutants have increased seed carotenoid content.31 Expression

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Transgenic Horticultural Crops: Challenges and Opportunities

of the carotenoid cleavage dioxygenase CCD4 was demonstrated to give chrysanthemum flowers white color instead of yellow.32 To date, this area of research is under-explored and could have considerable potential for development of new color phenotypes. Manipulating CCD expression also opens up the exciting possibilities of engineering both color and fragrance since many of the apocarotenoids are Â�important floral fragrance volatile compounds.33,34

PLANT VOLATILES Thousands of plant volatiles have been identified and are generally classified into four major groups including terpenes, phenylpropanoids, fatty acid derivatives, and amino acid derivatives (reviewed by Dudareva and Pichersky35). Only subsets of the thousands of plant volatiles identified are found in any given species, thus plant volatiles are generally thought of as secondary metabolites with their synthesis being highly intertwined with primary metabolism (reviewed by Pichersky et al.36). Plant volatile production occurs in many plant tissues including leaves, flowers, fruits, and roots with functions relating to pollinator attraction, seed dispersal, inter- and intra-plant communication, and defense. Many genes involved with plant volatile synthesis have been cloned and manipulated in transgenic plants resulting in altered volatile profiles. These studies demonstrate that plant volatile profiles can be genetically engineered and will be useful in improving floral fragrance and plant defense.

Floral Fragrance Floral fragrance has been a source of pleasure and reverence in human culture where fragrant flowers have had places in religious ceremonies and societal customs since ancient times. Modern technology has allowed flower fragrance profiles to be chemically copied for use in cosmetics, perfumes, home fragrances, aromatherapy items, cleaning products, and detergents, to name a few popular uses. Many of the compounds found in floral fragrance are documented to have positive effects on human behavior and therefore could be used as tools for improving the health and welfare of humanity.37–39 There are minimal publicly available marketing research data that investigate the value added by making new flower varieties with enhanced fragrance. One marketing report on Anthurium suggests that about 60% of consumers agreed fragrance would add value to the product.40 Floraculture International reported in November 2008 that the demand for fragrant roses was increasing but the number of “fragrant” roses available is minimal (http://www.floracultureinternational. com/index.php?option=com_content&task=view&id=125&Itemid=159&ed=25). Fragrance has historically not been the focus of ornamental plant breeding programs and flowers have lost their characteristic fragrance since breeders are primarily concentrating on flower color and longevity, plant growth habit, disease resistance, among other genetic factors. Genetic engineering could help overcome the hurdles of reintroducing this trait into crops where there is demand for fragrant flowers and would also provide testing material for determining the real market value of this trait. Over the past decade, our understanding of the biochemistry and physiology of floral scent has progressed rapidly. Floral scent is a highly variable, complex mixture

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of low molecular weight, volatile organic compounds that attracts insect and animal pollinators. Hundreds, perhaps thousands, of different volatile compounds are emitted from flowers in varying levels and mixtures.41 In many species, including Antirrhinum, Petunia, and Rosa, petals are the primary site of floral scent emission42–44 with fragrance compounds emitted from both the adaxial and abaxial petal epidermal layers of Rosa x hybrida ‘Anna’.44 Fragrance is a dynamic trait, with quantitative and qualitative changes occurring during flower development, in response to pollination, through day–night cycles, and in response to the environment (reviewed by Clark et al.45). Petunia x hybrida has emerged as a popular model system for characterizing floral volatile benzenoid/phenylpropanoid synthesis (Table 3.2) (reviewed by Clark et al.45). The floral perfume of Petunia cultivar ‘Mitchell’ is dominated by benzyl alcohol, benzaldehyde, methylbenzoate, benzylbenzoate, phenylacetaldehyde, 2-phenylethanol, phenylethylacetate, phenethylbenzoate, isoeugenol, and eugenol, TABLE 3.2 Published Reports of Genetically Engineered Plants with Modified Floral Fragrance Plant

Genetic Modification

Petunia x hybrida

35S::CbLIS Petunia CCD1 RNAi 35S::SAAT Petunia BSMT RNAi Petunia ODO1 RNAi Petunia PAAS RNAi Petunia BPBT RNAi 35S::RhAAT Petunia CFAT RNAi 35S::LePAR 35S::AtPAP

Dianthus caryophyllus Eustoma grandiflorum Nicotiana tabacum

35S::CbLIS 35S::antisenseF3H 35S::CbBEAT 35S::ClTER, 35S::ClLIM, 35S::ClPIN

Effect on Floral Volatiles

References

Accumulation of nonvolatile linalool conjugate Reduced β-ionone No effect Reduced methylbenzoate Reduced volatile benzenoids Reduced phenylacetaldehyde Reduced 2-phenylethanol Reduced benzylbenzoate Reduced phenethylbenzoate Higher benzyl acetate Higher phenethyl acetate Reduced isoeugenol Reduced phenylacetaldehyde Higher 2-phenylethanol Higher benzaldehyde Higher phenylacetaldehyde Higher benzaldehyde 4-hydroxy Higher benzaldehyde 3,4-dimethoxy Higher vanillin Linalool production Higher methyl benzoate No effect Plants with all three genes emit β-pinene, limonene, γ-terpinene, and side products

[153] [33] [55] [53] [47] [50] [51,52] [154] [49] [54] [60]

[57] [58] [155] [153]

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Transgenic Horticultural Crops: Challenges and Opportunities

which are compounds derived from the amino acid phenylalanine. Small amounts of the apocarotenoids β-ionone and 6-methyl-5-hepten-2-one are also detectable in the fragrance profile.33,46 Many of these compounds have been removed from the petunia volatile profile through silencing of regulatory and biosynthetic genes (Table 3.2). A reduction in multiple benzenoid compounds was achieved by silencing odorant1 (ODO1), a transcription factor that regulates transcription of genes important for supplying substrates for floral volatile production.47 β-Ionone emission was reduced by approximately 50% in transgenic petunias with RNA interference (RNAi)-suppressed CCD1.33 While emission of β-ionone is low, only detectable in picogram quantities, the published human odor threshold is low (0.007â•›n L L−1) compared to other more abundant volatiles in petunia.33,48 Therefore, β-ionone could potentially be important in the overall fragrance of petunia. The clove-like component of petunia scent was removed from the fragrance profile by suppression of coniferyl alcohol acyltransferase (CFAT) expression.49 CFAT catalyzes the acetylation of coniferyl alcohol to form coniferyl acetate, the precursor of isoeugenol and eugenol. The two volatile compounds imparting rosy, floral notes to petunia fragrance were reduced by silencing phenylacetaldehyde synthase (PAAS). Transgenic RNAi suppressed PAAS plants had reduced phenylacetaldehyde and 2-phenylethanol levels.50 Dexter et al.51 and Orlova et al.52 removed benzyl benzoate and phenethylbenzoate from the petunia fragrance by RNAi-induced silencing of benzyl CoA:benzyl alcohol/phenylethanol benzoyl transferase (BPBT). Silencing of benzoic acid/salicylic acid carboxyl methyltransferase1 and 2 (BSMT1 and BSMT2) led to a reduction in methylbenzoate, the most predominant volatile of petunia fragrance. Flowers lacking normal methylbenzoate levels had a distinct, detectable change in fragrance, and 80% of human olfactory panelists were able to distinguish them from control flowers.53 Increasing flux through existing biosynthetic pathways and introducing new fragrance pathways are a means to make novel fragrance profiles (Table 3.2). For example, petunia flowers that smell more like a rose could be developed by increasing 2-phenylethanol synthesis and decreasing levels of endogenous volatiles, which may mask the perception of rose aroma. Petunias that produced more intense roselike 2-phenylethanol were developed by introducing tomato phenylacetaldehyde reductase (LePAR) into the petunia genome.54 Introduction of a strawberry alcohol acyltransferase (SAAT) gene into petunia did not affect the volatile profile.55 Feeding isoamyl alcohol, a substrate of SAAT, to the transgenic tissue resulted in emission of acetyl ester products. Lücker et al.56 introduced the Clarkia breweri S-linalool synthase gene into petunia. However, plants did not produce linalool and instead accumulated a nonvolatile conjugated form. Linalool was detected in transgenic CbLIS Dianthus caryophyllus cultivar ‘Eilat’ plants.57 While these plants produced linalool and derivatives thereof, the transgenic flowers did not smell differently from control flowers in human olfaction tests. It will be of interest to introduce the LIS gene into a different background of carnation that has less fragrance since it is possible that there was masking by other volatile compounds. The reports discussed in this section unequivocally demonstrate that floral fragrance can be engineered. A number of important issues can be highlighted, including (1) altering fragrance profiles with heterologous genes is possible and will be

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useful, (2) substrate availability needs to be considered when altering volatile pathways, and (3) endogenous activities can convert products to nonvolatile forms. The opportunities for significant increases in floral volatile production most likely lie with controlled expression of genes early in the biosynthetic pathways and regulatory factors controlling gene expression of several biosynthetic pathways. Fragrance and Color Fragrance benzenoids and anthocyanin color metabolites are produced from phenylpropanoid metabolism. Therefore, altering fragrance metabolism could influence the production of color and vice versa. While there are no consistent trends for direct relationships between color and fragrance, there is evidence that this does occur in carnation. White flowers from antisense F3H carnation plants produced higher amounts of benzenoids compared to the red-orange control flowers.58 These transgenic flowers produced five- to sevenfold higher levels of methyl benzoate and 2-hydroxy methyl benzoate. Thus, in carnation, loss of F3H function may reduce flux through anthocyanin synthesis causing phenylpropanoid metabolism to be redirected toward benzenoid production. Interplay between color and fragrance production is less clear in petunia. Vivid pink color was introduced into a white flowered, nocturnally fragrant Petunia axillaris background by transformation with the AN2 flavonoid biosynthesis regulator.59 The transgenic colored flowers did not exhibit significant changes in benzenoid volatile production. Likewise, colored petunias with reduced expression of ODO1, a transcription factor thought to control substrate flux to benzenoid fragrance production in petunia, did not exhibit any visible differences in pigmentation compared to controls.47 Recently, Ben Zvi et al.60 showed that heterologous expression of the Arabidopsis thaliana PAP1 Myb transcription factor in Petunia hybrida cultivar ‘Blue Spark’ led to increased flower pigmentation and increased levels of certain fragrance volatiles. There were three to five times more benzaldehyde and higher internal pools of phenylacetaldehyde, benzyaldehyde 4-hydroxy, benzaldehyde 3,4-dimethoxy, and vanillin.60 The mechanism of how PAP1 is regulating these two processes in the ‘Blue Spark’ petunias remains to be elucidated.

Volatiles and Insect Plant Defense Plants produce a myriad of volatiles in response to herbivores that serve to intoxicate, attract predators and parasitoids, and/or elicit an avoidance response in the insect inflicting damage to the plant. There are only a handful of documented examples of engineering of plant defense-related volatiles. However, as this will likely be a useful method in the future for improving plant defense in both agronomic and ornamental crops, a few examples in model systems will be highlighted here. Increased parasitoid attraction and increased pest repellant properties have been achieved through introducing single genes into Arabidopsis. For example, increased aphid parasitoid attraction and aphid repellant behavior was induced in Arabidopsis plants expressing (E)-β-farnesene (Eβf) synthase cloned from Mentha x piperita.61 The Eβf expressing plants emitted high levels of Eβf, an aphid alarm pheromone. Arabidopsis plants overexpressing a strawberry linalool/nerolidol synthase (FaNES1) gene produced

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Transgenic Horticultural Crops: Challenges and Opportunities

high levels of linalool, which was a repellant to aphids.62 By targeting FaNES1 to the mitochondria instead of the cytosol, synthesis of 4,8-dimethyl-1,3(E),7-nonatriene ((E)-DMNT) and nerolidol was induced making transgenic plants more attractive to predatory mites.63 Both of these studies with FaNES1 report a growth retardation phenotype that could be a phytotoxic effect of the new volatiles being produced or the new pathway causing a metabolic drain on metabolites essential for normal growth and development. In the case of the latter, fine tuning expression through use of promoters conferring lower or more specific transgene expression may be useful for balancing metabolite flow between the newly introduced pathway and endogenous pathways.

FLOWER AND LEAF LONGEVITY Retention of high quality flowers and leaves for visual appearance is of utmost importance to the ornamental plant industry. Flowers that are short-lived or highly sensitive to postharvest handling conditions are limited in their marketability. Similarly, leaf yellowing or senescence negatively affects the consumer appeal of potted plants and bedding plants. Plant and cut flower quality has been improved through the use of proper postharvest handling procedures and chemical preservatives. Applying transgenic technology to ornamentals to improve their postharvest attributes presents the opportunity for saving on postharvest costs associated with labor, chemical, and environmental control, contributing to environmentally sustainable practices. With such great importance, there are now many examples where genetic engineering has been successfully employed to obtain flowers with longer vase life and plants with leaves that stay green.

Floral Longevity The plant hormone ethylene negatively affects flower longevity in many ornamentally important plant species.64,65 This gaseous phytohormone is produced in response to pollination, stress, and common postharvest handling procedures causing petal in-rolling, abscission, and senescence in many species of monocots and dicots, ultimately eliminating their marketability. Van Doorn et al.66 surveyed over 300 species of flowering plants from 50 different families and found that over 64% of these were highly ethylene sensitive. To address this issue, the molecular genetics and biochemistry of ethylene synthesis and perception has been the focus of much research and chemical and genetic methods for altering ethylene responses have been developed (Table 3.3). Flower longevity can be extended through the use of chemicals that inhibit ethylene biosynthesis and perception. Chemicals such as aminoethoxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) significantly reduce ethylene production and delay petal senescence and abscission of many species.67–70 However, the concern of potential toxic effects and lack of control of effects due to ethylene generated from outside sources halted their commercial use.67 Therefore, inhibiting ethylene perception is more desirable for preventing effects of exogenous ethylene that may occur during storage and transportation. Silver thiosulfate, Ag2S2O3 or STS, has

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TABLE 3.3 Publications Documenting Extended Flower Life through Genetic Engineering Ethylene Biosynthesis or Signaling Plant Petunia x hybrida

Genetic Modification 35S::Atetr1-1 35S::sense PhEIN2 RNAi PhEIN2

Nemesia strumosa

Antisense ACO Sense ACO Antisense and sense suppression of ACS PhFbp1::Atetr1-1 35S::Atetr1-1 Etr1::etr1-1 DcCmb2::Atetr1-1 Antisense and sense suppression of ACO 35S::Cmetr1

Campanula carpatica

Phfbp1::Atetr1-1

Kalanchoe blossfeldiana

Phfpb1::Atetr1-1

Dianthus caryophyllus

Torenia fournieri

Effect Flowers lasted ∼4× longer than controls Flowers lasted ∼4× longer than controls Flowers lasted ∼6× longer than controls ∼1.7× longer vase life ∼2× longer vase life ∼2× longer vase life ∼2.7× longer vase life ∼2–3× longer vase life ∼2–2.4× longer vase life ∼2× longer vase life Sense flowers lasted 0–3.5× longer than controls Flowers lasted 1–3 days longer than controls Variable. Lines ranging between 0–6× longer than control flowers Variable. 10× longer flower retention for 40%–50% of transgenic flowers in two lines

References [87] [88] [88] [77] [78] [79] [89] [89] [89] [80] [81] [90] [92] [93]

become widely used for blocking ethylene effects in the ornamental industry for several decades.67,71–73 Unfortunately, STS is a heavy metal pollutant, and in recent years commercial use has become more restricted because of environmental concerns. 1-Methylcyclopropene (1-MCP) is a more recently developed chemical found to block ethylene perception74 and has been commercialized and is effective in delaying flower senescence in many ornamentals (reviewed by Sisler and Serek75). Altering Ethylene Responses Genetically by Inhibiting Ethylene Biosynthesis The biosynthetic pathway of ethylene has been elucidated76 and ethylene biosynthetic genes have been altered in transgenic ornamental plants to reduce the effects of ethylene. Ethylene biosynthesis is a two-step process beginning with the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase (ACS). ACC is then converted to ethylene by ACC oxidase (ACO). Reducing expression of either of these enzymes through RNAi or cosuppression technology decreases ethylene production and extends vase life. Carnation plants with silenced ACS or ACO expression produce less ethylene from flowers and

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Transgenic Horticultural Crops: Challenges and Opportunities

have extended vase life.77–80 ACO cosuppressed carnation plants had approximately a twofold longer vase life than control flowers.78 Suppression of ACO in Torenia fournieri extended flower life up to 3.5-fold longer than controls.81 While these flowers produce less ethylene and have longer vase life, the flowers are still sensitive to exogenous sources of ethylene. Thus, much of the focus on engineering-improved flower life has centered on manipulations of ethylene receptor and signaling proteins. Inhibiting Ethylene Responses by Disrupting Ethylene Signal Transduction Ethylene is perceived by plants through transmembrane receptor proteins, which are encoded by the ethylene resistant (ETR) gene family, typically consisting of five or more genes in plants. The receptors have been identified in expressed sequence tag (EST) collections from more than 20 plant species, including monocots, dicots, and lower vascular plants.82 Binding of the hormone has been demonstrated in cyanobacteria, fungi, green alga, lower vascular plants, and angiosperms.83,84 While studies of receptor function at the molecular level have not been carried out in many plant species outside of Arabidopsis and tomato, the proteins are highly conserved in regions that are functionally important.82,84 It is therefore likely that the receptors act in a similar fashion in plant species other than tomato and Arabidopsis, that is, as negative regulators of ethylene responses. In the absence of ethylene, ethylene responses are repressed, and, in the presence of ethylene, repression of ethylene responses is lifted. Arabidopsis plants carrying the etr1-1 mutation are almost completely insensitive to ethylene.85 Another central component of the pathway, ethylene insensitive 2 (EIN2), is critical for ethylene signaling. Arabidopsis EIN2 loss-of-function mutants are unable to respond to ethylene.86 Both ETR and EIN2 are critical to plant ethylene responses, making them good candidates for manipulating ethylene responses at the molecular level. Floral longevity has been extended in transgenic plants with altered ethylene signaling. Petunias with longer-lasting flowers have been attained by overexpression of the mutant ethylene receptor allele, etr1-1, from Arabidopsis thaliana87 and by suppressing expression of EIN2 by RNAi or cosuppression.88 Petunia corollas expressing the Arabidopsis etr1-1 gene do not wilt after pollination or exposure to ethylene and often stay attached to the receptacle until the developing fruit detaches the corolla, up to 10 days after pollination compared to 2–3 days for control flowers. EIN2 RNAi and EIN2 cosuppression petunia plants also display delayed corolla senescence after pollination or exposure to exogenous ethylene. These flowers do not exhibit visual signs of senescence until more than 9–12 days after pollination.88 Similarly, constitutive expression of the Arabidopsis etr1-1 in carnation doubled the vase life of carnation flowers.80,89 Constitutive expression of a melon etr1-1-like mutant receptor in Nemesia strumosa resulted in flowers that lasted 1–3 days longer than controls.90 Transgenic plants expressing constitutive ethylene insensitivity not only prove the concept that genetically disrupting ethylene signaling will extend flower life but have also been useful as a tool for learning about the myriad of processes that ethylene is involved with in a plant life cycle. For example, 35S::etr1-1 petunias are unable to be vegetatively propagated due to an inability to form adventitious roots91 and exhibit higher mortality than control plants.88 Both of these issues are huge horticultural limitations. The problems likely can be overcome through the use of promoters that restrict

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expression to floral tissues. Use of flower-specific promoters to drive ethylene insensitivity has been demonstrated, but exhaustive physiological characterization and horticultural examination have not been documented for these transgenic lines. For example, flower-specific expression of the Arabidopsis etr1-1 in carnation with the petunia fbp1 promoter extended flower longevity to twice that of control flowers.89 Flower-specific expression of the Arabidopsis etr1-1 gene with the petunia fbp1 promoter extended floral display in Campanula carpatica.92 The petunia fbp1 promoter was also used to drive Arabidopsis etr1-1 expression in Kalanchoe blossfeldiana.93 While transgenic Kalanchoe plants had a longer flower display, expression of the transgene did not appear to be entirely flower specific with some expression in root tissue. There was no consistent, obvious difference in all transgenic ethylene-insensitive Kalanchoe lines for shoot dry weight, leaf area, or root length; however, exhaustive physiological and horticultural characterization was not performed. These results demonstrate that transgenic ethylene insensitivity can be engineered in specific plant tissues.

Leaf Longevity Leaf senescence is a highly ordered, deteriorative process where cellular components are broken down and nutrients are remobilized from dying tissue to other parts of the plant. Although this process involves macromolecular breakdown, synthesis of new proteins, from genes known as senescence associated genes (SAGs), is required.94 The initiation of the senescence process is known to involve alteration in the levels of the phytohormone cytokinin (reviewed by Sakakibara95). In addition to their role in senescence, cytokinins regulate a number of developmental processes in plants. These include promoting cell division, transducing nutritional signals, promoting shoot initiation, and increasing crop productivity96 (reviewed by Sakakibara95). The first cytokinin biosynthetic gene identified and characterized was the isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens.97 The IPT protein catalyzes synthesis of the cytokinin Δ2-isopentenyladenosine monophospate through a transferase reaction between dimethylallylpyrophosphate (DMAPP) and 5′AMP. The gene encoding IPT is located on the Ti-plasmid of A. tumefaciens and is integrated into the host genome after infection. The enzyme is active in plants; increased cytokinin production is observed in plants expressing ipt.98 Since the Agrobacterium ipt was cloned and characterized, cytokinin biosynthetic genes have been isolated from plants.99,100 Work to date has centered on heterologous use of the Agrobacterium ipt gene in transgenic plants. Increasing cytokinin levels in plants is a strategy for delaying leaf senescence, but most early attempts resulted in plants with irregular growth patterns.101–103 These reports made use of modified constitutive promoters, Cu2+ inducible, or heat shock promoters for driving expression of ipt. Gan and Amasino104 designed constructs for auto-regulated, senescence-specific expression of ipt using the SAG12 promoter from Arabidopsis. With this construct, expression of ipt is restricted until the plant initiates senescence. Expression of ipt is self-attenuating as leaf senescence ceases thereby preventing overproduction of cytokinins and eliminating abnormal growth. Tobacco plants expressing SAG12::ipt had longer lasting leaves and plants were morphologically normal.104

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Transgenic Horticultural Crops: Challenges and Opportunities

Petunia has been used as a model for engineering delayed leaf senescence using different SAG promoters and the ipt gene. The SAG12::ipt construct was introduced into ‘V26’ petunia and over 20 lines were screened by drought stress for delayed leaf senescence. Drought stress is a common problem encountered in commercial and home garden environments and induces the leaf senescence program, but does not induce SAG12 expression.105 Two lines out of 20 SAG12::ipt transgenic lines displayed a normal growth phenotype and had delayed leaf senescence.2,106–108 The numbers of chlorotic basal leaves on transgenic SAG12::ipt plants were about 14 times less that of control plants.2,106 To engineer plants for extended cold storage tolerance, Khodakovskaya et al.109 utilized cor15a, a cold-inducible promoter from Arabidopsis, to drive ipt expression in petunia and chrysanthemum. During horticultural propagation, plant parts are commonly stored in cool, dark environments. These conditions cause leaf senescence and ultimately increase mortality when plants are in prolonged storage. Plants transformed with cor15a::ipt had morphologically normal growth at a normal temperature (25°C), similar to that of controls. When a propagation environment was simulated with cold storage, the transgenic plants resisted senescence exhibiting greener leaves with elevated cytokinin levels and leaves with normal chlorophyll levels while control tissue was visibly senescing. Other approaches for delaying leaf senescence have been successful and have resulted in interesting phenotypes useful for manipulation in ornamental plants. The SAG12 promoter was used to drive expression of the maize Knotted1 gene. KN1 is a homeobox protein involved in meristem formation, cytokinin synthesis, and repression of senescence. Another SAG promoter, SAG13, was used to drive ipt expression in petunia. Plants expressing SAG12::KN1 or SAG13::ipt exhibited delayed leaf senescence after drought stress.107 Compared to controls, SAG13::ipt transgenic plants were more branched, leaves were thicker and larger, root growth was reduced, and flowering was delayed. These phenotypes were ascribed to “leakier” expression of the ipt transgene.107 Nutrient stress experiments showed that source–sink relationships were altered in SAG13::ipt and SAG12::KN1 lines as new growth after nutrient stress was chlorotic and necrotic.107 Interestingly, SAG13::ipt and SAG12::KN1 plants also showed enhanced tolerance to pathogens. In controlled greenhouse experiments in which Cercospora was spray-inoculated onto plants, control plants had sixfold more senescing leaves than SAG13::ipt or SAG12::Knotted1 plants, with transgenic lines exhibiting local hypersensitive responses to infection.107 These experiments highlight the value of exhaustive horticultural evaluations of multiple lines of transgenic plants. Selection of elite cultivars will be required, similar to conventional breeding.

PLANT ARCHITECTURE Plants have evolved to grow a fascinating array of floral and vegetative forms that are influenced by genetic and environmental factors. Plant height, branching, flower number, and floral morphology are just some of the readily apparent forms giving individual plants their unique architectural characteristics. Understanding how a plant develops its architectural attributes is of economic importance to both

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agronomic and ornamental industries since creating plants with more desirable architecture can increase yield, decrease the amount of input in growing a crop, and improve the overall aesthetic value of a plant.

Plant Height Plant height is routinely regulated during greenhouse crop production through the use of chemical growth regulators that control the synthesis of gibberellic acid (GA), a phytohormone that promotes stem elongation. Ideally, plants would produce internal growth regulators to decrease labor and chemical costs and create a product that would be easier to ship to the consumer and easier to maintain in the landscape. Transgenic control of plant height through manipulation of gibberellin synthesis has been proven as a successful means for controlling plant height. Plant height can be transgenically controlled by altering signaling of the phytohormone GA or lowering the levels of endogenous GA in the plant through inhibiting GA synthesis or increasing GA breakdown. GAs are chemically diverse with more than 120 different GAs identified; many are precursors or breakdown products and only a handful are biologically active in plants (reviewed by Yamaguchi110). GA-induced cell elongation has been attenuated in Chrysanthemum morifolium by using the Arabidopsis gibberellic insensitive (gai) gene. Transgenic gai chrysanthemum plants continually repress GA responses, exhibit dwarfism with transgenic plants growing to about 30% as tall as the control plants.111 Several GA oxidase enzymes that hydroxylate GA precursors to form inactive GA products have been identified in Arabidopsis.112 Two GA2-oxidases (AtGAox7 and AtGAox8) hydroxylate C20-GA precursors, but not active C19-Gas.112 Based on work by Schomburg et al.,112 petunias with reduced plant height were produced by overexpressing GA oxidase (35S::AtGAox7 and AtGAox8), thereby reducing endogenous levels of GA. Transgenic GA oxidase petunias have a range of dwarf phenotypes.2 The dwarf phenotype can be rescued by application of exogenous C19-Gas, a feature useful to commercial growers as a means of elongating cuttings for vegetative propagation.2

Flower Morphology Over the past two decades genes responsible for floral organ identity have been identified and models for flower development are rapidly progressing. In general, floral organ identity is controlled by the sole and combinatorial activity of multiple classes of organ identity genes encoding A, B, C, D, and E class transcription factors. The A class transcription factors are responsible for sepal formation. Interaction of A and B class factors specifies petals. B and C class together specify stamens, while the C class gives rise to carpels.113 Ovule development requires D class genes, possibly in combination with C class genes (reviewed by Angenent and Immink114). The E class functions in combination with A, B, and C factors to determine identity of petals, stamens, and carpels (reviewed by Angenent and Immink114). This mechanism of flower development, known as the ABC model, is generally conserved among flowering plants. Mutants with ectopic or repressed expression of these genes have altered floral form. Loss of function of PMADS3,

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an ortholog of the Arabidopsis AGAMOUS class C gene, in petunia gives rise to flowers with stamens converted into petal-like structures.115 Transgenic manipulation of floral organ identity genes has led to a greater understanding of the molecular mechanisms underlying flower development in diverse species, giving us the required knowledge for creating morphologically novel flowers with the tools of biotechnology. Mutation screens have been the primary means for identifying lines with altered floral morphology in many ornamental crops. For example, a transposon insertion into Duplicated (DP), a C class gene, caused a double flower phenotype in Japanese morning glory.116 Although it is possible to obtain altered flower morphology mutants, biotechnology will be useful in developing lines with novel floral appearance where mutant populations are difficult to generate and for understanding the basic molecular mechanisms underlying flower development. Sepals were converted into purple colored petal-like organs when green petal, a class  B function gene, was overexpressed in petunia.117 Antisense repression of the chrysanthemum AGAMOUS-like gene caused conversion of pistils to petal tissue and pistil-like Â�tissue.118 Interesting changes to petal floral morphology, such as changes in ray floret shape, have also been observed when expression of floral homeotic genes is altered in Gerbera (e.g., Yu et al.119). Altering petal position and number through manipulation of floral organ identity genes can result in a more colorful, showier flower and flowers with new morphology, both of which are traits of interest for consumers.

Fruit and Flower Size While larger flowers are often selected in ornamental breeding programs, only recently has this trait been modified through biotechnology. As mentioned in Leaf Longevity section, cytokinins are a class of phytohormones that promote cell division; therefore, increasing cytokinin biosynthesis is a strategy toward increasing cell numbers in target tissues. This was achieved by using the Arabidopsis thaliana AP3 promoter to drive flower-specific expression of the Agrobacterium tumefaciens ipt cytokinin biosynthetic gene in transgenic petunia.120 AP3 is a class B floral organ identity gene specifying petal and stamen identity. The AP3 promoter restricts expression to the petals and stamens in Arabidopsis121 and to floral organs in petunia.120 Five independent transgenic AP3::ipt petunia lines had increased flower diameter compared to control flowers. Flowers from these lines had a 20%–30% increase in the diameters of individual floral organs due to radial expansion. Transgenic lines had flowers with over 90% increase in fresh weight compared to controls. Some lines had >60% increase in overall fruit weight while others had fruit weights similar to those of controls.

Agrobacterium rhizogenes rol Genes The rol genes from Agrobacterium rhizogenes have been useful for changing Â�multiple aspects of plant architecture (reviewed by Casanova et al.122). There are four rol genes (rolA, B, C, and D), with rolC (cytokinin-β-glucosidase) having the most potential for transgenic enhancement of ornamental plants. In vivo the rol genes interfere with phytohormone metabolism and modify secondary metabolism, which

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is phenotypically manifested as a dwarf habit, increased rooting, darker leaves, increased lateral shoots, and increased flowering (reviewed by Casanova et al.122). The rol genes have been transformed into Antirrhinum,123 Begonia,124 Rosa,125 Petunia,126 Dianthus,127 Osteospermum,128 Angelonia,129 Lilium,130 and Pelargonium.131 In some instances, transgenic plants had smaller flowers and reduced fertility, which could be a limitation from a production standpoint, but may be useful in addressing environmental concerns since transgene flow would likely be more restricted.

BIOTIC STRESS TOLERANCE Engineering plants to tolerate biotic stresses has the potential for large cost savings in pesticide chemicals leading to more environmentally friendly production practices (reviewed by Redenbaugh and McHughen132). Pesticide use data collected from farmers growing insect resistant transgenic corn and cotton indicate biotech crops have decreased pesticide usage, increased yield, higher quality crops, and decreased labor.133 These are clear benefits that are currently only realized by producers of biotech food crops, but are applicable to ornamental crops. While there are no commercially available biotech ornamentals engineered for biotic stress tolerance, this trait is being engineered in some ornamentals and a few examples will be highlighted here. There are a number of strategies for genetic engineering plants to be more resistant to insect attack. Engineering plants to produce insect toxins has been the most successful means to date and is widely used today in corn and cotton production. Genes encoding insect control proteins from Bacillus thuringiensis, know as Cry genes, have been cloned and modified for efficient production in plants134 (reviewed by Schuler et al.135). These proteins bind to gut receptors in lepidopteran, dipteran, and coleopteran insects causing disfunction of the insect gut and lysis of gut epithelial cells leading to insect death (reviewed by Gill et al.136). The Bacillus cryIA(b) gene has been introduced into chrysanthemum causing feeding insects to die and significantly less consumption of leaf area.137 Insects feeding on transgenic plants consumed less than 5% of the leaf area while 95% or more of control leaf area was consumed. Similarly, transgenic petunias expressing the cryIA(c) gene caused significant reduction in insect fecundity.138 While the BT toxins have proven to be very successful for insect management, other biotech insecticidal strategies have been developed and will be important considerations for crop management to avoid insect resistance issues. As discussed in “Plant Volatiles” section, plants can be engineered to produce volatiles that modify insect behavior or attract predatory insects. Protease inhibitors, α-amylase inhibitors, lectins, and nonvolatile secondary metabolites, such as tryptamine, are also means for engineering insect defense in plants (reviewed by Llewellyn and Higgins139). However, examples of these methods for engineering insect defense in ornamental plants are few. While disease resistance in ornamental crops has largely been achieved through traditional breeding, this trait has been successfully genetically engineered using a number of approaches. Transgenic expression of genes commonly induced by disease infection (pathogenesis-related genes, PR) confers enhanced disease resistance. For example, chitinases degrade fungal cell walls into breakdown products

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that elicit plant defense responses and are often induced in response to pathogen attack (reviewed by Stintzi et al.140). Blackspot is a common, problematic disease in Rosa hybrida and is caused by the fungus Diplocarpon rosae. Transformation of rose via particle bombardment with a rice basic chitinase gene reduced blackspot severity by 13%–43% with lesion size reduced by up to half of those of control blackspot lesions.141 A PR gene encoding for a cysteine-rich antimicrobial protein (AMP) confers disease resistance in plants. Enhanced tolerance to powdery mildew was observed in transgenic rose plants constitutively expressing an AMP gene from onion.142 The onion AMP was also introduced into Pelargonium for enhanced resistance to Botrytis.143 In leaf disc assays, Botrytis sporulation was slightly reduced in transgenic lines. Greater disease protection is observed in transgenic plants expressing more than one PR gene and this should be considered in development of transgenic plants with enhanced disease resistance.144

CURRENT AND FUTURE ISSUES IN ORNAMENTAL BIOTECHNOLOGY Despite the documented technical successes, utility, and the enormous benefits genetic engineering rewards, there are only a limited number of commercialized transgenic ornamental plants on the market. Presently, the only commercialized ornamental plants on the market are carnations engineered for blue-violet color.145 This is in stark contrast to the rapid adoption of genetically engineered agronomic crops since their introduction in 1996 with transgenic plants accounting for well over 50%–90% of planted acreage of corn, soybean, and cotton in 2008 (http://www.ers. usda.gov/Data/BiotechCrops/). The lack of commercialized transgenic ornamentals is not due to lack of potential products, but is impeded by regulatory issues concerning safety, consumer acceptance, intellectual property, postcommercialization monitoring of transgenic crops, and the diversity of germplasm requiring development of a biotech trait.2 While ornamental crops are grown for aesthetic purposes and do not require human health safety testing, they are subject to strict handling procedures, field-testing, tracking, and postcommercialization stewardship. All of these increase the costs of developing a biotech crop to at least $1 million for one transgenic event per country,132 making it difficult to justify use of biotechnology in the ornamental market where crop values are significantly lower than agronomic crops. With turnover of new varieties in ornamental crops, sales are not high enough to generate a profit that would offset the costs to bring a transgenic plant to market. Regulatory issues pose a significant obstacle to the development and commercialization of genetically engineered crops. Any organism that has been modified through genetic manipulation technology is subject to regulation by overseeing agencies, such as the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Environmental Protection Agency (EPA), and Food and Drug Administration (FDA) in the United States, which oversees introduction, transport, risks to human health, and environmental risk assessment of the genetically or living modified organism (GMO or LMO, respectively). The regulations and procedures vary by country, creating additional complications in releasing a GMO internationally (reviewed by Chandler and Tanaka16), but the goals of regulating GMOs are

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unified in that negative environmental and health effects want to be avoided. A centralized, international resource, known as the Cartagena Protocol on Biosafety (CPB), has been developed for promoting the safe handling and use of GMOs internationally (http://www.cbd.int/biosafety/). However, the protocol is still being developed and ratified by many countries and countries such as the United States and Australia are not yet contributing to the development of this centralized protocol. Continued consumer education, outreach, and perception assessment are important avenues to continue pursuit of making commercialization of ornamental GMOs worthwhile. Reports indicate that consumer perception of GMOs is mixed, but most surveys are conducted with regard to the presence of GMOs in food and not ornamental plants. A survey of master gardeners revealed that ∼73% of survey respondents expressed interest in purchasing a GM plant product for the garden.146 This said, there were also clear opinions regarding how different sources of transgenes affected acceptability of the GMO, with plant-derived transgenes being more acceptable than those derived from bacteria, fungus, virus or animal sources. Respondents were also neutral about the effect of landscape GMOs. This latter point should be emphasized in education programs since many applications of biotechnology will result in decreased water use, decreased fossil fuel use, and decreased chemical use, all of which would have positive effects on our environment. For example, Redenbaugh and McHughen132 cite data showing declining pesticide sales with increased sales of biotech-based varieties of plants. Ornamental biotechnology will clearly benefit from the evolution of newly developing DNA sequencing technologies. New sequencing technologies including 454 sequencing and real time single molecule long read sequencing soon to be commercialized,147 will make it possible to quickly have genome data for most species of interest at very low costs. The limitation will no longer be on sequence availability but rather on data management and data mining. The availability of genome data will facilitate discovery of regulatory elements that will be useful for delivering more precise transgene expression patterns in plants, rapidly progress the discovery of enzymes involved in important biotechnological applications, and make development of transgenic ornamental plants more economically feasible.

ACKNOWLEDGMENTS The authors thank Dr. Thomas A. Colquhoun for critical reading of the manuscript and Dr. Bart Schutzman for assistance with figure preparation.

REFERENCES

1. Jerardo, A., Floriculture and Nursery Crops Handbook, USDA ERS Report September 2007. 2. Clark, D.G., Applications of plant biotechnology to ornamental crops, in Handbook of Plant Biotechnology, Vol. II, Cristou, P. and Klee, H., Eds., John Wiley & Sons, Ltd., West Sussex, U.K., 2004, pp. 863–879. 3. Shibuya, K. and Clark, D.G., Ethylene: Current status and future directions using transgenic plants to improve flower longevity of ornamental crops, J. Crop Improv. 18, 391, 2006.

76

Transgenic Horticultural Crops: Challenges and Opportunities

4. Grotewold, E., The genetics and biochemistry of floral pigments, Annu. Rev. Plant Biol., 57, 761, 2006. 5. Tanaka, Y., Sasaki, N., and Ohmiya, Y., Biosynthesis of plant pigments: Anthocyanins, betalains and carotenoids, Plant J., 54, 733, 2008. 6. Napoli, C., Lemieux, C., and Jorgensen, R., Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans, Plant Cell, 2, 279, 1990. 7. van der Krol, A.R. et al., Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression, Plant Cell, 2, 291, 1990. 8. Deroles, S.C. et al., An antisense chalcone synthase cDNA leads to novel color patterns in lisianthus (Eustoma grandiflorum) flowers, Mol. Breed, 4, 59, 1998. 9. Elomaa, P. et al., Agrobacterium-mediated transfer of antisense chalcone synthase cDNA to Gerbera hybrida inhibits flower pigmentation, Biotechnology, 11, 508, 1993. 10. Courtney-Gutterson, N. et al., Modification of flower color in Florist’s Chrysanthemum: Production of a white-flowering variety through molecular genetics, Biotechnology, 12, 268, 1994. 11. Welle, R. and Grisebach, H., Isolation of a novel NADPH-dependent reductase which coacts with chalcone synthase in the biosynthesis of 6′-deoxychalcone, FEBS Lett., 236, 221, 1988. 12. Davies, K.M. et al., Production of yellow colour in flowers: Redirection of flavonoid biosynthesis in Petunia, Plant J., 13, 259, 1998. 13. Lunau, K., Wacht, S., and Chittka, L., Colour choices of naïve bumble bees and their implications for colour perception, J. Comp. Physiol., A178, 477, 1996. 14. Ono, E. et al., Yellow flowers generated by expression of the aurone biosynthetic pathway, Proc. Natl. Acad. Sci. USA, 103, 11075, 2006. 15. Okuhara, H. et al., Molecular cloning and functional expression of tetrahydroxychalcone 2′-glucosyl-transferase genes, Plant Cell Physiol., 45, S133, 2004. 16. Chandler, S. and Tanaka, Y., Genetic modification in floriculture, Crit. Rev. Plant Sci., 26, 169, 2007. 17. Fukui, Y. et al., A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3′5′-hydroxylase gene, Phytochemistry, 63, 15, 2003. 18. Shiono, M., Matsugaki, N., and Takeda, K., Structure of the blue cornflower pigment, Nature, 436, 791, 2005. 19. Meyer, P. et al., A new petunia flower color generated by transformation of a mutant with a maize gene, Nature, 330, 677, 1987. 20. Meyer, P., Heidmann, I., and Niedenhof, I., Differences in DNA-methylation are associated with a paramutation phenomenon in transgenic petunia, Plant J., 4, 89, 1993. 21. Oud, J.S.N. et al., Breeding of transgenic orange Petunia hybrida varieties, Euphytica, 84, 175, 1995. 22. Lu, S. and Li, L., Carotenoid metabolism: The biosynthesis, regulation, and beyond, J. Int. Plant Biol., 50, 778, 2008. 23. Hirschberg, J., Carotenoid biosynthesis in flowering plants, Curr. Opin. Plant Biol., 4, 210, 2001. 24. Lewinsohn, E. et al., Carotenoid pigmentation affects the volatile composition of tomato and watermelon fruits, as revealed by comparative genetic analyses, J. Agric. Food Chem., 53, 3142, 2005. 25. Fray, R.G. and Grierson, D., Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression, Plant Mol. Biol., 22, 589, 1993. 26. Ronen, G. et al., Regulation of carotenoid biosynthesis during tomato fruit development: Expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant delta, Plant J., 17, 341, 1999.

Transgenic Ornamental Crops

77

27. Ye, X. et al., Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm, Science, 287, 303, 2000. 28. Paine, J.A. et al., Improving the nutritional value of Golden Rice through increased provitamin A content, Nat. Biotechnol., 23, 482, 2005. 29. Suzuki, S. et al., Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway, Plant Cell Rep., 26, 951, 2007. 30. Auldridge, M.E., McCarty, D.R., and Klee, H.J., Plant carotenoid cleavage oxygenases and their apocarotenoid products, Curr. Opin. Plant Biol., 9, 315, 2006. 31. Auldridge, M.E. et al., Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family, Plant J., 45, 982, 2006. 32. Ohmiya, A. et al., Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals, Plant Physiol., 142, 1193, 2006. 33. Simkin, A.J. et al., Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission of beta-ionone, a fragrance volatile of petunia flowers, Plant Physiol., 136, 3504, 2004. 34. Vogel, J.T. et al., The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions, J. Biol. Chem., 283, 11364, 2008. 35. Dudareva, N. and Pichersky, E., Metabolic engineering of plant volatiles, Curr. Opin. Biotechnol., 19, 181, 2008. 36. Pichersky, E., Noel, J.P., and Dudareva, N., Biosynthesis of plant volatiles: Nature’s diversity and ingenuity, Science, 311, 808, 2006. 37. Diego, M.A. et al., Aromatherapy positively affects mood: EEG patterns of alertness and math computations, Int. J. Neurosci., 96, 217, 1998. 38. Lehrner, J. et al., Ambient odor of orange in a dental office reduces anxiety and improves mood in female patients, Physiol. Behav., 71, 83, 2000. 39. Komiya, M., Takeuchib, T., and Harada, E., Lemon oil vapor causes an anti-stress effect via modulating the 5-HT and DA activities in mice, Behav. Brain Res., 172, 240, 2006. 40. Halloran, J. M. and Kuehnle, A.R., What do Anthurium buyers want in their flowers?  Results of a market survey, CTAHR Economic Fact Sheet, EFS-27, September 1998. 41. Knudsen, J.T., Tollsten, L., and Bergstrom, L.G., Review article number 76: Floral scents: A checklist of volatile compounds isolated by head-space techniques, Phytochemistry, 33, 253, 1993. 42. Kolosova, N. et al., Regulation of circadian methyl benzoate emission in diurnally and nocturnally emitting plants, Plant Cell, 13, 2333, 2001. 43. Verdonk, J.C. et al., Regulation of floral scent production in petunia revealed by targeted metabolomics, Phytochemistry, 62, 997, 2003. 44. Bergougnoux, V. et al., Both the adaxial and abaxial epidermal layers of the rose petal emit volatile scent compounds, Planta, 226, 853, 2007. 45. Clark, D. et al., Benzenoids dominate the fragrance of Petunia flowers, in Petunia, Gerats, T. and Strommer, J. Eds., Springer Publishing Company, New York, 2009, Chap. 3. 46. Underwood, B.A. and Clark, D.G., unpublished data, 2008. 47. Verdonk, J.C. et al., ODORANT1 regulates fragrance biosynthesis in petunia flowers, Plant Cell, 17, 1612, 2005. 48. Baldwin, E.A. et al., Flavor trivia and tomato aroma: Biochemistry and possible mechanisms for control of important aroma components, HortScience, 35, 1013, 2000. 49. Dexter, R. et al., Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol, Plant J., 49, 265, 2007.

78

Transgenic Horticultural Crops: Challenges and Opportunities

50. Kaminaga, Y. et al., Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation, J. Biol. Chem., 281, 23357, 2006. 51. Dexter, R.J. et al., Tissue-specific PhBPBT expression is differentially regulated in response to endogenous ethylene, J. Exp. Bot., 59, 609, 2008. 52. Orlova, I. et al., Murphy, A.S., Rhodes, D., Pichersky, E., and Dudareva, N., Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to Â�benzoic acid and enhancement in auxin transport, Plant Cell, 18, 3458, 2006. 53. Underwood, B.A. et al., Ethylene-regulated floral volatile synthesis in petunia corollas, Plant Physiol., 138, 255, 2005. 54. Tieman, D.M. et al., Tomato phenylacetaldehyde reductases catalyze the last step in the synthesis of the aroma volatile 2-phenylethanol, Phytochemistry, 68, 2660, 2007. 55. Beekwilder, J. et al., Functional characterization of enzymes forming volatile esters from strawberry and banana, Plant Physiol., 135, 1865, 2004. 56. Lücker, J. et al., Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-beta-D-glucopyranoside, Plant J., 27, 315, 2001. 57. Lavy, M. et al., Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene, Mol. Breed., 9, 103, 2005. 58. Zucker, A. et al., Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxlase gene, Mol. Breed., 9, 33, 2002. 59. Hoballah, M.E. et al., Single gene-mediated shift in pollinator attraction in Petunia, Plant Cell, 19, 779, 2007. 60. Ben Zvi, M.M. et al., Interlinking showy traits: Co-engineering of scent and colour biosynthesis in flowers, Plant Biotechnol. J., 6, 403, 2008. 61. Beale, M.H. et al., Prosser, I.M., Shewry, P.R., Smart, L.E., Wadhams, L.J., Woodcock, C.M., and Zhang, Y., Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior, Proc. Natl. Acad. Sci. USA, 103, 10509, 2006. 62. Aharoni, A. et al., Terpenoid metabolism in wild-type and transgenic Arabidopsis plants, Plant Cell, 15, 2866, 2003. 63. Kappers, I.F. et al., Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis, Science, 309, 2070, 2005. 64. Woltering, E.J. and van Doorn, W.G., Role of ethylene in senescence of petals— Morphological and taxonomical relationships, J. Exp. Bot., 39, 1605, 1988. 65. van Doorn, W.G., Effect of ethylene on flower abscission: A survey, Ann. Bot., 89, 689, 2002. 66. van Doorn, W.G., Categories of petal senescence and abscission: A re-evaluation, Ann. Bot., 87, 447, 2001. 67. Abeles, F.B., Morgan, P.W., and Saltveit, M.E., Ethylene in Plant Biology, 2nd edn., Academic Press, San Diego, CA, 1992. 68. Baker, J.E. et al., Delay of senescence in carnation by rhizobitoxin analog and sodium benzoate, HortScience, 12, 38, 1977. 69. Wang, C.J. et al., Effects of 2 analogs of rhizobitoxine and sodium benzoate on senescence of snapdragons, J. Am. Soc. Hort. Sci., 102, 517, 1977. 70. Wang, C.J. and Baker, J.E., Vase life of cut flowers treated with rhizobitoxine analogs, sodium benzoate, and isopentenyl adenosine, HortScience, 14, 59, 1979. 71. Mor, Y., Reid, M.S., and Kofranek, A.M., Pulse treatments with silver sulfate and sucrose improve the vase life of sweet peas, J. Am. Soc. Hort. Sci., 109, 866, 1984. 72. Veen, H. and van de Geijn, S.C., Mobility and ionic form of silver as related to longevity of cut carnations, Planta, 140, 93, 1978. 73. Reid, M.S. et al., Pulse treatments with the silver thiosulfate complex extend the vase life of cut carnations, J. Am. Soc. Hort. Sci., 105, 25, 1980. 74. Serek, M. et al., Inhibition of ethylene-induced cellular senescence symptoms by 1-Â�methylcyclopropene, a new inhibitor of ethylene action, Physiol. Plant., 94, 229, 1995.

Transgenic Ornamental Crops

79

75. Sisler, E.C. and Serek, M., Compounds interacting with the ethylene receptor in plants, Plant Biol., 5, 473, 2003. 76. Yang, S.F. and Hoffman, N.E., Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol., 35, 155, 1984. 77. Savin, K.W. et al., Antisense ACC oxidase RNA delays carnation petal senescence, HortScience, 30, 970, 1995. 78. Kosugi, Y. et al., Expression of genes responsible for ethylene production and wilting are  differently regulated in carnation (Dianthus caryophyllus L.) petals, Plant Sci., 158, 139, 2000. 79. Iwazaki, Y. et al., Generation and ethylene production of transgenic carnations harboring ACC synthase cDNA in sense or antisense orientation, J. Appl. Hort., 6, 67, 2004. 80. Chandler, S., Practical lessons in the commercialization of genetically modified plants— Long vase-life carnation, Acta Hort., 764, 71, 2007. 81. Aida, R. et al., Extension of flower Â�longevity in transgenic torenia plants incorporating ACC oxidase transgene, Plant Sci., 138, 91, 1998. 82. Mount, S.M. and Chang, C., Evidence for a plastid origin of plant ethylene receptor genes, Plant Physiol., 130, 10, 2002. 83. Chen, Y.F., Etheridge, N., and Schaller, G.E., Ethylene signal transduction, Ann. Bot. (Lond.), 95, 901, 2005. 84. Wang, W. et al., Identification of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis, Plant Cell, 18, 3429, 2006. 85. Chang, C. et al., Arabidopsis ethylene-response gene ETR1: Similarity of product to two-component regulators, Science, 262, 539, 1993. 86. Roman, G. et al., Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: Five novel mutant loci integrated into a stress response pathway, Genetics, 139, 1393, 1995. 87. Wilkinson, J.Q. et al., A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous plants, Nat. Biotechnol., 15, 444, 1997. 88. Shibuya, K. et al., The central role of PhEIN2 in ethylene responses throughout plant development in petunia, Plant Physiol., 136, 2900, 2004. 89. Bovy, A.G. et al., Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers, Mol. Breed., 5, 301, 1999. 90. Cui, M.-L., Takada, K., Ma, B., and Ezura, H., Overexpression of a mutated melon ethylene receptor gene Cm-ETR1/H69A confers reduced ethylene sensitivity in a heterologous plant, Nemesia strumosa, Plant Sci., 167, 253, 2004. 91. Clark, D.G. et al., Root formation in ethylene-insensitive plants, Plant Physiol., 121, 53, 1999. 92. Sriskandarajah, S., Mibus, H., and Serek, M., Transgenic Campanula carpatica plants with reduced ethylene sensitivity, Plant Cell Rep., 26, 805, 2007. 93. Sanikhani, M. et al., Kalanchoe blossfeldiana plants expressing the Arabidopsis etr1-1 allele show reduced ethylene sensitivity, Plant Cell Rep., 27, 729, 2008. 94. Gan, S., Mitotic and postmitotic senescence in plants, Sci. Aging Knowl. Environ., 38, RE7, 2003. 95. Sakakibara, H., Cytokinins: Activity, biosynthesis and translocation, Annu. Rev. Plant Biol., 57, 431, 2006. 96. Mok, D.W.S. and Mok, M.C., Cytokinins: Chemistry, Activity and Function, CRC Press, Boca Raton, FL, 1994. 97. Akiyoshi, D. et al., T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis, Proc. Natl. Acad. Sci. USA, 81, 5994, 1984. 98. Medford, J.I. et al., Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene, Plant Cell, 1, 403, 1989.

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99. Takei, K., Sakakibara, H., and Sugiyama, T., Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana, J. Biol. Chem., 276, 26405, 2001. 100. Zubko, E. et al., Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants, Plant J., 29, 797, 2002. 101. Faiss, M. et al., Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants, Plant J., 12, 401, 1997. 102. McKenzie, M.J., Mett, V., Reynolds, P.H.S., and Jameson, P.E., Controlled cytokinin production in transgenic tobacco using a copper-inducible promoter, Plant Physiol., 116, 969, 1998. 103. Smart, C.M. et al., Delayed leaf senescence in tobacco plants transformed with tmr, a gene for cytokinin production in Agrobacterium, Plant Cell, 3, 647, 1991. 104. Gan, S. and Amasino, R.M., Inhibition of leaf senescence by autoregulated production of cytokinin, Science, 270, 1986, 1995. 105. Weaver, L.M. et al., A comparison of the expression patterns of several senescenceassociated genes in response to stress and hormone treatment, Plant Mol. Biol., 37, 455, 1998. 106. Dervinis, C., Genetic transformation of Petunia x hybrida for delayed leaf senescence using PSAG12IPT, MS thesis, University of Florida, Gainesville, FL, 1999. 107. Jandrew, J., Nutritional and fungal stress responses of transgenic petunias with delayed leaf senescence, MS thesis, University of Florida, Gainesville, FL, 2002. 108. Chang, H. et al., Overproduction of cytokinins in petunia flowers transformed with PSAG12-IPT delays corolla senescence and decreases sensitivity to ethylene, Plant Physiol., 132, 2174, 2003. 109. Khodakovskaya, M. et al., Effects of cor15a-IPT gene expression on leaf senescence in transgenic Petunia x hybrida and Dendranthema x grandiflorum, J. Exp. Bot., 56, 1165, 2005. 110. Yamaguchi, S., Gibberellin metabolism and its regulation, Annu. Rev. Plant Biol., 59, 225, 2008. 111. Petty, L.M. et al., Manipulating the gibberellin response to reduce plant height in Chrysanthemum morifolium, Acta Hort., 560, 87, 2001. 112. Schomburg, F.M. et al., Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants, Plant Cell, 15, 151, 2003. 113. Coen, E.S. and Meyerowitz, E.M., The war of the whorls: Genetic interactions controlling flower development, Nature, 353, 31, 1991. 114. Angenent, G.C. and Immink, R.G.H., Combinatorial action of petunia MADS box genes and their protein products, in Petunia, Gerats, T. and Strommer, J., Eds., Springer Publishing Company, New York, 2009, Chap. 11. 115. Kapoor, M. et al., Role of petunia pMADS3 in determination of floral organ and meristem identity, as revealed by its loss of function, Plant J., 32, 115, 2002. 116. Nitasaka, E., Insertion of an En/Spm-related transposable element into a floral homeotic gene DUPLICATED causes a double flower phenotype in the Japanese morning glory, Plant J., 36, 522, 2003. 117. Halfter, U. et al., Ectopic expression of a single homeotic gene, the Petunia gene green petal, is sufficient to convert sepals to petaloid organs, EMBO J., 13, 1443, 1994. 118. Aida, R. et al., Chrysanthemum flower shape modification by suppression of chrysanthemum-AGAMOUS gene, Plant Biotechnol., 25, 55, 2008. 119. Yu, D., Kotilainen, M., Pöllänen, E., Mehto, M., Elomaa, P., Helariutta, Y., Albert, V.A., and Teeri, T.H., Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae), Plant J., 17, 51, 1999. 120. Verdonk, J.C. et al., Flower-specific expression of the Agrobacterium tumefaciens isopentenyltransferase gene results in radial expansion of floral organs in Petunia hybrida, Plant Biotechnol. J., 6, 694, 2008.

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121. Tilly, J.J., Allen, D.W., and Jack, T., The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects, Development, 125, 1647, 1998. 122. Casanova, E. et al., Influence of rol genes in floriculture, Biotechnol. Adv., 23, 3, 2005. 123. Handa, T., Genetic transformation of Antirrhinum majus L. and inheritance altered phenotype induced by Ri T-DNA, Plant Sci., 81, 199, 1992. 124. Kiyokawa, S. et al., Genetic transformation of Begonia tuberhybrida by Ri rol genes, Plant Cell Rep., 15, 606, 1996. 125. van der Salm, T.P.M. et al., Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L., J. Exp. Bot., 49, 847, 1998. 126. Winefield, C. et al., Alterations of Petunia plant form through the introduction of the rolC gene from Agrobacterium rhizogenes, Mol. Breed., 5, 543, 1999. 127. Ovadis, M. et al., Generation of transgenic carnation plants with novel characteristics by combining microprojectile bombardment with Agrobacterium tumefaciens transformation, in Plant Biotechnology and In Vitro Biology in the 21st Century, Altman, A., Izhar, S., and Ziv, M., Eds., Kluwer Academic Publishers, Dordrecht, the Netherlands, 1992, pp. 189–192. 128. Giovannini, A., Mascarello, C., and Allavena, A., Effects of rol genes on flowering in Osteospermum ecklonis, Flower. Newsl., 28, 49, 1998. 129. Koike, Y. et al., Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes, Plant Cell Rep., 21, 981, 2003. 130. Mercuri, A. et al., Agrobacterium-mediated transformation with rol genes of Lilium longiflorum Thunb., Acta Hort., 612, 129, 2003. 131. Boase, M.R. et al., Transgenic regal pelargoniums that express the rolC gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype, In Vitro Cell Dev. Biol., 40, 46, 2004. 132. Redenbaugh, K. and McHughen, A., Regulatory challenges reduce opportunities for horticultural biotechnology, Calif. Agric., 58, 106, 2004. 133. Fernando-Cornejo, J. and Caswell, M., The first decade of genetically engineered crops in the United States, Economic Information Bulletin Number 11, April 2006. 134. Perlak, F.J. et al., Modification of the coding sequence enhances plant expression of insect control protein genes, Proc. Natl. Acad. Sci. USA, 88, 3324, 1991. 135. Schuler, T.H. et al., Insect-resistant transgenic plants, Trends Biotechnol., 16, 168, 1998. 136. Gill, S.S., Cowles, E.A., and Pietrantonio, P.V., The mode of action of Bacillus thuringiensis endotoxins, Annu. Rev. Entomol., 37, 615, 1992. 137. Shinoyama, H. and Mochizuki, A., Insect resistant transgenic chrysanthemum [Dendranthema x grandiflorum (Ramat.) Kitamura], Acta Hort., 714, 177, 2006. 138. Omer, A.D. et al., Effects of transgenic petunia expressing Bacillus thuringiensis toxin on selected lepidopteran pests, Biocontrol Sci. Technol., 7, 437, 1997. 139. Llewellyn, D.J. and Higgins, T.J.V., Transgenic crop plants with increased tolerance to insect pests, in Plant Biotechnology and Transgenic Plants, Oksman-Caldentey, K.-M. and Barz, W., Eds., CRC Press, Boca Raton, FL, 2002. 140. Stintzi, A. et al., Plant ‘pathogenesis-related’ proteins and their role in defense against pathogens, Biochimie, 75, 687, 1993. 141. Marchant, R. et al., Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf), Mol. Breed., 4, 187, 1998. 142. Li, X. et al., Transgenic rose lines harboring an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced resistance to powdery mildew (Sphaerotheca pannosa), Planta, 218, 226, 2003. 143. Bi, Y.-M. et al., Resistance to Botrytis cinerea in scented geranium transformed with a gene encoding the antimicrobial protein Ace-AMP1, Plant Cell Rep., 18, 835, 1999.

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144. Zhu, Q. et al., Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco, Biotechnology, 12, 807, 1994. 145. Lu, C. et al., Florigene flowers: From laboratory to market, in Plant Biotechnology 2002 and Beyond, Vasil, I.K., Ed., Kluwer Academic Publishers, Dordrecht, the Netherlands, 2003. 146. Klingeman, W., Hall, C., and Babbit, B., Master gardener perception of genetically modified ornamental plants provides strategies for promoting research products through outreach and marketing, HortScience, 41, 1263, 2006. 147. Eid, J. et al., Real-time DNA sequencing from single polymerase molecules, Science, 323, 133, 2009. 148. van der Krol, A.R. et al., An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation, Nature, 333, 866, 1988. 149. Gutterson, N., Anthocyanin biosynthetic genes and their application to flower color modification through sense suppression, HortScience, 30, 955, 1995. 150. Katsumoto, Y. et al., Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin, Plant Cell Physiol., 48, 1589, 2007. 151. Suzuki, K. et al., Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes, Mol. Breeding, 6, 239, 2000. 152. Ueyama, Y. et al., Molecular and biochemical characterization of torenia flavonoid 3′-hydroxylase and flavone synthase II and modification of flower color by modulating the expression of these genes, Plant Sci., 163, 253, 2002. 153. Lücker, J. et al., Metabolic engineering of monoterpene biosynthesis: Two-step production of (+)-trans-isopiperitenol by tobacco, Plant J., 39, 135, 2004. 154. Guterman, I. et al., Generation of phenylpropanoid pathway-derived volatiles in transgenic plants: Rose alcohol acetyltransferase produces phenylethyl acetate and benzyl acetate in petunia flowers, Plant Mol. Biol., 60, 555, 2006. 155. Aranovich, D., Lewinsohn, E., and Zaccai, M., Post-harvest enhancement of aroma in transgenic lisianthus (Eustoma grandiflorum) using the Clarkia breweri benzyl alcohol acetyltransferase (BEAT) gene, Postharvest Biol. Technol., 43, 255, 2007.

4

Expression and Manufacture of Pharmaceutical Proteins in Genetically Engineered Horticultural Plants Qiang Chen

CONTENTS A Brief History.........................................................................................................84 Plants Provide a New Manufacturing Technology for Pharmaceutical Proteins......... 85 Protein Pharmaceutical Production Using Plant Systems......................................... 88 Monoclonal Antibodies........................................................................................ 89 Vaccines...............................................................................................................92 Glycosylation of Plant-Made Pharmaceuticals......................................................... 95 Downstream Processing of Plant-Derived Protein Pharmaceuticals........................96 Column Chromatography.....................................................................................97 Nonchromatographic Separations...................................................................... 101 Plant Species Used for PMP Production................................................................. 103 Leafy Crops........................................................................................................ 104 Seed Crops......................................................................................................... 105 Fruit and Tuber Crops........................................................................................ 106 Aquatic Plants.................................................................................................... 107 Plant Suspension Cell Cultures and Single-Cell Cultures of Algae................... 107 Regulatory Concerns and Public Acceptance......................................................... 108 Better Stewardship of the PMP Community...................................................... 109 Alternative PMP Production Technology and Risk Control.............................. 109 Nonbiological Containment.......................................................................... 109 Biological Containment................................................................................. 110 Regulatory Policies regarding PMP................................................................... 112 Conclusion.............................................................................................................. 112 References............................................................................................................... 113

83

84

Transgenic Horticultural Crops: Challenges and Opportunities

A BRIEF HISTORY Humans have been using plants as a natural factory of pharmaceutical molecules for many centuries. Many secondary metabolites of plants exhibit a diversity of therapeutic effects including anti-inflammatory, wound-healing, and antimicrobial properties. These natural plant medicines were widely used around the world by early civilizations to protect and maintain human and animal health. Even today, with our highly sophisticated pharmaceutical technologies, more than one-quarter of Â�prescription pharmaceutics are still derived from plants.1,2 These plant-based natural pharmaceuticals include single-molecule drugs such as the painkiller morphine from the opium poppy and botanicals that are a mixture of active plant molecules acting synergistically in the treatment of specific conditions. Traditionally, these plant molecules are extracted from either wild or cultivated species. Largescale production, however, is usually relying on modern advancement of plant tissue culture since the medicinal plants are sometimes rare in nature or are difficult to cultivate. Therefore, plants and plant tissue cultures have been serving as important bioproduction systems for traditional pharmaceutical bulk substances for a long time. With the advancement of modern molecular biology and biotechnology, it became possible to alter the plant genome by inserting expression cassettes including the target transgene and associated regulatory genetic elements. This technological breakthrough allowed the creation of transgenic plants that carry the transgene and produce recombinant proteins.3 The first recombinant pharmaceutical protein of plant origin was a human growth hormone produced in transgenic tobacco in 1986.4 Three years later, the success of expressing monoclonal antibodies (mAbs) in transgenic tobacco plants was published, demonstrating the ability of plant cells in expressing and assembling of complex functional multi-subunit proteins.5 Our research group directed by Dr. Charles Arntzen led the effort in plant-made vaccines and published our first finding in 1992. We demonstrated that transgenic tobacco could successfully produce hepatitis B virus surface antigen (HBsAg) with similar biochemical properties and antigenicity to that produced in human serum or recombinant yeast.6 Soon after, research from our group showed that E. coli heat-labile enteroxin (LT-B) derived from transgenic tobacco or potato had equivalent functionality to E. coli–derived LT-B and was orally immunogenic in mice, which provided the first proof of principle for plant-derived edible vaccines.7 The successes of these researches opened up a completely new application area for plants as factories for human and animal pharmaceutical production. In the last two decades, a broad range of functionally active vaccines and therapeutic proteins have been produced in an increasingly diverse species of crops with a variety of different plant expression systems. Recent improvements in plant expression vectors have allowed for significant strides in a variety of new plantbased expression systems and their broad application. Meanwhile, the importance of the downstream processing for extraction and purification of pharmaceutical proteins from plant materials has been realized, and increasing efforts have been applied to address this critical issue. The public opinion and regulatory landscape have been evolving to a more welcoming climate where scientists and regulatory agencies can work together to develop regulations and guidelines for plant-made

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85

recombinant pharmaceutical proteins. At present stage, plants provide a serious viable �alternative to the traditional mammalian or microbial cell culture technologies, allowing for cost-effective, highly scalable, and safe production of pharmaceutical proteins.

PLANTS PROVIDE A NEW MANUFACTURING TECHNOLOGY FOR PHARMACEUTICAL PROTEINS The majority of pharmaceutical proteins are currently produced in mammalian cell cultures, with a few exceptions produced in Escherichia coli, yeast, or insect cells.8 As one of the fundamental technological revolutions in the pharmaceutical industry, the development and optimization of mammalian cell culture allow for uniform production of pharmaceutical proteins with high efficacy. However, mammalian and other cell culture-based production systems rely on the fermentation of cells suspended in bioreactors, which requires a large initial capital investment and, therefore, severely constrains their economic effectiveness and scalability. Application of such technologies in the developing world will not only face intellectual property challenges, but undoubtedly will also encounter further financial and logistical barriers that are difficult to overcome. Meanwhile, the worldwide demand for proteinbased pharmaceuticals has greatly increased for the last two decades. If the demand for such products cannot be met or if they are too costly to produce for health care systems, all of the advances in recombinant pharmaceutical protein creation and optimization, as well as the vast therapeutic potential of these agents, are made irrelevant. This urgent situation calls for the development of new production platforms that are cost-effective, scalable, and safe. Plant-based protein production technology combines the innovations in medicine and plant biology and has a great potential to produce affordable pharmaceutical proteins at a large scale. Plants can produce large volumes of proteins efficiently and sustainably, and, under certain conditions, with significantly lower manufacturing costs than mammalian cell cultures.9–13 Plants are also far less likely to introduce human or animal pathogens to humans compared to mammalian cells or whole transgenic animal systems. Unlike bacterial and other prokaryotic systems, plants share a similar endomembrane system and secretory pathway with human cells.14 Therefore, plant cells are able to efficiently assemble multiple subunit proteins and perform necessary posttranslational modifications on transgenic pharmaceutical proteins. The low-cost, high-scalability, and safety characteristics of plant production system offer an attractive alternative technology platform for both commercial pharmaceutical production and for manufacturing products for the developing world. In fact, an increasing number of academic and industrial laboratories are investing in plant-made protein pharmaceuticals (PMPs). While much research is still required to optimize plant production of pharmaceuticals and to validate them in large-scale clinical trials, the results to date show a promising technology that justifies commercial development. Currently, three strategies are primarily employed for the expression of pharmaceutical proteins in plants including the stable transformation of the nuclear genome,

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Transgenic Horticultural Crops: Challenges and Opportunities

Pharmaceutical protein gene

Clone into plant transformation vector

Integrate into a viral coding sequence for expression as a “by product” of viral replication

Transient expression: infect plant to initiate viral replication

Stable expression: transgene integrates into chloroplast genome

Stable expression: transgene integrates into nuclear genome

Incorporate into deconstructed viral sequences as part of Agrobacterium transformation vector Transient expression: high-level protein expression without forming infectious viral particles

Stable expression: regulated release of viral replicon from a viral genome integrated into plant genome

Stable expression: integrate into nuclear genome, high-level protein expression

FIGURE 4.1â•… Strategies for the expression of pharmaceutical proteins in plants. (Adapted from Chen, Q., Biol. Eng., 1, 291, 2008. With permission.)

the stable transformation of the chloroplast genome, and transient transformation (Figure 4.1). In the first two strategies, an expression cassette harboring the gene of interest is used to transform plant cells to enable the insertion of transgene into the nuclear or chloroplast genomes. The stable integration of the cassette into either the nuclear or plastid genome allows the Mendelian inheritance of the transgene over generations and, in turn, the stable expression of transgenic proteins. The resulting transgenic lines can be propagated to establish a master seed bank for future largescale production of pharmaceutical proteins. For most plant species, exogenous transgene cassette can be delivered into their genome by using Agrobacterium tumefaciens, which inserts DNA into the plant genome in a somewhat random fashion. Alternatively, ballistic methods with DNA-coated microprojectiles can be used to bombard plant cells. The ballistic “gene gun” is especially useful for plant species that are difficult to transform efficiently by Agrobacterium.15 Up to today, the ballistic method with tungsten or gold particles remains to be the only efficient way for the transformation of the chloroplast genome. In the third plant expression strategy, transgenes are not integrated into one of the plant genomes, but instead they are present in the plant nucleus transiently while being transcribed, and later the transcripts are transported into the cytoplasm and the transgenic proteins are translated. While transient expression can be carried out with both nonviral and viral vectors, the latter are more widely used due to their robustness in replication, transcription, and translation.16 Plant viral vectors have been used in several major formats to transiently express PMPs. For example, many vaccine epitopes have been fused to the viral coat protein so that they will be displayed of on the surface of the virus.17,18 PMP genes can also be inserted into an

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87

expression cassette, and the target protein-coding sequences will be expressed as a by-product of the viral genome replication cycle under the control of subviral promoters.19,20 In a variation, recombinant viral genomes can be integrated into plant chromosomes via transformation. The subsequent transcription of viral RNA will initiate vial replication and, in turn, drive the expression of the target pharmaceutical protein.21 Overall, the robust transcription and amplified translation of the plant viral components allow the transient expression systems to yield high-level accumulation of recombinant proteins. Viral “deconstruction” is a recent application of using viral genomes to create transient transgene expression.22,23 This newly deconstructed viral vector system is based on in planta assembly of replication-competent tobacco mosaic virus (TMV) and potato virus X (PVX) genomes from separate provector modules.23,24 Agrobacterium tumefaciens is used to deliver DNA modules along with a separate construct that produces a recombination integrase. Upon its expression, the integrase assembles separate DNA modules into a replication-competent TMV or PVX genome under the control of a plant promoter. This assembled DNA construct is then transcribed and spliced to generate a functional infective replicon. This “Maginfection” system combines the advantages of three biological systems.22,24 First, the laborious in vitro process of generating RNA-based vectors is eliminated due to the use of Agrobacterium as viral vector delivery tool. Second, the deletion of viral coat protein genes in this system allows the speed and high protein yield of a viral system without the threat of creating functional infectious particles. In addition, “Maginfection” integrates the posttranslational processing capacity of eukaryotic plant cells for producing complex proteins. Peaks of PMP accumulation are usually detected between 7 and 14 days after vector delivery.24 This system thus gains the flexibility of nuclear gene expression with the speed and expression amplification of viral vectors. All three plant expression strategies are likely to have their place in the commercial realm of producing pharmaceutical proteins. Expression systems using stably transformed plants represent an inexpensive and permanent genetic propagation source for pharmaceutical protein production.25 However, this development of technology has been hindered by several scientific and regulatory issues. First, it requires a relatively long time frame to create and select the initial stable transgenic plants. In addition, the expression level of the transgene product can be fluctuated wildly or unstable not only between individual plants but also between different generations of the same plant line. These uncertainties are often caused by the randomness of transgene insertion into the plant nuclear genome (the “position effect”) and by the phenomenon of posttranscriptional or siRNA-dependent gene silencing. The latter may be triggered by the presence of a particular mRNA in high concentration, leading to both immediate and potential permanent production instability.26–31 The potential risk of unwanted transgene outflow from fields with genetically modified (GM) plants to neighboring fields with non-GM crops or their wild relatives has also raised regulatory and public acceptance issues for this technology. These scientific and regulatory hurtles have been addressed by a growing number of academic and industrial laboratories. For example, in response to regular concerns for potential transgene escape through outcrossing, transgenic

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Transgenic Horticultural Crops: Challenges and Opportunities

plants are increasingly being grown in containment or in areas with natural geographic barrier to isolate transgenic plants from their agricultural or wild relatives. Once the transgenic plant lines are selected and the “master seed banks” are established, however, stable transgenic plants will provide the most scalable technology when large amounts of pharmaceutical protein are required for commercial production. Since there are thousands of chloroplasts in each cell, the copies of transgene per cell in transplastomic plants are significantly higher than that of nuclear transformation, which, in turn, drive higher level of transgenic protein production.32–34 The prokaryotic origin of chloroplasts also supports facile homologous recombination and polycistronic transcript production. These features allow the production of multiple transgenic proteins from one polycistronic mRNA as well as better transgene targeting in chloroplast genome to avoid the “position effect” and transgene silencing, which cause inconsistent level of transgenic protein production. Chloroplasts are maternally transmitted in most crops, which exclude chloroplast transgene escape from pollen transmission. Therefore, transplastomic expression of transgene is considered a superb alternative for regulatory compliance in ensuring transgene containment and improving the biosafety of transgenic plants.35 Due to lack of posttranslational machineries in chloroplasts, however, plant lines with stably transformed chloroplast genomes will mostly be applicable to produce proteins, which do not require posttranslational modifications for their function or proteins for diagnostic purposes.36 The transient, viral-based expression systems21,23 are focused on production speed, and therefore, the most convenient technology for obtaining the initial research material (mg–g level) used in preclinical studies. These transient expression systems rely on Agrobacteria’s ability to deliver the “deconstructed” viral genome to majority of plant cells, therefore, eliminating the need for viral systemic spreading function. This characteristic not only eliminates the concern of transgene loss during systemic spreading but also allows the application of this technology to a diversity of plant species beyond the natural virus hosts. Since nontransgenic plant materials are used for viral vector delivery, they can be readily stocked up for semi-large-scale productions. Overall, a rapid evaluation of pharmaceutical candidates and transition to a large-scale commercial production platform can be accomplished by employing the combination of both transient and stable transgenic plant technologies.

PROTEIN PHARMACEUTICAL PRODUCTION USING PLANT SYSTEMS Unlike mammalian cell cultures or bacterial fermentation systems, which require capital-demanding bioreactors and expensive tissue culture media and operations, pharmaceutical protein production by transient or stable transgenic plants is more economical in both initial setup and subsequent scale-up. This is due to decrease in large hardware investment and expensive culture media regardless of whether it is produced in the field or greenhouses. As a result, a variety of pharmaceutical proteins have been expressed and characterized in plant systems. Here, we will focus on the production of the two most important classes of pharmaceuticals: mAbs and vaccines.

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89

Monoclonal Antibodies Monoclonal antibodies (mAbs) have a wide range of important pharmaceutical applications. MAb market was projected to reach $16.7 billion by 2010, accounting for over a quarter of the total protein therapeutics market.37,38 As a result, new expression systems have to demonstrate their effectiveness in producing mAbs in order to be considered seriously as contenders for commercial production of pharmaceutical proteins. MAbs are extremely expensive to produce in mammalian culture system and, therefore, are costly to patients and health care systems. Development of new production platforms is indeed necessary to reduce the cost and increase the scalability of mAb manufacture. MAbs have been successfully expressed in stable transgenic plants since the early stage of PMP development, despite they are complex heterotetrameric glycoproteins.5 Since then, a variety of mAbs and their derivatives, such as secretory IgAs, single-domain fragments, single-chain variable fragment (scFv), and diabodies and, more recently, recombinant immune complexes (RICs), have been successfully produced in many host plant species, with increasing number and type of mAb being produced each year (Table 4.1).39–62 Our group recently published the first report that demonstrated the efficacy of a plant-produced mAb against a potentially lethal infection several days after exposure in an animal challenge model.58 Several mAbs have been selected for clinical evaluation with two having reached Phase II clinical studies (Table 4.2).63,64 For example, a TMV-based vector was demonstrated to be an efficient vector to express a human scFv for the treatment of non-Hodgkin’s lymphoma (NHL) in tobacco plants.65,66 The tobaccoproduced scFv is an effective anti-idiotype vaccine candidate in a murine NHL tumor challenge model and has been further evaluated in human clinical trials.67 This patient-specific anti-idiotype vaccine requires a speedy system for rapid production and patient treatment evaluation. In contrast to mammalian cell cultures, the TMV-based plant expression system has successfully provided such speedy and versatile production platform. Similar to stable transgenic plants, transient expression systems have been very successful in producing single subunit therapeutics such as scFvs.68,69 However, until recently, viral vector-based transient expression systems are incapable of producing full-length mAbs or any other heterooligomeric proteins efficiently. This is due to the fact that viral vectors built on the same virus backbone are typically “competing” with each other, resulting in early segregation and subsequent preferential amplification of one of the vectors in one cell.23,70–72 The MagnICON™ system composed of two sets of noncompeting vectors (TMV and PVX) has successfully resolved this problem.22,73 TMV and PVX do not compete with, nor dominate over, each other, because they interact with different host factors for their movement and replication. As a result, the efficient co-expression of light chain and heavy chain of mAb in the same cells occurs when two vectors harboring the two mAb genes are co-delivered into plants.23 Extensive studies have shown that this transient system can rapidly (within 10 days of vector delivery) produce fully assembled mAbs in high levels, reaching up to 0.8â•›g mAb/kg of fresh tissue weight (FW).23,58 Therefore, MagnICON system provides a potential large-scale production platform for rapid manufacturing of mAbs and other oligomeric proteins.

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Transgenic Horticultural Crops: Challenges and Opportunities

TABLE 4.1 Representative Antibodies, Antibody-Fragments, and Antibody Fusions Produced in Plants Antigen

Antibody Type

Pharmaceutical Target

Crop

References

Streptococcal surface antigen SA I/II Herpes simplex virus 2 glycoprotein B Tobacco mosaic virus coat proteins Human creatine kinase-MM Human carcinoembryonic antigen (CEA)

Secretory IgA/G

Tooth decay

N. tobacum

[43]

IgG

Microbicide for genital herpes

G. max

[59]

BispecificscFv Fab

O. sativa N. tobacum

[242]

A. thaliana

[44]

N. tobacum

[60]

Human rhesus D

IgG1

A. thaliana

[61]

Human chorionic gonadotropin (HCG)

scFv, diabody and IgG1

N. tobacum

[69]

Tumor surface antigen Rabies virus Herpes simplex virus glycoprotein D Human epidermal growth factor receptor Hepatitis B surface antigen Protective antigen (PA) of Bacillus anthracis Lipopolysaccharide (LPS) of S. enterica Paratyphi B Tumor-associate antigen GA733

scFv

Model bispecificscFv molecule Neurone and rheumatic diseases Tumor imaging for colorectal, lung, breast, and pancreatic carcinomas Alloimmunization, haemolytic disease of new born Diagnostic and therapeutic for HCG-expressing cancers or as contraceptive Therapeutic vaccine for NHL Rabies virus Genital herpes diagnostics Therapeutics for refractory colorectal cancer Hepatitis B virus infections Therapeutics for anthrax exposure, antiterrorism agent Salmonella enterica diagnostic and therapeutic Colorectal carinomas

N. benthamiana

[66,67]

N. tobacum C. reinhardtii

[45] [248]

Z. mays (corn seeds)

[46]

N. tobacum BY2 cells N. benthamiana

[62]

N. tobacum

[48]

N. tobacum

[40]

Diabody

IgG LSC (IgA type) IgG

IgG IgG

scFv

IgG

[72]

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Expression and Manufacture of Pharmaceutical Proteins

TABLE 4.1 (continued) Representative Antibodies, Antibody-Fragments, and Antibody Fusions Produced in Plants Antigen

Antibody Type

Human epidermal growth factor receptor HER2 Tetanus toxin C fragment Botulinum toxin A

scFv

Tumor-associated antigen oligosaccharide Lewis Y HIV p24

IgG2a

CD30

TGEV Hepatitis A virus

RIC scFv

p24-IgA Hc fusion IgG

Minibody, Full IgA scFv-Fc

Pharmaceutical Target

References

N. tobacum

[49]

N. tobacum

[186]

N. tobacum

[50]

N. tobacum

[55]

HIV vaccine

N. tobacum

[185]

Immunotherapy for Hodgkin lymphoma (HL) and anaplastic large cell lymphoma Therapeutic for TGEV Hepatitis A virus infection Targeting agent for breast, ovary, prostate and lung cancer P. aeruginosa infection

Lemna minor

[136]

N. clevelandii

[51]

A. thaliana

[54]

N. tobacum

[52]

N. tobacum

[39]

N. tobacum and BY2 cell culture, Z. mays N. benthamiana, Z. mays, A. thaliana N. benthamiana N. benthamiana

[42,143]

Human epidermal growth factor receptor HER1 P. aeruginosa serotype O6ad PS O side chain HIV gp 41

scFv

IgG

HIV infection

HIV gp 120

IgG

HIV infection

Ebola GP1 West Nile Virus E DIII

IgG, RIC IgG

Therapeutic for Ebola Therapeutic for WNV infection

IgG

Crop

Diagnostic for breast, ovary, prostate and lung cancer Vaccine for tetanus toxin Antidote for botulinum toxin A, antibioterrorism agent Breast and colorectal cancer

[53,64,134,135]

[21,263] [58]

Source: Adapted from Chen, Q., Biol. Eng., 1, 291, 2008. With permission. Note: scFv, single-chain variable fragment; RIC, recombinant immune complex; LSC, large single chain antibody; NHL, non-Hodgkin’s lymphoma.

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Transgenic Horticultural Crops: Challenges and Opportunities

TABLE 4.2 Plant-Derived Human Pharmaceuticals That Have Reached Clinical Trial Stage Pharmaceutical Target

Product

Arizona State University

E. coli heat-liable toxin Hepatitis B virus surface antigen Norwalk virus capsid protein Alpha Interferon (Locteron) scFvs

Traveler’s diseases Hepatitis B

Potato Tobacco

Phase I Phase I

Norwalk virus

Phase I

Hepatitis C

Potato and tobacco Duckweeds

NHL

Tobacco

Phase I

Gastric lipase Lactoferrin

Cystic fibrosis Gastrointestinal diseases Tooth decay Gaucher’s disease

Corn Corn

Phase II Phase I Phase II Phase III

Diabetes Hepatitis B

Tobacco Carrot cell culture Carrot cell culture Safflower Lettuce

Rabies virus

Spinach

Phase I

Biolex Therapeutics Large Scale Biology Corporation Meristem Therapeutics

Planet Biotechnology Protalix Biotherapeutics SemBiosys

Thomas Jefferson University

sIgA (CaroRx) Glucocerebrosidase GCD Acetylcholinestrase Insulin Hepatitis B virus surface antigen Rabies glycoprotein

Nerve agents

Crop

Clinical Trial Stage

Organization

Phase II

Phase I Phases I/II Phase I

Source: Adapted from Chen, Q. et al., Subunit vaccines produced using plant biotechnology, in New Generation Vaccines, Levine, M.M. Ed., 4th edn., Informa Healthcare USA, Inc., New York, 2009, p. 77. Copyright 2009. With permission from Informa Healthcare USA, Inc.

Some heterooligomeric pharmaceutical targets such as secretory IgA, IgM, and certain viral-like particle (VLPs) vaccine candidates contain more than two heterosubunits.74,75 High-level co-expression and assembly of these types of proteins in plant systems have not been reported as of today. A Gemini virus-based expression system developed recently by our group and a system based on a disabled cowpea mosaic virus RNA-2, however, may provide the potential for expressing these heterooligomeric proteins.21,76 Successful production of mAbs by both transient and stable transgenic plants clearly indicates that a plant production system is a viable alternative technology in producing pharmaceutical proteins.

Vaccines The World Health Organization estimates that approximately 30 million children are born each year without adequate immunization, contributing to the majority of deaths. One cost-effective strategy for this incredible health concern is to expand the

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utilization of available and emerging vaccine manufacturing technologies. Therefore, there is a tremendous need for affordable vaccines in the world, especially in developing countries. An ideal vaccination program should thus not only contain the vaccine components that can generate a neutralizing antibody response, a long-term memory B cells stimulation, and a T cell-mediated immunity, but should also include a cost-effective production technology that can produce large quantities of vaccines affordable to people in all parts of the world. Plants offer unique advantages for the production of subunit vaccines in terms of scale, speed, costs, yield, and safety. Our first work in 1992, describing the expression of the hepatitis B surface antigen in transgenic tobacco, marked the beginning of developing low-cost strategies for the production and delivery of vaccines in plants.6 Since then, more than 80 vaccine candidates have been expressed in a variety of plant hosts and vector systems.13,77 For example, plant-produced vaccines for an Influenza A M2E epitope,78 a human immunodeficiency virus 1 (HIV-1) epitope,79,80 HBsAg,81,82 and the S protein of transmissible gastroenteritis coronavirus (TGEV)83,84 have successfully demonstrated their immunogenicity. Immunization with heat labile enterotoxin B (LTB),85,86 the FP1 epitope of foot and mouth disease virus (FMDV),87,88 and P. aeruginosa epitopes89,90 yielded successful challenge trials. In addition, vaccine candidates for the respiratory syncytial virus (RSV) G and F proteins,91 the VP6 protein of rotavirus,92,93 the measles virus (MV), hemagglutinin (H) protein,94,95 an epitope from the major surface antigen of Plasmodium falciparum (PfMSP1),96,97 the VP7 protein of rotavirus,98 F1 and V antigen of Yersinia peptis,99,100 E Domain III of dengue virus,101 ESAT6—Ag85B antigens of Mycobacterium tuberculosis,102 E7 protein of human papilloma virus,103 pB5 antigenic domain of smallpox,103 and PA and LF domains of Bacillus anthracis,105,106 have also been successfully expressed in plants. These data collectively demonstrated that (1) plants are capable to express a broad portfolio of diverse vaccines and (2) plant-derived antigens were able to induce active protective humoral and cell-mediated immune responses. For current licensed vaccines, they are the products of complicated and expensive downstream purification and their transport to final point of use and storage require continuous refrigeration “the cold-chain.” Both of these requirements add significant cost to the immunization program and are especially detrimental to developing countries. Fresh or dried plant parts containing subunit vaccines may present an ambient temperature-stable product similar to the storage and transport of fresh fruits or dehydrated food products, thereby providing a possible solution. This motivation and advances in our understanding of mucosal immunity and tolerance have facilitated further exploration of edible vaccines.107–112 Studies have found that when oral or other mucosal surfaces such as nasal, intestinal, rectal, and vaginal mucosal surface are exposed to vaccines, a strong mucosal (sIgA) as well as systemic (IgG) immune response could be elicited.113–115 Therefore, at least in theory, oral immunization can be achieved by simply eating edible parts of plants containing the expressed subunit vaccines (Figure 4.2). The edible vaccine strategy is very appealing because it allows plants to not only serve as a low-cost expression system but also as a novel delivery vehicle for vaccines. It will eliminate or reduce the need for downstream processing and, in turn, further reduce the overall vaccine production cost. In addition, the needle-free delivery method and natural preservation of

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FIGURE 4.2â•… (See color insert.) Human volunteers ate transgenic potatoes in a clinical trial study by Tacket et al.238 Transgenic potatoes expressing LTB antigen were peeled, diced, and consumed by human volunteers. Non-transgenic potatoes were used as negative controls.

vaccines in plant tissue will circumvent major infrastructural challenges in vaccine storage, distribution, and delivery and allow the realistic implementation of an immunization program in countries where the “cold-chain” and other medical supplies are limited.116 The optimal candidates for oral subunit vaccine development in plants are aggregated pathogen protein complexes as they are naturally recognized at mucosal sites. For example, viral surface proteins that assemble into VLPs spontaneously and bacterial toxins that naturally aggregate to form multimeric complexes have been explored as plant-derived oral vaccine candidates.117–119 These natural mucosally targeted protein complexes have also been studied as carriers for developing other plant-derived oral vaccines as they can be linked to other antigens by protein fusion technologies.99,120,121 So far, moderate success has been achieved in the development of plant oral vaccines as many vaccine candidates have been expressed in edible plant parts, and five human clinical trials have been performed with orally delivered plant-produced vaccines (Table 4.2).122–125 Results from clinical trials indicated that serum antibodies and, in some cases, mucosal antibodies can be successfully stimulated by plant-derived oral vaccines. Although

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protective efficacy in humans has yet to be determined for a plant-derived vaccine, challenge studies have yielded promising results in animal models. Commercial success of this strategy, however, has not been achieved due to challenges including the possible degradation of vaccines by the digestion system, poor recognition of certain antigen at mucosal immune effector sites, and persistent concerns about whether edible vaccines would cause inappropriate antigenic tolerance. Technical difficulties also contributed to the lack of commercial success. For example, challenges in controlling the fluctuation of antigen content among individual plants or plant tissue often cause inconsistent vaccine protein content per unit of plant tissue. This, in turn, complicates the dosage control process and ultimately hinders the commercial application of this strategy.115 This particular problem, however, is being resolved as progress in genetic element manipulations has allowed more precise control of vaccine expression in plants.126 While oral vaccines still offer an attractive option for vaccine delivery in developing countries, the application of this strategy in the United States will face regulatory obstacles, since a vaccine without a strictly controlled dose would have tremendous difficulty in getting approval by the Food and Drug Administration (FDA). As a result, increasing efforts have been shifted to downstream processing to identify innovative and low-cost ways to purify or partially purify vaccines from plant tissue (see below). Purified vaccines can be formulated with adjuvants and delivered through different immunization routes including oral and other mucosal surfaces. Thus, this new strategy is driven not only by the necessity to develop PMP vaccines with defined dosage content but also to address the needs of a creative formulation with adjuvants to enhance potency as well as creating multivalent and multicomponent vaccines.77,127–129 On going research by our group has allowed us to establish downstream processing standard operating procedures (SOPs) for extracting and purifying Norwalk virus capsid protein (NVCP) from N. benthamiana plants under stringent federal current Good Manufacturing Practices (cGMP) guidelines.13 We anticipate that our NVCP material produced under the cGMP regulation will be used in a human clinical trial as a vaccine candidate for sexually transmitted infections later in 2011.130 Due to its advantages in speed, scale, and cost for subunit vaccine production, the application of plant expression systems has recently been expanded to develop and produce vaccines for emerging and re-emerging diseases, cancers as well as agents of biological warfare. For example, vaccines for smallpox,104 anthrax,106 dengue virus,101 avian influenza A virus,131 and a personalized cancer vaccine for follicular B-cell lymphoma67 are some of these new vaccine candidates produced in plants.

GLYCOSYLATION OF PLANT-MADE PHARMACEUTICALS As with recombinant proteins produced in other systems, glycosylation of plantderived pharmaceutical proteins is a critical issue. Glycosylation is depending on a series of posttranslational modification steps by host cells, and the outcome can seriously impact pharmacokinetics, antigen/receptor binding, stability, effector functions,  and efficacy of the mAbs and other PMPs. As such, recombinant proteins produced by heterologous systems may have appreciable structural and functional differences from the native molecules and could be immunogenic in humans. In general, the glycosylation of proteins in plants is similar to that of mammalian cells. However,

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plants do have unique plant-specific β-1,2-xylose and core α-1,3-fucose residues on complex N-linked glycans and lack terminal β1,4-Gal and N-acetylneuraminic acid (Neu5Ac) residues.57 Extensive studies have shown that variations in glycosylation patterns do not always lead to loss of in vitro or in vivo function or cause side effects in humans.132 Despite this fact, a variety of strategies have been developed to circumvent the potential problems associated with the difference between plant and human glycosylation patterns. For example, glycoengineering, involving sophisticated genetic and metabolic engineering of plant with mammalian glycosylation enzymes, has recently emerged as the most promising approach to “humanized” glycosylation in plants.133 In one of these studies, a “humanized knockout” Arabidopsis thaliana plant line has been generated to express a mAb.134 Similar transgenic tobacco (N. benthamiana) lines with humanized glycans were generated later.135 In these plants, enzymes for the biosynthesis of plant-specific glycans are inactivated, and results have shown that the mAb was glycosylated with a mammalian-like pattern and was structurally equivalent to the same mAb derived from mammalian cells. In the last few years, the feasibility of glycoengineering at a biomanufacturing scale was demonstrated.136 For example, an RNA interference (RNAi) strategy was employed to suppress the expression of endogenous enzymes (α-1, 3-fucosyltransferase and β-1,2-xylosyltransferase) for plant-specific glycans. Results showed that mAbs produced in plant lines with mutated α-1,3-fucosyltransferase and β-1,2-xylosyltransferase had no detectable plant-specific N-glycans. More significantly, the plant-produced mAb contained only a single major N-glycan species and showed improved antibody-dependent cell-mediated cytotoxicity (ADCC) and effector cell-binding activities in comparison with the equivalent mAb produced in mammalian cells.135 For certain class of therapeutic proteins, the presence of the terminal Neu5Ac residues is required for their biological activities and stability. Adding the terminal Neu5Ac residues to plant-derived proteins is the last remaining challenge for the full humanization of plant N-glycosylation pathway. Recently, attempts were made to express the three key enzymes of the mammalian Neu5Ac biosynthesis pathway, namely, UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-N-acetylneuraminic acid synthetase, in plants. Results showed that significant amounts of Neu5Ac were generated when the three enzymes are simultaneously expressed in plants.137,138 Plant glycoengineering has demonstrated its ability in producing mAbs with better glycoform uniformity and enhanced functionality than mammalian cell cultures, therefore demonstrating its potential in producing pharmaceutical proteins with fully humanized glycans.

DOWNSTREAM PROCESSING OF PLANT-DERIVED PROTEIN PHARMACEUTICALS In the last decade, significant progress has been realized in boosting PMP expression levels through the enhancement of genetic elements for stable transgenic expression and the development of viral-based transient expression systems. As a result, PMP field has increasingly shifted its attention to developing technologies for efficient PMP recovery and delivery. While “immunization-by-eating” still presents a viable

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opportunity to deliver plant-derived vaccines in the developing world, considerations of licensure and regulatory issues have pointed to necessity of developing processing technologies to produce vaccines and therapeutics with defined unit dosage.139 Developing new technologies to minimize cost associated with downstream processing is crucial for the full realization of economic effectiveness of PMP technology, since it can account for up to 80% of the total cost in a therapeutic protein production.140 A well-developed downstream process will increase manufacturing productivity, reduce cost of goods, enhance scalability, and ensure the compliance of the manufacturing procedures with FDA’s cGMP regulations. Similar to other production systems, the goal of PMP downstream processing is to recover the maximal amount of highly purified target protein with minimal number of steps including tissue harvesting, protein extraction, purification, and product formulation (Figure 4.3). The unique properties of plant tissues, however, present both specific challenges and opportunities for each of the processing steps. Column chromatography has been extensively employed in PMP purification, while nonchromatographic methods are being explored aiming to provide alternatives for large-scale manufacture.

Column Chromatography Chromatography has been the method of choice for PMP purification due to its resolving power. As a result, many PMPs have been successfully purified from a diversity of host plants with a variety of chromatographic techniques tailored for each individual PMP protein based on its solubility, size, pI, charge,

Seed bank

(A)

–80 Master cell bank

(B)

Agrobacterium production Media

1

(C)

2 3 Working Inoculum Fermentation cell bank preparation

FIGURE 4.3â•… (See color insert.) General large-scale production steps for plant-made pharmaceuticals from green leafy materials using deconstructed viral vector transient expression strategy. (A) Plant cultivation. (B) Infiltration and growth. (C) Downstream processing. (Adapted from Chen, Q. et al., Subunit vaccines produced using plant biotechnology, in New Generation Vaccines, Levine, M.M. Ed., 4th edn., Informa Healthcare USA, Inc., New York, 2009, p. 77. Copyright 2009. With permission from Informa Healthcare USA, Inc.)

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hydrophobicity, and affinity to specific ligands and the parallel characteristics of plant host proteins.13,64,141–143 For mAb-based PMPs, Protein A or G affinity chromatography provides a superb purification step144 and has allowed successful purification of many plant-derived mAbs and their derivatives.21,23,64,145 Usually, a two-column purification scheme including a protein A capture step and an anion exchange polish step is sufficient to achieve high purity.58,146 However, direct loading of plant extracts onto Protein A (or G) columns often causes resin fouling and poor binding of target protein due to the fact that plants produce much more solid debris than other organisms and are rich in phenolics and alkaloids.142,147,148 For example, clarified tobacco extract was found to foul Protein A Sepharose and reduce the resin capacity.147 Ion exchange, gel filtration chromatography, and ultrafiltration methods have been employed to remove the interfering molecules, but with very little success.147 The solution for this problem came from a nonchromatographic method called aqueous two-phase partitioning system (ATPS). It was found that ATPS could effectively remove phenolics, toxic alkaloids and other plant compounds from tobacco extract. This extract could then be directly loaded to a Protein A column without causing resin fouling, which allowed efficient separation of target mAbs (anti-HIV mAb 2F5 and 2G12) from tobacco host proteins.149 ATPS has also been shown to be effective in removing interfering plant molecules from extracts of other plant species.150–152 Protein A is an expensive resin and requires extreme low pH (2.0–2.5) in the elution step. In large-scale productions, the resins are routinely recycled up to 50 times to maintain a reasonable cost.153 This practice, however, often leads to additional cost in cleaning and revalidation of the resins, which is necessary to prevent leaching of degraded ligands into products.145,149 Extensive researches have been conducted to identify alternative resins with equivalent or better affinity to antibodies or cheaper ways to produce protein A resin. One alternative is to create a single-use protein A-related resin with low production cost. For example, an attempt has been made to produce transgenic protein A or its fragments in plants to take advantage of the inexpensive nature of plant expression systems.145 Low-cost Protein A alternatives have also been developed in the form of fusion proteins. For instance, Protein A or its affinity components were genetically fused to molecules that are capable of polymerizing or binding to high molecular weight structures. Antibodies that bound to these Protein A-decorated large particles can then be separated from plant host proteins by simple centrifugation or ultrafiltration (see discussion of “affinity precipitation” on page 102). Examples of this strategy include fusion proteins of Protein A with self-polymerizing bacteriophage capsid proteins,154,155 high molecular weight bacterial S-layer proteins, cellulose-binding proteins, and starch-binding domains.156–160 Oleosins, a class of plant seed oil-body-associated small proteins, have also been explored as Protein A fusion partners.161–163 This technology has been developed and matured to the commercially ready stage by a Canadian Biotechnology company Sembiosys, in which the oleosin–Protein A fusion-mAbs complex can be purified by extracting with oil bodies (www.sembiosys.com). The yield of oleosin–Protein A fusion molecules in safflower plants is relatively low and needs further improvement.145 An immunoabsorbent nanoparticle technology based on a tobamovirus, displaying a 133-amino

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acid protein A fragment, has also been developed recently. These nanoparticles can be produced at high levels in plants, and the density of the Protein A fragment on the nanoparticle is high, satisfying the matrices of high mAb-binding capacity and lowcost economics required for a successful processing technology. A simple mAb purification process can be performed with these plant-derived nanoparticles to achieve >90% product purity.145 In addition to protein A, many conventional chromatographic resins have been investigated for mAb purification. These resins are less expensive, easier to scale up, and more resistant to chemical and biological degradations and, therefore, are more suitable for industrial manufacturing. It was showed that a simple S-Sepharose FF cation exchange chromatography followed by immobilized metal affinity chromatography (IMAC) on Zn-iminodiacetic acid (IDA)-agarose can efficiently purify an anti-HIV mAB (2G12) from corn seeds with 90% purity.64 Histamine Sepharose was also used as an affinity resin in purifying mAbs from plant extracts.143 A >95% purity of a mAb can be achieved by this resin in a single purification step when used with a maize seed extract. When the same resin was used to purity the mAb from tobacco leaf extracts, however, the binding capacity and the purification efficiency were slightly reduced due to the interference of tobacco-specific phenolics, alkaloids, and host proteins.143 “Pseudobiospecific ligands” represent another class of non-Protein A resins for mAb purification. They include biologically engineered peptides and protein domains as well as synthetic mimetics exhibiting different degree affinity to antibodies. Comparative studies showed that the mAb affinity of these ligands (including hydrophobic, thiophilic, hydroxyapatite, chelating metal ions, and mixed mode affinity ligands) is generally lower than that of Protein A, but high enough for mAb selectivity.164 Overall, under optimized conditions, the mAb-binding specificity and capacity of these resins are comparable to those of the Protein A, but vary depending on the source and the isotype of mAbs.165–167 Advantages of pseudobiospecific ligands include reduced cost of production, mild elution condition, facile sterilization and validation, resistance to chemical and biological degradation, and lower toxicity, all of which makes them favorable as large-scale mAb purification alternatives. Furthermore, chemical modifications allow these affinity ligands to acquire new affinities to mAb-derivatives that traditional Protein A lacks, therefore broadening the range of application. For example, artificial PpL is able to purify a Fab to >90% purity with 77% recovery.168 MEP Hypercel, IDA-Ni, epitope peptides, Mabsorbent, affibodies, and artificial Protein A are some examples of pseudobiospecifc ligands.146 Not all artificial affinity ligands have been tested for plant-derived mAb purification. However, there is no doubt that they will contribute substantially to the development of economical Protein A alternatives for large-scale manufacturing of plant-derived mAbs. For purification of non-mAb-based vaccines and therapeutics, there are no platforms based on a universal affinity resin equivalent to Protein A. Usually, the purification is carried out by multiple steps of conventional chromatographic methods and has to be developed individually based on the properties such as the pI, size, hydrophobicity, and stability of the target protein and the contaminating host molecules.142 This time-consuming and challenging process calls for the

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need to develop more “universal” or versatile purification methods. Technologies employing on affinity tags provide possibilities for such more universal solution.169–173 In this strategy, instead of attempting to identify a specific ligand to the target protein, a highly selective affinity tag with known ligand is fused to the target protein by genetic engineering. Successful examples of this strategy include pharmaceutical proteins tagged with biotin, histidine, glutathione S-transferase (GST), and c-Myc.174–179 In addition, tags developed for mAb purification (see antibody purification discussed above) such as bacteriophage capsid proteins, S-layer proteins, cellulose-binding domains, starch-binding domains, oleosins, and tobamovirus nanoparticle have also been successfully used to purify vaccines and other transgenic proteins.156,162,180–183 The high affinity of the Fc fragment of immunoglobulin to Protein A provides an excellent tag for PMP purification. As a result, many antigen proteins have been fused to the heavy chain of antibodies and purified by Protein A affinity chromatography.184,185 In fact, genetic fusions of antigens to mAbs against them have created a new class of highly potent vaccines called RICs.186 The mAb component in RICs allows not only facile purification but also enable them to induce significantly stronger immune responses in host animals without the help of adjuvants. In addition to providing a convenient purification tool, affinity tag often adds several other benefits to the target protein such as enhancing its yield, solubility, stability, and promoting its correct folding.184,187 However, no individual affinity tag alone is ideal in delivering all and every possible benefit. Consequently, the tandem affinity tag purification (TAP) or combinatorial tagging strategy was developed to deliver the maximum possible benefit.175,188–193 For example, a dual affinity His6-MBP affinity tag vector was developed, in which the MBP moiety improves the yield and enhances the solubility of the recombinant protein while the His tag facilitates its purification.194,195 Since all tags have the potential to interfere with the structure and biological activity of the target recombinant protein, for certain applications, they have to be removed from the purified protein by site-specific protease. Challenges still remain in identifying proteases with both high efficiency and precision for tag removal.196,197 Some endoproteases such as thrombin, TEV protease, and 3C protease often leave one or two extra amino acid residues at their cleavage sites and produce unnatural N-terminus on the target protein.198 In contrast, proteases like enterokinase and factor Xa have better precision and leave target proteins with their native N-termini.197 However, these enzymes have relatively low efficiency and routinely require high enzyme concentrations and long incubation time,197 which often lead to nonspecific cleavage at cryptic sites. Regardless, the inclusion of proteases in the manufacturing process should be avoided whenever it is possible as it would require extra step(s) to eliminate the protease from the final product. This additional step(s) would, in turn, increase the overall processing cost and raise additional regulatory concerns.196,197 Consequently, nonenzymatic affinity tag removal techniques have been explored to avoid such problems.200–202 For example, intein, a self-splicing protein element, has been used as a fusion partner of affinity tags to replace protease cleavage sites.202 The preliminary results of this strategy are promising. However, further testing in high-throughput settings is necessary before it can be applied for large-scale PMP manufacturing.

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One of the most critical strategies for PMP purification is to take early measures to prevent undesirable host proteins, microorganisms, and other contaminants from entering purification feed streams. For example, bioburden, environmental contaminants, and certain plant host proteins should be reduced as early as possible prior to the chromatographic purification steps. These early measures are not only important in simplifying the subsequent purification process, but are also essential in ensuring the regulatory compliance of the final product. For example, whole leaf homogenization should be avoided for isolating apoplast-targeted proteins with molecular weight <50â•›k Da. Instead, they should be extracted directly into the extraction buffer by a specialized centrifugation technique.203 Since this technique allows the extraction of target protein without breaking plant cells, the purification process is greatly simplified as a result of a reduction in host protein contaminants. The carbon assimilation enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo) is the major contaminating protein in plant leaves, and it should be removed from the extract by centrifugation under low-pH buffer conditions (∼pH 5.3) prior to the chromatographic steps.117 Phenolics, alkaloids, and other plant specific molecules should also be eliminated by nonchromatographic separation methods (see below) prior to column chromatography to prevent resin fouling.149 Ongoing research by our group has established a purification process for vaccine candidate NVCP based on low-pH precipitation and conventional chromatographic steps.130 SOPs based on this purification scheme have been established and used in a cGMP production of NVCP for human clinical trials.

Nonchromatographic Separations The promise of PMP production on an agricultural scale demands a downstream processing platform with extraordinarily large-scale capabilities. Despite the widespread use of chromatographic separations in PMP purification, there are growing concerns that this approach cannot be scaled up sufficiently for very large-scale production. As a result, alternative technologies have been explored for very largescale downstream processing of PMPs, aiming to not only enhance manufacture scalability, but also to provide separation power similar or better than that of column chromatography, while reducing separation steps and the manufacture cost. These technologies include ATPS, precipitation, and membrane chromatography.204 ATPS has tremendous scalability potential and is able to handle large biomass load compared to other separation methods.205,206 It relies on the incompatibility of certain polymer mixtures in forming homogeneous solutions, but instead forming two immiscible phases under specific concentrations.207 As discussed earlier, ATPS is an effective method in eliminating phenolics, toxic alkaloids, pigments, and other compounds from plant extracts.149,150 ATPS has been successfully used in purifying tobacco-derived anti-HIV mAbs and protein subunit vaccine candidates as well as nanoparticles including VLPs.149,208–210 ATPS offers high separation rates and, therefore, minimizes the contact time between PMP protein and deleterious plant extract. Inclusion of this system in downstream processing will facilitate plant extract clarification, removal of phenolics, alkaloids and other harmful compounds, and partial purification of target proteins. Overall, the advantages in low-cost, scalability, high

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biocompatibility, and ability for continuous processing qualify this system as an initial fractionation strategy for large-scale processing. Precipitation is a simple and robust method to separate a precipitable protein from other soluble protein contaminants. One of the strategies to make a target protein precipitable is to fuse it to a stimulus-responsive polymer. Since this type of polymer can transit reversibly between water-soluble and water-insoluble state depending on temperature, pH, or other environmental conditions, the target protein can be easily separated from host proteins by a simple centrifugation or filtration step while the polymer is in a water-insoluble state.211 Both centrifugation and filtration work well in separating precipitated proteins from soluble ones; the latter, however, has the advantage of the possibility of being operated in a continuous process.212 Several PMPs including human interleukin-4, 10,213 scFv214 and anti-HIV mAbs215 have been purified by precipitation using an elastinlike peptide (ELP). A temperature-induced “inverse transition cycling” allowed ELP-fusion proteins to transition to an insoluble state. As a result, the fusion proteins were easily purified by centrifugation.216 Affinity precipitation is a special case of precipitation methods and has been suggested as one of the most promising nonchromatographic techniques for largescale processing.146 The power of affinity precipitation is derived from the unique combination of the specificity and selectivity of biorecognition with the simplicity, robustness, and high concentration factors associated with precipitation. This method does not require the fusion between target protein and the precipitation carrier. Instead, an affinity ligand is covalently linked to a stimulus-responsive polymer or other molecules capable of forming high molecular weight particles. Since there is no fusion between the PMP and the precipitation carrier, structural and functional integrity of the target protein is better preserved, and the need for proteases to remove fused precipitation carriers is eliminated. Affinity precipitation has been extensively tested with plant-derived mAbs. As discussed earlier, protein A or its affinity components have been fused to bacteriophage capsid proteins, bacterial S-layer proteins, oleosins, cellulose, and starch-binding domains, and recently to tobamovirus nanoparticles. Binding of mAbs to these Protein A-precipitation carrier fusions has allowed efficient purification of a variety of mAbs from plants with comparable purity and recovery as mAbs purified by Protein A chromatography, but with much superior cost effectiveness and scalability.145 Similarly, other affinity ligands have also been successfully used to create agents for affinity precipitation of affinity-tagged vaccines and non-mAb therapeutics.162,183 Another variation of affinity precipitation is magnetic separation, in which magnetic nanoparticles are decorated with affinity ligands, and nanoparticle-bound target-protein can be then separated from other soluble proteins by using magnets.217,218 Plant-derived mAbs have been successfully purified by such method.24 In addition to using magnetic crystals, magnetic particles can also be produced by certain bacteria, which can be engineered to display Protein A or other affinity ligands on their surface.219,220 Another attractive alternative to column chromatography is membrane separation  or membrane chromatography. The effectiveness of these membrane-based separation techniques has been successfully demonstrated in purifying mAbs and other therapeutic proteins.221,222 For example, an anion-exchange membrane efficiently removed impurities from the feed stream when used in a flow-through mode.223,224

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The binding capacity of this membrane is much higher than that of the equivalent anion-exchange resins. One of the attractive features of membranes is that they especially have higher binding capacity for large-size impurities, which are abundant in feed streams and a major cause for column clotting.225 Since plant feed streams have unusually abundant high-molecular-weight impurities, membrane separation may provide a promising noncolumn chromatographic solution for problems associated with large chromatographic columns, such as low flow rate and expensive operations in column packing, cleaning, and validation. In fact, preliminary tests have shown that this technique can be successfully applied in purifying PMPs. For example, separations with a combination of cation-exchange and protein A membranes have successfully purified a tobacco-derived antipseudomonas human mAb (06ad).226 The high binding capacity and scalability of this technique make it highly suitable for very large-scale manufacturing operations. Many speculated that membrane separation is likely to be the first nonchromatographic method used in production-scale processing of biopharmaceuticals.164 Regardless of the purification method used or the nature of a specific target protein, the overall extraction and purification design has to be robust, scalable, costeffective, and compliant to cGMP regulations. An ideal purification scheme should have no more than three processing steps in order to achieve high product purify while maintaining a practical recovery rate. In addition, the PMP purification and manufacturing process must be governed by an independent quality management system (QMS) similar to regulations used by other expression systems.227,228 Accordingly, materials and processes widely accepted by the pharmaceutical industry should be considered first during process-development to minimize future regulatory expenditure. Furthermore, in spite of the lack of animal viruses and other infectious agents in plants, it must be validated that the purification process is adequate in removing endotoxin, phenolics, and alkaloids as well as potential herbicide and insecticide contaminations from the final product.

PLANT SPECIES USED FOR PMP PRODUCTION A diversity of plant species and expression systems has been used to express pharmaceutical proteins. The choice of a system for a particular transgenic protein production depends not only on specific biological properties of the target protein/host plant species but also on economical, regulatory, and social factors. The optimal plant expression system for a target pharmaceutical protein must have (1) one or more expression cassettes with genetic elements that can drive the high-level expression of the transgene, (2) the ability to target the transgenic protein to a cellular compartment where it is stable, and (3) technologies to isolate and purify the target protein from host plants. The optimal plant species coupled to the optimal expression system must also (1) produce high-yield biomass, (2) have facile scalability, (3) have sufficient biomass stability during the required storage period, and (4) have a reasonable land and labor requirement for plant growth and harvesting. Other economic factors may include, for example, the value of the protein product, which will dictate the affordability of investment in new equipment and labor as well as in converting existing farming and food processing infrastructure for molecular pharming.

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Other  factors  such  as  the distance between sites of biomass production and processing will determine the required period for biomass storage, which, in turn, will determine the choice between leafy and seed production system. In addition, considerations have to be in place in choosing an expression system/species to address regulatory compliance and public concerns on issues of transgene containment, product biosafety, and others related to GM plants in general.

Leafy Crops Tobacco and its related Nicotiana species have been one of the earliest crop systems for successful transgenic plant pharmaceutical expression, in which target proteins can be extracted directly from harvested leaves. Tobacco is one of the easiest plants to transform, and, therefore, the molecular tools for transgene expression in this species are well established. Biomass yield for tobacco is higher than most of other leafy crops. Tobacco is also a prolific seed producer allowing rapid scalability. The existence of large-scale processing infrastructures in traditional tobacco industry provides the advantage of quick adaptation for processing pharmaceutical-containing biomass. Since tobacco is a nonfood, nonfeed crop, the risk of contamination of human food chain or animal feed chain by transgenic tobacco is relatively low. All these advantages of tobacco make it one of the most advanced candidates for molecular farming in commercial production of pharmaceutical proteins. Other green leaf expression systems also include alfalfa, soybean, Arabidopsis thaliana, lettuce, and spinach. They share many common advantages with tobacco including easy to engineer and high biomass yield. One of the most advantageous properties of the leaf expression systems is that it allows the flexibility to choose from all three plant expression technology platforms including stable nuclear, chloroplast transformation, and viral vector-based transient expressions. This flexibility increases the chance of success for many target proteins, since multiple options are available to identify an optimal expression platform based on the property of the target protein or the nature of the particular project. Alfalfa and soybean are legumes and, therefore, can obtain organic nitrogen through nitrogen fixation. This may provide some advantages in reducing the consumption of nitrogen-containing chemicals. Alfalfa also can be harvested up to nine times a year, adding to a higher biomass yield. Arabidopsis thaliana has been a model plant for plant molecular genetics and molecular biology research. It has been used to produce a variety of pharmaceutical proteins. However, the small size of this plant will limit its scalability. Lettuce and spinach are palatable without cooking and, therefore, have been particularly targeted to produce edible vaccines for oral delivery.124 Because they are food crops, the biosafety and containment of the transgene are major concerns for their commercial application. One of the drawbacks for leaf expression systems is the potential interference of expressed transgenic proteins on plant growth and development. For this type of proteins, inducible promoters may be advantageous as transgenic proteins are not expressed during plant growth under the control of such promoters but will only be produced upon application of specific inducer agent post-biomass harvest.229 Leaves also tend to contain more pigments, alkaloids, and polyphenols, which sometimes may be difficult to remove from the target protein.

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The amount of these compounds varies between plant species. Tobacco and related Nicotiana plant leaves generally contain high levels of phenolics and toxic alkaloids. However, tobacco cultivars with low-alkaloid and other toxic metabolites have been developed for pharmaceutical protein production.230 Protein stability is one of the major issues for leafy crops. Transgenic proteins expressed in leaves accumulate in a more aqueous environment and thus tend to be unstable. This translates into a short target protein shelf life in harvested green biomass due to proteolysis. They, therefore, must be quickly transported to a processing facility at a controlled temperature to be extracted for target protein or to be processed by freezing, drying, or other means for short-term storage. In contrast, pharmaceutical proteins produced in cereal seeds can remain stable for a long period at ambient temperature (see below). Regulatory concerns for nonfood, nonfeed pharmaceutical producing leafy crops include the potential leaching of recombinant products into the environment and the exposure of herbivores to transgenic proteins. Again, this type of risks can be reduced by using inducible promoters to drive the expression of the transgene so that transgenic proteins can only be synthesized in a controlled environment post biomass harvest.229,231

Seed Crops Expression of pharmaceutical protein has been explored in seeds of several crops including maize, rice, wheat, barley, tobacco, oilseed rape, and a variety of beans.232–234 For certain transgenic proteins that required long-term storage in biomass before processing, expression in seeds driven by seed-specific promoters is preferred over leafy expression systems. In contrast to the “watery” leaves, seeds are natural desiccators, which preserve functional proteins by a drying process during their maturation and, therefore, allow long-term protein stability during storage. Studies have shown that mAbs produced in corn seeds remain stable after 3 years of storage at room temperature and retain their biological activities.235 Phenolic and alkaloid compounds are not present in cereal crop seeds, thus simplifying downstream purification process and reducing the overall production cost accordingly. Cereal seeds do contain higher levels of lectins that could be difficult to remove from the final product especially if the target proteins are glycosylated. Legumes and other oil crops such as oilseed rape are useful alternative systems for pharmaceutical production, because their oil bodies can be exploited to facilitate target protein purification. For example, SemBioSys Genetics has developed an oleosin-fusion technology to target pharmaceutical proteins to oil bodies. As a result, these proteins can be purified by a simple extraction process.236 The level of expression of recombinant proteins in seeds is generally lower than that in tobacco leaves. This is mostly due to the fact that many of the seed expression systems are still at early developing stage and have ample rooms for further optimization.234 An inherent disadvantage of producing protein in seeds is that it can only be evaluated until seeds are produced after a long growth cycle. Within the three expression platforms, only nuclear stable transformation is feasible for seed expression. Transient expression techniques for seed expression have not been well established, therefore, hindering the quick assessment of expression feasibility for a given product. Seed crops offer both biosafety advantages and drawbacks for expression of recombinant proteins. On one hand, it reduces the

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risk of product exposure to herbivores and other nontarget organisms. On the other hand, however, the necessity of pollination for seed formation presents a new risk for potential transgene escape into the environment. Maize was one of the first seed crops used for recombinant protein production.237 Compared with other cereal plants, it has several advantages including high biomass yield, ease of transformation, and the availability of large-scale processing technology. Since maize has been a model plant for genetic studies, there is a vast genetic and molecular knowledge base available for its genetic manipulation. Maize has been a successful seed crop for producing a variety of recombinant proteins including mAbs, biotech reagents, even commercial grade b-glucuronidase and avidin.10 For example, mAb (2G12), a promising anti-HIV microbicide candidate, was produced in transgenic maize.64 This research showed that maize-produced mAb neutralized HIV as effectively as the same antibody produced in Chinese hamster ovary (CHO) cells. Despite these advantages, disastrous transgene outflow incidents happened in this crop have raised serious regulatory and public concerns on using maize or other food crops for recombinant protein production (see “Regulatory Concerns and Public Acceptance” section of this chapter). A comparative study was conducted to examine the yield of a scFv in different seed and leafy crops including wheat, rice, pea, and tobacco. Results showed that the target protein yield per unit of biomass was in the order of rice seeds > tobacco leaf > wheat seeds = pea seeds.234 The highest yield per unit of land, however, belongs to tobacco plants because of its high biomass production. Since different promoters were used for seed and leaf expression, the results have to be evaluated with caution and need further validation. This study did demonstrate that choosing a optimal production crop was relying on multiple factors such as target protein yield per unit biomass and biomass yield per acre and can be only determined on a protein-by-protein basis.

Fruit and Tuber Crops Pharmaceutical protein production in fruit and tuber crops provides the opportunity for a novel approach of pharmaceutical delivery through oral route by ingesting palatable crop organs. This unique advantage makes them ideal hosts for the production of oral vaccines or topical therapeutics. For example, potato plants have been explored as one of the earliest plant systems for expression of a diversity of vaccines.81,238,239 Our group and others also conducted at least three clinical trials by administering uncooked transgenic potato tubers expressing exterotoxic E. coli LT-B or VLPs of NVCP to human volunteers. Fruits such as tomatoes and strawberries are more edible than raw potatoes and thus are more feasible for edible vaccine development. Tomatoes have been used for both vaccine and therapeutic mAb expression due to its high biomass yield and ease of production in greenhouses for transgene containment.240 Bananas have been envisioned for a long time as hosts for edible vaccine production and delivery especially in Africa where vaccination for infectious diseases is urgently needed. However, difficulties in generating transgenic banana plants and the long time frame needed to grow fruit-bearing crops still present hurdles for its practical application. Since fruits and edible tubers are food crops, one of the biggest obstacles for their commercialization is the regulatory and public concern for biosafety and transgene containment.

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Aquatic Plants One of the most innovative and the simplest whole-plant transgenic expression systems may be that of employing the common duckweed, Lemna. Largely developed by Biolex Therapeutics (Pittsboro, NC), Lemna-based LEX Systems expression technology allows rapid pharmaceutical product development and potential seamless transition to large-scale manufacture under GMP conditions. This technology is enabled by several advantages of the duckweed plants including small genome for facile molecular manipulation, ease of transformation and regeneration, rapid clonal propagation, and biomass growth requiring only water and inorganic nutrients. Furthermore, smaller pharmaceutical proteins produced in duckweed can be directly secreted into the culture medium. This would eliminate the need for homogenization of plant tissue and significantly simplify target protein purification and, in turn, reduce the overall processing cost. Since Lemna plants produce neither pollen nor seeds, but are propagated vegetatively in simple containers, the system also offers high degree of containment. The clonal nature of Lemna plants and the feasibility of growing them in a controlled environment also allow better batchto-batch product reproducibility. Like any other bioreactor or container-based production systems, the scalability of the duckweed system could be limited compared with those of other crops. Biolex Therapeutics has produced 35 proteins with their LEX  Systems and their leading drug candidate, Locteron®, an α-interferon for the treatment of hepatitis C, is being examined in a Phase 2b human clinical trial in Europe (http://www.biolex.com, 2011). Duckweeds have also been shown as an excellent host system for therapeutic mAb production. Because of its small genome, its glycosylation pathway can be manipulated with relative ease. This added advantage allows Lemna-derived mAbs to have uniform and “humanized” glycans.136 It was shown that Lemna-produced CD30â•›mAb contained only a single major N-glycan species and showed more superior ADCC and effector cell-binding activities than those of equivalent mAb produced in mammalian cells. In addition to Lemna minor used by Biolex, other species in the Lemnaceae family such as Pirodela oligorhiza and several Wolffia are also being exploited as a source for recombinant protein production.241

Plant Suspension Cell Cultures and Single-Cell Cultures of Algae Plant suspension cells are liquid culture of cells or cell aggregates derived from callus tissue. Transgenic plant cell cultures can be established by transforming suspension cells with Agrobacterium or by using stable transgenic plant to produce the callus tissue. Since these suspension-cultured plant cell lines can be grown in a controlled environment, they allow better product reproducibility, a high level of containment, and the potential to manufacture protein under cGMP similar to mammalian cell culture practices. Even though these culture systems share similar limitations in scalability with current bacterial, yeast, and mammalian cell cultures, they still provide useful alternatives in producing pharmaceutical proteins owing to the fact that plant cell lines require less expensive culture media to grow. Moreover, pharmaceutical proteins smaller than 20â•›k Da can be directly secreted into the culture

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medium readily for purification. As a result, many pharmaceutical proteins including mAbs, scFv, and their fusion proteins have been produced in tobacco or ricecell-suspension cultures.242 In fact, the first plant-made pharmaceutical approved by the FDA was a poultry vaccine against Newcastle disease produced in tobacco suspension cells.243 In addition to tobacco cells, rice and carrot cells are also being developed for pharmaceutical production. For example, carrot cells are being used by Protalix Biotherapeutics, a biotechnology company in Israel, to produce the enzyme glucocerebrosidase (GCD) as an enzyme replacement therapy agent to treat the Gaucher disease. They have shown that GCD produced in cultured carrot cells has several advantages over GCD derived from CHO cells, including increased glycan efficacy and consistency and longer product half-life in blood serum.244 Single-cell cultures of algae such as chlorella and chlamydomonas offer short generation time, fast vegetative biomass growth, and require inexpensive cultivation in water and inorganic nutrients with sunlight. The biased alga-specific genetic codon usage has been problematic for expressing human or animal genes in these algae, but has been resolved by using alga-codon optimized synthetic genes.245 It was initially proposed that the algae-based protein expression systems could be easily maintained in ponds or larger water bodies for large-scale biomass production. However, biosafety and environmental concerns have limited the use of these cultures in controlled simple containers or more sophisticated flow-through transparent “reactors.” The green alga Chlamydomonas reinhardtii has been a successful model alga in expressing several pharmaceutical proteins including foot-and-mouth disease virus VP1-CTB fusion protein and a scFv for herpes simplex virus glycoprotein D with transgenes transformed into either its nuclear or chloroplast genomes.246–247 Other algae species such as Chlorella ellipsoidea and Dunaliella salina have also been explored for PMP production.246,249

REGULATORY CONCERNS AND PUBLIC ACCEPTANCE PMP is a potentially revolutionary technology for providing vast affordable pharmaceutical proteins to the world. However, as other transformative technological developments, PMP is also plagued with controversy and has stimulated significant public debate. One of the major issues confronting commercial PMP production is the public perception that the bioproduction of pharmaceuticals with food or feed crops could potentially contaminate the human or animal food chains. Some in the agriculture and food industry also share such concerns for reasons that any potential contamination could negatively impact their businesses.250 Many of these concerns are caused by several contamination incidents that have special resonance for the PMP field.251 The most publicized episode was the one involving a Texas-based biotech company, ProdiGene Corp. In 2002, corn and soybean harvested in Nebraska and Iowa were found contaminated with ProdiGene’s transgenic corn seeds intend to express a transgene of TGEV for a candidate pig diarrhea vaccine. As a result of the contamination, more than 12,000 tons of soybeans have to be destroyed, and ProdiGene was penalized by the U.S. government with a fine of $250,000.251 In addition to the potential contamination of human food supplies, other concerns among the general public and scientists include the potential escape of transgene into the environment

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through outcrossing with conventional crops or their related wild species, exposure of microorganisms and insects or other herbivores to transgene and transgenic products, and possible immunological tolerance caused by edible vaccines. To ultimately address these public concerns, it requires the diligent stewardship by the PMP community, scientific and technical advancement in PMP production platforms, and a close collaboration between different regulatory agencies to establish clear and effective policies and guidelines for transgenic crops and PMPs.

Better Stewardship of the PMP Community One of the embarrassing lessons learned from the ProdiGene incident by the PMP community was the need to improve their stewardship on PMPs. Practices without consideration and common sense caused several incidents and controversies, which are unnecessary and avoidable. For example, when Ventria Bioscience planned to test their PMP carrying transgenic rice in field plots, instead of growing them in containment or in a location far away from areas of major food rice production, it proposed to grow them in California Central Valley where rice production for human food is a major industry.250 This proposal angered some local farmers, and the controversy forced Ventria to abandon its original plan and moved its testing plots to Kansas. The ProdiGene and Ventria controversies have alarmed PMP community to develop stricter self-disciplinary guidelines on PMP containment. As a result, the Biotechnology Industry Organization established a “Reference Document for Confinement and Development of Plant-Made Pharmaceuticals in the United States” to express their commitments in ensuring the safety of PMPs during all stages of development and production, including full compliance with all applicable laws, regulations and guidance, and the regulatory framework from regulatory agencies that oversee the PMPs.250 This document also provided guidelines for practical measures in preventing inadvertent human exposure to the transgenic plants and their expression product(s) through food and feed and in minimizing occupational and environmental exposure. This has led the PMP community to reconsider the risks associated with using food crops as PMP hosts and to develop new expression platforms to reduce such risks.

Alternative PMP Production Technology and Risk Control Nonbiological Containment To prevent human and environmental exposure of transgene or PMP products, the simplest form of containment would be having a physical barrier between PMP host plants and their surroundings. The barrier can be green houses or geographic distances. For example, SemBioSys, a Canadian PMP company, has adopted this approach and decided to grow its insulin-producing transgenic safflower in an isolated geographical area of Chile far away from any food or feed safflower crops.250 Crops such as tomato, which is routinely grown in large-scale greenhouses for food production, are being explored as expression platforms for PMPs as they can be easily adapted for PMP production in containment.240 Other physical measures can

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also be used to prevent the exposure of environment to the transgene and transgenic products. For example, PMP tobacco plants are routinely harvested through mowing before reproductive organs are formed, and transgenic corn is detasseled mechanically to prevent transgene flow to the environment through pollen outcrossing.252 In addition to meeting the traditional requirements such as high PMP yield and easy scalability, many new expression systems were developed with the added advantage for containment. For example, since duckweeds produce no pollen or seeds, but propagate vegetatively and can grow in closed containers, the duckweeds-based LEX Systems offers a higher degree of containment than other systems. Similarly, plant suspension cultures are also grown in controlled containers, allowing better biosafety control. Furthermore, the feasibility of growing aquatic plant and plant cell culture in a controlled environment also allows better batch-to-batch product reproducibility and the ability of protein production under cGMP. In fact, these advantages have enabled them to be operated similarly to conventional bacterial or mammalian cell cultures and to achieve considerable commercial success ahead of other PMP systems. These successes include that tobacco suspension cells produced the first FDA-approved plant-made poultry vaccine and that PMPs produced by Lemna or carrot cell cultures are being examined in Phase 2 or Phase 3 human clinical trials (http://www.biolex.com, 2011). The transient expression technology based on “deconstructed” viral vectors also provides advantages in biosafety and containment because it does not involve the production of transgenic plants. Since high level of PMP product can be obtained easily with this system, the amount of biomass required for the production of a given quantity of PMP is reduced, which provides the possibility of performing the processes in contained facilities. This, in turn, will eliminate the need for field-grown plants, and the environmental concerns for the use of “deconstructed” viral vectors. Furthermore, this system does not require fermenters or containers to grow biomass, giving it an easier scalability than fermenter-based duckweed or plant tissue cultures. This transient expression technology, therefore, represents one of the most promising PMP platforms for commercialization. Biological Containment It is necessary in certain circumstances to use biological containment, which is less dependent on human oversight than physical containment as an additional measure to ensure the biosafety of PMP production. Biological containment usually employs molecular or genetic techniques to either create a biological barrier against transgene flow into other plants or organisms or to prevent the production of transgenic products before host plants being harvested or moved into a controlled environment. Transgenes can flow from PMP transgenic plants in two major forms—pollen and seeds carried by various vectors. Correspondingly, molecular genetic strategies for containing transgene outflow have been focused on the areas of male sterility, maternal inheritance, transgene mitigation, and seed sterility (also called “terminator” technology).253 Male sterility is one of the early strategies of biological containment and is the only strategy that has been applied to commercial transgenic plant (rape seeds) production (Plant Genetic systems, Ghent, Belgium). Male sterile plants are unable to produce mature, viable pollen, therefore preventing

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transgene flow via pollen.254 Examples of such systems include pollen sterility in tobacco and oilseed rape with a barnase gene driven by a tapetum-specific promoter255 and male sterility in tobacco with constitutive expression of the Agrobacterium rhizogenes rolC sterility gene.256 These systems have been shown to be successful in preventing transgene spread from GM flowering plants into other related species.254 Since most crop chloroplasts genes are inherited maternally, transgenes inserted into the plastid genomes are unlikely to be transmitted by pollen transmission.35 As discussed earlier, transplastomic plants have been extensively used for PMP expression partially due to their advantage in ensuring transgene containment. To further reduce the risk of transgene flow-through pollination, a double fail-safe strategy was developed using combinations of both male sterility and transplastomic genomes.257 One of the handicaps of the male sterile or transplastomic plants is that they can still be pollinated by nontransgenic varieties, and the resulting hybrids can serve as pollen donors to spread transgene into the environment. Transgene mitigation is a strategy to address the leakage and unidirectionality handicap of male sterility and maternal inheritance. In this technology, a mitigation gene, which encodes a trait detrimental to wild plants, is genetically linked to the transgene.258 If the transgene is escaped and transmitted to an unintended host plant, the mitigation gene will co-escape with the transgene, and its expression would prevent the hybrid from competing with wild plants and setting seed. For example, this strategy based on the Arabidopsis gibberellic acid insensitive gene (Δgai) was able to induce dwarfism in tobacco plants. When Δgai transgenic plants or hybrids are grown together with wild-type tobacco, the dwarf transgenic or hybrid plants cannot compete effectively for sunlight with the taller wild-type plants for photosynthesis and rarely survive to set seed, therefore, preventing the establishment of transgene in the population.258 Furthermore, Δgai gene also causes male sterility in tobacco plants. The combination of male sterility and mitigation effect of Δgai makes it a superb gene for preventing transgene outflow and for mitigating the flow if sterility containment failure or reverse gene influx occurred.254 In addition to pollens, seeds are also major routes of transgene and transgenic product spreading by human errors or animal activities. To resolve this problem, several “genetic use restriction technologies” (GURT) including the “terminator” (US patent 5,723,765) and its alternatives have been developed to produce “conditional-sterilized” seeds.259 Seeds from plants engineered with these “terminator”-like genes are usually nonviable, unless the plants are intentionally exposed to specific activating molecules or environmental stimulus. These chemical or environmental conditions will induce the expression of a repressor gene, which, in turn, will inactivate the terminator gene. Several such technologies have been shown to be effective.260,261 For example, transgenic plants carrying the barnase gene routinely produce non-viable seeds, but can become viable only after an exposure to a heat treatment at 40°C.261 Such seed sterility technologies will allow effective blocking of transgene flowthrough volunteer seeds and will prevent recurrence of incidents similar to the ones caused by ProdiGene. Inducible expression systems have been developed to prevent human, animal, and environmental exposure to plant transgenic protein products. In these systems, transgene are driven by an inducible promoter instead of a constitutive one. As a result, transgene

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transcription and translation remain being switched off during plant growth until upon the application of the specific inducer molecules or stimulus. Therefore, no transgenic products are produced in the field, and they are only made after being harvested and transported into a controlled environment. Such inducible systems have been applied to both nuclear and chloroplast transgenic plants.229,231 It is important to note that none of the above biological containment is absolute or is working in all circumstances. Novel containment strategies with high level of reliability and simplicity are still highly desired for PMP plants.

Regulatory Policies regarding PMP In the United States, the regulatory responsibilities of PMPs are distributed among three relevant agencies: the US Department of Agriculture (USDA), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). The regulatory environment has evolved tremendously since the infancy of the PMP development.227,262 At the beginning, the novelty and complexity of this technology caused confusions on which regulatory agencies should regulate it or where the PMP would fit into the regulatory agencies’ structured framework for biotechnology pharmaceuticals. This uncertain regulatory environment contributed to the inertia of large pharmaceutical companies toward PMPs and other new production systems. With the great stride of PMP field in the last few years, however, the regulatory environment has also evolved from a chaotic situation in which the regulatory bodies could not coordinate with each other to a more welcoming climate where the regulatory agencies have a more defined role in regulating PMPs and coordinate with each other and begin to work with scientists to establish more clear policies and guidelines specifically for PMPs.262,264

CONCLUSION Enormous strides have been made in the past decade with respect to PMP expression and optimization. A wide spectrum of pharmaceutical proteins has been successfully produced with active functionality in a variety of crop and noncrop plant systems. Plant expression strategies based on the combination of viral vector transient expression and stable transgenic plants have the greatest potential to provide a technology with the speed and scalability required for producing affordable high-quality pharmaceutical proteins to meet the increasing global demand. New technologies for downstream processing have received increasing attention and are being developed to realize the full potential of the PMP platform. However, like any biotechnology innovation, the ultimate commercial success of this technology cannot solely rely on its scientific and technical merits. Before reaching its full commercial potential, extensive work has to be done to overcome challenges in areas of its social acceptance and in attracting the interest of capital investment and big pharmaceutical companies. Ultimately with technical optimization and careful implementation under regulatory guidelines, plant expression technology will support the production of pharmaceutical proteins with scalability, speed, efficiency, cost-effectiveness, and safety at a commercial scale.

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REFERENCES

1. Farnsworth, N.R. et al., Medicinal plants in therapy, Bull World Health Organ, 63, 965, 1985. 2. Duke, J., Medicinal plants and the pharmaceutical industry, in New crops, Janick, J. and Simon, J. Eds., Wiley, New York, 1993, 664. 3. Ma, J., Drake, P., and Christou, P., The production of recombinant pharmaceutical proteins in plants, Nat Rev Genet, 4, 794, 2003. 4. Barta, A. et al., The expression of a nopaline synthase—Human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue, Plant Mol Biol, 6, 347, 1986. 5. Hiatt, A., Cafferkey, R., and Bowdish, K., Production of antibodies in transgenic plants, Nature, 342, 76, 1989. 6. Mason, H.S., Lam, D.M., and Arntzen, C.J., Expression of hepatitis B surface antigen in transgenic plants, Proc Natl Acad Sci USA, 89, 11745, 1992. 7. Haq, T.A. et al., Oral immunization with a recombinant bacterial antigen produced in transgenic plants, Science, 268, 714, 1995. 8. Yin, J. et al., Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes, J Biotechnol, 127, 335, 2007. 9. Kostandini, G., Mills, B.F., and Norton, G.W., The potential impact of tobacco biopharming: The case of human serum albumin, Am J Agr Econ, 88, 671, 2006. 10. Hood, E.E., Woodard, S.L., and Horn, M.E., Monoclonal antibody manufacturing in transgenic plants—Myths and realities, Curr Opin Biotechnol, 13, 630, 2002. 11. Giddings, G., Transgenic plants as protein factories, Curr Opin Biotechnol, 12, 450, 2001. 12. Nandi, S. et al., Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain, Transgenic Res, 14, 237, 2005. 13. Chen, Q., Expression and purification of pharmaceutical proteins in plants. Biol Eng, 1, 291, 2008. 14. Vitale, A. and Pedrazzini, E., Recombinant pharmaceuticals from plants: The plant endomembrane system as bioreactor, Mol Interv, 5, 216, 2005. 15. Hansen, G. and Wright, M.S., Recent advances in the transformation of plants, Trends Plant Sci, 4, 226, 1999. 16. Lico, C., Chen, Q., and Santi, L., Viral vectors for production of recombinant proteins in plants, J Cell Physiol, 216, 366, 2008. 17. Porta, C. et al., Cowpea mosaic virus-based chimaeras. Effects of inserted peptides on the phenotype, host range, and transmissibility of the modified viruses, Virology, 310, 50, 2003. 18. Lico, C. et al., Peptide display on Potato virus X: Molecular features of the coat proteinfused peptide affecting cell-to-cell and phloem movement of chimeric virus particles, J Gen Virol, 87, 3103, 2006. 19. Scholthof, H.B., Morris, T.J., and Jackson, A.O., The capsid protein gene of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes, Mol Plant–Microbe Interact, 6, 309, 1993. 20. Musiychuk, K. et al., A launch vector for the production of vaccine antigens in plants, Influenza, 1, 19, 2007. 21. Huang, Z. et al., High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system, Biotechnol Bioeng, 106, 9, 2010. 22. Gleba, Y., Klimyuk, V., and Marillonnet, S., Magnifection—A new platform for expressing recombinant vaccines in plants, Vaccine, 23, 2042, 2005. 23. Giritch, A. et al., Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors, Proc Natl Acad Sci USA, 103, 14701, 2006. 24. Marillonnet, S. et al., In planta engineering of viral RNA replicons: Efficient assembly by recombination of DNA modules delivered by Agrobacterium, Proc Natl Acad Sci USA, 101, 6852, 2004.

114

Transgenic Horticultural Crops: Challenges and Opportunities

25. McCarthy, A.A., Biolex, Inc.: Pharmaceutical production in plants, Chem Biol, 12, 141, 2005. 26. Baulcombe, D., Overview of RNA interference and related processes, Curr Protoc Mol Biol, 26, 1, 2003. 27. Baulcombe, D., RNA silencing in plants, Nature, 431, 356, 2004. 28. Baulcombe, D., RNA silencing, Trends Biochem Sci, 30, 290, 2005. 29. Baulcombe, D.C., Short silencing RNA: The dark matter of genetics? Cold Spring Harb Symp Quant Biol, 71, 13, 2006. 30. Baulcombe, D.C., Molecular biology: Amplified silencing, Science, 315, 199, 2007. 31. Baulcombe, D.C. and Molnar, A., Crystal structure of p19—a universal suppressor of RNA silencing, Trends Biochem Sci, 29, 279, 2004. 32. Daniell, H., Chloroplast genetic engineering, Biotechnol J, 1, 31, 2006. 33. Daniell, H., Production of biopharmaceuticals and vaccines in plants via the chloroplast genome, Biotechnol J, 1, 1071, 2006. 34. Daniell, H. et al., Chloroplast-derived vaccine antigens and other therapeutic proteins, Vaccine, 23, 1779, 2005. 35. Ruf, S., Karcher, D., and Bock, R., Determining the transgene containment level provided by chloroplast transformation, Proc Natl Acad Sci USA, 104, 6998, 2007. 36. Daniell, H., Khan, M., and Allison, L., Milestones in chloroplast genetic engineering: An environmentally friendly era in biotechnology, Trends Plant Sci, 7, 84, 2002. 37. Pavlou, A.K. and Belsey, M.J., The therapeutic antibodies market to 2008, Eur J Pharm Biopharm, 59, 389, 2005. 38. Monocolonal Antibodies 2007: Competitive Landscape & Pipeline Insight. Market Research Report, January 2007, 158 pp. Arrowhead Publishers, Minneapolis, MN. 39. Mclean, M.D. et al., A human anti-Pseudomonas aeruginosa serotype O6ad immunoglobulin G1 expressed in transgenic tobacco is capable of recruiting immune system effector function in vitro, Antimicrob Agents Chemother, 51, 3322, 2007. 40. Ko, K. et al., Inhibition of tumor growth by plant-derived mAb, Proc Natl Acad Sci USA, 102, 7026, 2005. 41. Kelley, B., Very large scale monoclonal antibody purification: The case for conventional unit operations, Biotechnol Prog, 23, 995, 2007. 42. Sack, M. et al., Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures, FASEB J, 21, 1655, 2007. 43. Ma, J. et al., Generation and assembly of secretory antibodies in plants, Science, 268, 716, 1995. 44. Peeters, K., De Wilde, C., and Depicker, A., Highly efficient targeting and accumulation of a Fab fragment within the secretory pathway and apoplast of Arabidopsis thaliana, Eur J Biochem, 268, 4251, 2001. 45. Ko, K. et al., Function and glycosylation of plant-derived antiviral monoclonal antibody, Proc Natl Acad Sci USA, 100, 8013, 2003. 46. Ludwig, D. et al., Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed, Human Antibodies, 13, 81, 2004. 47. Hull, A.K. et al., Human-derived, plant-produced monoclonal antibody for the treatment of anthrax, Vaccine, 23, 2082, 2005. 48. Makvandi-Nejad, S. et al., Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scFv) antibody against Salmonella enterica serotype paratyphi B, Transgenic Res, 14, 785, 2005. 49. Galeffi, P. et al., Expression of single-chain antibodies in transgenic plants, Vaccine, 23, 1823, 2005. 50. Almquist, K.C. et al., Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco, Vaccine, 24, 2079, 2006.

Expression and Manufacture of Pharmaceutical Proteins

115

51. Alamillo, J.M. et al., Use of virus vectors for the expression in plants of active fulllength and single chain anti-coronavirus antibodies, Biotechnol J, 1, 1103, 2006. 52. Semenyuk, E.G. et al., Expression of single-chain antibody-barstar fusion in plants, Biochimie, 89, 31, 2007. 53. Jin, C. et al., A plant derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits, Glycobiology, 18, 235, 2008. 54. Van Droogenbroeck, B. et al., Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds, Proc Natl Acad Sci USA, 104, 1430, 2007. 55. Brodzik, R. et al., Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells, Proc Natl Acad Sci USA, 103, 8804, 2006. 56. Pujol, M. et al., Fighting cancer with plant-expressed pharmaceuticals, Trends Biotechnol, 25, 455, 2007. 57. Gomord, V. et al., Production and glycosylation of plant-made pharmaceuticals: The antibodies as a challenge, Plant Biotechnol J, 2, 83, 2004. 58. Lai, H. et al., Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice, Proc Natl Acad Sci USA, 107, 2419, 2010. 59. Zeitlin, L. et al., Comparison of an anti-HSV-2 monoclonal IgG and its IgA switch variant for topical immunoprotection of the mouse vagina, J Reprod Immunol, 40, 93, 1998. 60. Vaquero, C. et al., A carcinoembryonic antigen-specific diabody produced in tobacco, FASEB J, 16, 408, 2002. 61. Bouquin, T. et al., Human anti-rhesus D IgG1 antibody produced in transgenic plants, Transgenic Res, 11, 115, 2002. 62. Yano, A., Maeda, M., and Takekoshi, M., Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen, J Med Virol, 73, 208, 2004. 63. Fischer, R., Twyman, R.M., and Schillberg, S., Production of antibodies in plants and their use for global health, Vaccine, 21, 820, 2003. 64. Ramessar, K. et al., Cost-effective production of a vaginal protein microbicide to prevent HIV transmission, Proc Natl Acad Sci USA, 105, 3727, 2008. 65. Mccormick, A.A. et al., Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants, Proc Natl Acad Sci USA, 96, 703, 1999. 66. Mccormick, A.A. et al., Individualized human scFv vaccines produced in plants: Humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig, J Immunol Methods, 278, 95, 2003. 67. Mccormick, A.A. et al., Plant-produced idiotype vaccines for the treatment of nonHodgkin’s lymphoma: Safety and immunogenicity in a phase I clinical study, Proc Natl Acad Sci USA, 105, 10131, 2008. 68. Vaquero, C. et al., Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves, Proc Natl Acad Sci USA, 96, 11128, 1999. 69. Kathuria, S. et al., Efficacy of plant-produced recombinant antibodies against HCG, Hum Reprod, 17, 2054, 2002. 70. Dietrich, C. and Maiss, E., Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants, J Gen Virol, 84, 2871, 2003. 71. Diveki, Z., Salanki, K., and Balazs, E., Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement, Biochimie, 84, 997, 2002. 72. Hull, R. and Plaskitt, A., Electron microscopy on the behavior of two strains of alfalfa mosaic virus in mixed infections, Virology, 42, 773, 1970. 73. Marillonnet, S. et al., Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants, Nat Biotechnol, 23, 718, 2005.

116

Transgenic Horticultural Crops: Challenges and Opportunities

74. Latham, T. and Galarza, J.M., Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins, J Virol, 75, 6154, 2001. 75. Pushko, P. et al., Evaluation of influenza virus-like particles and Novasome adjuvant as candidate vaccine for avian influenza, Vaccine, 25, 4283, 2007. 76. Sainsbury, F. and Lomonossoff, G.P., Extremely high-level and rapid transient protein production in plants without the use of viral replication, Plant Physiol, 148, 1212, 2008. 77. Chen, Q. et al., Subunit vaccines produced using plant biotechnology, in New Generation Vaccines, Levine, M.M. Ed., 4th edn. Informa Healthcare USA, Inc., New York, 2009, p. 306. 78. Denis, J. et al., Development of a universal influenza A vaccine based on the M2e peptide fused to the papaya mosaic virus (PapMV) vaccine platform, Vaccine, 26, 3395, 2008. 79. Yusibov, V. et al., Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1, Proc Natl Acad Sci USA, 94, 5784, 1997. 80. Marusic, C. et al., Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1, J Virol, 75, 8434, 2001. 81. Richter, L.J. et al., Production of hepatitis B surface antigen in transgenic plants for oral immunization, Nat Biotechnol, 18, 1167, 2000. 82. Gao, Y. et al., gd T cells provide an early source of IFN-g in tumor immunity, J Exp Med, 198, 433, 2003. 83. Gomez, N. et al., Oral immunogenicity of the plant derived spike protein from swinetransmissible gastroenteritis coronavirus, Arch Virol, 145, 1725, 2000. 84. Tuboly, T. et al., Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants, Vaccine, 18, 2023, 2000. 85. Chikwamba, R. et al., A functional antigen in a practical crop: Maize synthesizes LT-B protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT), Transgenic Res, 11, 479, 2002. 86. Streatfield, S.J. et al., Development of an edible subunit vaccine in corn against enterotoxigenic strains of Escherichia coli, In Vitro Cell Dev Biol Plant, 38, 11, 2002. 87. Carrillo, C. et al., Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants, Viral Immunol, 14, 49, 2001. 88. Dus Santos, M.J. et al., Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen, Vaccine, 23, 1838, 2005. 89. Gilleland, H.E. et al., Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection, FEMS Immunol Med Microbiol, 27, 291, 2000. 90. Staczek, J. et al., Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa, Vaccine, 18, 2266, 2000. 91. Yusibov, V. et al., Peptide-based candidate vaccine against respiratory syncytial virus, Vaccine, 23, 2261, 2005. 92. Saldana, S. et al., Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.) fruit by expression of capsid proteins VP2 and VP6 and immunological studies, Viral Immunol, 19, 42, 2006. 93. Matsumura, T., Itchoda, N., and Tsunemitsu, H., Production of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants, Arch Virol, 147, 1263, 2002.

Expression and Manufacture of Pharmaceutical Proteins

117

94. Webster, D.E. et al., Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralising antibodies in mice following mucosal vaccination, Vaccine, 24, 3538, 2006. 95. Webster, D.E. et al., The development of a plant-based vaccine for measles, Vaccine, 23, 1859, 2005. 96. Ghosh, S. et al., Expression of Plasmodium falciparum C-terminal region of merozoite surface protein (PfMSP119), a potential malaria vaccine candidate, in tobacco, Plant Sci, 162, 335, 2002. 97. Wang, L. et al., Immunogenicity of Plasmodium yoelii merozoite surface protein 4/5 produced in transgenic plants, Int J Parasitol, 38, 103, 2008. 98. Li, J.-T. et al., Immunogenicity of a plant-derived edible rotavirus subunit vaccine transformed over fifty generations, Virology, 356, 171, 2006. 99. Santi, L. et al., Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system, Proc Natl Acad Sci USA, 103, 861, 2006. 100. Mett, V. et al., A plant-produced plague vaccine candidate confers protection to monkeys, Vaccine, 25, 3014, 2007. 101. Saejung, W. et al., Production of dengue 2 envelope domain III in plant using TMVbased vector system, Vaccine, 25, 6646, 2007. 102. Dorokhov, Y.L. et al., Superexpression of tuberculosis antigens in plant leaves, Tuberculosis, 87, 218, 2007. 103. Massa, S. et al., Anti-cancer activity of plant-produced HPV16 E7 vaccine, Vaccine, 25, 3018, 2007. 104. Golovkin, M. et al., Smallpox subunit vaccine produced in Planta confers protection in mice, Proc Natl Acad Sci USA, 104, 6864, 2007. 105. Phelps, J.P., Dang, N., and Rasochova, L., Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis, J Virol Methods, 141, 146, 2007. 106. Chichester, J.A. et al., Immunogenicity of a subunit vaccine against Bacillus anthracis, Vaccine, 25, 3111, 2007. 107. Mor, T.S., Gomez-Lim, M.A., and Palmer, K.E., Perspective: Edible vaccines—A concept coming of age, Trends Microbiol, 6, 449, 1998. 108. Ogra, P.L., Faden, H., and Welliver, R.C., Vaccination strategies for mucosal immune responses, Clin Microbiol Rev, 14, 430, 2001. 109. Czerkinsky, C. et al., Mucosal immunity and tolerance: Relevance to vaccine development, Immunol Rev, 170, 197, 1999. 110. Mestecky, J. et al., Routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans, Behring Inst Mitt, 98, 33, 1997. 111. Zivny, J.H. et al., Mechanisms of immune tolerance to food antigens in humans, Clin Immunol, 101, 158, 2001. 112. Hathaway, L.J. and Kraehenbuhl, J.P., The role of M cells in mucosal immunity, Cell Mol Life Sci, 57, 323, 2000. 113. De Magistris, M.T., Mucosal delivery of vaccine antigens and its advantages in pediatrics, Adv Drug Delivery Rev, 58, 52, 2006. 114. Eriksson, K. and Holmgren, J., Recent advances in mucosal vaccines and adjuvants, Curr Opin Immunol, 14, 666, 2002. 115. Streatfield, S.J., Mucosal immunization using recombinant plant-based oral vaccines, Methods, 38, 150, 2006. 116. Nochi, T. et al., From the cover: Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination, Proc Natl Acad Sci USA, 104, 10986, 2007. 117. Santi, L. et al., An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles, Vaccine, 26, 1846, 2008.

118

Transgenic Horticultural Crops: Challenges and Opportunities

118. Moravec, T. et al., Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine, Vaccine, 25, 1647, 2007. 119. Dong, J.-L. et al., Oral immunization with pBsVP6-transgenic alfalfa protects mice against rotavirus infection, Virology, 339, 153, 2005. 120. Choi, N.-W., Estes, M.K., and Langridge, W.H.R., Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response, J Biotechnol, 121, 272, 2006. 121. Shin, E.-A. et al., Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants, Protein Expr Purif, 47, 99, 2006. 122. Walmsley, A.M., Krik, D.D., and Mason, H.S., Passive immunization of mice pups through oral immunization of dams with a plant-derived vaccine, Immunology, 86, 71, 2003. 123. Tacket, C.O. et al., Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn, Vaccine, 22, 4385, 2004. 124. Kapusta, J. et al., A plant-derived edible vaccine against hepatitis B vaccine, FASEB J, 13, 1796, 1999. 125. Yusibov, V. et al., Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine, Vaccine, 20, 3155, 2002. 126. Streatfield, S.J., Approaches to achieve high-level heterologous protein production in plants, Plant Biotechnol J, 5, 2, 2007. 127. Takagi, H. et al., From the cover: A rice-based edible vaccine expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2-mediated IgE responses, Proc Natl Acad Sci USA, 102, 17525, 2005. 128. Takaiwa, F., A rice-based edible vaccine expressing multiple T-cell epitopes to induce oral tolerance and inhibit allergy, Immunol Allergy Clin North Am, 27, 129, 2007. 129. Yu, J. and Langridge, W.H., A plant-based multicomponent vaccine protects mice from enteric diseases, Nat Biotechnol, 19, 548, 2001. 130. Herbst-Kralovetz, M., Mason, H.S., and Chen, Q., Norwalk virus-like particles as vaccines, Expert Rev Vaccines 9, 299, 2010. 131. Nemchinov, L.G. and Natilla, A., Transient expression of the ectodomain of matrix protein 2 (M2e) of avian influenza A virus in plants, Protein Expr Purif, 56, 153–159, 2007. 132. Chargelegue, D. et al., A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice, Transgenic Res, 9, 187, 2000. 133. Saint-Jore-Dupas, C., Faye, L., and Gomord, V., From planta to pharma with glycosylation in the toolbox, Trends Biotech, 25, 317, 2007. 134. Schahs, M. et al., Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern, Plant Biotechnol J, 5, 657, 2007. 135. Strasser, R. et al., Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure, Plant Biotechnol J, 6, 392, 2008. 136. Cox, K.M. et al., Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor, Nat Biotechnol, 24, 1591, 2006. 137. Castilho, A. et al., Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis, Plant Physiol, 147, 331, 2008. 138. Castilho, A. et al., In planta protein sialylation through overexpression of the respective mammalian pathway, J Bio Chem, 285, 15923, 2010. 139. Kaiser, J., Is the drought over for pharming? Science, 320, 473, 2008. 140. Roque, A.C.A., Lowe, C.R., and Taipa, M.A., Antibodies and genetically engineered related molecules: Production and purification, Biotechnol Prog, 20, 639, 2004. 141. Nikolov, Z.L. and Woodard, S.L., Downstream processing of recombinant proteins from transgenic feedstock, Curr Opin Biotechnol, 15, 479, 2004.

Expression and Manufacture of Pharmaceutical Proteins

119

142. Menkhaus, T.J. et al., Considerations for the recovery of recombinant proteins from plants, Biotechnol Prog, 20, 1001, 2004. 143. Platis, D. and Labrou, N.E., Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine, J Sep Sci, 31, 636, 2008. 144. Langone, J.J., Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci, Adv Immunol, 32, 157, 1982. 145. Werner, S. et al., Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A, Proc Natl Acad Sci USA, 103, 17678, 2006. 146. Low, D., O’leary, R., and Pujar, N.S., Future of antibody purification, J Chromatogr B, 848, 48, 2007. 147. Valdes, R. et al., Large-scale purification of an antibody directed against hepatitis B surface antigen from transgenic tobacco plants, Biochem Biophys Res Commun, 308, 94, 2003. 148. Bai, Y. and Glatz, C.E., Bioprocess considerations for expanded-bed chromatography of crude canola extract: Sample preparation and adsorbent reuse, Biotechnol Bioeng, 81, 775, 2003. 149. Platis, D. and Labrou, N.E., Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract, J Chromatogr A, 1128, 114, 2006. 150. Balasubramaniam, D. et al., Tobacco protein separation by aqueous two-phase extraction, J Chromatogr A, 989, 119, 2003. 151. Vilter, H., Aqueous two-phase extraction of plant enzymes from sources containing large amounts of tannins and anionic mucilages, Bioseparation, 1, 283, 1990. 152. Miller, K., Gao, J., and Hooker, B.S., Initial clarification by aqueous two-phase partitioning of leaf extracts from Solanum tuberosum plants expressing recombinant therapeutic proteins, Bioprocessing J, 47, 2004. 153. Hahn, R. et al., Comparison of protein A affinity sorbents III—Life time study, J Chromatogr A, 1102, 224, 2006. 154. Kushwaha, A. et al., Construction and characterization of M13 bacteriophages displaying functional IgG-binding domains of Staphylococcal protein A, Gene, 151, 45, 1994. 155. Djojonegoro, B.M., Benedik, M.J., and Willson, R.C., Bacteriophage surface display of an immunoglobulin-binding domain of Staphylococcus aureus protein A, Biotechnology, 12, 169, 1994. 156. Moll, D. et al., S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays, Proc Natl Acad Sci USA, 99, 14646, 2002. 157. Vollenkle, C. et al., Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification, Appl Environ Microbiol, 70, 1514, 2004. 158. Ramirez, C. et al., A bifunctional affinity linker to couple antibodies to cellulose, Nat Biotech, 11, 1570, 1993. 159. Shpigel, E. et al., Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain, Biotechnol Appl Biochem, 31 197, 2000. 160. Ji, Q. et al., Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis, Plant Mol Biol, 51, 789, 2003. 161. Van Rooijen, G., and Moloney, M.M., Plant seed oil-bodies as carriers for foreign proteins, Nat Biotechnol, 13, 72, 1995. 162. Capuano, F. et al., Properties and exploitation of oleosins, Biotechnol Adv, 25, 203, 2007. 163. Ling, H., Oleosin fusion expression systems for the production of recombinant proteins, Biologia, 62, 119, 2007. 164. Roque, A.C.A., Silva, C.S.O., and Taipa, M.Â., Affinity-based methodologies and ligands for antibody purification: Advances and perspectives, J Chromatogr A, 1160, 44, 2007.

120

Transgenic Horticultural Crops: Challenges and Opportunities

165. Schwartz, W. et al., Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies, J Chromatogr A, 908, 251, 2001. 166. Ghose, S., Hubbard, B., and Cramer, S.M., Evaluation and comparison of alternatives to protein A chromatography: Mimetic and hydrophobic charge induction chromatographic stationary phases, J Chromatogr A, 1122, 144, 2006. 167. Ghose, S. et al., Antibody variable region interactions with Protein A: Implications for the development of generic purification processes, Biotech Bioeng, 92, 665, 2005. 168. Roque, A.C.A., Taipa, M.Â., and Lowe, C.R., An artificial protein L for the purification of immunoglobulins and Fab fragments by affinity chromatography, J Chromatogr A, 1064, 157, 2005. 169. Arnau, J. et al., Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins, Protein Expr Purif, 48, 1, 2006. 170. Lichty, J.J. et al., Comparison of affinity tags for protein purification, Protein Expr Purif, 41, 98, 2005. 171. Tagwerker, C. et al., A tandem affinity tag for two-step purification under fully denaturing conditions: Application in ubiquitin profiling and protein complex identification combined with in vivocross-linking, Mol Cell Proteomics, 5, 737, 2006. 172. Lawrence, S., Novak, N., and Slack, J., Epitope tagging: A monoclonal antibody specific for recombinant fusion proteins in plants, Biotechniques, 35, 488, 2003. 173. Terpe, K., Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems, Appl Microbiol Biotechnol, 60, 523, 2003. 174. Kwon, S. et al., Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach, Mol Cells, 24, 268, 2007. 175. Witte, C.-P. et al., Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope, Plant Mol Biol, 55, 135, 2004. 176. Maxim, G. et al., Smallpox subunit vaccine produced in planta confers protection in mice, Proc Natl Acad Sci USA, 104, 6864, 2007. 177. Pérez Filgueira, D.M. et al., Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8 produced in plants using a TMV-based vector, Arch Virol, 149, 2337, 2004. 178. Spiegel, H. et al., Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells, Plant Sci, 149, 63, 1999. 179. Peschen, D. et al., Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen, Nat Biotechnol, 22, 732, 2004. 180. Shpigel, E. et al., Production and purification of a recombinant human hsp60 epitope using the cellulose-binding domain in Escherichia coli, Protein Expr Purif, 14, 185, 1998. 181. Shpigel, E. et al., Immobilization of recombinant heparinase I fused to cellulose-binding domain, Biotechnol Bioeng, 65, 17, 1999. 182. Shani, Z. and Shoseyov, O., Process of expressing and isolating recombinant proteins and recombinant protein products from plants, plant derived tissues or cultured plant cells, U.S. Patent, 6, 331, 416, 2001. 183. Smith, M.L. et al., Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications, Virology, 348, 475, 2006. 184. Schriebl, K. et al., Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells, Protein Expr Purif, 49, 265, 2006. 185. Obregon, P. et al., HIV-1 p24-immunoglobulin fusion molecule: A new strategy for plant-based protein production, Plant Biotechnol J, 4, 195, 2006. 186. Chargelegue, D. et al., Highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants, Infect Immun, 73, 5915, 2005. 187. Waugh, D.S., Making the most of affinity tags, Trends Biotechnol, 23, 316, 2005. 188. Puig, O. et al., The tandem affinity purification (TAP) method: A general procedure of protein complex purification, Methods, 24, 218, 2001.

Expression and Manufacture of Pharmaceutical Proteins

121

189. Rohila, J.S. et al., Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants, Plant J, 38, 172, 2004. 190. Van Leene, J. et al., A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana, Mol Cell Proteomics, 6, 1226, 2007. 191. Rohila, J. et al., Protein–protein interactions of tandem affinity purification-tagged protein kinases in rice, Plant J, 46, 1, 2006. 192. Rubio, V. et al., An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation, Plant J, 41, 767, 2005. 193. Abe, M. et al., Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification, Plant Cell Physiol, 49, 420, 2008. 194. Nallamsetty, S. et al., Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli, Protein Sci, 14, 2964, 2005. 195. Donnelly, M.I. et al., An expression vector tailored for large-scale, high-throughput purification of recombinant proteins, Protein Expr Purif, 47, 446, 2006. 196. Kenig, M. et al., Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins, J Chromatogr A, 1101, 293, 2006. 197. Arnau, J., Lauritzen, C., and Pedersen, J., Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags, Nat Protoc, 1, 2326, 2006. 198. Pedersen, J. et al., Removal of N-terminal polyhistidine tags from recombinant proteins using engineered aminopeptidases, Protein Expr Purif, 15, 389, 1999. 199. Feeney, B. et al., Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker, Protein Expr Purif, 47, 311, 2006. 200. Rais-Beghdadi, C. et al., Purification of recombinant proteins by chemical removal of the affinity tag, Appl Biochem Biotechnol, 74, 95, 1998. 201. Wood, D.W. et al., A genetic system yields self-cleaving inteins for bioseparations, Nat Biotechnol, 17, 889, 1999. 202. Wood, D.W. et al., Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor, Biotechnol Prog, 16, 1055, 2000. 203. Lohaus, G. et al., Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species, Physiol Plantarum, 111, 457, 2001. 204. Thommes, J. and Etzel, M., Alternatives to chromatographic separations, Biotechnol Prog, 23, 42, 2007. 205. Albertsson, P.-Å. et al., Partition of cell particles and macromolecules in polymer twophase dystems, Adv Protein Chem, 24, 309, 1970. 206. Kula, M.R. et al., Technical aspects of extractive enzyme purification, Ann NY Acad Sci, 369, 341, 1981. 207. Albertsson, P.-Å., Partition between polymer phases, J Chromatogr A, 159, 111, 1978. 208. Zhang, C. et al., Purification and stabilization of ricin B from tobacco hairy root culture medium by aqueous two-phase extraction, J Biotechnol, 117, 39, 2005. 209. Benavides, J. et al., Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems, J Chromatogr B, 842, 48, 2006. 210. Negrete, A., Ling, T.C., and Lyddiatt, A., Aqueous two-phase recovery of bionanoparticles: A miniaturization study for the recovery of bacteriophage T4, J Chromatogr B, 854, 13, 2007. 211. Kim, J.Y. et al., Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay, Anal Chem, 77, 2318, 2005. 212. Ge, X. et al., Purification of an elastin-like fusion protein by microfiltration, Biotech Bioeng, 95, 424, 2006. 213. Patel, J. et al., Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves, Transgenic Res, 16, 239, 2007.

122

Transgenic Horticultural Crops: Challenges and Opportunities

214. Scheller, J., Leps, M., and Conrad, U., Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides, Plant Biotechnol J, 4, 243, 2006. 215. Floss, D.M. et al., Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants, Plant Biotechnol J, 6, 379, 2008. 216. Lin, M. et al., Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: Purification via inverse transition cycling, Biochem J, 398, 577, 2006. 217. Franzreb, M. et al., Protein purification using magnetic adsorbent particles, Appl Microbiol Biotechnol, 70, 505, 2006. 218. Safarik, I. and Safarikova, M., Magnetic techniques for the isolation and purification of proteins and peptides, Biomagn Res Technol, 2, 7, 2004. 219. Lang, C., Schüler, D., and Faivre, D., Synthesis of magnetite nanoparticles for bioand nanotechnology: Genetic engineering and biomimetics of bacterial magnetosomes, Macromol Biosci, 7, 144, 2007. 220. Yoshino, T. and Matsunaga, T., Efficient and stable display of functional proteins on bacterial magnetic particles using Mms13 as a novel anchor molecule, Appl Environ Microbiol, 72, 465, 2006. 221. Boi, C., Membrane adsorbers as purification tools for monoclonal antibody purification, J Chromatogr B, 848, 19, 2007. 222. Zhou, J.X. and Tressel, T., Basic concepts in Q membrane chromatography for largescale antibody production, Biotechnol Prog, 22, 341, 2006. 223. Knudsen, H.L. et al., Membrane ion-exchange chromatography for process-scale antibody purification, J Chromatogr A, 907, 145, 2001. 224. Zhou, X. et al., Viral clearance using disposable systems in monoclonal antibody commercial downstream processing, Biotech Bioeng, 100, 488, 2008. 225. Etzel, M. and Riordan, W., Membrane chromatography: Analysis of breakthrough curves and viral clearance, in Process Scale Bioseparations for the Biopharmaceutical Industry, Shukla, A., Etzel, M., Gadam, S. Eds., Taylor & Francis, Boca Raton, FL, 2006, p. 277. 226. Yu, D. et al., Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes, J Chromatogr A, 1187, 128, 2008. 227. Mchughen, H. and Smyth, S., US regulatory system for genetically modified organism, rDNA or transgenic crop cultivars, Plant Biotechnol J, 6, 2, 2008. 228. Crosby, L., Commercial production of transgenic crops genetically engineered to produce pharmaceuticals: Agriculture technology already exists to address most GMP issues, Biopharm Int, 16, 60, 2003. 229. Lossl, A. et al., Inducible trans-activation of plastid transgenes: Expression of the R. eutropha phb operon in transplastomic tobacco, Plant Cell Physiol, 46, 1462, 2005. 230. Menassa, R. et al., A self-contained system for the field production of plant recombinant interleukin-10, Mol Breeding, 8, 177, 2001. 231. Martinez, A. et al., Ecdysone agonist inducible transcription in transgenic tobacco plants, Plant J, 19, 97, 1999. 232. Lamphear, B.J. et al., A corn-based delivery system for animal vaccines: An oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine, Vaccine, 22, 2420, 2004. 233. Lamphear, B.J. et al., Delivery of subunit vaccines in maize seed, J Control Release, 85, 169, 2002. 234. Stoger, E. et al., Practical considerations for pharmaceutical antibody production in different crop systems, Mol Breed, 9, 149, 2002. 235. Stoger, E. et al., Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies, Plant Mol Biol, 42, 583, 2000. 236. Moloney, M.M., Oil bodies and associated proteins as affinity matrices, U.S. Patent 6924363, 2003.

Expression and Manufacture of Pharmaceutical Proteins

123

237. Ramessar, K. et al., Maize plants: An ideal production platform for effective and safe molecular pharming, Plant Sci, 174, 409, 2008. 238. Tacket, C.O. et al., Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato, Nat Med, 4, 607, 1998. 239. Tacket, C.O. et al., Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes, J Infect Dis, 182, 302, 2000. 240. Mcgarvey, P.B. et al., Expression of the rabies virus glycoprotein in transgenic tomatoes, Nat Biotechnol, 13, 1484, 1995. 241. Kruse, C. et al., Transient transformation of Wolffia columbiana by particle bombardment, Aquat Bot, 72, 175, 2002. 242. Fischer, R. et al., Towards molecular farming in the future: Using plant-cell-suspension cultures as bioreactors, Biotechnol Appl Biochem, 30, 109, 1999. 243. Vermij, P. and Waltz, E., USDA approves the first plant-based vaccine, Nature 24, 233, 2006. 244. Shaaltiel, Y. et al., Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher’s disease using a plant cell system, Plant Biotechnol J, 5, 579, 2007. 245. Fuhrmann, M. and Hegemann, W., A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii, Plant J, 19, 353, 1999. 246. Sun, M. et al., Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast, Biotechnol Lett, 25, 1087, 2003. 247. Griesbeck, C., Kobl, I., and Heitzer, M., Chlamydomonas reinhardtii, Mol Biotechnol, 34, 213, 2006. 248. Mayfield, S.P., Franklin, S.E., and Lerner, R.A., Expression and assembly of a fully active antibody in algae, Proc Natl Acad Sci USA, 100, 438, 2003. 249. Kim, D.-H. et al., Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea, Mar Biotechnol, 4, 63, 2002. 250. Murphy, D.J., Improving containment strategies in biopharming, Plant Biotechnol J, 5, 555, 2007. 251. Fox, J.L., Puzzling industry response to ProdiGene fiasco, Nat Biotechnol, 21, 3, 2003. 252. Stevens, W.E. et al., Optimizing pollen confinement in maize grown for regulated products, Crop Sci, 44, 2146, 2004. 253. Rotteveel, T., Al-Ahmad, H., and Gressel, J., Assessing risks and containing or mitigating gene flow of transgenic and non-transgenic phytoremediating plants, in Phytoremediation Rhizoremediation, Mackova, Martina; Dowling, David; Macek, Tomas, Eds., Springer, Dordrecht, the Netherlands, 2006, p. 259. 254. Al-Ahmad, H. and Gressel, J., Transgene containment using cytokinin-reversible male sterility in constitutive, gibberellic acid–insensitive (Δgai) transgenic tobacco, J Plant Growth Regul, 24, 19, 2005. 255. Mariani, C. et al., Induction of male-sterility in plants by a chimeric ribonuclease gene, Nature, 347, 737, 1990. 256. Schmülling, T. et al., Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants, Mol Gen Genet, 237, 385, 1993. 257. Wang, T. et al., Low frequency transmission of a plastid-encoded trait in Setaria italica, Theor Appl Genet, 108, 315, 2004. 258. Al-Ahmad, H., S. Galili, and Gressel, J., Tandem constructs to mitigate transgene persistence: tobacco as a model, Mol Ecol, 13, 697, 2004. 259. Oliver, M. et al., Seed-based strategies for transgene containment, in Proceedings 8th International Symposium on the Biosafety of Genetically Modified Organisms, Montpellier, France, p. 154, 2004. 260. Kuvshinov, V., Anissimov, A., and Yahya, B.M., Barnase gene inserted in the intron of GUS-a model for controlling transgene flow in host plants, Plant Sci, 167, 173, 2004.

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261. Kuvshinov, V. et al., Molecular control of transgene escape from genetically modified plants, Plant Sci, 160, 517, 2001. 262. Spök, A. et al., Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants, Trends Biotechnol, 26, 506, 2008. 263. Phoolcharoen, W. et al., Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana, Plant Biotechnol J, In press, 2011. 264. Chen, Q. et al., Geminiviral vector based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Human Vaccines, 7, 3, 2011.

5

Transgenic Fruit Crops in Europe Henryk Flachowsky and Magda-Viola Hanke

CONTENTS Importance of Fruit Crops in Europe...................................................................... 125 Field Trials with GM Fruit Crops in Europe........................................................... 127 Apple.................................................................................................................. 127 Citrus.................................................................................................................. 131 Cherry................................................................................................................. 132 Grapevine........................................................................................................... 133 Kiwifruit............................................................................................................. 134 Pear..................................................................................................................... 135 Plum................................................................................................................... 135 Raspberry........................................................................................................... 136 Strawberry.......................................................................................................... 137 Focus of Research on GM Fruit Crops in Europe................................................... 137 Improvement of Agronomically Important Traits.............................................. 137 Use of Early Flowering GM Plants to Accelerate Breeding Cycles.................. 137 Development of a Rapid Assay for Gene Function Analysis............................. 138 Development of New Systems for GM Fruit Crop Production.......................... 139 References............................................................................................................... 142

IMPORTANCE OF FRUIT CROPS IN EUROPE The popular adage “an apple a day keeps the doctor away” is only one example that illustrates the importance of fruits for human’s diet. A grain of truth is behind the adage because it is a matter of common knowledge that a diet rich in fruits and vegetables and low in saturated fats protects against a number of diseases, such as cardiovascular diseases and certain cancers.1–3 The World Health Organization (WHO) therefore recommends an intake of more than 400â•›g of vegetables and fruits per capita per day (http:// faostat.fao.org). The mean daily consumption of one person in Europe is 561â•›g of fruits and vegetables, which is more than recommended (http://faostat.fao.org). However, the daily intake ranges from about 350â•›g in Slowakia to more than 1100â•›g in Greece. In southern Europe (Greece, Italy, Portugal, Albania, and Spain), more fruits and vegetables are consumed (∼700–1100â•›g per capita per day) than in middle and northern countries (Finland, Iceland, Belarus, Latvia, Ukraine, the Czech Republic, Poland, and the Russian Federation) with about 400–450â•›g per person per day. 125

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Fruit crop production is of particular economic importance in Europe. In 2007, a total of 77.5 million metric tons of fruit crops were produced in Europe on approximately 8.7 million hectares of land (http://faostat.fao.org). Italy, Spain, and France are the main producers with more than 60% of the overall production. The entire spectrum of fruit crops produced in Europe consists of more than 30 different fruit species with grapes, apples, and oranges as the three most important. An overview of the cultivated fruit crops is given in Table 5.1. TABLE 5.1 European Fruit Crop Production in 2007 Fruit Crop Grapes Apples Oranges Watermelons Peaches, nectarines Pears Tangerines, mandarins, clementines Plums, sloes Other melons Lemons, limes Strawberries Currants Cherries Apricots Sour cherries Kiwifruit Bananas Carobs Figs Gooseberries Avocados Quinces Grapefruit Persimmons Dates Pineapples

Production Quantity a 29 14 6.2 5.1 4.3 3.1 3 2.5 2.3 1.5 1.5 0.9 0.8 0.8 0.8 0.6 0.4 0.14 0.12 0.12 0.1 0.08 0.06 0.05 0.013 0.003

Area Harvestedb 4140 1319 295 310 301 209 177 580 110 88 177 144 191 118 201 35 12 83 130 22 22 11 3 3 1 0.3

Number of Countries

Leading Countriesc

28 37 11 20 24 35 9

IT FR SP DE PT GR RU IT FR PL DE UK SP SP IT GR PT RU SP RO GR IT UK IT SP GR FR IT SP NE FR BE AU PT SP IT GR

35 14 8 36 27 34 23 21 8 4 5 12 15 4 22 6 2 2 1

SE FR RO SP IT UK BO RU SP IT FR GR U SP IT GR RU SP PL DE RU PL UK IT BE FR SP RU UK GR IT FR GR SP RU HU UK PL RU UK SE HU IT FR GR SP PT SP SP GR IT PT RU PL UK SP PT SP SE RU RO SP PT GR IT IT SL AL SP PT

Source: http://faostat.fao.org Production quantity in million metric tones. b Area harvested in thousand hectares c AL—Albania, AU—Austria, BE—Belgium, BO—Bosnia, FR—France, DE—Germany, GR— Greece, HU—Hungary, IT—Italy, NE—the Netherlands, PL—Poland, PT—Portugal, RO—Romania, RU—Russian Federation, SE—Serbia, SL—Slovenia, SP—Spain, UK—Ukraine. a

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The high level of demand for fruit and fruit products requires the availability of highly productive cultivars to satisfy such a demand. Therefore, breeders are always anxious to develop new cultivars with improved traits. The breeding of new cultivars of woody plants is time consuming and very expensive.4 Genetic engineering offers an exciting tool today to overcome several of the existing problems. On this account, many European scientists have started to develop and to establish methods for the development of trans-, intra-, or cisgenic fruit crops. Most of these studies have been performed under laboratory or glasshouse conditions. Only a few studies, which will be reviewed here, have been performed in the open field.

FIELD TRIALS WITH GM FRUIT CROPS IN EUROPE In Europe, deliberate releases of genetically modified (GM) plants into the environment require notification according to Directive 2001/18/EC (http://gmoinfo.jrc. ec.europa.eu). Directive 2001/18/EC regulates the release of GM plants for the performance of field trials as well as for introduction into the market. A petition for a deliberate release of GM plants into the field must be submitted for approval by the national authorities of the member state in which the release will be conducted. At that time, the other European Union (EU) member states are informed by sending them a SNIF (summary notification information format). All SNIFs that have been submitted to the national authorities of the individual EU member states since 2001 are available at the European Web site, http://gmoinfo.jrc.ec.europa.eu/. This Web site is managed by the Joint Research Centre of the European Commission on behalf of the Directorate General for the Environment. However, GM fruit crops are still quite far away from commercial use in Europe. In recent years, a total of 44 SNIFs for a deliberate release of GM fruit crops into the field have been submitted. In several countries, GM plants of apple, citrus, pear, plum, strawberry, grapevine, raspberry, and kiwifruit should have been planted in the field, but in the end, only a few field trials have been initiated.

Apple For apple, a total of nine summary notifications can be found: four from the Netherlands, two from Belgium, two from Sweden, and one from Germany (Table 5.2). All notifications had the goal to release GM plants for scientific investigations. The intended field trials were focused on transgenic plants of different apple scion and rootstock cultivars with improved resistance to fungal or bacterial diseases or on plants with a better rooting ability (Table 5.2). Only two field trials with GM apples have been performed in Europe until now. The others were refused by the national authorities or by the regional minister or they could not be started because of the lack of consent given by the competent authority. One field trial has been performed in the Netherlands (notification numbers B/NL/02/03, B/NL/04/02). This field trial has a long history as it was far from easy to obtain the planting approval. The field trial proposal was first submitted in the Netherlands. After rejection of the notification by the Dutch minister in 2001, it

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TABLE 5.2 Summary Notifications for the Deliberate Release of GM Fruit Crops in Europe Species Apple

Carrizo citrange Cherry

Grapevine

Kiwifruit

Lemon Pear Plum

Leading Countries

Notification Number

Species/Cultivar

NL

B/NL/98/06

Gala, Elstar

NL

B/NL/00/04

Paradise apple

NL

B/NL/02/03

Gala, Elstar

NL

B/NL/04/02

Gala, Elstar

BE

B/BE/02/V1

Gala, Elstar

BE SW SW DE

B/BE/03/V1 B/SE/99/1644 B/SE/04/1227 B/DE/03/140

SP SP SP IT IT IT FR FR FR

B/ES/06/43 B/ES/08/03 B/ES/08/21 B/IT/98/27 B/IT/98/28 B/IT/98/29 B/FR/99/03/10 B/FR/96/03/14 B/FR/94/11/04-CON

FR IT DE

B/FR/04/05/01 B/IT/99/26 B/DE/98/100

Elstar M26, M9 M26, M9 Pinova, Pilot, Reka, Remo, Elstar, Royal Gala, AU 56-83 C. sinensis × P. trifoliata Carrizo citrange Carrizo citrange P. avium P. avium P. avium V. × berlandieri V. vinifera V. berlandieri × riparia, V. berlandieri × rupestris V. vinifera × berlandieri V. vinifera × berlandieri 41B Thompson seedless (Sultana) Dornfelder, Seyval, Riesling

IT IT IT IT SW SP SP CZ RO PL

B/IT/98/24 B/IT/98/25 B/IT/98/26 B/IT/04/03 B/SE/04/1227 B/ES/96/16 B/ES/05/14 B/CZ/06/03 B/RO/07/04 DOPgmo 4301/02-4/2002

A. deliciosa A. deliciosa A. deliciosa Citrus sp. BP10030 Stanley C5 Stanley C5 Stanley C5 Stanley C5 Stanley C5

Improved Trait Fungal, bacterial resistance Fungal, bacterial resistance Fungal, bacterial resistance Fungal, bacterial resistance Fungal, bacterial resistance Self-fertility Rooting ability Rooting ability Fungal, bacterial resistance Plant architecture Plant architecture Early flowering Root formation Root formation Root formation Virus resistance Virus resistance Virus resistance

Virus resistance Parthenocarpic fruits Fungal resistance, gene flow Root formation Root formation Fungal resistance Fungal resistance Rooting ability Virus resistance Virus resistance Virus resistance Virus resistance Virus resistance

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TABLE 5.2 (continued) Summary Notifications for the Deliberate Release of GM Fruit Crops in Europe Species Raspberry Strawberry

Sweet orange

Leading Countries

Notification Number

Species/Cultivar

IT IT IT IT IT IT

B/IT/99/25 B/IT/98/32 B/IT/98/33 B/IT/99/23 B/IT/99/24 B/IT/02/11

R. idaeus F. × ananassa F. × ananassa F. × ananassa F. vesca F. × ananassa

SP

B/ES/98/06

Fresa

SP

B/ES/98/10

Andalucía

GB SP

B/GB/95/R23/2 B/ES/96/15

SP

B/ES/08/02

F. virginiana × chiloensis Orange cv. ‘Pineapple’, lime cv. ‘Mexicana’, citrange cv. ‘Carrizo’ Navelina

SP

B/ES/08/04

Pineapple

SP

B/ES/08/05

Pineapple

Improved Trait Fruit ripening Fungal resistance Root formation Development Fruit ripening Parthenocarpic fruits Fruit development, fruit quality Pollen flow, out-crossing Insect resistance Pollen flow, transgene stability Modified fruit aroma Resistance to Phytophthora Flowering time

Note: NL—the Netherlands, BE—Belgium, SW—Sweden, DE—Germany, IT—Italy, FR—France, CZ—Czech Republic, RO—Romania, PL—Poland, SP—Spain, GB—United Kingdom.

was decided to try to obtain the permit for a field trial in Belgium. The government bodies and advisory boards to the Belgian king advised in favor of approving the permit, but the Flemish Minister for the Environment used its veto right to refuse the trial. Subsequently, the scientists went back to the Netherlands submitting a modified version of the notification, and after a change of government the new minister granted the permit. The field trial (notification number B/NL/02/03) was planted in 2003, but the permit was challenged by Greenpeace and revoked, and, subsequently, a temporary permit was issued by the minister. This permit was later replaced by a new permit (B/NL/04/02). The new permit was challenged again, but remained in force and was confirmed by the High Court in 2005. This field trial was performed with GM plants of the apple cultivars ‘Gala’ and ‘Elstar’ overexpressing the hth gene of barley. The hth gene encodes for a type 1 alpha-hordothionin and the GM plants were tested in the field (Figure 5.1) for their resistance to apple scab (Venturia inaequalis), powdery mildew (Podosphaera leucotricha), and fire blight (Erwinia amylovora). This field trial ended in 2008 (Frans Krens, personal communication).

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FIGURE 5.1â•… (See color insert.) Dutch field trial with GM apples overexpressing the hth gene from barley. GM apple plants of the cultivars ‘Gala’ and ‘Elstar’ were tested for their resistance to apple scab, fire blight, and powdery mildew. The field trial was finished in 2008. The maximum area of the site was 3850â•›m2. (The figure was kindly provided by Frans Krens from PRI Wageningen, the Netherlands.)

Another field trial has been carried out in Sweden (notification numbers B/SE/99/1644 and B/SE/04/1227). This field trial was performed with GM plants of the apple rootstocks M26 and M9/29 transformed with the rolB gene of Agrobacterium rhizogenes to improve rooting ability. The GM rootstocks were characterized,5–8 planted into the field in 2001, and budded with different apple cultivars including ‘Aroma’, ‘Jonagold’, ‘Elstar’, ‘Discovery’, and ‘Elise’ in 2002.9 The transformed rootstocks were compared to non-GM rootstocks for traits such as tree growth, tree anchorage, tree size, branching, leaf form flowering, fruit set, fruit quality, transgene stability, and systemic transport of transgene products.9,10 In order to accelerate classical breeding, molecular techniques can be applied to enrich existing commercial apple cultivars with functional alleles from sexually compatible plants, preventing genetic drag and keeping the genetic makeup of the commercial cultivar. This concept is termed ‘cisgenesis’.11 Cisgenesis is defined as the genetic modification of a recipient plant with natural gene(s) from a sexually compatible plant.12,13 In cisgenesis, no foreign genes are allowed in the

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final product. If no novel traits are introduced compared to classical breeding, it can be argued that such plants are as safe as plants from classical breeding, and should be exempted from the GMO regulations.12–15 In the near future, several releases of cisgenic apple plants are expected in Europe. Scientific groups in the Netherlands, Italy, and Switzerland are working on the development of cisgenic apple plants with improved resistance to apple scab using the HcrVf2 scab resistance gene originating from the crab-apple Malus floribunda 821. The HcrVf2 is one of four genes clustered at the Vf-region in the M. floribunda 821 genome. All four genes encode for receptor-like proteins with similarity to the Cladosporium fulvum resistance gene family of tomato and were therefore named HcrVfs (homologues of C. fulvum resistance genes of the Vf-region).16 Later Barbieri et al.17 and Belfanti et al.18 confirmed that the HcrVf2 gene induces scab resistance in transgenic plants of susceptible apple cultivars (for review see Gessler et al.19). This gene has become one of the most promising genes for biotechnological approaches on apple in the world. As cisgenesis allows stacking of desired genes, multiple traits can be brought together. The HcrVf2 gene is race specific, and there is much interest for stacking with other scab resistance genes, providing resistance to other isolates of the pathogen. There are also several new resistance genes for apple scab that are nearly isolated and functionally analyzed, like Vr2 and Vm. By stacking these genes, a broad spectrum of resistance to apple scab can be realized leading to an increase in durability of resistance.11 The Plant Research International (PRI) in Wageningen, the Netherlands, the private fruit breeding company Inova Fruit BV, and two other partners have set their joint goal to release the first cisgenic scab-resistant apple cultivar in Europe in 2012.20 Further objectives of ongoing projects are the development of low allergenic GM apple cultivars by silencing the major apple allergen Mal d121 and cisgenic apple cultivars with an increased amount of health-promoting compounds by overexpressing the recently identified MYB transcription factors (MdMYB1 and/or MdMYB10), which are known to upregulate genes of the flavonoid biosynthesis.22–24

Citrus Eight summary notifications can be found for the release of GM citrus plants in Europe (Table 5.2): four for sweet orange (B/ES/96/15, B/ES/08/02, B/ES/08/04, B/ES/08/05), three for citrange (B/ES/06/43, B/ES/08/03, B/ES/08/21), and one for lemon (B/IT/04/03). All summary notifications for GM citrus plants have been submitted in Spain, except for lemon which was submitted in Italy. The first field trial with GM citrus plants in Europe (notification number B/ES/96/15) was planted in 1997. This field trial was notified by the Instituto Valenciano de Investigaciones Agrarias (IVIA) in Spain and spanned an area of 1638â•›m2. A total of 48â•›GM trees, 16 orange (cv. ‘Pineapple’), 16 lime (cv. ‘Mexicana’), and 16 citrange (cv. ‘Carrizo’), containing the nptII and the uidA gene were planted. These plants were used to study transgene stability, the inheritance of the transgenic traits, and the rate of gene flow over a number of years. The second field trial with GM citrus plants in Europe (notification number B/ES/06/43) was initiated 3 years ago by the same notifier. A semidwarf transgenic

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citrange rootstock expressing a GA20 oxidase gene from citrange in antisense orientation was planted. The notifiers are investigating the effect of GM rootstock plants on nontransgenic scions. The main question is whether the roostock confers compact size to the non-GM scion without negative effects on yield and fruit quality. In 2008, IVIA submitted five additional summary notifications for the release of GM citrus plants. These field trials will be performed in a 1â•›ha area at the experimental fields of IVIA in the STA of Villareal (Castellón, Spain) with a total of more than 800 plants. The first notification (B/ES/08/02) was submitted for a field trial with GM sweet orange plants of the cultivar ‘Navelina’ expressing a limonene synthase gene from citrus in sense and antisense orientation. The GM plants will be tested for the effect of the altered endogenous content of limonene on fruit aroma, on phenological and morphological characteristics of the transgenic trees, and on possible new interactions of transgenic fruits with herbivore insects, as Ceratitis capitata, and predators of herbivores. Furthermore, a new field trial (notification number B/ES/08/03) for GM citrange plants expressing a GA20 oxidase gene from citrange in antisense orientation was initiated. This field trial has the same goals as the second (notification number B/ES/06/43) described above. Another notification (B/ES/08/04) was submitted for GM sweet orange plants of the cultivar ‘Pineapple’. The GM plants overexpress the PR P23 gene of tomato. The PR P23 gene encodes for an osmotin-like protein, which led to a higher tolerance to Phytophthora citrophthora. The last two field trials (notification numbers B/ES/08/05 and B/ES/08/21) were initiated for sweet orange and citrange plants overexpressing the flowering genes apetala1 (AP1), suppressor of overexpression of constans 1 (SOC1), and flowering locus t (FT). The plants will be evaluated for early flowering, as shown for AP1 plants in Figure 5.2, and for effects on tree growth, flower and fruit development, and fruit quality characteristics. The notifiers are currently planting the five notified new field trials (Leandro Pena, personal communication). In Italy, one summary notification has been submitted for GM lemon plants of the commercial cultivar ‘Femminello siracusano’ (B/IT/04/03). This notification is still in progress.25 The GM lemon plants were transformed with the chit42 gene (encoding for an endochitinase) of the microparasite Trichoderma harzianum CECT  2413. The transgenic clones overexpressing the chit42 gene showed an increased resistance to the phytopathogenic fungi Phoma tracheiphila and Botrytis cinerea.25 This project is promising because the cultivar ‘Femminello siracusano’ is the most important lemon cultivar in Italy, and the transgenic clones could theoretically be cultivated after the field evaluation. However, whether GM lemon plants will be cultivated in Europe within the next few years or not cannot be stated at the moment due to strict GMO regulations and low consumer acceptance.

Cherry There are three summary notifications for the release of GM cherries in Italy (Table 5.2). The GM plants of the cherry rootstock ‘Colt’ carrying the transferred DNA (T-DNA) of Agrobacterium rhizogenes were planted in 1999.26 The aim of this study was to improve the rooting ability of the rootstock. However, GM cherries are still quite a long way from commercial use in Europe.

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FIGURE 5.2â•… (See color insert.) Juvenile control (left) and overexpressing APETALA1 juvenile Carrizo citrange plants showing flowering 8 months after transferring to the greenhouse. The juvenile period of this genotype is usually 5–7 years. (The figure was kindly provided by Leandro Pena from the Department of Plant Protection and Biotechnology at the Instituto Valenciano de Investigaciones Agrarias [IVIA] in Spain.)

Grapevine A total of six summary notifications (B/FR/94/11/04-CON, B/FR/96/03/14, B/FR/99/03/10, B/FR/04/05/01, B/IT/99/26, B/DE/98/100) can be found for the deliberate release of GM grapevine plants in Europe. Petitions for the release of GM grapevine plants were submitted in France, Italy, and Germany (Table 5.2). However, GM grapevine plants are still quite a long way from commercial use in Europe. The first field trial with GM grapevine plants (notification number B/FR/94/11/04CON) was carried out in a vineyard in the Champagne region of France. Plants of 18 transgenic grapevine lines of the rootstocks 41B (V. vinifera × V. berlandieri) and SO4 (V. berlandieri × V. riparia) overexpressing the coat protein gene of the Grapevine fanleaf virus (GFLV) were established in spring 2006.27 Nontransformed scions of V. vinifera cv. Chardonnay were subsequently grafted onto these rootstocks and tested for GFLV-type symptoms for several years. Furthermore, the plants were used to study the occurrence of recombination events between viral transgene transcripts and ribonucleic acids (RNAs) from indigenous virus populations. The study published by Vigne et al.27 provides evidence that the transgenic grapevines did not promote the emergence of viable GFLV recombinants. Another grapevine field trial (notification number B/IT/99/24) has been performed in Italy (Figure 5.3). Transgenic plants of the grapevine cultivars ‘Thompson Seedless’ and ‘Silorca’ overexpressing the chimeric gene construct DefH9-iaaM were established in the field at the Experimental Farm of the Marche Polytechnic University in Agugliano in March 2001.28 These plants were evaluated for the

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Transgenic Horticultural Crops: Challenges and Opportunities

FIGURE 5.3â•… (See color insert.) DefH9-iaaM transgenic grapevines grown in a field trial at the Experimental Farm of the Marche Polytechnic University in Agugliano (Italy). The GM grapevine plants were planted in March 2001 and tested for plant vegetative development, fecundity, and fruit nutritional quality from 2003 to  2005. (The  figure was kindly provided by Bruno Mezzetti from the Marche Polytechnic University Ancona, Italy.)

effects of DefH9-iaaM on plant vegetative development, fecundity, and fruit nutritional quality from the third to the fifth production cycles (2004, 2005, and 2006). Costantini et al.28 found that the expression of DefH9-iaaM in grapevine resulted in enhanced fecundity. The berry number per bunch was increased in both transgenic cultivars whereas the quality and the nutritional value of the GM berries were comparable to non-GM control fruits. In Germany, one field trial (notification number B/DE/98/100) has been performed at the Institute for Grapevine Breeding Geilweilerhof of the Federal Research Centre for Cultivated Plants in Siebeldingen. GM grapevine plants of the cultivars ‘Dornfelder’, ‘Seyval’, and ‘Riesling’ overexpressing glucanase, chitinase, or ribosomal inactivating protein encoding genes were planted in the field and used to investigate the distance of pollen (gene) flow.

Kiwifruit A total of three summary notifications (B/IT/98/24, B/IT/98/25, and B/IT/98/26) can be found for GM kiwifruit plants (Table 5.2). These notifications were submitted by the Università degli Studi della Tuscia Dipartimento di Produzione Vegetale in Italy for a period from April 1998 to April 2007. The field trials were performed in the province Viterbo on areas of 650â•›m2 (B/IT/98/24), 150â•›m2 (B/IT/98/25), and

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900â•›m2 (B/IT/98/26). The GM kiwifruit plants expressed either the rol genes of Agrobacterium rhizogenes or an osmotin gene expected to confer resistance to fungal pathogens. The field trials were focused on the effect of transferred genes to fungal diseases’ resistance or to the rooting ability of the transformed plants. The commercial use of GM kiwifruits in Europe is not to be expected in the near future.

Pear In Europe, only one summary notification was found for a pear rootstock. The intended field trial (B/SE/04/1227) was carried out in Sweden. The GM plants containing the rolB gene of Agrobacterium rhizogenes were planted at the same place as the transgenic apples. The GM pear plants have an improved rooting ability. The purposes of the field trial were the evaluation of the effects of the transgenic rootstocks on growth, flowering, fruit set and fruit quality of grafted cultivars, the stability of the modified trait (rooting ability), and the rooting ability of the transgenic rootstocks themselves by stooling. GM pears are still quite a long way from commercial use.

Plum The most important agronomic trait in plum is virus resistance, especially to Plum pox virus (PPV), the etiological agent of sharka disease, which is one of the most devastating pathogens. Sharka disease can cause extensive economic losses.29,30 Furthermore, the virus has quarantine status in many countries.31 In Europe, there are about 100 million stone fruit trees currently infected with the virus.32 Breeders are always anxious to develop resistant cultivars, but breeding of sharka-resistant plums is difficult. PPV resistance is often polygenic and in addition may be strain specific. The long juvenile period of seedlings hampers the success of traditional breeding programs considerably. Genetic engineering offers an exciting tool to overcome some of the existing problems. Several recent studies have focused on overexpression of coat proteins of the PPV.33,34 The transgenic line C5 (named ‘HoneySweet’) was selected because of its high level of resistance. This line contains a multicopy insert of the cpPPV gene that acts as a single locus. The expression level of this gene is reduced in HoneySweet, a result of posttranscriptional gene silencing (PTGS) (reviewed in Scorza and Ravelonandro31). Based on inoculation studies, it was found that HoneySweet is highly resistant to the major serotypes of PPV. The stability and durability of the PTGS-based PPV resistance of HoneySweet was tested in field trials in different countries for a number of years.35–37 Since the early 1990s, field trials with HoneySweet have been performed in Europe and the United States. In Europe, a total of five summary notifications can be found for GM plums (http://bgmo.jrc.ec.europa.eu/deliberate/dbplants.asp). Field trials with HoneySweet were performed in Spain (two), in Poland (one), in the Czech Republic (one), and in Romania (one). Based on the results obtained with HoneySweet, a petition for deregulation was approved in the United States in June 2007.38 The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA)

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excluded the GM plum line C5 HoneySweet from the regulations at 7 CFR part 340 (http://www.isb.vt.edu/cfdocs/fieldtests1.cfm). It has also been cleared by the U.S. Food and Drug Administration (FDA). Approval by the U.S. Environmental Protection Agency was granted in 2010, and subsequent commercialization of HoneySweet in the United States is expected within the next few years. However, GM plums are still quite a long way from commercial use in Europe, although the results obtained on HoneySweet are quite promising.

Raspberry Only one summary notification (B/IT/99/23) can be found for the release of GM raspberry plants in Europe. The field trial has been performed at the Experimental Farm of the Marche Polytechnic University in Agugliano, Italy (Figure 5.4). Forty transgenic plants expressing the chimeric DefH9-iaaM gene construct and 40 control plants were planted in four plots and evaluated for different fruit traits.34 The transgenic plants showed a significant increase in fruit number, fruit size, and total yield. Furthermore, they had more flowers per inflorescence and an increased number of inflorescences per plant. It is interesting to note that the DefH9-iaaM gene construct has led to parthenocarpic fruits on emasculated flowers.34

FIGURE 5.4â•… (See color insert.) DefH9-iaaM transgenic raspberry plants grown in a field trial at the Experimental Farm of the Marche Polytechnic University in Agugliano (Italy). The GM raspberry plants were planted in 2001 and tested on different fruit parameters in the field for two consecutive years (2002 and 2003). (The figure was kindly provided by Bruno Mezzetti from the Marche Polytechnic University Ancona, Italy.)

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137

Strawberry Eight summary notifications can be found for GM strawberries. Field trials with GM strawberry plants have been performed in Spain, Great Britain, and Italy (Table 5.2). In Great Britain, transgenic plants with improved resistance to insects were planted whereas the GM plants planted in Spain were evaluated on fruit development, fruit quality, pollen flow, and out-crossing. Unfortunately, information on the results of these studies is scarce. More information is available for the Italian field trial. This field trial was performed at the Experimental Farm of the Marche Polytechnic University in Agugliano. Transgenic plants of Fragaria vesca cv. ‘Alpina W. Original’ and Fragaria × ananassa breeding selection AN93.231.53 expressing the chimeric DefH9-iaaM gene construct and nontransgenic control plants of each were planted in the field and evaluated for fruit traits.34 The results obtained on strawberry were nearly identical to that of DefH9iaaM transgenic raspberries. Transgenic plants showed a significant increase in fruit number, fruit size, and yield. They had more flowers per inflorescence, an increased number of inflorescences per plant, and parthenocarpic fruits on emasculated flowers.39 However, GM strawberries are still quite a long way from commercial use in Europe.

FOCUS OF RESEARCH ON GM FRUIT CROPS IN EUROPE Improvement of Agronomically Important Traits In recent times, the European focus of research on GM plants has switched from the development of efficient regeneration and transformation protocols, including the selection of suitable marker genes to the improvement of selected agronomically important traits. Whereas earlier studies were mostly aimed at the method of gene transfer itself, newer studies are focused on the development of products with improved traits for introduction into the market. For apple and pear, traits like resistance to insects, bacterial (fire blight caused by Erwinia amylovora is the main disease in Europe) and fungal diseases (scab and mildew), stress tolerance (cold, heat), precocity, color and health properties, and reduced allergenic potential are of particular importance. For Prunus species the improvement of resistance to the PPV, the most dangerous pathogen in Europe, is the primary goal. The studies on small fruits like strawberry are mainly aimed at the improvement of resistance to insects, viruses, and fungal diseases (e.g., Verticillium dahliae, Botrytis cinerea, and Colletotrichum acutatum). Other traits such as salt or freezing tolerance, reduced softening, sugar content, fruit color, flavor, ripening, and yield are also of importance.

Use of Early Flowering GM Plants to Accelerate Breeding Cycles Recently, a study was published by a German group which describes the use of transgenic apple plants with a reduced juvenile phase for applied breeding. The transgenic plants were used to accelerate the breeding process because fruit tree breeding is time consuming, requires substantial space, and is therefore very

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Transgenic Horticultural Crops: Challenges and Opportunities

expensive.4 The production of a new apple cultivar takes at least 15–20 years and can cost in the range of €400,000 (Ken Tobutt, personal communication cited in Fenning and Gershenzon40). The breeding effort is mostly hampered by the long juvenile phase in which plants are not able to flower and fruit. In contrast to annual plants which flower within a few months, juvenility in tree species can last 5–40 or more years.4 A full evaluation of a progeny, the selection of the best seedlings and therefore all further breeding activities must wait until the plants complete the juvenile phase. Therefore, shortening the juvenile phase is always an important breeding objective for fruit crops. In recent years much effort has been made using biotechnological methods. Leandro Pena and coworkers were the first to describe the successful induction of early flowering in a fruit tree species after overexpression of the flower inducing genes LFY and AP1 from Arabidopsis thaliana.41 Since that time several studies on different fruit crop species using different genes have been published which were focused on the reduction of the juvenile phase (for review see Hanke et al.42). However, recently Flachowsky et al.4 were the first to report that transgenic early flowering apple plants were successfully used for practical breeding (Figure 5.5). Transgenic apple plants overexpressing the BpMADS4 gene of silver birch43 were pollinated with pollen of the apple wild species M. fusca. Transgenic seedlings flowered within the first season (Figure 5.6). They were pollinated with pollen of the apple cultivar ‘Topaz’ and the first fruits were harvested in the end of the first growing season.4 This work demonstrated that one crossbred generation per year is feasible and represents a dream come true for breeders. Several scientific groups in Europe are currently evaluating this system. However, the GM apple plants used in this study were not optimal. The transgenic plants constitutively overexpressing the BpMADS4 gene are often malformed and the fruit yield and seed set is very low.4 Therefore, new plants expressing the gene driven by an inducible promoter are under development.

Development of a Rapid Assay for Gene Function Analysis Functional genome analysis in fruit crops, especially for genes expressed in the fruit, is often very laborious. Genes to be characterized must either be overexpressed or silenced in transgenic plants. The production of stably transformed plants is time consuming and expensive. A full evaluation of the effects coming from the transgene is not possible before the plant enters the adult phase, and this can sometimes take up several years. Much more complicated is the characterization of genes, which are members of a multigene family. Several transgenic lines for each gene are necessary. This requires time, space, and the associated expenses. Recently an ingenious method was described by Hoffmann et al.44 on strawberry, which is based on transient gene silencing through the infiltration of Agrobacterium tumefaciens carrying an engineered plasmid into ripening fruits. Young fruits, which were still attached to the plants, were injected with an Agrobacterium suspension by using a sterile 1â•›m L hypodermic syringe. The bacteria containing a hairpin gene construct were evenly distributed throughout the entire fruit. Using this method it was possible to silence the FaCHS gene in nearly

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Transgenic Fruit Crops in Europe

P

× t

goi

F1

× t

goi

t+goi

BC΄x t

goi

t+goi

FIGURE 5.5â•… (See color insert.) Schema of the breeding program using BpMADS4 transgenic early flowering apple plants (in accordance to Flachowsky et al.4). t—transgene (red mark), induces early flowering; goi—gene of interest (yellow mark), for example, resistance gene for scab or fire blight resistance, green color—number of positive traits is high (top cultivar, e.g., ‘Pinova’, ‘Elstar’, ‘Gala’); brown color—number of negative unwanted traits is high (apple wild species used as resistance donor, e.g., Malus × robusta 5 for fire blight resistance); blue arrow—genotype useful for further breeding steps, white arrow—genetically improved, but nontransgenic (transgene and gene of interested must be unlinked to obtain such genotypes); P—parental generation; F1—first filial generation, seedlings contain 50% genome (genes/alleles) of each parent; BC′x—generation after numerous pseudo-backcrosses of ‘t+goi’ genotypes by high-quality cultivars, the percentage of wild species genome was reduced by backcross breeding.

the entire fruit (Figure 5.7). The functionality of the Agrobacterium infiltration silencing method was confirmed by Griesser et al.,45 who silenced a glycosyltransferase encoding gene (FaGT1) in strawberry fruits in the same way. The method published by Thomas Hoffmann and colleagues provides a powerful tool for functional gene analysis in strawberry. The knowledge obtained from strawberry can then be easily transferred to other Rosaceae crops, so that strawberry can be used as a model species in the future.

Development of New Systems for GM Fruit Crop Production The development of GM fruit crops in Europe is primarily based on Agrobacterium tumefaciens mediated transformation. Different tissues, such as leaf blades, stem internodes, axillary shoot-meristems, cotyledons, hypocotyl slices, or shoot

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Transgenic Horticultural Crops: Challenges and Opportunities

FIGURE 5.6â•… (See color insert.) BpMADS4 transgenic apple seedling. First flowers were obtained approximately 4 months after seed planting. The seedling was obtained after crossing a F1 plant of the cross T1190 (BpMADS4 transgenic line of the apple cv. ‘Pinova’, published by Flachowsky et al.43) by M. fusca (fire blight-resistant apple wild species) and the scab-resistant apple cv. ‘Topaz’.

apices have been tested to determine the best starting material for each species. However, the most effective and reproducible method for plant regeneration has remained through adventitious shoot formation and in most cases leaves from in vitro shoots are used for transformation. The selection of transgenic regenerants was and is still done by using selectable marker genes conferring resistance to selective chemical agents, such as antibiotics or herbicides. The presence of marker gene sequences in GM crops has been critically discussed in recent years. Public opinion in Europe demands that genetically engineered plants released into the environment be marker-free or at least with marker genes not conferring resistance to antibiotics used for medical or veterinary purposes. This and other demands are considered in the 2001 passed European Union directive 2001/18/EC. The directive 2001/18/EC forbids the release of GM crops containing antibiotic marker genes “which may have adverse effects on human health and the environment” beginning in December 2008. Although the European Food Safety Authority (EFSA) stated in 2004 and again in 2007 that “the use of the nptII gene as selectable marker in GM plants does not pose a risk to human or animal

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Transgenic Fruit Crops in Europe

ihp- CHS-RNAi

Control

FIGURE 5.7â•… (See color insert.) Transient silencing of the chalcone synthase (CHS) gene in strawberry fruits (left) after agroinfiltration using the ihp-CHS-RNAi gene construct (according to Hoffmann et al.44). (The figure was kindly provided by Wilfried Schwab, Technical University Munich, Biomolecular Food Technology, Germany.)

health or to the environment,” there remains a reluctance to use this important tool for GM plant selection. Therefore, it is considered essential by many research groups to establish methods which allow the production of marker-free plants. Clean vector technologies are the most promising strategy because other methods, which are based on sexual outcrossing of the marker gene, are not usable for fruit crops. Self-incompatibility and a high degree of heterozygozity make such methods often unsuitable. The first successes in the establishment of clean vector technologies have recently been reported for apple,46 sweet oranges,47 strawberry,48 and apricot.49,50 However, the development of highly effective strategies for the production of marker-free plants is still one of the most important goals in many countries. For apple, there is a large program in progress at the moment. Scientists of several countries have joined into Working Group 4 “Biotechnological Approaches for Pome Fruit Trees” of the European COST-Action 864 “PomeFruitHealth.” They evaluate various vector systems (an example is shown in Figure 5.8) to find the most usable system for apple transformation. Other technologies such as transformation without the use of marker genes have also been tested on apple.12,13 While the results are promising, no molecular evidence exists to date confirming these results. All of these technologies offer the possibility to transfer a gene of interest from one to another genotype from the same or a crossable species without DNA from noncrossable organisms. In Europe the first commercial GM plant will likely contain

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Transgenic Horticultural Crops: Challenges and Opportunities

FIGURE 5.8â•… (See color insert.) Heat stress induced removal of the nptII marker gene on apple. Transgenic apple plants containing the nptII marker gene and an Flp recombinase gene between two FRT recombination sites were produced. The expression of the Flp recombinase is controlled by a heat-stress inducible promoter. Both genes (nptII and Flp) are located between the CaMV 35S promoter and the uidA marker gene. In transgenic plants containing nptII and Flp no GUS gene expression is possible (left), because the CaMV 35S::uidA reading frame is interrupted. The reading frame is restored by excision of nptII and Flp. GUS expression is only possible in cells in which both genes have successfully been removed (right). (The figure was kindly provided by Katja Herzog, Julius Kühn-Institut, Federal Research Centre for Cultivated Plants (JKI), Institute for Breeding Research on Horticultural and Fruit Crops Dresden, Germany; unpublished.)

a gene transferred from a crossable species with its native introns and flanking regions, such as native promoter and terminator in a sense orientation. Such “cisgenic” plants11 will probably be sooner accepted than GM plants containing marker genes and genes of noncrossable species.

REFERENCES

1. Block, G., Patterson, B., and Subar, A., Fruit, vegetables and cancer prevention: A review of the epidemiological literature. Nutrition and Cancer, 18, 1, 1992. 2. Ferro-Luzzi, A. et al., The Mediterranean diet revisted: Focus on fruit and vegetables. International Journal of Food Science and Nutrition, 45, 291, 1994. 3. Morris, D.M., Kritchevsky, S.B., and Davis, C.E., Serum carotenoids and coronary heart disease: The lipid research clinics coronary primary prevention trial and follow-up study. The Journal of the American Medical Association, 274, 1439, 1994. 4. Flachowsky, H. et al., A review on transgenic approaches to accelerate breeding of woody plants. Plant Breeding, 128, 217, 2009.

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5. Zhu, L.H. and Welander, M., Adventitious shoot regeneration of two dwarfing pear rootstocks and the development of a transformation protocol. Journal of Horticultural Science & Biotechnology, 75, 745, 2000. 6. Zhu, L.H. et al., Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth. Plant Science, 160, 433, 2000. 7. Zhu, L.H., Ahlman, A., Li, X.Y., and Welander, M., Integration of the rolA gene into the genome of the vigorous apple rootstock A2 reduced plant height and shortened internodes. Journal of Horticultural Science & Biotechnology, 76, 758, 2001. 8. Zhu, L.H. et al., Detection of transgenes in apple rootstocks using anchored PCR. Acta Horticulturae, 625, 331, 2003. 9. Welander, M., Zhu, L.H., and Li, X.Y., Transformation of dwarfing apple and pear rootstocks with the rolB gene and its influence on rooting and growth. Acta Horticulturae, 663, 437, 2004. 10. Zhu, L.H. et al., Improvement of rooting and reduction in plant height in apple and pear through gene transfer. Acta Horticulturae, 738, 353, 2007. 11. Joshi, S. et al., Approaches for development of cisgenic apples. Transgenic Plant Journal, 3, 40, 2009. 12. Schouten, H.J., Krens, F.A., and Jacobsen, E., Cisgenic plants are similar to traditionally bred plants. EMBO Reports, 7, 750, 2006. 13. Schouten, H.J., Krens, F.A., and Jacobsen, E., Do cisgenic plants warrant less stringent oversight? Nature Biotechnology, 24, 753, 2006. 14. Jacobsen, E. and Schouten, H.J., Cisgenesis strongly improves introgression breeding and induced translocation breeding of plants. Trends in Biotechnology, 25, 219, 2007. 15. Schouten, H.J. et al., Cisgenesis is a promising approach for fast, acceptable and safe breeding of pip fruit. Acta Horticulturae, 814, 199, 2009. 16. Vinatzer, B.A. et al., Apple contains receptor-like genes homologous to the Cladosporium fulvum resistance gene family of tomato with a cluster of genes cosegregating with Vf apple scab resistance. Molecular Plant–Microbe Interactions, 14, 508, 2001. 17. Barbieri, M. et al., Progress of map-based cloning of the Vf-resistance gene and functional verification: Preliminary results from expression studies in transformed apple. Hortscience, 38, 329, 2003. 18. Belfanti, E. et al., The HcrVf 2 gene from a wild apple confers scab resistance to a transgenic cultivated variety. Proceedings of the National Academy of Sciences of the United States of America, 101, 886, 2004. 19. Gessler, C. et al., Venturia inaequalis resistance in apple. Critical Reviews in Plant Sciences, 25, 473, 2006. 20. Bethge, P., Verbotene Frucht. Der Spiegel, 50, 164, 2008. 21. Gilissen, L.J.W.J. et al., Silencing the major apple allergen Mal d 1 by using the RNA interference approach. Journal of Allergy and Clinical Immunology, 115, 364, 2005. 22. Allan, A.C., Hellens, R.P., and Laing, W.A., MYB transcription factors that colour our fruit. Trends Plant Science, 13, 99, 2008. 23. Espley, R.V. et al., Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant Journal, 49, 414, 2006. 24. Takos, A.M. et al., Light induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiology, 142, 1216, 2006. 25. Gentile, A. et al., Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene. Plant Breeding, 126, 146, 2007. 26. Rugini, E. and Guiterrez-Presce, P., Transgenic Prunus fruit species (almond, apricot, cherry rootstocks, sour and sweet cherry, peach and plum), in: Biotechnology in Agriculture and Forestry, Bajaj, Y.P.S. (Ed.), Springer, Berlin, Germany, vol. 44, p. 245, 1999, chap. Transgenic trees.

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27. Vigne, E., Komar, V., and Fuchs, M., Field safety assessment of recombination in transgenic grapevines, expressing the coat protein gene of Grapevine fanleaf virus. Transgenic Research, 13, 165, 2004. 28. Costantini, E. et al., Auxin synthesis-encoding transgene enhances grape fecundity. Plant Physiology, 143, 1689, 2007. 29. Németh, M., History and importance of plum pox in stone-fruit production. OEPP/ EPPO Bulletin, 24, 525, 1994. 30. Roy, A.S. and Smith, I.M., Plum pox situation in Europe. OEPP/EPPO Bulletin, 24, 515, 1994. 31. Scorza, R. and Ravelonandro, M., Control of Plum pox virus through the use of genetically modified plants. OEPP/EPPO Bulletin, 36, 337, 2006. 32. Kegler, H. and Hartmann, W., Present status of controlling conventional strains of plum pox virus, in: Plant Virus Disease Control. Hadidi, A., Khetarpal, R.K., and Koganezawa, H. (Eds.), APS Press, St. Paul, MN, pp. 616–628, 1998. 33. Laimer da Câmara Machado, M. et al., Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus. Plant Cell Reports, 11, 25, 1992. 34. Scorza, R. et al., Transgenic plums (Prunus domestica L.) express the plum pox coat protein gene. Plant Cell Reports, 14, 18, 1994. 35. Fuchs, M. et al., Safety assessment of transgenic plums and grapevines expressing viral coat protein genes: New insights into real environmental impact of perennial plants engineered for virus resistance. Journal of Plant Pathology, 89, 5, 2007. 36. Hily, J.M. et al., Stability of gene silencing-based resistance to Plum pox virus in transgenic plum (Prunus domestica L.) under field conditions. Transgenic Research, 13, 427, 2004. 37. Malinowski, T. et al., Field trials of plum clones transformed with the Plum pox virus coat protein (PPV-CP) gene. Plant Disease, 90, 1012, 2006. 38. Scorza, R. et al., Deregulation of plum pox resistant transgenic plum ‘Honey Sweet’. Acta Horticulturae, 738, 669, 2007. 39. Mezzetti, B. et al., The def H9-iaaM auxin-synthesizing gene increases plant fecundity and fruit production in strawberry and raspberry. BMC Biotechnology, 4:4 doi:10.1186/1472-6750-4-4, 2004. 40. Fenning, T.M. and Gershenzon, J., Where will the wood come from? Plantation forests and the role of biotechnology. Trends in Biotechnology, 20, 291, 2002. 41. Peña, L. et al., Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nature Biotechnology, 19, 263, 2001. 42. Hanke, M.V. et al., No flower no fruit—Genetic potentials to trigger flowering in fruit trees. Genes, Genomes and Genomics, 1, 1, 2007. 43. Flachowsky, H. et al., Overexpression of BpMADS4 from silver birch (Betula pendula Roth.) induces early flowering in apple (Malus x domestica Borkh.). Plant Breeding, 126, 137, 2007. 44. Hoffmann, T., Kalinowski, G., and Schwab, W., RNAi-induced silencing of gene expression in strawberry fruit (Fragaria × ananassa) by agroinfiltration: A rapid assay for gene function analysis. Plant Journal, 48, 818, 2006. 45. Griesser, M. et al., Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit. Plant Physiology, 146, 1528, 2008. 46. Krens, F.A. et al., Clean vector technology for marker-free transgenic fruit crops. Acta Horticulturae, 663, 431, 2004.

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47. Ballester, A., Cervera, M., and Pena, L., Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by sitespecific recombination. Plant Cell Reports, 26, 39, 2007. 48. Schaart, J.G. et al., Effective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene. Plant Biotechnology Journal, 2, 233, 2004. 49. López-Noguera, S., Petri, C., and Burgos, L., Production of marker-free transgenic plants after transformation of apricot cultivars. Acta Horticulturae, 717, 225, 2006. 50. López-Noguera, S., Petri, C., and Burgos, L., Using MAT vector system to produce marker-free transformed apricot plants. Acta Horticulturae, 738, 607, 2007.

6

Transgenic Horticultural Crops on the African Continent Idah Sithole-Niang

CONTENTS Introduction............................................................................................................. 147 South Africa............................................................................................................ 149 Bt Potato............................................................................................................. 149 Fruits.................................................................................................................. 150 Egypt....................................................................................................................... 150 Squash, Melon, Watermelon, and Cucumber..................................................... 150 Kenya...................................................................................................................... 150 Sweet Potato....................................................................................................... 150 Uganda.................................................................................................................... 151 Transgenic Banana............................................................................................. 151 Ongoing Projects..................................................................................................... 151 Status of Biosafety Regulation in Selected African Countries............................... 152 Expected Economic Effects of Introducing Transgenic Horticultural Crops in Ghana.................................................................................................................. 152 Way Forward: Synergies and Lessons Learned...................................................... 153 References............................................................................................................... 153

INTRODUCTION Globally, the area planted to genetically modified crops has continued to increase. In 2008, the area planted to genetically modified organisms (GMOs) was 125 million hectares, representing 13.3  million farmers in 25 countries made up of 15 developing countries and 10 developed countries.1 Of these countries, only three African countries have commercialized GMOs, namely, South Africa, Egypt, and Burkina Faso, with the latter two being in their first year of commercialization. South Africa first commercialized Bacillus thuringiensis (Bt) cotton in 1997, and in subsequent years, six other crop/trait combinations would be commercialized: Bt maize in 1998, round-up ready (RR) soybean in 2000, RR cotton also in 2000, herbicide tolerant (HT) maize in 2002, the stacked traits HT and Bt cotton in 2005,

147

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Transgenic Horticultural Crops: Challenges and Opportunities

and HT and Bt maize in 2007.2 Three countries are still a small number of countries in Africa but nevertheless a welcome improvement on previous years, as the spread now covers all regional blocks on the continent that could result in viable synergies. For the first time, each block has a regional lead country, Egypt to the north, Burkina Faso to the west, and South Africa to the east and south. The signing into law of the draft Biosafety bill in Kenya in January 2009 as well as the fact that Kenya already has a number of GM crops in the pipeline clearly places Kenya in the driving seat to take the lead in the east. In 2004, the International Food Policy Research Institute (IFPRI) published a study on the development of transgenic crop varieties in Africa.3 The study showed that there were 37 events being developed on the continent, half of which were virus and insect resistance and the rest representing agronomic performance, fungal resistance, product quality, herbicide tolerance, and bacterial resistance.4 South Africa had the widest diversity of crops under study, and the events spanned seven traits. Twenty-one out of the 28 events had been developed independent of public private partnerships using local germplasm. Up until 2007, South Africa was the only country to have commercialized GM crops on the continent.5 These were seven in all, maize with insect resistance, both white and yellow, HT soybean, insect resistant cotton, and cotton with stacked gene traits, made up of insect resistance and herbicide tolerance. To date, both Egypt and Burkina Faso have commercialized Bt maize and Bt cotton, respectively.1 Globally, the transgenic horticultural crops commercialized to date include tomato, sweet corn, potato, squash, papaya, sweet pepper, and biotech carnation, with the United States and China leading in research and development (R&D).6 In Africa, while having a wide range of horticultural crops (vegetable, fruits, nuts, and ornamentals), there are a few transgenic horticultural crops that have been developed, and these include squash, melon, watermelon, grapes, apples, potato, sweet potato, groundnut, and banana. These crops are limited to four countries only: South Africa, Egypt, Kenya, and Uganda (Table 6.1). Some of the opportunities for developing transgenic horticultural products desired by consumers and producers include7 • • • •

Novel genetic methods for disease and insect protection Weed control Longer-lived flowers Slower-growing grass

In terms of transgenic horticultural crops, the limited market distribution and R&D efforts are not unique to Africa. The first transgenic horticultural crop, Flavr Savr, developed to have an extended shelf life, and another version with higher viscosity intended for easier processing was commercialized in 1994 in the United States but has since been withdrawn. Subsequently, sweet corn, potato, squash, and papaya varieties made to resist viruses were also developed but failed to garner a significant market share. To date, only the virus-resistant papaya enjoys a significant market share with 70% of the Hawaiian crop shipped to mainland United States annually being from this product.7

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TABLE 6.1 Transgenic Horticultural Crops and Traits under Commercialization and Status of NBFs in Four African Countries Country

Transgenic Hort Crops and Traits

Egypt

Kenya South Africa

Uganda

Watermelon-VR Squash-VR Melon-VR Cucumber-VR Potato-IR Sweet potato-VR Grapes-FR Potato-IR Sweet potato-VR Strawberries-RR Banana-FR

Biosafety Law

CPB

Commercialization

Yes

✓

Bt maize 2008

2009 1997, 2003

✓ ✓

None Bt maize-yellow Bt maize-white Bt cotton RR-soybean Stacked gene traits

Draft bill

✓

None

Banana-AP Banana-BR Banana-NE Note: AP, agronomic performance; BR, bacterial resistance; CPB, Cartagena Protocol on Biosafety; FR, fungal resistance; IR, insect resistance; NE, nutritional enhancement; RR, round-up ready; and VR, virus resistance; NBFs, national biosafety frameworks.

The major challenges, on the other hand, have been summarized by Bradford and Alson7 as encompassing technical, economic, regulatory, and market-related factors. While a number of products have been developed and tested, unfortunately they have not reached commercialization.8 Consequently, there has been a drastic decrease in R&D efforts going into horticultural products with investors choosing to invest in breeding and the use of molecular markers.9,10

SOUTH AFRICA Bt Potato The case studies on Bt potato in both Egypt11 and South Africa have been extensively covered12 and will not be described in detail here. South Africa has had 11 years of experience working with Bt potato, and five of those years have been spent on confined field trials (CFTs). The Agricultural Research Council (ARC) has now applied for approval of larger-scale field trials in a number of selected areas and that approval is still awaited. Just as was found in Egypt, the Bt technology for resistance to potato tuber moth works extremely well. The outstanding safety concerns have to do with stewardship issues, seeing that South Africa’s neighbors, except for Zimbabwe, do not have functioning biosafety regulations in place to safeguard the transboundary movement of potato, a vegetatively propagated crop.

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Fruits The ARC-Infruitec has requested approval for a CFT of fungal resistant grapes to see if the reporter gene works well. Similarly, transgenic apples have undergone CFTs for the last 5–7 years to see if the novel genes are expressed. The first GM strawberry with tolerance to glyphosate was developed locally and tested in a field trial in the early 1990s. The RR gene worked well, but the project did not go commercial. Meanwhile, the landscape for transgenic horticultural crops might still change given that Monsanto Company acquired Seminis (a seed company), a year ago.

EGYPT Squash, Melon, Watermelon, and Cucumber Egypt produces an estimated 46,000 hectares (ha) of watermelon, 28,000â•›ha of squash, pumpkin and gourds, 20,000â•›ha cantaloupes and other melons, and 18,000â•›ha of cucumber annually. Production constraints are due to both biotic (viruses) and abiotic (drought and salinity) stresses. The most economically important viruses being zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), the watermelon strain of papaya ringspot virus (PRSV-W), and cucumber mosaic virus (CMV). The ZYMV coat protein gene was cloned and sequenced at Michigan State University and used to develop transgenic cucurbits. In collaboration with Egyptian scientists at the Agricultural Genetic Engineering Research Institute (AGERI), transgenic cucurbits/melon and squash were produced. The transformation technology for melon together with the coat protein gene were transferred to AGERI and used to produce local transgenic melons and squash. The technology also resulted in local scientists developing transformation and regeneration protocols for local varieties of both melon and watermelon.11 While all these efforts have worked well, the actual challenge for Egypt is the limited biosafety data that are available to support commercialization of these products. Furthermore, the Egyptian government might have been reluctant to tackle the anti-GM activism that surrounded the Bt potato, although it is interesting to note that in 2008, Egypt did indeed commercialize Bt maize, a food crop.1

KENYA Sweet Potato The average yield of sweet potato in Kenya is 6 ton/ha, whereas in China it is 18 ton/ha. While sweet potato is widely grown in Kenya, the yields continue to decline due to nematode and viral diseases with the sweet potato feathery mottle virus (SPFMV) being the major virus. Transgenic sweet potato carrying the coat protein gene from SPFMV was developed by Kenyan and Monsanto scientists using materials from Kenya. When technical problems developed with this material, the material from Papua New Guinea was used instead. When transgenic sweet potatoes were field tested in Kenya, the resistance broke down and a whole negative publicity ensued.13 The development of virus-resistant sweet potato still continues and this

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time in collaboration with scientists at the Vegetable and Ornamental Plant Institute of the Agricultural Research Council (VOPI-ARC) in South Africa. While negative publicity surrounded the Kenyan VR-sweet potato, a lot of the effort in developing a biosafety regulatory framework in Kenya came as result of this project. It was used at every step to fine tune and streamline the process. The fact that certain experiments fail is precisely the reason why field trials are required to see whether the material holds up under field conditions and that practice must be encouraged. It must be encouraged as long as the right questions are asked at the right stage of development, that is, not asking for food safety data when all an application is seeking to address is a performance under field conditions. In January 2009, President Kibaki signed the Biosafety Bill into law. This heralds a new era for Kenya, which already boosts a number of GM-products in the pipeline and two crops under CFTs, Bt cotton, and VR-cassava.

UGANDA Transgenic Banana Bananas are a major staple food in the Great Lakes regions with per capita consumption estimated at 450â•›kg/year. Major production constraints are due to weevils (Cosmopilities sordidud), nematodes (Pratylenchus sp., and Helicotylenchus sp.) and the fungal (Black Sigatoka, fusarium wilt), viral (Banana buchy-top virus and Banana streak virus), and bacterial (banana bacterial wilt) diseases.14 The national banana research program has designed both short- (germplasm evaluation for resistance, importation of hybrids, and propagation of clean planting materials) and longterm (development of transgenic materials) strategies to address these problems. Through an Agricultural Biotechnology Support Project II (ABSPII) partnership with, the Catholic University of Leuven (KUL), the University of Leeds, the United Kingdom, and the Ugandan National Agricultural Research Organization (NARO) transgenic banana carrying the rice antifungal chitinase resistance gene, RCG3, to control Black Sigatoka, and a maize cystatin gene and other nematicidal genes have also been developed and are being evaluated. To date, a CFT for banana with fungal resistance to Black Sigatoka is being conducted at the Kawanda Agricultural Research Institute, National Agricultural Research Laboratory (NARL). The NARL also has other transgenic bananas in the pipeline, namely, banana biofortified with provitamin A, zinc, and iron, banana with a cell cycle regulatory gene for rapid growth, and banana with bacterial wilt- or weevil-resistance.

ONGOING PROJECTS The ABSPII located at Cornell University, United States, in collaboration with Malian scientists engineered a gene for resistance to potyviruses from pepper (Pvr1) into tomato (http://www.absp2.cornell.edu/projects). Furthermore, they are breeding tomato with resistance to the tomato yellow leaf curl virus (TYLCV), with the goal of backcrossing the resistance genes into locally preferred varieties with the final product containing both conventionally bred resistance to TYLCV and genetically engineered resistance to the potyvirus.

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The African Agricultural Technology Foundation (AATF) has acquired a royaltyfree technology license agreement with Academia Sinica of Taiwan for their plant ferredoxin-like protein (pflp) from sweet peppers for use in conferring bacterial wilt resistance in banana. The AATF has in turn granted a sublicense to the International Institute of Tropical Agriculture (IITA) for this purpose. The pflp gene has already been used successfully in rice, tobacco, potato, tomato, and orchids. Current efforts on transgenic banana have already gone beyond the proof-of-concept stage and shown to be resistant when tested against inoculum of Xanthomonas campestris pv musacearum. The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in India has developed groundnuts resistant to groundnut rosette virus, and these plants are currently being evaluated in South Africa. A gene for enhanced proline production to confer drought tolerance was incorporated into groundnut in South Africa and is currently being evaluated.

STATUS OF BIOSAFETY REGULATION IN SELECTED AFRICAN COUNTRIES To date, there are 11 African countries with fully developed national biosafety frameworks (NBFs). Some laws are explicit, such as the ones in South Africa and Zimbabwe, whereas others such as those for Egypt and Tanzania are implicit and embedded in other national laws. There are 13 countries with Interim NBFs, 15 that are described as a work in progress, and 16 that have no NBFs. Of these countries, only three have commercialized transgenic crops: South Africa, Egypt, and Burkina Faso. Uganda launched two Bt cotton CFTs in 2009: one at Serere and the other at Kasese. Kenya has conducted a number of CFTs while Nigeria has given approval for two CFTs on Bt-cowpea and VR-cassava.

EXPECTED ECONOMIC EFFECTS OF INTRODUCING TRANSGENIC HORTICULTURAL CROPS IN GHANA In 2007, IFPRI15–18 conducted an economic impact assessment study on the potential of introducing transgenic tomato, cabbage, garden egg, and cassava in Ghana. The study looked at the feasibility, cost-effectiveness, and long-term impact on productivity and yield stability. The study showed that the benefits of using these transgenic crops would accrue to both the individual as well as the entire Ghanaian society. Since the cost of pesticides for use on tomato, cassava, and garden egg is minimal, the investments in pesticides as a percentage of total production costs would be low; however, in the case of cabbage, the costs would be significant and were likely to influence adoption. In the long term, any technology that was likely to reduce yield variability was also likely to contribute positively to poverty reduction. Since the export levels to Europe, of the crops under study, are low, the benefits at the farm level were likely to be higher than the potential losses in trade. These findings have policy implications and highlight the role that government, the public, and private sectors could play if the farmers are to realize maximum

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benefits from the technology. The public sector could be involved in the development of the GM varieties, and those varieties should meet both consumer and producer demand and preference. The private sector could lead in developing a viable vegetable seed multiplication and distribution sector, while the government could play a role in the provision of an effective agricultural extension service.

WAY FORWARD: SYNERGIES AND LESSONS LEARNED Biosafety regulatory frameworks are still highly fragmented. Even the CPB has not managed to get countries to work around the regional economic communities (RECs). Approaches are not aligned regionally nor are the limited skills ever shared giving rise to even more stringent regulations.2 Because some multinational companies have set such a high regulatory bar, it will be difficult for African countries, most of them having limited investments in the area to fulfill such a regulatory package. The first Bt cotton that was developed by China is already being marketed in India. This is an interesting case of south–south collaboration that African countries could emulate. The notion that biotechnology is not for the small-scale farmer is far from the truth. Indian farmers are already reaping massive welfare benefits from the technology.1 Meanwhile, African countries still operate under fear of losing markets even when the evidence points to a nonexistent threat. The Common Market for Eastern and Southern Africa (COMESA) commissioned a study that analyzed the value and volume of agricultural food and feed exports by six African countries to various regions of the world including the EU. Of the countries under study, especially for Kenya and Uganda only 1.1% and 6.5% of total export value was at risk of being rejected, respectively.19 Meanwhile, the emergence of an enabling environment such as the Open Forum on Agricultural Biotechnology (OFAB) represents a public platform that is fast gaining political mileage. Under the auspices of the AATF and with patronage of the Kenyan Minister for Science and Technology, OFAB was formed in 2006 to provide a monthly platform for the exchange of factual information amongst stakeholders in agricultural biotechnology. The Uganda chapter, which is equally active, was formed in 2008, while the Nigerian chapter was launched in April 2009, and the Tanzanian chapter was launched in May 2009.20 With six countries outside South Africa that have conducted CFTs for a number of crops, perhaps, the time has come when African countries can do away with ambivalence toward the technology and begin to reap the benefits.

REFERENCES

1. James, C., Global Status of Commercialized Biotechâ•›/GM Crops: 2008, ISAAA Brief No. 39, ISAAA: Ithaca, NY, 2008. 2. Van der Walt, W., Final report on the collaborative RABSAC-IFPRI-FANRPAN-PBS II project report for SADC with focus on Malawi, Mauritius and South Africa, Report to FANRPAN, 2009. 3. Sithole-Niang, I., Cohen, J., and Zambrano, P., Putting GMO technologies to work: Public research pipelines in selected African countries, African Journal of Biotechnology, 3 (11), 564, 2004.

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4. Glover, D., Agricultural biotechnology in sub-Saharan Africa: Facts and figures, SciDev. Net. June 5, 2007, http://rssww.scidev.net/en/features/agri-biotech-in-sub-saharanafrica-facts-and-figur.html, January 17, 2010. 5. Gain Report, Republic of South Africa, Biotechnology Annual Report, 2007. 6. Huang, J. and Rozelle, S., China aggressively pursuing horticultural and plant biotechnology, California Agriculture, 58 (2), 112, 2004. 7. Bradford, K.J. and Alston, J.M., Diversity of horticultural biotech crops contributes to market hurdles, California Agriculture, 58 (2), 84, 2004. 8. Fuchs, M. and Gonsalves, D., Safety of virus-resistant transgenic plants two decades after their introduction: Lessons from realistic field assessment, Annual Review of Phytopathology, 45, 173, 2008. 9. Morris, M., Edmeades, G., and Pehu, E., The global need for plant breeding capacity: What roles for the public and private sectors? HortScience, 41(1), 31, 2006. 10. Pehu, E. and Ragasa, C., Agricultural biotechnology: Transgenics in agriculture and their implication for developing countries, Background paper to the World Development Report, 2007. 11. Brenner, C., Telling Transgenic Technology Tales: Lessons from the Agricultural Biotechnology Support Project (ABSP) Experience, ISAAA Briefs No. 31, ISAAA, Ithaca, NY, 2004. 12. Eicher, C. K., Maredia, K., and Sithole-Niang, I., Crop biotechnology and the African farmer, Food Policy, 31, 504, 2006. 13. Odame, H., Kameri-Mbote, P., and Wafula, D., Innovations and policy process: Case of transgenic sweet potato in Kenya, Economic and Political Weekly, July 6, 2002, pp. 2770–2777. 14. deVilliers, S. and Ferguson, M., Inventory of agricultural biotechnology in southern Africa, Prepared by IITA for USAID-RCSA, 2004. 15. Horna, D., Smale, M., and Falck-Zepeda, J., Assessing the economic impact of genetically modified crops in Ghana: A methodological framework, IFPRI Policy Brief, 1 of 5, 2007. 16. Horna, D., Smale, M., and Falck-Zepeda, J., Assessing the economic impact of genetically modified crops in Ghana: Virus resistant tomato, IFPRI Policy Brief, 2 of 5, 2007. 17. Horna, D., Smale, M., and Falck-Zepeda, J., Assessing the economic impact of genetically modified crops in Ghana: Insect resistant cabbage, IFPRI Policy Brief, 3 of 5, 2007. 18. Horna, D., Smale, M., and Falck-Zepeda, J., Assessing the economic impact of genetically modified crops in Ghana: Insect resistant garden egg, IFPRI Policy Brief, 4 of 5, 2007. 19. Wafula, D., Persley, G., and Karembu, M., GMOs and Exports: Demystifying concerns in Africa, Biosafety Policy Brief, June 2008. 20. Partnerships: A quarterly newsletter of the African Agricultural Technology Foundation 2009, vol. 3.

7

Transgenic Horticultural Crops in Asia Desiree M. Hautea, Von Mark Cruz, Randy A. Hautea, and Vijay Vijayaraghavan

CONTENTS Introduction............................................................................................................. 155 Horticulture Crop Supply and Demand in Asia...................................................... 156 Overview of Current Adoption of Transgenic Crops in Asia.................................. 159 Public Acceptance and Perception of Biotech Crops in Asia................................. 161 Transgenic Horticultural Crops in the Pipeline in Asia.......................................... 162 Development and Delivery of Transgenic Horticultural Crops: Experiences and Lessons Learned from South and Southeast Asia............................................ 165 Development of Transgenic PRSV-Resistant Papaya for Southeast Asia.......... 166 Development and Commercialization of Fruit and Shoot Borer–Resistant Eggplant in South and Southeast Asia............................................................... 168 Conclusion.............................................................................................................. 170 Acknowledgments................................................................................................... 171 References............................................................................................................... 171

INTRODUCTION Asia is home to more than half of the world’s population and many of the countries situated in the region are developing countries. According to the United Nations, the world’s population reached 6.82 billion in 2009 with the population in Asia at approximately 4.12 billion. By the year 2050, this is projected to increase to 6.01 billion, which is about 54% of the projected world total of 11.03 billion.1 The Asian population growth coupled with increases in per capita incomes caused by economic growth and urbanization has rapidly expanded the demand for food and other resources.2 Most notably, the change in consumption patterns and diet due to increasing incomes in Asia can be seen as a shift from basic staples to a more diversified diet of meat, vegetables, fruits, and processed foods.3 On the other hand, Asia continues to have the highest absolute incidence of hunger, accounting for about twothirds of the world’s hungry people.4 The large food market and demand in Asia create challenges on ways to ensure increased and sustainable food production. In the past, the Green Revolution has helped avert widespread food shortages in the region, most notably in the Indian subcontinent and countries in Southeast Asia.5,6 The continuing challenge to provide 155

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adequate and affordable food in Asia is daunting as the key production resources of available arable land and water are more constrained today than in the past. National and regional research and development programs for crop improvement and management remain essential to meet the demands for staples and horticultural products such as vegetables and fruits. The food price hikes of 2008 provided additional impetus not just on ensuring food security but also food self-sufficiency. Numerous published studies and experiences have asserted that biotechnology applications, specifically genetic engineering and transgenesis, could make a significant contribution in helping conventional systems increase yields while providing more efficient use of resources and products that benefit human health and the environment.7 Studies have also suggested that increasing crop yields through applications of agricultural biotechnology is essential to increasing food production to meet future needs.8 Since biotech crops were first commercialized, there has been a steady increase in adoption of the four major transgenic crops in the market with around 134 million ha planted in 25 countries in 2009.9 However, from the time the first transgenic vegetable (the Flavr Savr tomato) was put out to market in 1994, there has been only a handful of transgenic horticultural crops that followed.10 Among these are the virus resistant squash and papaya in the United States and the virus-resistant sweet pepper and papaya in China. Several reasons on the slow development and commercialization of products of horticultural biotechnology include the technical challenge in developing genotype-independent transformation procedures despite the fact that protocols for genetic transforÂ�mation have been developed with success and reported in horticultural crops such as Brassica, cucumber, chilli pepper, eggplant, lettuce, apple, and tomato.11–14 Where Agrobacterium transformation method is used, technical challenges remain on designing strategies to avoid its persistence in plant issues especially those on vegetatively propagated fruit trees.12 Other than the technical constraints, economic, regulatory, and market factors pose additional hurdles to developing commercial biotech horticultural crops compared to biotech field crops such as corn or soybean. For example, the relatively small market and the limited hectarage of horticultural crops in general have been identified as major factors that limit the recovery of product development costs.15–18 These hurdles limit the application and deployment of biotech traits and products that can potentially provide broader direct benefits to consumers such as those related to improving nutritional quality or enhancing aesthetic quality. Moreover, the fact that most horticultural crops are consumed as food, in raw or semi-raw form, apparently elicits a more extensive and demanding food safety and regulatory requirements to ensure consumer confidence and market acceptance.

HORTICULTURE CROP SUPPLY AND DEMAND IN ASIA In Asia, horticulture crops mean food, income, nutrition, and well-being, particularly for women. Asia contributes a substantial proportion to the global horticultural production, particularly vegetables and fruits. According to the FAO, the area harvested in the region in 2007 is equivalent to about 73% and 48% of the world’s vegetable and fruit production areas, respectively. The cultivation area of vegetables is about

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800 700 600 500 400 300 200 100 0 1993 1995 1997 1999 2001 2003

Millions

1999

2001

2003

2005

2007

450 400 350 300 250 200 150 100 50 0 1993

1995

1997

1999

2001

2003

600 500 400 300 200 100 0 1997

1999

2001

2003

2005

2007

1999

2001

2003

2005

2007

1050

175

1000

170 165

950 900

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850

155 1997 1999 2001 2003 2005 2007

800 1997

Asia

Fruits

60 50 40 30 20 10 0 1997

180

Hundreds

Yield (hg/ha) Thousands

Production (ton) Millions

1000 900 800 700 600 500 400 300 200 100 0 1997 1999 2001 2003 2005 2007

Millions

Vegetables 60 50 40 30 20 10 0 1997 1999 2001 2003 2005 2007

Millions

Consumption (ton) Millions

Area harvested (ha) Millions

1.47 times larger than that of fruits. China alone accounts for about 60% of the world’s production and 50% of the world’s vegetable production area. India is second to China in vegetable production in the region primarily because majority of the population are vegetarian.19 About 14.9% of the country’s total farm area in India is being utilized for vegetable growing.20 Figure 7.1 shows an increasing trend in the area harvested, volume of production, consumption, and yield of vegetables and fruits in Asia, as well as globally. The yield of vegetables in Asia remains higher than the world’s average, while the yield of fruit species though lower in the past decade has also surpassed the world’s average in 2007. Northeast Asian countries like China, Japan, North Korea, South Korea, and Taiwan in particular have higher per capita consumption and production of horticultural crops than other countries in the region.

World

FIGURE 7.1â•… Selected indicators on production and consumption of vegetables and fruits in Asia and the World, 1997–2007. (Data from Food and Agriculture Organization of the United Nations, Rome, Italy, FAOSTAT database, http://faostat.fao.org, accessed October 28, 2009.)

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The substantial growth in both volume and variety of fruits and vegetables traded globally has been noted due to rising incomes, falling transportation costs, improved technology, and evolving international agreements.21 However, within Asia, trading of horticultural products still occurs mostly among neighboring countries or those within the region.22,23 In 2007, the total import value of fruits and vegetables in Asia was more than $26 billion with banana and apple contributing about $1.3 and $1.0 billion, respectively. The total export value of fruits and vegetables on the other hand was higher, at about $30 billion. The trade difference (export–import) in many developing Asian countries (Table 7.1) has remained relatively low, an observation also reported by Lee in 1998,22 which he attributed to a well-established self-supply system in these countries. The top export vegetables and fruits from the region include onions, tomatoes, bananas, garlic, and apples. China, Thailand, India, and

TABLE 7.1 Trade Statistics of Fruits and Vegetables in Selected Asian Countries, 2007 Bangladesh Brunei China Hong Kong, SAR India Indonesia Iran Israel Japan Korea, DPR Korea, Republic of Malaysia Myanmar Nepal Pakistan Philippines Singapore Sri Lanka Thailand Turkey Vietnam United Kingdom United States

Import

Export

2007 Difference (Export − Import)

285,214 31,957 2,928,422 1,743,593 2,283,659 738,506 100,679 372,776 6,853,672 14,036 1,639,434 782,266 15,186 84,517 440,813 402,062 848,485 189,687 496,378 453,776 181,908 12,643,526 17,633,830

70,487 29 11,529,843 586,395 1,741,764 496,539 918,254 1,154,544 182,395 3,557 339,802 270,666 355,102 4,338 194,341 1,540,489 210,626 84,078 2,542,711 3,558,332 1,017,798 1,195,303 13,373,194

−214,727 −31,928 8,601,421 −1,157,198 −541,895 −241,967 817,575 781,768 −6,671,277 −10,479 −1,299,632 −511,600 339,916 −80,179 −246,472 1,138,427 −637,859 −105,609 2,046,333 3,104,556 835,890 −11,448,223 −4,260,636

Source: FAOSTAT database, http://faostat.fao.org, accessed October 28, 2009. Note: Data from the United Kingdom and the United States are included for comparison (in thousands of dollars).

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the Philippines are the largest exporter of fruits and vegetables while Japan, India, Hong Kong, South Korea, and Singapore are the top importers.24 It is evident that horticultural crops in Asia represent an enormous opportunity for appropriate agricultural biotechnology applications that can help develop and commercially deploy a broad range of improved horticultural products that meet national and regional needs. This mostly explains the extensive level of biotech research and development on horticultural crops in the region as elaborated in the succeeding sections.

OVERVIEW OF CURRENT ADOPTION OF TRANSGENIC CROPS IN ASIA Of the 25 countries that reportedly planted transgenic (or biotech) crops in 2009, three mega-biotech countries (countries that are growing 50,000â•›ha or more) are developing countries in Asia. These are India (8.4 million ha), China (3.7 million ha), and the Philippines (0.5 million ha). Their combined areas accounted for 9.4% of the total global area planted to biotech crops in 2009.9 While China, India, and the Philippines are the only countries in Asia cultivating biotech crops, other countries in the region have granted regulatory approvals for their importation and direct use for food, feed or processing only. However, there are still many countries in the region that have no official record of granting approval for both planting and for direct use of any transgenic crops although trade of commodities between these Asian countries and countries growing transgenic crops are on going realities. Many of these countries in Asia are parties to the Cartagena Protocol but are still in various stages of approval or in very early phases of implementation of their national biosafety framework, laws, and guidelines. Table 7.2 presents a summary of number of regulatory approvals of the various transgenic crops and traits in the region as of 2009. Asian countries with most number of regulatory approvals are Japan, South Korea, Philippines, and China. Only China and the Philippines have planted biotech food crops in the region. Japan has the most number of approvals, second only to the United States but does not have any commercial planting of any of the approved transgenic crops due to market factors and consumer acceptance. Interestingly, India, which ranks fourth in global production of transgenic crops, has only approved Bt cotton. It is also evident in Table 7.2 that the majority of regulatory approvals granted are for agronomic or field crops, which was not surprising. Of the total number of approvals granted, those given for horticultural crops accounted for only 18% of planting and 12% for direct use for food and feed. A closer inspection of Table 7.2 shows that the only horticultural food crops approved for planting are all grown in China. Japan has given approval for ornamentals but these are not planted in the field. The Philippines has only approved transgenic horticultural food crop for direct use for food and feed such as potato. In terms of traits, single event “input traits” led by herbicide tolerance, dominate the approvals. Overall, Asia’s total number of approvals for commercial cultivation of transgenic crops is still quite few. This scenario is expected to change dramatically within the next 5 years in light of recent developments in crop biotechnology in the

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TABLE 7.2 Regulatory Approvals Granted for Transgenic Crops and Traits for Planting and Direct Use for Food, Feed, or Processing (FFP) in Selected Countries in Asia through 2009 Number of Eventsb Approved Country China

India Indonesia Japan

Malaysia Pakistan Philippines

Singapore South Korea

Taiwan Thailand

Crop Group (Crops) Vegetables (tomato, green and sweet pepper) Fruit (papaya) Ornamental (petunia) Others (canola, cotton, maize, poplar, rice, soybean) Others (cotton) Others (cotton) Ornamental (carnation, rose) Vegetables (tomato, potato) Others (alfalfa, canola, cotton, maize, poplar, rice, soybean, sugar beets) Others (soybean) Others (cotton) Vegetable/root (potato) Others (alfalfa, canola, cotton, maize, soybean, sugar beet) Others (cotton, maize, sugar beet) Vegetable/root (potato) Others (canola, cotton, maize, soybean, sugar beet) Others (maize, soybean) Others (maize, soybean) Horticultural crops Agronomic/field/other crops Total

Traita

Planting

FFP

DR, VR

4

4

VR FC HT, IR, HT + IR, high phytase IR IR FC, HT, flav path, FC DR, IR, IR + VR HT, IR, HT + F, HT + IR, Lys, Lys + IR, CPP, OC, OC + HT, high cellulose HT IR, IR/IR IR, IR + VR HT, IR, HT + IR, Lys, Lys + IR, plant qual, IR/HT IR, HT IR, IR + VR HT, IR, HT + IR, IR/HT

1 1 4

1

HT, IR, HT + IR, IR/HT HT

21

6 0 9 0 52

6 1 21 101

0 0 0 5

1 2 8 46

0 0 0

4 4 53

0 0 15 67 82

26 2 38 263 301

Source: James, C., Global Status of Commercialized Biotech/GM Crops: 2009, ISAAA Briefs 41, International Service for the Acquisition of Agri-biotech Applications, Ithaca, New York, 2009. Trait legend: HT, herbicide tolerance; IR, insect resistance; DR, delayed ripening/altered shelf life; VR, virus resistance; FC, modified flower color; Lys, enhanced lysine content; OC, modified oil content; F, fertility restored; CPP, cedar pollen peptide; plant qual, modified amylase; flav path, flavonoid pathway. a A transgenic event refers to a unique product resulting from stable transformation—incorporation of foreign DNA into a living cell. b Approvals for planting have been granted, but no actual planting in the country has been done.

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region. The  most significant of these developments are China’s approval of transgenic rice and maize, India’s Genetic Engineering Approval Committee (GEAC) recommendation for commercial release of Bt brinjal (eggplant), Japan’s approval for commercial planting of transgenic blue rose, and the significant number of transgenic food crops that are already in advanced stages of development and commercialization in India and China.

PUBLIC ACCEPTANCE AND PERCEPTION OF BIOTECH CROPS IN ASIA Public acceptance of biotech crops and products ultimately decides whether the products are commercially deployed and can deliver on their potential benefits. Stakeholders in Asian developing countries particularly in the Philippines, Malaysia, India, Vietnam, and China were reported to have, in general, positive perceptions on biotechnology applications.25–27 Consumers in the industrial Asian countries of Japan and South Korea, on the other hand, were reported to be more conservative in outlook or negative in perception toward agricultural biotechnology compared to others in the region.26,28 The underlying differences among these country groups were partly attributed to the relative importance of agriculture in these societies. Japan and South Korea are food importing countries where the generally affluent consumers are rather unsure about the potential contributions of the technology to their individual or household food status.28 However, if made aware about potential contributions of the technology to sustainability, consumers in both countries show a marked positive shift in their perception and acceptance of the technology, suggesting that they easily identify themselves with the higher value and goal of sustainability. The availability of the biotech blue rose recently unveiled in Japan and the apparent market acceptance and excitement about the product further suggest a positive consumer response to a product with perceived added value. The rising hectarage planted to biotech crops that are commercialized in some Asian countries and the increase in the number of regulatory approvals for importing countries suggest a growing confidence and acceptance of biotech crops and products. In Malaysia, Bangladesh, and the Philippines, surveys indicated that biotechnology is perceived to be highly important and would be instrumental in future economic development. Farmers generally accept biotech crops if they can derive benefits from its production, as the preliminary survey on biotech rice in Bangladesh revealed.29 Similar perspectives of farmers and consumers were also reported in Malaysia and the Philippines.25,30,31 In India, Krishna and Qaim32 reported that 60% of urban consumers will likely adopt Bt eggplant when commercialized even when marketed at the regular vegetable prices, indicating a relatively high acceptance level of the eggplant technology. Among the stakeholders, farmers’ perception on biotech crops and biotechnology in general is very important since they are the direct principal beneficiaries. In the Philippines, the positive experience of farmers on Bt corn since it was commercialized starting in 2003 averted what could have been continuing opposition from sectors that were initially against the technology.31 In most cases, ambivalence

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to biotech crops and biotechnology were attributed to critically low awareness and knowledge on the subject by the public.33–35 This knowledge gap has also been stressed in Taiwan where there is public disconnect on the topic though there are many ongoing research on biotechnology in the country.34 The effect of increasing the public’s knowledge of biotechnology is a decrease in their perceived risks of various gene technology applications in food production and in agriculture. Collectively, enhancing the awareness of the public will help them make informed decisions and hopefully create a favorable attitude toward products of the technology. These factors are important in spelling success or failure during the commercialization process on biotech crops.36,37 There were recommendations that early consultation and dialogs with various stakeholder groups should be considered during product development, regulatory review of those in the pipeline, and prior to final genetically modified (GM) crop approvals to ensure product success.36,38 Overall, the huge economic benefits and positive experience on biotech products already being grown and/or used in Asia is expected to help pave the way for future success and consumer uptake of other biotech horticultural crops.

TRANSGENIC HORTICULTURAL CROPS IN THE PIPELINE IN ASIA Since biotech crops were first commercialized, there has been a steady increase in adoption of the four major transgenic crops in the market with more than 130 million ha planted in 2009.9 However, despite current and potential benefits offered by the technology, development and commercialization of transgenic horticultural crops in the United States has almost ground to a halt.17 This is in marked contrast to what is happening in Asia where transgenic horticultural crops have taken center stage particularly in India and China. Led by China, Asia has significantly increased its investments in agricultural biotechnology. In 1999, China’s investment was estimated to be $112 million39 and in 2008, the government announced to spend up to $3.5 billion for a special project to develop new transgenic crop varieties.40 Other countries in the region that identified agricultural biotechnology as important for their future economic development have also allocated part of their national agricultural Research and Development (R&D) budget to investments in biotechnology including about $10.2 billion in South Korea and $115 million annually in India. Even the small developing country like the Philippines allocated 5% of its national agriculture budget (Agriculture and Fisheries Modernization Act) to biotechnology. Overall, the investment of the public sector to respective national agricultural R&D ranges from 90.7% to 100% in various Asian countries.41 As a result of this favorable investment climate, the development of transgenic crops has begun in several countries in Asia. FAO database shows that about 603 transgenic crop events are under various phases of development and commercialization pipeline in the region (Figure 7.2) although majority of these are in the experimental phase. India has the highest number of transgenic horticultural crops in the research pipeline and many of these are horticultural crops. In 2009, a significant number of transgenic horticultural crops have reached advanced R&D pipeline, led not surprisingly by India (Table 7.3). Advanced R&D pipeline refers to the stage

163

Transgenic Horticultural Crops in Asia Bangladesh China India Indonesia Malaysia Pakistan Philippines South Korea Sri Lanka

Experimental phase Field trial Commercialization

Thailand Vietnam 0

10

20

30

40 50 Number

60

70

80

90

FIGURE 7.2â•… Relative number of transgenic horticultural crops among Asian countries at different stages of product development. (Data from FAO Bio-Dec, Food and Agriculture Organization Biotechnology for Developing Countries database (FAO-BioDeC), 2009, http:// www.fao.org/biotech/inventory_admin/dep/default.asp)

when a transgenic event is not yet in the regulatory process but at late stages of development, that is, in large scale, multi-location field trials, and generation of data for the review process.42 Table 7.3 and Figure 7.3 present the summary of the current (2009) and possible numbers of expected transgenic horticultural crops and traits in Asia by 2015. The numbers presented were obtained from the worldwide survey of the transgenic crop pipeline42 and other sources.9,39,43–45 Figure 7.3 shows the relative proportion of GM crops by trait. Table 7.3 also shows that at least 30 transgenic horticultural events (approximately 60% of Asia’s total) are predicted to reach the market by 2015. From 2009 to 2015, the total number of transgenic horticultural crops and traits grown in the region is expected to increase by almost fivefold (from 7 to 33); approximately 70% (22 out of 33) of these are transgenic vegetables from India and China. The most dramatic increase (from 0 to 16 events) is expected to happen in India if the current situation on the pending market release of Bt brinjal (eggplant) in India will be resolved soon. It is also evident in Table 7.3 that majority of the efforts have been focused on crops and traits identified to be important to national and regional needs. India’s main focus on vegetables and “input” traits, particularly pest and disease resistance, is understandable given the vegetarian diet of its people, the high incidence of pest and diseases that limits production, and problems with abiotic stresses. What is not shown in the table, but available from the sources listed, is the high proportion (16/39 or 41%) of private (local and multinational) companies involved in the development of transgenic crops in India.9,42 This is in sharp contrast with the situation in China, where most of the developers are public institutions funded by the Chinese government.39,42 It remains to be seen which between China’s public-sector-dominated or India’s public–private investment strategy will prove to be more effective and sustainable in the long term. Notwithstanding the difference in their strategies, what

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TABLE 7.3 Number of Current and Possible Transgenic Horticultural Crops and Traits in Selected Asian Countries through 2009 and Expected to Reach the Market by 2015 Country China

Horticulture Crop Group

Crops

Vegetables and melons

Cabbage, chili, melon, potato, sweet pepper, tomato

Fruits Ornamental

Papaya Petunia

Others

Pogostemon

Vegetables and melons

Cabbage, cauliflower, eggplant, okra, tomato, watermelon

Japan

Others Ornamental

Philippines

Fruits

Mustard Rose, carnation Papaya

Bangladesh Thailand

Vegetables Vegetables Fruits

Eggplant Eggplant Papaya

India

Trait(s) Virus resistance, fungal resistance, quality improvement, abiotic stress tolerance, shelf life Virus resistance Modified flower color Bacterial resistance Insect resistance, abiotic stress tolerance, fungal resistance, plant stature, quality improvement, virus resistance Male fertility Modified flower color Shelf life, virus resistance Insect resistance Insect resistance Virus resistance Total

Commercial in 2009

Advanced R&D

Total by 2015

3

7

10

1 1

0 0

1 1

0

1

1a

0

15

15

0 2a

1 0

1 2

0

2

0 0 0 7

1 1 1 29

(1)b (1)b 30–33

Sources: James, C., Global Status of Commercialized Biotech/GM Crops: 2009, ISAAA Briefs 41, International Service for the Acquisition of Agri-biotech Applications, Ithaca, New York, 2009, Annex 1; Stein, A. and Rodriguez-Cerezo, E., Nat. Biotechnol., 28, 23, 2010, Supplementary data; Huang, J. et al. Science, 295, 674, 2002; Huang, J. and Rozelle, S., Calif. Agric., 58, 112, 2004; Authors’ firsthand knowledge. a A shrub used to extract oil for fragrance or medicinal purposes.45 b Same transformation event used in India but bred into local varieties in Bangladesh and the Philippines.

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Transgenic Horticultural Crops in Asia AB 9% DR 11%

VR 31%

OO 6%

SL 6%

IR 20% QI 11%

FR 6%

FIGURE 7.3â•… (See color insert.) Total events distributed by traits. AB, abiotic stresses; DR, disease resistance including bacterial resistance; FR, fungal resistance; IR, insect resistance; OO, others; QI, quality improvement; SL, shelf life; VR, virus resistance.

seems to be certain is that transgenic horticultural crops will become widespread in China and India, with high potential to be spread in their neighboring countries considering their physical proximity and the nature and size of trade among the countries in the region.

DEVELOPMENT AND DELIVERY OF TRANSGENIC HORTICULTURAL CROPS: EXPERIENCES AND LESSONS LEARNED FROM SOUTH AND SOUTHEAST ASIA The development pipeline of transgenic crops from discovery to commercialization could be a long, demanding, and expensive undertaking. Depending on the crop and/ or trait, it has been estimated that it would take between 7 and 15 years at an estimated cost ranging from hundreds of thousand to millions of dollars to successfully bring to market a transgenic crop. A number of published papers have identified the possible barriers which limit the successful development and commercialization of transgenic crops, including horticultural crops in the United States16,17,31,46 and in developing countries.36,47 These potential barriers fall mainly into the following categories: (1) technical/technology, (2) intellectual property rights and freedom to operate, (3) regulation, (4) market considerations, and (5) Â�consumer acceptance. Technical/technology barriers include challenges in developing efficient transformation procedures and designing vector constructs, choosing the crop and trait, designing critical product concept, and trait efficacy in the field. Intellectual property protection and lack of freedom to operate present hurdles in accessing proprietary technologies that are owned mostly by the private sector and concerns on potential for liability claims associated with unwanted transgenes (low level presence) in local crops.42 The high cost of compliance and uncertainties in regulatory approval

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Transgenic Horticultural Crops: Challenges and Opportunities

process are major hurdles particularly for horticultural crops because of small market and limited hectarage that could limit the potential for recovery of production costs or developing countries could simply not afford. Market reluctance particularly by actors in the supply chain (food manufacturers, processors, and retailers) also presents an enormous challenge even in transgenic crops that have been approved for market release due to worries about consumer acceptance and higher cost and potential for liability associated with identity preservation and product stewardship.46 In this section, we discuss our own experiences and the lessons we learned in dealing with many of the barriers similar to the ones described above, in the course of pursuing the development and commercialization of two important horticultural crops in the region—the transgenic fruit and shoot borer–resistant eggplant (Bt eggplant) in South and Southeast Asia and the papaya ringspot virus (PRSV) resistant papaya in Southeast Asia. We hope that by sharing these experiences, we can contribute by sharing knowledge on and insights into some approaches adopted by a developing country’s national R&D programs to address or overcome the identified constraints to biotech crop development and deployment. Table 7.4 summarizes the identified principal barriers to biotech crop development and deployment of Bt eggplant and PRSV-R papaya and some of the approaches that the two regional projects adopted to address the specific constraints as discussed in the succeeding sections below.

Development of Transgenic PRSV-Resistant Papaya for Southeast Asia The development of the transgenic PRSV resistant papaya was undertaken through the Papaya Biotechnology Network of Southeast Asia (the Network). The Network was established in 1998 with the assistance of the International Service for the Acquisition of Agri-biotech Applications (ISAAA) with funding support from the governments of the five member countries and other public and private sector donors. An early consultation and planning process among the five countries of Southeast Asia—Indonesia, Malaysia, Philippines, Thailand, and Vietnam—made possible the exchange of research updates and strategies among the five countries and identified common constraints and opportunities. It became evident during the consultation process that it would be highly beneficial to establish partnerships among the countries to enable a regional collaboration that would capitalize on their respective strengths and build a regional critical mass of expertise that would otherwise take time to develop in each individual country. The network requested and designated Malaysia Agricultural Research and Development Institute and Thailand (National Center for Genetic Engineering and Biotechnology and Kasetsart University) to serve as the two regional hubs. Malaysia and Thailand, which were the furthest advanced in the development of transgenic papaya, provide advanced technical assistance and training support to other members of the network in molecular virology, tissue culture and transformation, molecular analyses, among others. Among the early identified common constraints were: (1) intellectual property restrictions to research and development tools, (2) technical skill limitations,

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Transgenic Horticultural Crops in Asia

TABLE 7.4 Barriers to Biotech Crop Development and Deployment and Examples of Some Approaches from Two Regional Projects in Asia Strategic Approach Barrier Technical/ technology: event generation/ selection

Intellectual property (IP) and freedom to operate

Bt Eggplant Access and use a common, single near-market transformation event and transfer by conventional breeding to local varieties “Shuttle breeding” through internship of scientists from partner institutions at Mahyco’s Research Center Royalty-free sublicensing agreement through public–private partnership (PPP) Capacity building on IP management thru workshops and trainings

Regulation

Market

Public acceptance

Build and share food and safety data package of the common transformation event to spread the cost of food safety package Design and generate an insect resistance management plan Early discussion with regulators on regulatory data requirements Technical assistance on DNA detection Design product stewardship, monitoring and seed distribution plan Capacity building of partners on product stewardship and monitoring Outreach and communications to actors in the supply chain Proactive and continuous information, communication, and education (IEC) activities for various sectors of society implemented through a network of partners; activities include Workshops, seminars, public forums Publications Regular conduct of public perception surveys Study visits to project sites Interaction with the media thru interviews; news sources, guest in radio and TV broadcast, television features

PRSV-Resistant Papaya Country-specific transformation using local virus strain to address virus diversity and trait efficacy Internship of scientists from partner institutions at Monsanto Laboratory Royalty-free licensing agreement through public– private partnership (PPP) Capacity building on IP management thru workshops and trainings Capacity building for scientists and regulators Workshop to harmonize regional regulatory needs for papaya

Market analyses and design of seed distribution plan

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(3) anticipated regulatory compliance requirements coupled with lack of regulations and guidelines in most countries at the time the network was established, and (4) anticipated public/consumer acceptance issues. The network facilitated a technology donation agreement with a private sector technology provider, Monsanto, for all virus resistance technologies over which Monsanto has effective intellectual and tangible proprietary rights. Beyond the technology donation, Monsanto also hosted and provided key technical training to papaya scientists from all five countries on genetic engineering for virus resistance and provided continuing technical assistance in product development and regulatory data development. In addition to the public–private technology transfer, Thailand and Malaysia provided key technical training and assistance to other member countries of the network, and Malaysia actually served as a research laboratory for the Philippines for the initial transformation work at the time that existing Philippine regulations did not allow Agrobacterium-mediated transformation of papaya. Recognizing the need at the time to simultaneously facilitate product development with the evolving regulations and guidelines, the regional network also broadened the capacity building activities to include intellectual property management, biosafety, and food safety, to include not just the national technology developers but the emerging corps of national regulators. Undertaking the capacity building activities at a regional basis enabled a level of harmonization in the science-based risk assessment among countries without intruding into the sovereign rights of member countries to formulate their respective regulations. Finally, the network has been proactive in education and information activities to reach out to various sectors and the general public by raising awareness and understanding of modern biotechnology including transgenic crops. The network is considered as among the first of regional initiatives in Asia that served as a model of North–South and South–South technology transfer partnership in agricultural biotechnology. It facilitated the transfer of proprietary technologies, enhanced regional and national capacity in biotechnology including the associated policy and regulatory areas, and helped shift the focus of national R&D from mostly academic work toward developing tangible, commercializable products that meet farmers’ needs and compliant to best practices and standards.

Development and Commercialization of Fruit and Shoot Borer–Resistant Eggplant in South and Southeast Asia The development and commercialization of transgenic fruit and shoot borer–resistant eggplant containing cry1Ac gene from Bacillus thuringiensis (Bt eggplant) is a public–private partnership project initiated through the initiative of the Agricultural Biotechnology Support Project II (ABSPII), a consortium of public and private sector institutions led by Cornell University and funded by the U.S. Agency for International Development (USAID). Bt eggplant was chosen through a two-stage priority setting process. In the first stage, a consultation meeting was conducted with the various stakeholders (scientists, policy makers, regulators, industry representatives, and consumers) in our focus countries of India, Bangladesh, and the Philippines. Bt eggplant

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was given high priority in each country based on SWOT (strengths, weaknesses, opportunities, and threats) analyses performed. In the second step, ex-ante analysis on the socioeconomic impacts of Bt eggplant was conducted by a team of local and international economists working in the project. The results showed that eggplant is a very important food for the people of India, Bangladesh, and the Philippines and a highly profitable cash crop for small farmers in the three countries. Farmers who grow eggplant in both regions share the same problem—need for a cost-effective and safe method to control the most damaging insect pest of eggplant—the eggplant fruit and shoot borer (EFSB). Eggplant yields and incomes are drastically reduced by the damage caused by EFSB. Farmers resort to frequent application and excessive amounts of pesticide to control the pest, raising the level of pesticide residue in the environment and the eggplant consumed by the public. Conventional breeding of insect-resistant varieties has failed because no resistant eggplant varieties have been found that can effectively control EFSB. The results of the ex-ante analyses revealed that substantial economic and environmental benefits are projected if Bt eggplant is commercialized in all three countries.48,49 Other important considerations such as freedom to operate, favorable regulatory approval and likelihood of gaining market acceptance and public support were deemed feasible. Considering the cost of product development and regulation, ABSPII made a strategic decision to use one common transformation event owned by Maharashtra Hybrid Seed Company (Mahyco), Mahyco eggplant event EE-1, in all three geographies. The strategy presented opportunities to spread the regulatory cost and contribute to the regional harmonization of regulatory systems by building a common safety package for the event, which was proposed to be submitted to the regulators in each partner country. In addition, the trait stability and efficacy of EE-1 has already been proven and was determined to be technologically a near-market product. The choice of EE-1 also presented an opportunity to explore the opportunities of a public–private partnership. From the very outset of the Bt eggplant project, ABSPII in cooperation with Hyderabad-based Sathguru Management Consultants Pvt. Ltd. worked with Mahyco to devise a system whereby all farmers in India as well as Bangladesh and the Philippines could gain access to the Bt eggplant technology. The dialogue led to a mutually beneficial arrangement that addressed freedom-to-operate issues via appropriate licensing arrangements. The Bt eggplant transformation event developed by Mahyco with support from Monsanto (which licensed the use of Cry1Ac expression vector) was thus cleared of potential intellectual property (IP) barriers, paving the way for further development and commercialization. Mahyco sublicensed the technology on a royalty-free basis to the public sector partner institutions in India, Bangladesh, and the Phiilippines.50 A specific product stewardship and monitoring strategy to be implemented by the licensee to safeguard the interest of the licensors was also devised during the sublicensing process. The issue of market share among the different Bt varieties in India was also addressed by segmenting the market between the Bt eggplant hybrid of Mahyco and the Bt varieties of the public partners. The regulatory file development of Bt eggplant benefited tremendously from the expertise and experience of Mahyco, the first Indian company to commercialize Bt cotton hybrids in India. In addition to access to data for the cry1Ac gene construct

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Transgenic Horticultural Crops: Challenges and Opportunities

used in both Bt cotton and Bt eggplant, Mahyco led the partners in streamlining the development of a comprehensive environmental and food safety regulatory package for Bt eggplant event EE-1. The regulatory file complied by Mahyco for event EE-1 was made available in the Ministry of Environment website (http://www.envfor.nic. in/divisions/csurv/geac/information brinjal.htm). The same dossier was provided to all partners for submission and review of relevant information by their respective regulatory bodies, thus accelerating the approval process in these two countries. Proactive and frequent interactions with the regulatory bodies in the three countries also helped in accelerating the approval process. A proactive information, communication, and education strategy was also developed very early in the project implementation to address anticipated public and market perception issues. The communication design followed the product development pathway so that the communication and outreach activities and messages were focused on the needs of specific stakeholders as the product moved from the development stage toward regulatory clearance, precommercial stage, and eventually toward the commercial stage. An important feature of the communication strategy was a regular survey of perception, attitude, and level of knowledge and information needs of stakeholders about biotechnology in general and the Bt eggplant in particular.

CONCLUSION Asia is home to more than half of the world’s population as well as to the highest number of poor people suffering from hunger and malnutrition. Ensuring the food security of Asia is a formidable challenge not only because of the enormous demand brought about by a growing population with rising incomes, but because the additional food will have to be produced amidst decreasing land and water resources and increasingly severe and frequent biotic and abiotic stresses attendant to climate change. The region accounts for about 75% and 50%, respectively, of the global hectarage growing vegetables and fruits. Horticultural crops thus comprise a major part of the region’s and the world’s agricultural production and, being high value cash crops, contribute substantially to the income and well-being of millions of farmers and consumers especially in the developing countries. Modern biotechnology applications, including genetic engineering, are a powerful tool to complement the conventional methods of crop improvement. Asia currently has three countries cultivating biotech/transgenic crops—China, India, and the Philippines, but only China commercially grows a transgenic fruit crop, a papaya engineered for virus resistance. Biotech-improved blue rose is also cultivated in Japan and as an ornamental crop would qualify as a new commercial transgenic horticultural product in the region. Genetic engineering of various horticultural crops is extensively pursued by most countries in the region that have developed the basic capacity to undertake modern biotechnology. Timely development and commercial deployment of biotech horticultural crops in the region are hampered by several constraints, including (1) access to proprietary biotechnology applications, (2) regulations (or the absence thereof) and costs of regulatory compliance, (3) technical capacity, and (4) market and public acceptance. Overcoming these constraints are key to effective development and use of a broader

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range of biotech crop products that meet national/local needs. Innovative approaches such as public–private and multicountry partnerships and networks, product-focused capacity building activities and proactive and dynamic engagement with various stakeholders may facilitate overcoming some of these hurdles and expedite commercial adoption of new, improved products. Two examples of such regional initiatives, working on biotech vegetable (eggplant) and fruit (papaya) products, are cited in this chapter to draw some lessons and insights. The biotech pipeline for horticultural crops in the Asian region, coming mostly from public sector institutions, is a rich array of key crops and traits that meet the identified priority needs of producers and consumers. It is essential, for the present and future well-being of Asian farmers and consumers, for these products to be commercially deployed in the most appropriate and timely manner for their benefit.

ACKNOWLEDGMENTS The authors gratefully acknowledge ABSPII, a United States Agency for International Development (USAID)-funded consortium led by Cornell University and the Papaya Biotechnology of Southeast Asia facilitated by ISAAA for allowing us the use of their project strategies and experiences in Asia as examples of regional biotech initiatives. We wish to thank Dr. Lourdes D. Taylo and Ms. Zabrina J. Bugnosen for their valuable assistance in proofreading and formatting of the manuscript.

REFERENCES





1. U.N., World Population Prospects: The 2008 Revision, Highlights. United Nations, Department of Economic and Social Affairs, Population Division, Working Paper No. ESA/P/WP.210. New York, 2009. http://www.un.org/esa/population/publications/ wpp2008/wpp2008_highlights.pdf (accessed on August 19, 2009). 2. DFAT, Asian Agrifood Demand Trends and Outlook to 2010, Australia Department of Foreign Affairs and Trade Subsistence to Supermarket II Series, 2004. http://www.dfat. gov.au/PUBLICATIONS/agrifoodasia/vIII.html (accessed on August 29, 2009). 3. Hautea, R. and Escaler, M., Plant biotechnology in Asia, AgBioForum, 7(1&2), 2, 2004. 4. FAO, Press Release: More people than ever are victims of hunger, Food and Agriculture Organization of the United Nations, Rome, Italy, 2009. http://www.fao.org/fileadmin/user_ upload/newsroom/docs/Press%20release%20june-en.pdf (accessed on August 19, 2009). 5. Karplus, V.J. and Deng, X.W., Eds., Agricultural Biotechnology in China: Origins and Prospects, Springer, New York, 2008. 6. Borlaug, N.E, Sixty-two years of fighting hunger: Personal recollections, Euphytica, 157, 287, 2007. 7. Brookes, G. and Barfoot, P., GM Crops: Global Socio-Economic and Environmental Impacts 1996–2007, P.G. Economics Ltd, Dorchester, U.K., 2009. http://croplife.intraspin com/Biotech/gm-crops-global-socio-economic-and-environmental-impacts-1996-2007 (accessed on August 23, 2009). 8. McGloughlin, M.M., Ten reasons why biotechnology will be important to the developing world, AgBioForum, 2(3&4), 163, 1999. 9. James, C., Global Status of Commercialized Biotech/GM Crops: 2009, ISAAA Briefs 41, International Service for the Acquisition of Agri-biotech Applications, Ithaca, NY, 2009. 10. Bruening, G. and Lyons, J.M., The case of the FLAVR SAVR tomato, California Agriculture, 54(4), 6, 2000.

172

Transgenic Horticultural Crops: Challenges and Opportunities

11. Yu, T.A., Yeh, S.D., and Yang, J.S., Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue, Plant Cell, Tissue and Organ Culture, 74, 169, 2003. 12. Petri, C. and Burgos, L., Transformation of fruit trees. Useful breeding tool or continued future prospect? Transgenic Research, 14, 15, 2005. 13. Wang, K., Ed., Agrobaterium protocols, in Methods in Molecular Biology, 2nd edn. Vol. 1., Humana Press, Totowa, NJ, 2006, p. 343. 14. Seong, E.S. and Song, K.J., Factors affecting the early gene transfer step in the development of transgenic ‘Fuji’ apple plants, Plant Growth Regulation, 54, 89, 2008. 15. Bradford, K.J. et al., Challenges and opportunities for horticultural biotechnology, California Agriculture, 58(2), 68, 2004. 16. Bradford, K.J. and Alson, J.M., Diversity of horticultural biotech crops contributes to market hurdles, California Agriculture, 58(2), 84, 2004. 17. Clark, D., Klee, H., and Dandekar, A., Despite benefits, commercialization of transgenic horticultural crops lags, California Agriculture, 58(2), 89, 2004. 18. Sumner, D.A., World trade rules affect horticultural biotechnology, California Agriculture, 58(2), 77, 2004. 19. Dalal, M., Dani, R.G., and Kumar, P.A., Current trends in genetic engineering of vegetable crops, Scientia Horticulturae, 107, 215, 2006. 20. Johnson, G.I., Weinberger, K., and Wu, M.H., The Vegetable Industry in Tropical Asia: An Overview of Production and Trade, with a Focus on Thailand, Indonesia, the Philippines, Vietnam, and India (Explorations series; no. 1), AVRDC—The World Vegetable Center, Shanhua, Taiwan, 2008. 21. Huang, S.W., Global trade patterns in fruits and vegetables, USDA-ERS Agriculture and Trade Report No. WRS-04-06, 2004. 22. Lee, J.M., Unique feature of horticulture in Asia and its global impact, presented at World Conference on Horticultural Research, Rome, Italy, June 17–20, 1998. http:// www.agrsci.unibo.it/wchr/wc1/lee.html (accessed October 26, 2009). 23. Chatterjee, S. et al., Food consumption, trade reforms and trade patterns in contemporary India: How do Australia and NZ fit in? Centre for Applied Economics and Policy Studies Working Paper 2/06, Massey University, New Zealand, 2006. 24. FAO, Selected indicators of food and agricultural development in the Asia-Pacific region 1998–2008. Food and Agriculture Organization of the United Nations, Regional Office for Asia and the Pacific, Bangkok, Thailand, 2009. http://www.fao.org/docrep/012/ i1020e/i1020e00.htm (accessed on October 28, 2009). 25. Amin, L. et al., Uncovering factors influencing Malaysian public attitude towards modern biotechnology, Pacific Journal of Molecular Biology and Biotechnology, 14(2), 33, 2006. 26. AFIC, Food Biotechnology: Consumer Perceptions of Food Biotechnology in Asia, Asian Food Information Center, Bangkok, Thailand, 2008. 27. ISAAA, Public understanding, perceptions, and attitudes towards biotechnology in Vietnam. Country Monograph, International Service for the Acquisition of Agri-biotech Applications (ISAAA) and the University of Illinois at Urbana-Champaign, http://isaaa. org/kc/inforesources/publications/perception/Vietnam.pdf (accessed on August 19, 2009). 28. Hoban, T., Public attitudes toward agricultural biotechnology, ESA Working Paper No. 04-09. Agricultural Economics and Development Division, Food and Agriculture Organization of the United Nations, 2004. http://www.fao.org/docrep/007/ae064e/ ae064e00.htm (accessed on August 24, 2009). 29. Gruere, G.P. et al., Potential of transgenic crops in Bangladesh: Findings from a consultation of Bangladeshi scientific experts, Plant Cell Tissue and Organ Culture, 86, 411, 2006. 30. Aerni, P. and Bernauer, T., Stakeholder attitudes toward GMOs in the Philippines, Mexico, and South Africa: The Issue of Public Trust, World Development, 34(3), 557, 2006.

Transgenic Horticultural Crops in Asia

173

31. Cabanilla, L.S., Socio-economic and political concerns for GM foods and biotechnology adoption in the Philippines, AgBioForum, 10(3), 178, 2007. 32. Krishna, V.V. and Qaim, M., Consumer attitudes towards GM food and pesticide residues in India. Review of Agricultural Economics, 30(2), 233, 2008. 33. Torres, C.S. et al., Public Understanding and Perception of and Attitude towards Agricultural Biotechnology in Indonesia. International Service for the Acquisition of Agri-biotech Applications (ISAAA), SEAMEO Regional Center for Graduate Study and Research in Agriculture (SEARCA) and College of Development Communication, University of the Philippines Los Baños (CDC-UPLB), Los Baños, Philippines, 2006. 34. Fan, M.F., Stakeholder perceptions and responses to GM Crops and foods: The case of Taiwan, Sustainable Development, 17, 391, 2009. 35. Chen, M.F. and Li, H.L., The consumer’s attitude toward genetically modified foods in Taiwan, Food Quality and Preference, 18, 662, 2007. 36. Gregory, P. et al., Bioengineered crops as tools for international development: Opportunities and strategic considerations, Experimental Agriculture, 44, 277, 2008. 37. Rommens, C.M., Barriers and paths to market for genetically engineered crops, Plant Biotechnology Journal, 8, 101, 2010. 38. Cohen, J.I. and Paarlberg, R., Unlocking crop biotechnology in developing countries–– A report from the Field, World Development, 32(9), 1563, 2004. 39. Huang, J. et al., Plant biotechnology in China, Science, 295, 674, 2002. 40. Lin, X. and Li, J., Agricultural biotechnology and its management in China: Country Report, presented at FAO Regional Biosafety Training Workshop, Rama Gardens Hotel, Bangkok Thailand, November 30 to December 4, 2009. 41. Beintema, N.M. and Stads, G.J., Agricultural R&D capacity and investments in the Asia–Pacific region. IFPRI Research Brief No. 11. International Food Policy Research Institute, Washington, DC, 2008. 42. Stein, A. and Rodriguez-Cerezo, E., International trade and the global pipeline of new GM crops, Nature Biotechnology, 28, 23, 2010. 43. Cohen, J.I., Poorer nations turn to publicly developed GM crops, Nature Biotechnology, 23(1), 27, 2005. 44. FAO Bio-Dec, Food and Agriculture Organization Biotechnology for Developing Countries database (FAO-BioDeC), 2009. http://www.fao.org/biotech/inventory_ admin/dep/default.asp (accessed on September 3, 2009). 45. Huang, J. and Rozelle, S., China aggressively pursuing horticulture and plant biotechnology, California Agriculture, 58, 112, 2004. 46. Alston, J.M., Bradford, K.J., and Kalaitzandonakes, N., The economics of horticultural biotechnology, in Plant Biotechnology in Ornamental Horticulture, Li, Y. and Pei, Y., Eds., Haworth Food and Agricultural Products Press, an imprint of The Haworth Press, Inc., Binghamton, NY, 2006. 47. Delmer, D.P., Agriculture in the developing world: Connecting innovations in plant research to downstream applications, Proceedings of the National Academy of Sciences of the United States of America 102(44), 15739, 2005. 48. Ramasamy, C. et al., Economic and Environmental Benefits and Costs of Transgenic Crops: Ex-Ante Assessment, TNAU, Coimbatore, India, 2007. 49. Norton, G.W. and Hautea, D.M., Eds., Projected Impacts of Agricultural Biotechnologies for Fruits and Vegetables in the Philippines and Indonesia. ISAAA and SEARCA, Los Banos, Laguna, Philippines, 2009. 50. Medakker, A. and Vijayaraghavan, V., Successful commercialization of insect-resistant Â�eggplant by a public–private partnership: Reaching and benefiting resource-poor farmers, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices, Krattiger, A. et al., Eds., MIHR, Oxford, U.K. and PIPRA, Chap. 17.25.

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The Economic and Marketing Challenges of Horticultural Biotechnology Steven Sexton and David Zilberman

CONTENTS Introduction............................................................................................................. 175 High Fixed Costs of R&D....................................................................................... 176 Consumer Acceptance............................................................................................. 178 Regulation............................................................................................................... 180 Bans on GMOs................................................................................................... 181 Identity Preservation, Segregation, and Labeling.............................................. 182 Directed R&D and Role for Public Sector: Public–Private Partnerships............... 183 Predictions and Policy Recommendations.............................................................. 186 References............................................................................................................... 187

INTRODUCTION In the 13 years that followed the commercial introduction of genetically engineered field crops in 1996, agricultural biotechnology became the most quickly adopted crop technology in history. More than 14 million farmers in 25 different countries planted a combined 134 million hectares of genetically modified (GM) crops in 2009.1 Virtually, none of this land, however, was planted to horticultural crops, even though agricultural biotechnology was born with the Flavr Savr tomato in 1994. While the introduction of agricultural biotechnology in major field crops has been a success and marked by a tremendous acceptance by farmers and a strong empirical record of productivity growth, the development of horticultural biotechnology has been slow. The Flavr Savr tomato, intended to resist damage in shipping, is no longer produced, nor is a strawberry engineered to resist frost damage or an insect-resistant potato. Only the GM papaya has achieved any degree of commercial success, with 70% of the U.S.-bound Hawaiian crop planted with GM seeds. No GM horticultural crop has been deregulated since 1999, and the number of field trials has declined in recent years.2

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In spite of the commercial failure of horticultural biotechnology, there exists ample opportunity for welfare-improving innovation in specialty crops. In fact, given the agronomic demands of specialty crops and the quality demands of those who consume them, the capacity for genetic improvements in horticultural crops may exceed that of major field crops. Farmers of horticultural crops could benefit not just from the herbicide-tolerant and insect-resistant traits introduced into major field crops like cotton, maize, soybean, and canola, but also from traits to protect against plant disease.3–5 Traits that extend the shelf life of produce or boost their nutrient content would also be valuable, as would traits to control growth in ornamental plants and make flowers last longer.2 In addition, because plant breeding is particularly slow and cumbersome for specialty crops, the capacity to introduce an advantageous trait into an elite cultivar without backcrossing is highly beneficial.2 In spite of the serious potential for gains to producers and consumers, horticultural crops have been largely overlooked by the biotechnology revolution, and research and development (R&D) for these crops is declining. The failure of horticultural biotechnology innovation to proceed at the same pace as agricultural biotechnology is largely a matter of economics. In this chapter, we explain how high fixed costs for biotechnology research, diversity in horticultural crops, market size, consumer doubts about GM foods, consolidation in food processing and marketing, and government regulation of genetic plant engineering constrain the development of new specialty crops. We further articulate policies to help spur socially beneficial innovation in horticultural biotechnology and offer predictions for where horticultural biotechnology may succeed.

HIGH FIXED COSTS OF R&D In Schmookler’s seminal book, Invention and Economic Growth,6 he observed that “invention is largely an economic activity, which, like other economic activities, is pursued for gain.” The role of profits in determining the pace and direction of technological change is a subject that has motivated vast theoretical and empirical literatures. Profits are central not just to endogenous growth models but also to theories of induced innovation and directed technical change.7–10 In his assessment of the adoption of hybrid corn seed in the United States, Griliches provided early evidence that profitability and market size are closely tied to innovation and technology adoption.11 More recently, Kremer argued that investment in cures to Third World diseases is too low because markets for such cures are too small.12 The literature on adoption and innovation finds that adoption and innovation activities require making risky investments.13,14 The likelihood, timing, and scale of adoption and innovations are affected by risk considerations. Increased variability and riskiness tend to reduce economic activity.15,16 The challenges to innovation in horticultural biotechnology are fundamentally economic, and they are derived from the profitability and risk management decisions made by firms throughout the supply chain, which affect the rate and direction of innovation. They are not altogether different from the challenges the medical world faces in overcoming Third World diseases: The potential for welfare gains is tremendous, and yet the relevant R&D is substantially lacking. As we will see in subsequent

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sections of this chapter, the potential to recoup costly investments in horticultural R&D is limited by consumer acceptance and regulation. Against these expectations of low benefits, firms must weigh the costs of innovation, which are mostly fixed. While the costs of commercializing GM crops are independent of the size of the crop, the benefits are directly proportional to the size of crops, which means that the development of GM horticulture is less likely to be profitable than the development of GM field crops. Apart from the costs of research, the regulatory approval process for new biotech crop varieties is slow and expensive, imposing barriers to the development and commercialization of new technologies and creating orphan crops.17,18 The cost of commercializing a single transgenic variety can exceed $100 million.18 Once a new trait has been developed, it must undergo extensive testing for environmental and human safety in order to be approved for production. In the United States, no fewer than three federal agencies are responsible for approving various aspects of GM technologies—the U.S. Environmental Protection Agency, the U.S. Department of Agriculture (USDA), and the U.S. Food and Drug Administration (FDA). The registration process relies on ex ante testing of new technologies, followed by continual monitoring and inspection in the field, with the technology being subject to cancellation if adverse events occur. The outcomes of research efforts, the regulatory costs, and time of approval are uncertain, and investors require extra gains to take these risks. Each transgenic event must undergo the same rigors of testing, regardless of whether the same trait was previously approved for a different plant species or variety.19 Registration of each GM variety is estimated to cost as much as $15 million. Testing at the varietal level can be costly and causes firms to introduce new traits into a smaller number of varieties than would be optimal absent the costly testing.20 Evidence suggests that crop biotechnology innovation has slowed because of high costs of regulatory compliance.17,21,22 The diversity of horticultural crops causes biotechnology regulatory costs to be disproportionately burdensome relative to field crops. Horticulture comprises hundreds of species and thousands of cultivars each produced in relatively small quantities. It can be costly to introduce a GM trait into any specific crop and cultivar, even if the trait has previously been used in other species.2 In many horticultural crops, several different varieties are commercially important. If introgression of the new trait via backcrossing is not an option, such as may be the case for clonally propagated varieties that do not breed true, each variety must be separately transformed in the lab, and each must be separately tested and approved. Regulatory costs would add up, but they could not be spread out over nearly as large a market as could row crops.19 A regulatory regime based on testing at the trait level rather than the event or varietal level would reduce compliance costs and encourage development of traits that could be introduced into closely related specialty crops, like watermelon and cantaloupe.19 Another cost associated with the development of horticultural biotechnology is the cost of gaining access to complimentary intellectual property rights (IPRs). The transaction costs for gaining the freedom to operate (FTO) in relevant IP space are considerable—as high as $100,000 per contract. And these costs are independent

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of market size. The costs also increase as firms seek to export to foreign markets because additional FTO agreements must be negotiated. A large share of horticultural crop output is exports, making these costs an important consideration in investment decisions. The costs of FTOs are higher for horticultural crops than field crops because the relevant IP is more secured and distributed across a greater number of IP holders.23 Due to IP congestion and the difficulty in managing IPRs and navigating IP spaces, several leading U.S. public-sector agricultural research organizations established the Public Intellectual Property Resource for Agriculture (PIPRA), which provides IP management solutions for public sector and small private sector players in horticulture. PIPRA seeks to coordinate the disparate portfolios of its member organizations to support specialty crop applications. For instance, PIPRA attempts to better specify licensing agreements to distinguish the “fields of use” so that a technology could at once be licensed for use on major row crops as well as for uses in smaller markets, including those in developing countries. PIPRA also is developing an IP clearinghouse and working to pool patents to be licensed as a bundle for uses in specific crops or specific regions. Such efforts can help to mitigate some of the barriers to entry in horticultural biotechnology. In spite of efforts like PIPRA, the high fixed costs of biotechnology R&D, particularly, the regulatory burden, require a substantial market in order for innovating firms to recoup their investments. Many of California’s specialty crops have become technological orphans because agricultural chemical companies have concluded that the potential market is too small to warrant high fixed costs to develop pesticides for specialty crops. By a number of measures, it appears that seed companies share the sentiment of the chemical companies, preferring to invest in conventional breeding of horticultural crops, rather than genetic engineering. Bradford et al. argue that the marked decline in R&D intensity for lower market crops is a consequence of high registration costs.2

CONSUMER ACCEPTANCE In the 11,000 years since the domestication of plants, crop improvement has been widely hailed as a necessity for overcoming human suffering and promoting economic growth. Plant breeding has advanced considerably since the days of weak selection. It was not until genetic plant engineering was introduced that sophisticated breeding techniques elicited consumer opposition.24 The application of the same genetic processes to pharmaceutical production has been widely accepted by consumers, whereas there remain considerable health and environmental concerns associated with biotechnology in food crops. In the 13 years since the commercial introduction of GM field crops, the technology has largely proven to be safe to human health and the environment and to provide considerable benefits in terms of increased food output and reduced use of chemical pesticides and tilling operations.25 Shelton et al. reviewed the risks of insect-resistant GM crops and determined the risks to humans and the environment to be lower than the risks posed by alternative pest-control technologies.26 Nevertheless, surveys of public attitudes routinely find large portions of the population are skeptical of biotechnology in general and food applications in particular.27,28

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Moschini traced current doubts about biotechnology to the introduction of recombinant DNA and the surrounding controversy in the 1970s.27 Twenty years later, and with accumulated evidence from millions of experiments, the debate about the safety of recombinant DNA was settled—at least to the satisfaction of the scientific community.29 By 2003, following a comprehensive review of existing evidence, the International Council for Science had also declared consumption of foods containing GM ingredients to be safe. The safety of commercialized GM food products was also certified by a number of national science academies and governmental agencies, including the USDA. Environmental concerns center on the impact of biotechnology on nontarget species, including the risk of gene flow beyond the farm gate. While such risks surely exist and some isolated cases of gene flow between GM crops and proximal non-GM crops have been documented, governments have guarded against contamination of conventional crops by requiring buffer crops between transgenic and conventional crops. Against these risks, including the yet undocumented risk of gene flow to wild flora and fauna, weigh environmental benefits associated with the adoption of agricultural biotechnology, including reduced use of chemical pesticides, substitution to less toxic and less persistent herbicides, and adoption of low-till and no-till farming practices that reduce soil erosion and carbon emissions. Not withstanding these assurances by scientific and regulatory agencies, biotechnology in food crops remains controversial, perhaps owing to heterogeneity with regard to scientific literacy among the public and misperceptions about how inserted traits alter plant development and food production.27 Miranowski et al. and Josling and Tangermann reported differing opinions on biotechnology between scientists and the public at large.30,31 Despite a spirited debate about the safety of biotechnology in academic and policy circles, surveys consistently show that consumers have little information about the risks and benefits associated with GM foods.32,33 Much of the controversy surrounding consumption of GM foods may stem from the fact that the first commercialized transgenic crops contained input traits designed to improve agronomic performance and food production, rather than output traits intended to boost food quality.34 The former presumably benefits consumers in the form of lower food prices, though these benefits may be less discernable to consumers than products with new attributes, like additional nutrients or extended shelf lives. Agronomic traits can be perceived as benefiting farmers and processors rather than consumers. Based on a survey of more than 2000 consumers, however, Bredahl found broad rejection of GM technology overall, regardless of trait.35 A number of studies have found that consumers are willing to pay a premium for food products that are free from GM ingredients.36–39 Opposition to GM foods declines, however, when consumers are provided information about the health or environmental benefits of genetically engineered traits.37,38,40,41 Frewer et al. and Mucci and Hough found evidence that consumers are more willing to accept genetic modification that provides nutritional or health benefits but not traits that reduce cost or extend shelf life.42,43 Chern and Rickertsen reported results from a cross-national survey that showed acceptance of GM technology was broader among consumers in the United States than in Norway.44 As many as 70% of

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Americans consumed products with GM traits that either reduced chemical applications on crops or increased the nutritional value of foods. Golden rice—fortified with beta-carotene—elicits a positive willingness to pay (WTP). Any WTP for reduced pesticide residual in food, however, is offset by the perceived risks associated with GM technology. Low consumer acceptance of crop biotechnology reverberates up the supply chain, providing a disincentive for innovation. Food processors have difficulty in marketing products made with GM ingredients, so they contract with farmers for non-GM crops. Without contracts for GM crop output, farmers are disinclined to plant GM seeds. And if farmers will not purchase the seed, seed companies and biotechnology firms will not invest in the substantial R&D costs necessary to produce the seeds. The backlash against the GM potato serves as a cautionary tale about the limits of consumer willingness to accept GM foods. McDonald’s, the largest consumer of potatoes in the United States, began using an insect-resistant potato in its French fries in order to reduce costs. Consumer opposition was intense, however, and the company and other major food processors stopped sourcing GM potatoes in early 2000. One year later, Monsanto, which had developed the insect-resistant potato 6 years before, stopped marketing it altogether. The extent to which the experience of the GM potato slowed innovation in horticultural biotechnology is not clear, but it is likely no coincidence that R&D directed toward food crops declined sharply after Monsanto withdrew its GM potato. In 1999, 374 field test notifications were submitted for GM horticultural crops. By 2003, that number had fallen to 97.2 In contrast, the number of field-test notifications for major GM field crops rose from 506 in 1999 to 520 in 2003. Consumers have generally been more tolerant of GM crops destined for animal feed rather than human consumption, which may explain why the most rapid innovation is occurring in the development of drought-tolerant varieties for major crops.

REGULATION Consumer acceptance of GM foods certainly limits the returns to agricultural and horticultural R&D. But government regulation further constrains the market and deters commercialization of GM horticultural crops that would be demanded, at least in niche markets. Ex ante testing and registration requirements for new transgenic events increase the fixed costs of commercialization and create a disincentive for investment in horticultural biotechnology research, as was discussed previously. However, two other elements of government policy also limit the profitability of GM horticulture throughout the supply chain. First, some governments ban imports of foods containing GM ingredients and deny registrations for domestic GM crop production. These policies not only restrict market access for GM output but also limit demand for seed technologies themselves. Second, where GM products are commercialized, processors often face strict rules for segregating and labeling GM foods and derivatives of GM ingredients. Liability associated with adventitious comingling of GM and non-GM ingredients creates a deterrent to GM-food provision by food processors.

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Bans on GMOs The United States was an early and aggressive adopter of agricultural biotechnology; the E.U. was not. In fact, it adopted a precautionary approach that emphasized a zero-risk tolerance and resulted in a de facto ban on GM technologies for a decade, ending only in 2008. The typical explanation for the stark divergence in policies is that European consumers are more risk averse than American consumers, in part because of their history with major food scares.45–48 Graff and Zilberman, however, rejected this conventional wisdom because risk preferences for agricultural chemicals and intra-E.U. trade reveal a level of acceptance that is inconsistent with the zero-risk tolerance that defined GM food policy.49 They instead offered an explanation for policy divergence based on political economy considerations in which Europeans sought to protect the agrochemical industry in which they had a comparative advantage from the agricultural biotechnology industry dominated by American firms. Graff et al. formalized this hypothesis in a model in which interested parties, like chemical companies, environmental activists, and some farming cohorts, persuaded consumers to be wary of GM foods.50 Consumer sentiment then influenced policy, resulting in regulations that may not serve the interests of consumers in the E.U. or elsewhere. The European ban on GM foods has influenced policies in Africa, which has largely banned agricultural biotechnology in spite of the potential for substantial gains.51 African countries largely rely on European countries for export markets, so they are unlikely to deviate from European policy. Still, there is evidence that existing technologies could generate cost savings in agricultural production of 10% and that yields would climb.52 Subsequent generations of agricultural biotechnology could yield even greater benefits for Africa, providing drought-tolerant varieties and boosting the nutrient content of staple crops. The European ban on GM food imports has impacted the adoption decisions of farmers beyond Africa.48 For instance, Europe is a major destination for U.S. farm production and vice versa. Following the institution of the GM food moratorium in Europe, U.S. corn exports to the region essentially halted out of fear of comingling GM and non-GM corn.53 A consequence of GMO bans around the world—and especially in Europe—is that the market for GM traits is artificially constrained. Even though the adoption of agricultural biotechnology has been rapid by historical standards, only about one-fourth of the world’s countries planted GM seeds in 2009—and half of it was planted in the United States.1 Farmers in nonadopting countries cannot use the new technologies even if they want to use them, and those in adopting countries may not want to use them if adoption would jeopardize export markets. Without a robust market for new seed technologies, the R&D pipeline shrinks, stranding traits that have already been created and slowing the creation of new traits. Graff and Zilberman show the fall in patent applications, investment dollars, and firm start-ups that occurred during the European ban.49 While stringent regulation may have been intended to protect against risks associated with damagecontrolling GM traits, they also surely delayed the introduction of quality-enhancing traits. The introduction of the second-generation GM crops is long overdue. The European moratorium, then, may prove to have significant and lasting social

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welfare  costs in terms of delayed or foregone innovation in nutrition, production efficiency, and environmental mitigation.54 The E.U. lifted its moratorium on GM products in 2008 and by 2010 had deregulated its first transgenic event in more than a decade. Nevertheless, the adoption of GM crops in Europe declined from 2008 to 2009, and Germany instituted a ban on the production of GM crops. Spain remains the only consistent and significant adopter of GM technologies in Europe, though the land it plants to GM seed constitutes a little more than a rounding error in the total GM land base. Adoption in Africa continues to lag.1

Identity Preservation, Segregation, and Labeling Despite broad consumer opposition to GM foods, there are niche markets that can be served by particular genetically engineered traits. These niches may be underserved, however, because of demands from consumers and regulators that foods derived from GM crops be segregated. The provision of GM foods, then, may require separate transportation, shipping, and labeling facilities. Identity preservation of GM and non-GM food is consistent with broader trends in agriculture and food production. Agricultural commodities are increasingly becoming differentiated products in response to wealthier and more sophisticated consumers who demand foods that offer environmental and health benefits relative to conventional production. In order to serve niche markets for organic, free-range, hormone-free, and local foods, processors increasingly contract for farm output in order to ensure quality attributes and traceability. Identity preservation of GM foods is likely to be more difficult than in other contexts for a number of reasons. First, policies on GM foods vary by country. Some GM varieties, produced in the United States, for instance, have not been approved in the E.U. Heterogeneous policies require processors and distributors to maintain a high level of food purity or risk access to foreign markets. More than 40 countries have adopted labeling regulations. The United States is most lax, providing voluntary labeling guidelines for GM-free food, but imposing no requirement. Only foods that are substantially different from their conventional counterparts, with respect to nutritional content, for instance, require labels. The E.U. is most strict, requiring labeling of GM foods with a 0.9% tolerance for adventitious presence of GM crops.55 Australia, Japan, South Korea, and China have adopted stringent regulations, while Canada, like the United States, has no labeling requirement. Strict mandatory labeling creates a disincentive for use of GM ingredients. Following the adoption of labeling requirements, GM food disappeared from store shelves in the E.U. and Japan.56 As Lapan and Moschini noted, the European regulation requires food produced from a GM ingredient to carry a GM label regardless of whether the final product contains DNA or protein of GM origin.57 Against such a standard, the cost of avoiding commingling throughout the supply chain is likely to be enormous and prohibitive, prompting firms to source from suppliers who do not handle any GM products. Such regulations, with low tolerance thresholds while intended to improve consumer welfare by allowing consumers to choose between GM and conventional products, practically kill the market for GM foods.

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To avoid comingling, GM and non-GM crops must be separated on the farm by a sufficient distance to prevent cross-pollination. They must also be kept separate in transportation to grain elevators and from elevators to processors. The challenge of isolating crop varieties within the grain marketing system and preventing unwanted comingling is illustrated by the 2000 discovery in the human food supply of a GM corn variety approved only for animal and industrial uses. The StarLink corn was discovered first in taco shells marketed at retail grocery outlets, including the Taco Bell brand marketed by Kraft. The discovery ultimately led to the recall of 200 food products and a temporary ban on U.S. corn imports in Japan until corn purity could be verified. The incident was estimated to cost U.S. producers as much as $290 million.58 Aventis, the maker of StarLink corn, reportedly budgeted $1 billion for costs associated with recalling contaminated corn and food and for compensating farmers.59 A number of lawsuits were also filed against Aventis, which finally settled for $125 million. It took nearly half a decade for the contaminated corn to exit the food supply chain despite efforts to destroy it quickly. The contamination likely occurred via gene flow across buffer crops to conventional crops destined for human consumption. The StarLink episode demonstrates that without reasonable thresholds in labeling and trade import regulations for the adventitious presence of GM DNA or proteins, the risk to food processors is high while the benefits are low, particularly in small markets. Given low consumer acceptance and strict and heterogeneous labeling requirements, firms face considerable risk to serve available markets. Adverse events could not only impose significant liability but also jeopardize brand reputation, which could spread to non-GM operations of the firm. Niche markets for GM food will be underserved by risk-averse firms, further constraining the market for biotechnology in horticultural crops.

DIRECTED R&D AND ROLE FOR PUBLIC SECTOR: PUBLIC–PRIVATE PARTNERSHIPS Funding for horticultural biotechnology research has lagged investment in transgenic research for major field crops, even controlling for relative market size.23 By 2004, investment in horticultural biotechnology had all but died off, with leading innovators focusing their horticultural R&D efforts on conventional plant breeding instead of genetic engineering. Theory predicts that the investment in R&D is suboptimally low because of free-rider problems, except where strong IPRs are vigorously enforced. Where IPRs are strong, however, innovating firms are rewarded with monopoly power for the terms of their patents. Monopoly power permits patent holders to earn supra-normal profits, but high technology prices lead to adoption rates that are suboptimally low from a social welfare perspective. The traditional response to underprovision of agricultural innovation is to invest public dollars in research. Historically, the government has been a leader in agricultural research, and horticulture has been more dependent on public support than major crops. Since the 1990s, however, public research has begun to decline as new R&D paradigms emerge.

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Since the mid-nineteenth century, governments around the world have supported agricultural research, which has historically produced publicly available crop variety improvements that left little room for private returns.60 The rise of genetic plant engineering at the close of the twentieth century, however, dramatically changed the agricultural R&D landscape. Major advances in crop science are no longer mostly in the public domain. Instead, due to the nature of the technologies and the development of strong IPRs, the benefits of innovation are excludable. Since the 1980s, governments began adopting policies, like IPRs, to motivate a greater share of agricultural research investment from the private sector. Consequently, most agricultural biotechnology R&D is undertaken by biotechnology start-ups and established seed and agrochemical companies. Because the costs of horticultural biotechnology are immense, including the fixed costs of commercialization, the costs of identity preservation, and the risks associated with consumer acceptance and comingling, the private sector is expected to invest scarce R&D dollars elsewhere, like GM applications for large market crops that do not directly enter the food supply. The declining investment in horticultural biotechnology has been documented by a number of authors.54,61 Horticultural research has typically been undertaken in the public sector, but biotechnology research has been dominated by the private sector. While there are some examples of public–private partnerships for research in biotechnology, none relates directly to horticultural biotechnology.62 Horticultural biotechnology research would benefit from public–private partnerships because the traditional paradigm of one-way information flows from public research institutions to private firms is outdated. Policy changes designed to push research expenses toward the private sector, the consequent stagnation of public funding for crop science research, the rise of large life sciences firms, and the alignment of public and private incentives for long-term R&D created an imperative for a new formulation of the R&D pipeline that involved closer collaboration between industry and university.62,63 Universities are considered to have a comparative advantage in basic research and industry in applied research.64–66 The public-good attributes of basic research make it an appropriate fit for the university environment.67–70 Applied research, with its focus on solving particular problems, can be better motivated by a profit incentive, making it a better fit for industry. The division of research labor between industry and academia, therefore, allows both the university to pursue its traditional mission of providing an intellectual commons and the firm to pursue its profit motive. The complementarities between university and industry research have been fully appreciated only in recent decades.66,71–73 In line with their mission of maintaining “intellectual commons,” universities have historically placed innovations in the public domain.74 Administrators now recognize, however, that they can increase the benefit to society and enhance university prestige and revenue if they license knowledge to firms that, by virtue of having rights to technologies, will invest in their development and deployment.75 Firms have little incentive to commercialize a technology in the public domain because competing firms can freely capitalize on the R&D investments of others.76 While the outlook for investment in new horticultural biotechnologies appears dim based on trends in public funding and the diversion of private funds to

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higher valued investments, the induced innovation hypothesis offers a glimmer of hope that new traits in horticultural crops can be introduced. A considerable literature on agricultural R&D has subjected the induced innovation hypothesis proposed by Hicks77 and formalized by Hayami and Ruttan78 to considerable scrutiny. It has emerged as both an intuitive explanation for innovation in the industry and one that explains observed phenomena of the past century or more.9,79–82 As first proposed by Hicks, it holds that “a change in relative prices of the factors of production is itself a spur to invention, and to invention of a particular kind—directed to economizing the use of a factor which has become relatively expensive.” More generally, induced innovation holds that firms’ R&D effort is directed toward technical changes, for which there is demand, typically because of scarcity. More generally, the theory predicts that innovation responds to scarcity and crisis. It is not surprising that one of the few GM horticultural crops on the market today was developed at a time when the industry was severely threatened. By 1994, the papaya ringspot virus, which had forced the relocation of Hawaii’s papaya industry from two other islands, reached the final refuge of papaya farmers in the Puna district of Hawaii Island. The industry was in crisis. Fortunately, as crop damage spread, an effort to commercialize a papaya variety with genetically engineered resistance moved forward. GM papaya was commercialized in 1998, effectively saving the industry.83 Today, more than 70% of Hawaiian papaya is produced from the GM seed. The underprovision of biotechnology solutions for horticulture is analogous to the problem of provision for developing countries and mirrors problems in medicine regarding the development of treatments and cures for rare diseases and those that plague the poor. Strong patent protections in medicine have motivated R&D in global illnesses like AIDS and cardiovascular disease, but relatively little public or private research is directed at diseases specific to developing countries, like tuberculosis and malaria, even though millions are afflicted by these diseases every year.84 The incentive problems in these areas of research are the same as that which causes the orphan drug problem—too small markets for firms to undertake risky R&D investments. But the 1983 Orphan Drug Act in the United States has provided a jolt to research on rare illnesses like Wilson’s disease and Huntington’s disease. The Act provided tax incentives for research into illnesses that caused less than 200,000 deaths per year, guaranteed exclusivity for 7 years (in addition to patent protection), provided expedited reviews by the FDA, and introduced grants for qualifying research. Twelve times as many biotechnology-based treatments for rare diseases were introduced annually in the decade following the enactment of the legislation compared to the decade before. Mortality from rare illnesses also declined after the bill, both in absolute terms and relative to more common diseases. Deaths from rare illnesses had been growing relative to deaths from other diseases.84 In agriculture, PIPRA constitutes a response to the “orphan drug” problem related to the development of GM crop traits for horticultural crops. Researchers in both the medical and agricultural fields also endeavor to develop international partnerships to

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mimic the successes for orphan drugs and orphan crops for the purpose of delivering valuable technologies to poor parts of the world.

PREDICTIONS AND POLICY RECOMMENDATIONS Since 1998, there has been a downward trend in R&D intensity for crops with low market potential. Technology has marched forward, but it seems to have left horticultural crops behind. The pace of innovation has been slowed by market resistance and regulatory hurdles, but it seems to have slowed disproportionately for specialty crops.2 Despite comparable market size and time to market, pharmaceutical R&D investment has far outpaced crop biotechnology investment.22 Despite the decline in R&D effort, there are reasons to be optimistic that rising demand for horticultural biotechnology could provide a jolt to lagging R&D. Though no plant breeding technology had ever elicited consumer opposition before agricultural biotechnology, other revolutionary technology in the food industry has caused concerns that were eventually diminished. Huffman and Rousu reported that it took 40 years for consumer opposition to pasteurization of milk to diminish.24 At first, the opposition was widespread, but after considerable experience with pasteurized milk and long after the emergence of a scientific consensus on its benefits, full acceptance was attained. The slow path to acceptance, however, was not without costs as Pirtle estimated thousands of avoidable deaths occurred because acceptance of pasteurized milk was not more immediate.85 General acceptance of agricultural and horticultural biotechnology can be expected to grow with accumulating experiences. Regardless, clear trends in food demand suggest that an increasingly wealthy, educated, and discriminating public will demand more and more differentiated products so that they can accomplish heterogeneous health, environmental, and ethical objectives. As they learn to distinguish organic, low-fat, low-carbohydrate, and farm fresh foods, for instance, they can also be expected to identify quality-enhancing GM traits and separate irrational concerns from legitimate risks. In other words, niche markets for GM products should continue to grow. To the extent there is a consensus view on consumer acceptance, it holds that the right quality-enhancing attributes will be warmly received. As consumers become more accepting and demanding of GM foods, retailers should become less averse to stocking them, sending appropriate signals up the supply chain to seed companies. The regulatory environment for horticultural biotechnology is also changing and possibly improving with growing evidence of its deficiencies. Perhaps, no policy change has broader implications than Europe’s decision in 2008 to lift its moratorium on GM foods. Though the market in Europe remains constrained, the liberalizing of biotechnology should have implications for policymakers in political capitals around the world, especially in Africa, and should provide an inducement to innovating firms. Costs associated with regulatory compliance should decline as policymakers work to remove socially inefficient barriers to entry. Governments have also undertaken to improve identity preservation infrastructure throughout their food systems in response to lapses in food safety and risks of terrorist attacks on the food supply. As this infrastructure improves, it should be

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easier for food processors to serve niche markets for GM foods without risking liability and reputational effects associated with comingling. And while registrations for horticultural crops in the United States and other developed countries have stagnated, China is aggressively moving ahead with horticultural biotechnology applications. Perhaps, competition will induce other countries to follow. The rise of public–private partnerships like PIPRA that intended to reduce barriers to entry is also likely to provide increasingly valuable services to start-ups and horticultural crop operations as they develop. A number of changes to IPR and regulatory policy could also improve the outlook for horticultural biotechnology. First, governments should move from a system of event and variety-based testing to one that is trait-based. That is, innovators should not be required to undertake the same testing and compliance effort to introduce an already-approved trait into a new variety or species. To require such redundancy in testing regimes ignores the reduced risks associated with moving a probably safe trait into a new plant. Second, governments should adopt tolerances for adventitious GM content in foods that respect consumer choice but do not cause the market for GM foods to collapse. The tolerances adopted in the E.U. are too strict to permit the development of GM food markets. They should be increased. Governments should also pursue standardized policies that reduce the compliance costs to firms that rely on exports to many jurisdictions. Finally, in recognition of the private sector’s insufficient incentives to invest in horticultural crops at the socially optimal level, the public sector should step up its effort to reduce the orphan-crop problem. Horticulture has historically been publicly supported. Even though policy changes have provided private sector inducements for agricultural biotechnology, it is clear by revealed preference that firms are not sufficiently motivated to provide biotechnology applications for small markets. An orphan crop act, like the Orphan Drug Act, could increase the incentive for research on GM specialty crops.

REFERENCES

1. James, C., Global Status of Commercialized Biotech/GM Crops: 2009, ISAAA Brief 41, Ithaca, NY, ISAAA, 2009. 2. Bradford, K., Alston, J., and Kalaitzandonakes, N., Regulation of biotechnology for specialty crops, in Regulating Agricultural Biotechnology: Economics and Policy, eds. R. E. Just, J. M. Alston, and D. Zilberman, Springer, Berlin, Germany, 2006, p. 683. 3. Clark, D., Klee, H., and Dandekar, A., Despite benefits, commercialization of transgenic horticultural crops lags, California Agriculture, 58(2), 89, 2004. 4. Gianessi, L. P., Economic and herbicide use impacts of glyphosate-resistant crops, Pest Management Science, 61(3), 241, 2005. 5. Gianessi, L., Sankala, S., and Reigner, N. 2004, Potential impact for improving pest management in European agriculture: Maize case study, available at: http://www.ncfap.org. Accessed on January 2007. 6. Schmookler, J., Invention and Economic Growth, Harvard University Press, Cambridge, MA, 1966. 7. Aghion, P. and Howitt, P., A model of growth through creative destruction, Econometrica: Journal of the Econometric Society, 60(2), 323, 1992. 8. Grossman, G. M. and Helpman, E., Innovation and Growth in the Global Economy, The MIT Press, Cambridge, MA, 1993.

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9. Hayami, Y. and Ruttan, V. W., Agricultural Development: An International Perspective, Johns Hopkins University Press, Baltimore, MD, 1985. 10. Acemoglu, D., Labor-and capital-augmenting technical change, Journal of the European Economic Association, 1(1), 1, 2003. 11. Griliches, Z., Hybrid corn: An exploration in the economics of technological change, Econometrica, 25(4), 501, 1957. 12. Kremer, M., Pharmaceuticals and the developing world, The Journal of Economic Perspectives, 16(4), 67, 2002. 13. Feder, G., Just, R. E., and Zilberman, D., Adoption of agricultural innovations in developing countries: A survey, Economic Development and Cultural Change, 32(2), 255, 1985. 14. Sunding, D. and Zilberman, D., The agricultural innovation process: Research and technology adoption in a changing agricultural sector, in Handbook of Agricultural Economics, Volume 1A Agricultural Production, eds. B. L. Gardner and G. C. Rausser, Elsevier Science B. V., Amsterdam, the Netherlands, 2001, p. 207. 15. Sandmo, A., On the theory of the competitive firm under price uncertainty, The American Economic Review, 61(1), 65, 1971. 16. Dixit, A. K. and Pindyck, R.S., Investment under Uncertainty, Princeton University Press, Princeton, NJ, 1994. 17. Bradford, K. et al., Challenges and opportunities for horticultural biotechnology, California Agriculture, 58(2), 68, 2004. 18. Alston, J. M., Horticultural biotechnology faces significant economic and market barriers, California Agriculture, 58(2), 80, 2006. 19. Zilberman, D., The economics of biotechnology regulation, in Regulating Agricultural Biotechnology: Economics and Policy, eds. R. E. Just, J. M. Alston, and D. Zilberman, Springer, New York, 2006, p. 243. 20. Ameden, H., Qaim, M., and Zilberman, D., Adoption of biotechnology in developing countries, in Agricultural Biodiversity and Biotechnology in Economic Development, eds. J. Cooper, L. M. Lipper, and D. Zilberman, Springer, New York, 2005, pp. 329–357. 21. Jaffe, G., Regulatory slowdown on GM crop decisions, Nature Biotechnology, 24, 748, 2006. 22. McElroy, D., Sustaining agbiotechnology through lean times, Nature Biotechnology, 21, 996, 2003. 23. Graff, G. D. et al., Access to intellectual property is a major obstacle to developing transgenic horticultural crops, California Agriculture, 58(2), 120, 2006. 24. Huffman, W. and Rousu, M., Consumer attitudes and market resistance to biotech products, in Regulating Agricultural Biotechnology: Economics and Policy, eds. R. E. Just, J. A. Alston, and D. Zilberman, Springer, New York, 2006, pp. 201–225. 25. Qaim, M., The economics of genetically modified crops, The Annual Review of Resource Economics, 1, 665, 2009. 26. Shelton, A. M., Zhao, J. Z., and Roush, R. T., Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants, Annual Review of Entomology, 47(1), 845, 2002. 27. Moschini, G. C., Biotechnology and the development of food markets: Retrospect and prospects, European Review of Agricultural Economics, 35, 331, 2008. 28. Gaskell, G. et al., Europeans and biotechnology in 2005: Patterns and trends, A report to the European Commission’s Directorate-General for Research, London School of Economics and Political Science, London, U.K., 2006. 29. Berg, P. and Singer, M. F., The recombinant DNA controversy: Twenty years later, Proceedings of the National Academy of Sciences of the United States of America, 92, 9011, 1995. 30. Miranowski, J. A. et al., Economic perspectives on GMO market segregation, Staff Paper No. 298, Department of Applied Economics, University of Iowa, Ames, IA, 1999.

The Economic and Marketing Challenges of Horticultural Biotechnology

189

31. Josling, T. and Tangermann, S., Implementation of the WTO agreement on agriculture and developments for the next round of negotiations, European Review of Agricultural Economics, 26(3), 371, 1999. 32. Pew Initiative on Food and Biotechnology, Mellman Group/Public Opinion Strategies, Public sentiment about genetically modified food, 2005. Available online: http://www.pewtrusts.org/news_room_detail.aspx?id=32804 (accessed on February 2, 2011). 33. Eurobarometer 55.2, Europeans, science and technology, European Commission, Public Opinion Analysis, Brussels, 2001 [cited 20 December 2002], 62pp. Available from Internet: http://ec.europa.eu/research/press/2001/pr0612â•›en-report.pdf (accessed on February 2, 2011). 34. Huffman, W. E. et al., The effects of prior beliefs and learning on consumers’ acceptance of genetically modified foods, Journal of Economic Behavior & Organization, 63(1),193, 2007. 35. Bredahl, L., Determinants of consumer attitudes and purchase intentions with regard to genetically modified food—Results of a cross-national survey, Journal of Consumer Policy, 24(1), 2001. 36. Lusk, J. L., Effects of cheap talk on consumer willingness-to-pay for golden rice, American Journal of Agricultural Economics, 85(4), 840, 2003. 37. Lusk, J. L. and Rozan, A., Consumer acceptance of biotechnology and the role of second generation technologies in the USA and Europe, Trends in Biotechnology, 23(8), 386, 2005. 38. Moon, W. and Balasubramanian, S. K., Willingness to pay for non-biotech foods in the US and UK, Journal of Consumer Affairs, 37(2), 317, 2003. 39. Chen, H. and Chern, W. S., Consumer acceptance of genetically modified foods, paper presented at Annual Meeting of the American Agricultural Economics Association, Long Beach, CA, 2002. 40. Onyango, B., Govindasamy, R., and Nayga Jr., R. M., An application of choice modeling to measure US consumer preferences for genetically modified foods, paper presented at the American Agricultural Economics Association Annual Meeting, Denver, CO, 2004. 41. Lusk, J. L. et al.,  Effect of information about benefits of biotechnology on consumer acceptance of  genetically modified food: Evidence from experimental auctions in the United States, England, and France, European Review of Agricultural Economics, 31(2), 179, 2004. 42. Frewer, L. J., Howard, C., and Shepherd, R., The influence of realistic product exposure on attitudes towards genetic engineering of food, Food Quality and Preference, 7(1), 61, 1996. 43. Mucci, A. and Hough, G., Perceptions of genetically modified foods by consumers in Argentina, Food Quality and Preference, 15(1), 43, 2004. 44. Chern, W. S. and Rickertsen, K., A comparative analysis of consumer acceptance of GM foods in Norway and the United States, paper presented at 6th International Conference on Agricultural Biotechnology: New Avenues for Production, Consumption, and Technology Transfer, Ravello, Italy, 2002. 45. Bernauer, T., Genes, Trade, and Regulation, Princeton University Press, Princeton, NJ, 2003. 46. Hoban, T. J., Trends in consumer attitudes about agricultural biotechnology, AgBioForum, 1(1), 3, 1998. 47. Runge, C. F., Bagnara, G. L., and Jackson, L. A., Differing US and European perspectives on GMOs: Political, economic and cultural issues, Estey Centre Journal of International Law and Trade Policy, 2(2), 221, 2001. 48. Sheldon, I. M., Regulation of biotechnology: Will we ever “freely” trade GMOs? European Review of Agricultural Economics, 29(1), 155, 2002. 49. Graff, G. D. and Zilberman, D., Explaining Europe’s resistance to agricultural biotechnology, Agricultural and Resource Economics Update, 7(5), 1, 2004.

190

Transgenic Horticultural Crops: Challenges and Opportunities

50. Graff, G. D., Hochman, G., and Zilberman, D., The political economy of agricultural biotechnology policies, AgBioForum, 12(1), 34, 2009. 51. Paarlberg, R. L., Starved for Science: How Biotechnology Is Being Kept Out of Africa, Harvard University Press, Cambridge, MA, 2008. 52. Evenson, R. E., Status of agricultural biotechnology: An international perspective, in Regulating Agricultural Biotechnology: Economics and Policy, eds. R. E. Just, J. A. Alston, and D. Zilberman, Springer, New York, p. 103, 2006. 53. Hochman, G. et al., Agricultural biotechnology in California and the EU, UC Berkeley: Institute of Governmental Studies, retrieved from: http://www.escholarship.org/uc/ item/7kv3s4mg, 2008 (accessed on February 2, 2011). 54. Graff, G. D., Zilberman, D., and Bennett, A. B., The contraction of agbiotech product quality innovation, Nature Biotechnology, 27(8), 702, 2009. 55. Gruère, G. P., Carter, C. A., and Farzin, Y. H., Explaining international differences in genetically modified food labeling regulations, Review of International Economics, 17(3), 393, 2009. 56. Carter, C. A. and Gruère, G. P., Mandatory labeling of genetically modified foods: Does it really provide consumer choice? AgBioForum, 6(1&2), 68, 2003. 57. Lapan, H. and Moschini, G. C., Grading, minimum quality standards, and the labeling of genetically modified products, American Journal of Agricultural Economics, 89(3), 769, 2007. 58. Schmitz, T. G., Schmitz, A., and Moss, C. B., Did StarLink reduce import demand for corn? Journal of Agricultural & Food Industrial Organization, 2(2), Article 6, 2004. 59. Smyth, S., Khachatourians, G. G., and Phillips, P. W. B., Liabilities and economics of transgenic crops, Nature Biotechnology, 20(6), 537, 2002. 60. Alston, J. M., Pardey, P. G., and Taylor, M. J., Agricultural Science Policy: Changing Global Agendas, Johns Hopkins Press, Baltimore, MD, 2001. 61. Alston, J. M., Bradford, K. J., and Kalaitzandonakes, N., The economics of horticultural biotechnology, Journal of Crop Improvement, 18(1), 413, 2006. 62. Rausser, G., Private/Public research: Knowledge assets and future scenarios, American Journal of Agricultural Economics, 81(5), 1011, 1999. 63. Alston, J. M. and Pardey, P. G., Making Science Pay: The Economics of Agricultural R&D Policy, American Enterprise Institute, La Vergne, TN, 1996. 64. Nelson, R. R., The simple economics of basic scientific research, The Journal of Political Economy, 67(3), 297, 1959. 65. Bush, V., Science: The endless frontier, Transactions of the Kansas Academy of Science, 48(3), 231, 1945. 66. Rausser, G. and Ameden, H., Public–private partnerships needed in horticultural research and development, California Agriculture, 58(2), 116, 2006. 67. Arrow, K., Economic welfare and the allocation of resources for invention, in The Rate and Direction of Inventive Activity, ed. R. Nelson, Princeton University Press, Princeton, NJ, 1962, pp. 609–626. 68. Griliches, Z., The search for R&D spillovers, The Scandinavian Journal of Economics, 94, 29, 1992. 69. Stiglitz, J. E. and Wallsten, S. J., Public private technology partnerships: Promises and pitfalls, American Behavioral Scientist, 43(1), 52, 1999. 70. Salter, A. J. and Martin, B. R., The economic benefits of publicly funded basic research: A critical review, Research Policy, 30(3), 509, 2001. 71. Peters, L. and Fusfeld, H., Current US University–Industry Research Relationships, National Science Board, Washington, DC, 1983, pp. 1–161. 72. Fairweather, J. S., Entrepreneurship and Higher Education: Lessons for Colleges, Universities, and Industry, ASHE-ERIC Higher Education, Report No. 6, Association for the Study of Higher Education, Washington, DC, 1988.

The Economic and Marketing Challenges of Horticultural Biotechnology

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73. Geisler, E. and Rubenstein, A. H., University-industry relations: A review of major issues, in Cooperative Research and Development: The Industry, University, Government Relationship, eds. A. N. Link and G. Tassey, Kluwer Academic Publishers, Norwell, MA, 1989. 74. Hofstadter, R., Academic Freedom in the Age of the College, Transaction Publishers, New Brunswick, NJ, 1995. 75. Etzkowitz, H. et al., The future of the university and the university of the future: Evolution of ivory tower to entrepreneurial paradigm, Research Policy, 29(2), 313, 2000. 76. Graff, G. D. et al., The public–private structure of intellectual property ownership in agricultural biotechnology, Nature Biotechnology, 21(9), 989, 2003. 77. Hicks, J. R., The Theory of Wages, Macmillan, London, U.K., 1932. 78. Hayami, Y. and Ruttan, V. W., Agricultural productivity differences among countries, The American Economic Review, 60(5), 895, 1970. 79. Boserup, E. and Kaldor, N., The Conditions of Agricultural Growth: The Economics of Agrarian Change under Population Pressure, G. Allen and Unwin, London, U.K., 1965. 80. Binswanger, H. P., A microeconomic approach to induced innovation, The Economic Journal, 84(336), 940, 1974. 81. Binswanger, H. P. and Rosenzweig, M. R., Behavioral and material determinants of production relations in agriculture, Journal of Development Studies, 22(3), 503, 1986. 82. Olmstead, A. L. and Rhode, P., Induced innovation in American agriculture: A reconsideration, Journal of Political Economy, 101(1), 100, 1993. 83. Gonsalves, D., Transgenic papaya in Hawaii and beyond, AgBioForum, 7(1&2), 36, 2004. 84. Grabowski, H., Patents, innovation and access to new pharmaceuticals, Journal of International Economic Law, 5(4), 849, 2002. 85. Pirtle, T. R., Handbook of Dairy Statistics, U.S. Department of Agriculture, Washington, DC, 1922.

9

Consumer Acceptance of Genetically Modified Foods: Traits, Labels, and Diverse Information Wallace E. Huffman

CONTENTS Early Development of GM Crops........................................................................... 194 New Development of GM Vegetable Crops............................................................ 197 Food Labels and Information.................................................................................. 198 Experiments Designed to Assess Consumer Willingness to Pay for GM Foods....... 200 Experimental Evidence on Key Bid-Price Differences...........................................204 Simple Differences in Mean Bid Prices.............................................................204 Individual Bid-Price Differences and Diverse Information...............................204 Individual Differences in Bid Prices and Prior Information..............................206 Other Related Results.........................................................................................207 Discussion of GM Technology and Mixed Messages.............................................207 Conclusions and Predictions for the Future............................................................208 Appendix A:â•… Information Injected into the 2001 Des Moines and St. Paul Experiments........................................................................................209 Appendix B:â•… Information Injected into the 2007 Des Moines and Harrisburg Experiments................................................................................... 212 References............................................................................................................... 215 The United States has more than a decade of experience with commercially marketed, genetically modified (GM) horticultural and field crops and is the dominant player in GM crops. The first GM commercial crops were the Flavr-Savr tomato and the Russet Burbank New Leaf potato, both deregulated in 1994 and marketed commercially shortly thereafter. At about the same time, field crop varieties possessing “input traits” were also developed and first marketed commercially. GM papaya was developed by the public sector and successfully marketed a little later. Some of the economic issues facing biotech horticultural crops have been summarized.1,2 In this chapter, new experimental economic methods are described and used to assess consumer willingness to pay for food products that might be made with new transgenic and intragenic GM traits. Participants in my lab auctions are randomly 193

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chosen adult consumers in major U.S. metropolitan areas. Food labels are kept simple and focus on key attributes of experimental goods. Diverse private information from the agricultural biotech industry (largely Monsanto and Syngenta), environmental groups (largely Greenpeace and Friends of the Earth), and independent third-party information (scientifically objective at the time of the experiments) is used to construct the information treatments. Willingness to pay is determined by experimental lab auctions under random food label and information treatments. Auctions are best described as sealed bid random nth price and not a first price of Vickery 2nd price auction. I show that participants in these experiments respond both to food labels and information treatments, but no single type of information is dominant. The first section of the chapter reviews early development of GM crops that were largely input traits of herbicide tolerance and insect resistance that have now spread unevenly across the world, and the second section describes some new methods for developing GM vegetable crops with enhanced consumer attributes. The third section summarizes the development of U.S. food-label policy and requirements. The fourth section briefly describes experimental evidence on consumer willingness to pay for GM foods using lab auctions of experimental commodities. In the final section, some conclusions and predictions about likely future developments in commercial horticultural crops are presented.

EARLY DEVELOPMENT OF GM CROPS The first commercial GM crops were the Flavr-Savr tomato, developed for sale in the United States by Calgene, and the Russet Burbank New Leaf potato developed by Monsanto. These two products were the first whole foods produced with biotechnology that were approved by the U.S. Food and Drug Administration (FDA) for retail sale.* The Flavr-Savr tomato, “a delayed-ripening tomato,” was the product of more than a decade of research to develop a tomato that could be picked when ripe and transported without bruising (pp. 256–257).3 The claim was that it would have a longer shelf-life than conventional tomatoes and would provide consumers and processors with tastier tomatoes because the fruit had been left to mature on the vine.† This was accomplished

* A potential food safety concern was raised by the fact that in creating the GM tomato, a marker gene for the antibiotic kanamycin was inserted. The marker gene is helpful for identifying which plants have been affected by a target gene.3 When the marker is an antibiotic, technicians test for presence of successfully transferred genes of interest by applying the antibiotic. Only those cells that contain the antibiotic resistance will survive this treatment, and thereby indicate that an accompanying target gene is present. The FDA, however, found no food safety concerns due to insertion of foreign antibiotics into plant cells. † Because U.S. winter fresh market tomato production is concentrated in Florida, which means shipping the fruit long distances to the U.S. retail markets in the East, Midwest, and South, it is important that the fruit does not perish on its journey to market because of its soft skin. (Winter tomatoes for the West are generally supplied by Mexico.) The conventional solution has been for tomato farmers to pick the fruits while they are green, i.e., “mature greens,” transport them to the location of the retail market and then spray them with ethylene, a natural ripening agent, to artificially ripen and redden the fruit. However, the artificially ripened tomatoes have an inferior flavor relative to vine-ripened tomatoes.

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by gene-silencing, through an antisense RNA that interfered with translation and reduced the production of specific proteins that cause ripening (pp. 256–257).3 The first Flavr-Savr tomatoes were sold in U.S. grocery stores in the summer of 1994 and were marketed as GM. They sold relatively well at first and were in about 2500 stores by June 1995, but it became apparent that their performance did not match expectations. First, the genes for delayed ripening were inserted into a tomato variety that was best suited for processing, not direct eating, and that bruised relatively easily, contrary to its development objective. Second, contrary to expectations, it had a bland taste relative to conventional winter tomatoes. Third, the new tomato variety was suited to California’s dry summer growing conditions, but not to the humid winter tomato growing regions of Florida and, as a result, was susceptible to Florida’s tomato fungal diseases. Fourth, the retail price was more than two times higher than conventional fresh market tomatoes. Hence, a number of factors contributed to the failure of the Flavr-Savr tomato in the U.S. market (pp. 256–257).3,4 At the same time, Zeneca produced a related high-solid GM tomato for use in purees and soups, obtained approval for sale in the United Kingdom, and began marketing in 1996 under the brand names Safeway Double Concentrated Tomato Puree and Sainsbury’s California Tomato Puree. These products were sold at a lower per unit price than purees from conventional tomatoes and were marketed in larger containers to make the product appear to consumers as a “better value.” By 1999, the GM puree had captured up to 60% of the processed tomato market share in the United Kingdom. However, when unrelated food scares (e.g., BSE in sheep and cattle, dioxin in livestock feed) started to unfold in the United Kingdom in the late 1990s, Zeneca’s GM high-solid tomato varieties were a casualty, and they were withdrawn from the market.4 Monsanto engineered the Russet Burbank New Leaf potato to be resistant to the Colorado potato beetle, a major potato pest, and this potato was deregulated in 1994. This new variety offered growers the advantage of significantly reducing the need for chemical pesticide applications, and initially gained favor in the fresh potato market. However, under pressure from consumer groups, the fast-food industry (e.g., McDonalds) and grocery store chains would not purchase it or halted early purchases, and this prompted Monsanto to withdraw the GM potato variety from the retail market in 1999.5 To date, the most commercially successful GM crops in the United States have been those engineered with so-called input traits, namely, insect resistance (Bt) and herbicide tolerance (HT or RR/RoundUp Ready). These GM traits were obtained by transferring genes largely from soil bacteria into selective species of commercial field crops to induce resistance or tolerance to target organisms, thereby creating a so-called transgenic crop variety (pp. 250–256).3 The target of these traits has been canola, soybean, cotton, and corn, with commercial GM varieties of these four crops first introduced in the mid-1990s. These GM field crops, whose products are largely destined for oil, feed, and fiber, have had very rapid grower adoption (Figure 9.1). For instance, HT soybeans were first marketed to farmers in 1996 and now account for more than 90% of U.S. soybean acreage. Canola with HT has also been successful, although it is a relatively small acreage crop in the United States. Bt and HT cotton

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HT soybeans

80 HT cotton 60

40

Bt cotton HT corn Bt corn

20

0 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

FIGURE 9.1â•… (See color insert.) Adoption of genetically engineered crops: United States, 1996–2007. Note: Data for each crop category include varieties with both HT and Bt (stacked) traits. (Data for 1996–1999 are from Femandez-Cornejo and McBride (2002); data for 2002–2007 are available in the ERS Data Product, adoption of genetically engineered crops in the United States, Tables 1–3.)

also got off to a fast start in 1996. By 2005, about 60% of U.S. cotton carried HT or Bt genes, with the most recent varieties carrying both traits as a “stacked gene” variety. GM field corn got off to a slow start, and in 2005 about 30% of the corn acreage was planted to Bt and 15% to HT.* In corn, recent stacking of multiple Bt genes, imparting resistance against European corn borer and rootworm, often together with HT, has pushed GM corn acreage up in the last 2 years and is anticipated to become the “gold standard” in the future.† Genetic improvement of papaya remains the one bright example of successful public sector bioengineering of a horticultural food crop. Starting in the 1940s, the Hawaiian papaya fruit industry was ravaged by papaya ringspot virus and, by the 1980s, Hawaii’s papaya production had fallen significantly and was concentrated in the Puna district of the Big Island. However, by the early 1990s, ringspot virus was invading that area, too. The University of Hawaii at Mãnoa, Cornell University and the U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS) then initiated new research and developed a transgenic papaya variety that was resistant to ringspot, labeled Rainbow, and it was released freely to farmers in * Zilberman has argued that rational regulation of transgenic products should compare their risks and benefits with the risks and benefits of alternative technologies. Current regulations ignore the alternatives, and this is costly to society.6 † Although herbicide-tolerant wheat varieties have been developed for the United States by Monsanto, they have not been marketed widely, nor are they currently available to farmers because of consumer resistance due to the use of wheat largely for food such as breads, pastas, etc.

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1998.7,8 Since its introduction, the Rainbow variety of papaya has been used strategically to create a virus-free ring as a buffer to slow down the spread of papaya ringspot virus in the Puna area and to slow down or reduce resistance of consumers and/or some growers to the new GE technology.7,9 Papaya is the only U.S. crop in which public sector scientists have pioneered the development of a commercially successful GM crop variety. Overall, the early commercial successes with GM crops were not with horticultural crops, but rather with a small set of field crops. The most likely reason is that consumers failed to personally see enhanced value from many of the new GM traits that were scientifically possible. In fiber crops and crops that are used heavily, but not exclusively, for livestock feed, consumer acceptance was less important. However, the image of GM crops with consumers has been damaged by the fact that new GM products with unique enhanced consumer attributes have been slow to develop.

NEW DEVELOPMENT OF GM VEGETABLE CROPS As transgenic GM technology has been developed and marketed for a small set of field crops, a new line of research has recently emerged around intragenic GM horticultural crops.10,11 Prompted by continued consumer resistance to transgenic food crops, these new methods introduce new traits into a crop variety by using only DNA from the same species, thereby yielding an “intragenic” genetic modification. This research was made possible by a small set of scientists recognizing that a huge range of genetic diversity exists within horticultural crops that have been grown for a long period of time under diverse environments and human needs, perhaps in relative isolation. For example, major genetic diversity exists in potato and tomato, stretching from very old primitive or landrace varieties to the modern commercial varieties of today. However, in the case of the potato, inbreeding depression, tetraploid genetics, and clonal propagation conspire to make traditional breeding difficult and slow. By taking a new intragenic bioengineering approach, genomic and metabolic pathway discoveries can be quickly introduced into established commercial varieties to fasttrack the breeding process without introducing foreign DNA or antibiotic markers. Economists have shown recently that consumer acceptance of GM food crops is intimately linked to the type of traits engineered into the crops, the types of food labels on retail food products, and the information environment. For example, the GM food market has been subjected to diverse and conflicting information, and this makes informed decision making by consumers and producers difficult.12 Although the first commercial GM crop was a horticultural crop (the Flavr-Savr tomato) with “enhanced consumer attributes,” all of the other commercially successful GM crops in the United States have possessed input traits—traits that reduce either the cost of production or the variance in the cost of production to farmers and, hence, have only benefited consumers to the extent that they have lowered food prices or increased food availability. Although Falck-Zepeda et al.13 and Moschini et al.14 show that consumer surplus benefits from these technologies have been sizeable, these benefits have not registered effectively with consumers. With new intragenic potatoes and tomatoes that are engineered for dramatically

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enhanced antioxidants and vitamins (e.g., vitamin C, A, or E), improved starch content, and/or reduced bruising becoming scientifically possible, U.S. consumers may, for the first time, see GM crops as having direct positive value to them over conventionally bred crops.*

FOOD LABELS AND INFORMATION Economists have shown that food labels and information are important factors conditioning consumer response to GM foods (e.g., see Rousu et al.,12 Huffman and Rousu,15 and Moschini and Lapan16). Food products in the United States can be labeled for nutritional claims and for safety. The 1990 Nutrition Labeling and Education Act dramatically changed nutrition labels on packaged foods sold in U.S. supermarkets.17,† This law requires packaged foods to display nutrition information prominently in a new label format, namely, the Nutrition Facts panel. It also regulates serving size, health claims (that link a nutrient to a specific disease), and descriptor terms, for example, “low fat,” on food packages. The goal of this legislation was to improve consumer welfare by providing nutrition information that would assist consumers in making healthy food choices. As an indication of the costliness of effective food nutrient labeling, it is estimated that the U.S. food industry spent $2 billion to comply with the 1990 Nutrition Labeling and Education Act (NLEA) (Public Law 101-535).18 However, some attributes, such as enhanced calcium and vitamins A and C, are viewed positively by consumers, that is, more is better in the case of positive consumer attributes. But other food attributes, such as salt, fat, and pesticide residue, are negative, and then the consumer views less to be better. Food labels before the Nutrition Labeling and Education Act had a seeming emphasis on negative labeling. Balasubramanian and Cole17 suggest that this tendency can be explained by consumers having an asymmetric value function, weighing a dollar of loss more heavily than a dollar of gain, which is Tversky and Kahneman’s19 prospect theory. The policies under the Nutrition Labeling and Education Act also tend to emphasize negative rather than positive labeling. First, permissible health claims are ones that associate specific nutrients with reduced risk of specific diseases. Of the seven health claims approved by the FDA at the onset of the new nutrient labeling act, three linked negative attributes exclusively with deadly diseases, that is, dietary fat with cancer, sodium with hypertension, and dietary saturated fat with high cholesterol and heart disease, and only one claim featured a positive attribute, that is, calcium and osteoporosis. Later claims have, however, been more balanced. Second, regulations on nutrient-content claims tend to focus more heavily on negative attributes (calories, sugar, sodium, fat, fatty acids, and cholesterol) than on positive attributes such as fiber and vitamins. * Antioxidants are substances that may protect human cells from the damage otherwise caused by unstable molecules known as free radicals. Free radical damage over time is believed to cause some types of cancer. Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might otherwise cause. Antioxidants include beta-carotene, lycopene, vitamins C, E, and A, and other substances. These compounds are sometimes called phytonutrients and are naturally occurring in at least low levels in most fruits and vegetables. † Unpackaged foods—for example, fresh fruits and vegetables, are not affected.

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GM content has not been shown to have negative human health consequences. Nevertheless, with foods made currently from crop varieties that contain GM input traits, adding a label for GM content would be an example of labeling a negative food attribute. Genetically engineered products used for food, however, do have to pass a food safety test. In 1992, the FDA announced its landmark decision that GM food and food products will be regulated the same as those created by conventional means. This policy allows new GM foods to be treated as conventional foods as long as they meet three conditions: their nutritional value has not been lowered; they incorporate new substances that are already a part of the human diet; and they contain no new allergenic substances. In January 2001, the FDA issued a “Guidance for Industry” statement reaffirming this policy. In this statement, the FDA stated to the biotech industry that the only GM foods that need to be labeled are foods that have different characteristics from the non-GM version, for example, elevated vitamin A levels. In the United States, labeling food for GM content is not otherwise required. Firms, however, are to notify the FDA at least 4 months before putting a new GM food product on the market, and the scientific description of the product is posted on the Internet for review during this time.20 Only minor changes have been made in these guidelines since 2001. Hence, the GM-labeling policy in the United States can be classified as being voluntary. If a voluntary label is affixed, the FDA has mandated that it cannot use the phrase “genetically modified.” The FDA prefers the phrase “genetically engineered” or “made through biotechnology.”* Effective GM labeling, however, involves real costs, especially the costs of testing for the presence of GM content, segregating GM and non-GM products, variable costs of monitoring for truthfulness of labeling and enforcement of the regulations that exist, and risk premiums for being out of contract.21,22 An effective GM-labeling policy includes effective segregation of GM from non-GM commodities. If one or the other of these products could be inexpensively color coded, segregation might not be very expensive. If, however, identity preservation through the production, marketing, and processing chain was required, this system would be substantially more costly.21 To the extent that there is a market for non-GM products, buyers would be expected to specify in their purchase contracts some limit on GM content and/or precise prescriptions regarding production/marketing/handling processes. One can envision a marketplace of buyers with differentiated demand according to their aversion to GM content. To make this differentiation effective, new costs and risks are incurred. Additional testing involves costs of conducting the tests, for which there are several technologies of varying accuracy. The risk is that GM products will be commingled with non-GM products, so the detection system must test to see that customers’ shipments are within contract limits for GM content. This is a serious * In contrast, the European Commission adopted GM food labels in 1997. The Commission requires each member country to enact a law requiring labeling of all new products containing substances derived from GM organisms. Japan, Australia, and many other countries have also passed laws requiring GM labels for major foods. The international environmental lobby has frequently argued that “consumers have the right to know whether their food is GM or not.”23

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economic problem, as agents seek to determine the optimal strategy for testing and other risk mitigation strategies.* While private sector handlers routinely segregate and blend grains and beans as a primary function of their business, new risks arise when handling GM and nonGM products, due to the added risk of adventitious commingling. When GM is the inferior product, growers and handlers of GM products have an incentive to mix GM with non-GM products. For U.S. grains, Wilson and Dahl suggest that this risk may be about 4% at the grain elevator level.21 Farmer-processor contracting in horticultural or specialty crops, however, could reduce this margin by specializing in the product being delivered, such as non-GM or a positive GM trait. Another source of risk is testing, because no test is 100% accurate. Testing risk, however, varies with the technology, tolerance, and variety of products handled, and seems likely to fall over time, as the technology of testing advances. In markets where there is imperfect information due to one or more parties having private information, private parties have an incentive to use their information to enhance their private goals.24,25 Highly conflicting information has been injected into the GM food market by interested parties. These vested parties are the agricultural biotech industry (pp. 153–183),26 including Monsanto, DuPont/Pioneer Hi-Bred, Dow, Syngenta, and BASF, that have disseminated information that is very favorable to GM technologies, crops and food products, and environmental groups, including Greenpeace, Friends of the Earth, Action Aid, and Earth Watch, that have disseminated information that is very negative about GM crops, such as calling it “Frankenfood.”27 This diverse information has undoubtedly contributed to the GM food controversy and may be one factor explaining differences across Western countries in their acceptance of GM crops. Also, consistent with consumer education, independent third-party or verifiable information about agricultural Â�biotechnology may have considerable value if available and disseminated to consumers.12,28,29 Verifiable information provides an objective assessment of the benefits and costs, including environmental risks, of GM crop varieties and the foods made from these raw materials. Hence, society can avoid losses due to the strategic behavior of interested parties toward new technologies and products if decision makers have access to and use independent third-party or verifiable information.

EXPERIMENTS DESIGNED TO ASSESS CONSUMER WILLINGNESS TO PAY FOR GM FOODS Because GM foods are relatively new, my research team chose to use an auction market setting30 to collect information about consumer willingness to pay for (or demand) GM foods.12,31 This reflects the reality that GM food products are not * “Tolerances” are an important issue in segregation and identity preservation. GM tolerance refers to the maximum impurity level for GM content that is tolerated in a product that still carries the non-GM label. There are two levels where tolerances apply: one is defined by regulatory agencies such as the FDA, and the other is commercial tolerance. Individual firms can and seem likely to adopt different tolerance levels, subject to any regulation. Moreover, different countries are likely to have different tolerance levels, and this increases the risks and costs of segregation or identity preservation.

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generally labeled in the United States, so grocery store purchases are not informative on this issue. Some scientists have used contingent value or stated preference surveys of consumer willingness to pay for new products. These surveys are known to contain hypothetical bias; participants in these surveys are not required to execute their stated preferences, that is, “participants don’t have to pay what they say.”32–34 In contrast, in our auction market settings, consumers were expected to execute their winning bids by purchasing one unit of the auctioned commodity. Also, in contrast to most economics experiments that use university undergraduate students from the investigator’s class as auction participants, we used randomly chosen adult consumers in major metropolitan areas that were identified by an independent Â�survey agency and told that a university project was being undertaken to obtain consumers’ assessments of food and new household products. In particular, screened individuals were not told that they would be assessing genetically modified organisms (GMOs) or even would be participating in an experimental auction. Economists frequently choose a Vickery 2nd price auction (http://en.wikipedia. org/wiki/Vickrey_auction) for valuing goods. However, it is well known that individuals who anticipate that they are far from placing the margin bid will bid randomly and insincerely. These participants have a real sense that their bid is not pivotal in determining the market price. We chose the random nth price auction.35 In this auction, the winning bidders are chosen from a uniform distribution over 1 to n, the total number of bidders in a session. For example, if there are 15 participants in a session, the bids are first ranked from 1 to 15, and the randomly drawn n is 5; then the four highest bidders pay the fifth highest price. With this type of auction mechanism, all bidders are engaged because they sense that their bid will help determine the Â�market price, or bidding their true willingness to pay is a weakly dominate strategy.36 Moreover, our auction is best described as being a sealed-bid random nth price auction, because no information about willingness to pay for experimental products is released before all bids are placed. Individuals who agreed to participate came to a central location, signed a personal consent form, were paid $40 for their participation, and completed a short questionnaire on their social-demographic-economic characteristics and beliefs about a few technologies, including GMOs. They received instruction in the mechanics of a random nth price auction, and participated in an auction practice session. Next, they took a short test on their understanding of the auction mechanism and any questions were answered. The auctioning of experimental commodities followed. After winning bids were determined, the participants completed another short survey, and then were told to execute winning binds by completing purchases of auctioned commodities in an adjacent stock room. Otherwise, they were told that they were free to leave. In all of our experiments, we used three sets of diverse information about genetic modification and GM foods to construct information treatments: (1) the industry (pro-biotech) perspective—a collection of statements and information on genetic modification provided by a group of leading biotechnology companies, including Monsanto and Syngenta; (2) the environmental group (anti-biotech)

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perspective—a collection of statements and information on genetic modification from Greenpeace, a leading environmental group; and (3) the independent, thirdparty (verifiable information) perspective—a statement on genetic modification approved by a third-party group, consisting of a variety of people knowledgeable about GM goods, including scientists, professionals, religious leaders, and academics, who do not have a financial stake in GM foods. We limited the information statements to one page and organized the information under five different headings, General Information, Scientific Impact, Human Impact, Financial Impact, and Environmental Impact, to reduce the information load on participants (Appendices A and B). Information treatments, consisting of one-to-three of the above information types, were injected into each of the sessions or experimental trials. For example, in the first set of experiments, the information treatments were: (1) only the industry perspective; (2) only the environmental group perspective; (3) industry and environmental perspectives; (4) environmental and thirdparty perspectives; (5) industry, environmental, and third-party perspectives. Information treatments were randomly assigned sessions without replacement. When a session received industry and environmental perspectives, the order was randomized. When a session received the third-party perspective, it was always displayed last. In the 2001 Des Moines and St. Paul experiments, each experimental unit (or session) of 13–16 individuals/consumers participated in only two rounds of bidding on experimental food items. The rounds were differentiated by the food label. In one round, which could be round 1 or 2 depending on the experimental unit, participants/consumers bid on three food products, each with a conventional food label that stated only the type of food and weight. In the other round, participants bid on the same three food products with a GM label, which differed from the conventional food label by the inclusion of only one extra sentence: “This product is made using genetic modification (GM)” (Figure 9.2). Each session or experimental unit received an information treatment chosen randomly from the six available treatments. A total of 172 individuals participated in these experiments. In the 2007 Des Moines and Harrisburg experiments, each experimental unit (or session) of 13–16 individuals/consumers participated in four rounds of bidding on experimental food items. The rounds were differentiated by the composition of the food label, which had seven variants including a baseline with no information (with a maximum of four used in any session) (Figure 9.3). Although all bidders in a session or round of bidding saw the same food labels, they received different information treatments in the Des Moines and Harrisburg experiments. The Des Moines and Harrisburg experiments were unique in not only distinguishing the type of GMO, but also for injecting a treatment with “No Information.” Also, the exact wording of the three types of information was modified to be appropriate to the emphasis of these experiments; for example, the industry perspective and third-party perspective described the key differences between “Transgenic” and “Intragenic GM Products” (Appendices A and B).

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Russet potatoes

Russet potatoes

Net weight 5 lb.

Net weight 5 lb.

This product is made using genetic modification (GM).

Tortilla chips Net weight 16 oz. fresh made Thursday April 5th This product is made using genetic modification (GM).

Tortilla chips Net weight 16 oz. fresh made Thursday April 5th

Vegetable oil

Vegetable oil

Net weight 32 fl. oz.

Net weight 32 fl. oz.

This product is made using genetic modification (GM).

FIGURE 9.2â•… GM and Plain Food Labels for 2001 Des Moines and St. Paul Experiments.

Potato (5 lbs.)

Potato (5 lbs.)

GM free product

Potato (5 lbs.)

Potato (5 lbs.)

Intragenic GM product

Transgenic GM product

Potato (5 lbs.)

Potato (5 lbs.)

Potato (5 lbs.)

Enhanced levels of antioxidants and vitamin C

Enhanced levels of antioxidants and vitamin C

Enhanced levels of antioxidants and vitamin C

GM product

Intragenic GM product

Transgenic GM product

FIGURE 9.3â•… Food Labels for Potato in the 2007 Des Moines and Harrisburg Experiments.

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EXPERIMENTAL EVIDENCE ON KEY BID-PRICE DIFFERENCES37 Simple Differences in Mean Bid Prices We first examine simple bid-price differences for consumer willingness to pay for food items labeled as being genetically modified versus having a plain/conventional food label, specifying only the type and weight of the food. The 2001 Des Moines and St. Paul data showed that bidders discounted the GM-labeled product by about 15% relative to its plain-labeled counterpart.* Turning to the 2007 Des Moines and Harrisburg samples, bidders were, on average, willing to pay a sizeable premium for food products containing the label “Enhanced Levels of Antioxidants and Vitamin C—Intragenic GM Product” relative to a plain/conventionally labeled food product. The premium ranged from 39 to 45 cents per unit on the three food products, or 19%–26% higher. The mean price for each of the three commodities—broccoli, tomatoes, and potatoes—with enhanced attributes relative to products with a plain/conventional label, was different from zero at the 5% significance level.† Hence, when consumers bid on GM products containing input traits, the GM product was weakly inferior to the plain/conventionally labeled product. However, when consumers bid on fresh intragenic GM horticultural products containing enhanced levels of antioxidants and vitamin C, they were willing to pay a premium and, hence, the intragenic products were economically and statistically superior to a plain/conventionally labeled product.‡ These results imply a dramatic difference in the incentives for private industry to label new GM products with enhanced consumer attributes relative to those derived from raw materials that contain input traits.22

Individual Bid-Price Differences and Diverse Information37 When bidders received both pro-biotech and anti-biotech information, the bid price difference was reduced, reflecting the opposing forces of the two types of information in this information treatment. Moreover, this information treatment did not have a statistically significant effect on bid price differences. From these results, we conclude that in those sessions where bidders received only anti-biotech or both probiotech and anti-biotech information, they bid differently than when they received only pro-biotech information. When bidders were in sessions that received the pro-biotech and verifiable information treatment, the impact of this combination was not statistically significant. * All three differences are significantly negative at better than the 0.07 significance level. † The mean bid prices where the biotech method was switched from intragenic to transgenic, but otherwise containing enhanced consumer attributes, were larger than for the plain label but 14%–22% lower than for the intragenic label. ‡ We have not undertaken sensory tests to determine whether consumers can perceive any differences in the conventional product relative to one with intragenic enhanced consumer attributes. Given that no “foreign” DNA nor antibiotic marker is present in the intragenic product, my hypothesis is that no perceived sensory differences exist. Moreover, Zhao et al. found no difference in a sensory analysis of conventional and organically grown fruits and vegetables, except that conventionally grown tomatoes scored slightly higher for ripeness, which is positively correlated with flavor intensity.38

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When bidders were in sessions that received anti-biotech and verifiable information, bid-price differences are reduced (less negative) and the difference is significantly different from zero. Hence, those who received a treatment of anti-biotech information and verifiable information discounted GM foods less than those who received only a treatment of anti-biotech information. When bidders were in sessions that received a treatment that contained all three types of information (pro-biotech, anti-biotech, and verifiable), the impact of this treatment on bid-price differences was small and not statistically significant. Hence, in this complex setting, verifiable information did not have a distinguishable effect. Bidders who had larger household incomes discounted GM by a larger amount than those with less household income. This result is statistically significant at the 5% level and is consistent with non-GM products being viewed, on average, by bidders as a superior product. Participants coming into our experiments were asked about how well-informed they were about genetic modification. This subjective information was then coded into a dichotomous variable. Those bidders who considered themselves to be at least “somewhat informed about GM foods” discounted GM-labeled foods more than did other bidders. This effect is statistically significant (at the 10% level). Moreover, this result suggests that bidders in our experiments who were “GM-informed” had, on average, acquired/received negative information about GM foods prior to the experiment.* Bids also were affected by the labeling sequence. Bidders in sessions that bid on the GM-labeled food products in round one (and the plain-labeled food products in round two) discounted GM-labeled foods by less than those who were in sessions that bid on the products in the opposite order. This result reinforces the importance of randomized assignments of treatments to sessions in experimental auctions, which is an innovation in our methodology. Next, we turn to the 2007 sample of individuals from Des Moines and Harrisburg. In these results, the base case with no-information treatment gives a bid-price difference of intragenic GM- over plain-labeled food products by a statistically significant 52 cents per unit of product. When pro-biotech information treatment was injected into the experiments, the bid-price difference was a statistically significant 73 cents per unit or 21 cents more than for the no-information treatment, suggesting a net positive influence of industry-provided biotech information on willingness to pay for intragenic GM products. The injection of the anti-biotech information treatment gives a bid-price difference of only 25 cents per unit, which is 27 cents per unit lower than for the no-information treatment. However, this coefficient is not different from zero at the 5% or 10% significance levels, suggesting that environmental groups do not distinguish between intragenic and transgenic biotech methods for engineering new crops. The injection of a pro-biotech and anti-biotech treatment (where the order is random across participants in a session) increases the bid price difference by a statistically significant 56 cents per unit. The impact of this information treatment on bid-price differences is slightly higher (4 cents per unit) than for the no-information treatment, which is a reflection of the opposing forces of pro-biotech and anti-biotech information, but with the edge going to the pro-biotech information. When the * See Huffman et al.39 for an analysis of the impact of bidders’ prior beliefs about GM technology and food products on their willingness to pay for food items that are potentially GM.

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injected information treatment includes all three types of information (with verifiable information always last), the bid-price difference is larger by a statistically significant 44 cents per unit, which is 8 cents less than for the no-information treatment. Comparing this result to the previous one, verifiable information seems to be a moderating force relative to the pro-biotech industry perspective. Consumers have expressed some skepticism of transgenic GM food products, and our 2007 Des Moines and Harrisburg data set shows that consumers bid significantly higher prices for intragenic than transgenic food products that contain enhanced levels of antioxidants and vitamin C. However, the differences in these bid prices may be affected by the information environment in which consumers are bidding. To test this hypothesis, we examined the differences in bid prices for products with an intragenic GM label versus one with a transgenic GM label. Bid-price differences for the baseline case of the no-information treatment are 18 cents per unit higher for intragenic than transgenic, but this number is not significantly different from zero at the 10% or 5% levels, suggesting that this information is truly uninformative about these biotech methods. In contrast, an injection of the pro-biotech information treatment gives a bid-price difference by a statistically significant 67 cents per unit, suggesting that, on net, the industry perspective favors intragenic over transgenic. In contrast, an injection of anti-biotech information treatment gives a bid-price difference of only 17 cents per unit, which is slightly lower than the point estimate for the no-information treatment and 50 cents per unit lower than for the pro-biotech information treatment. Injection of the pro-biotech and anti-biotech information treatment gives a bid-price difference of a statistically significant 42 cents per unit, which is in the middle of the estimates for the probiotech and anti-biotech treatment estimates. The injection of the information treatment containing all three information types gives a bid price difference of 38 cents, which suggests that objective information moderates the positive impact of the pro-biotech information on bid-price differences. The estimated coefficient for the dummy variable denoting that the food product has enhanced antioxidants and vitamin C is a positive 12 cents per unit and significant at the 10% level. This result suggests that the presence of enhanced consumer attributes increases the value to bidders of the intragenic over transgenic methods. Our results also showed a significant “bidding round or order effects” in the data, which supports methodological advances using randomization. Household income has no impact on bid-price differences in these results. In conclusion, I can say that the information setting affects consumer discounting of transgenic relative to intragenic horticultural products.

Individual Differences in Bid Prices and Prior Information New food products using GM crops appeared in U.S. supermarkets starting in 1996, and consumers perceived some risks. Because consumers are exposed to diverse and sometimes conflicting perspectives about GM technologies and foods, they form subjective beliefs. Huffman et al. examined the role of these prior beliefs on consumer willingness to pay for foods that might be genetically modified.39 The data are from the 2001 Des Moines and St. Paul experiments, where participants

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were asked before the experimental auction how well-informed they were about genetic modification—extremely, well, somewhat, not very well, or not informed at all. Huffman et al. show that participants who had informed prior beliefs about genetic modification discounted GM-labeled food products by a larger amount than those who had uninformed prior beliefs. Also, uninformed participants were especially susceptible to information from interested and third parties. In contrast, informed participants were generally not affected significantly by new information. These results contradict some earlier psychological studies that claimed that individuals tend to base rates.19 The results show how both skeptics and proponents of new technologies might try to manage information to achieve private or group-wide, but not social, objectives.

Other Related Results Rousu et al. examine the impact of tolerance levels, or the impact of the minimum level of GM contamination that will pass as GM-free.40 Using the 2001 Des Moines and St. Paul data, they examine bids on three food products that have different tolerance labels. In one trial, all consumers bid on foods with a non-GM label, certified to be completely free of genetically engineered material, and in the other trial, consumers bid on foods with a non-GM label indicating that a certain percentage of GM material, either 1% or 5% percent, was tolerated. Consumers in these treatments did not receive any information on GM food products. This experiment contained three experimental units/sessions with a total of 44 participants. Rousu et al. found evidence that consumers preferred foods that were 100% non-GM, relative to food products with small amounts of GM material (1% or 5%). Consumers bid approximately 10% less for the GM-tolerant food products than they did for the certified GM-free products. However, they found that once GM content was present, no difference existed in bids between foods that contained 1% versus 5% GM content.40 Thus, while these findings indicate that a significant percentage of consumers will pay more for GM-free labeled food products relative to conventionally labeled food products, it does not appear that 1% or higher tolerance levels for GM material matters.

DISCUSSION OF GM TECHNOLOGY AND MIXED MESSAGES Although consumers in the United States are relatively tolerant of alternative production methods for their food, they do respond adversely to some risks. For example, when genetic modification refers to input traits, consumers in our experiments discounted GM food products by 15%, relative to a plain- or conventionally labeled alternative. This seems to arise from environmental, biodiversity, or health concerns from introducing foreign DNA into food crops. To circumvent these concerns, new methods have been developed for intragenic genetic modification, where no foreign DNA is introduced in the GM varietal development process. Our results from the 2007 data support the hypothesis that consumers have a more favorable perspective about these genetic modifications, and that they are, in fact, willing to pay a premium for enhanced levels of antioxidants and vitamin C by intragenic GM methods.

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However, when we were experimenting with food traits to consider including enhanced food products, we experimented with “low pesticide residual” as a food quality attribute. Consumer reaction to this trait was complex, because the label raised a dormant issue that, yes, there is pesticide residue in our food. Mentioning that insect-resistant (Bt) traits could be introduced so as to reduce the need for farmers to apply commercial pesticides, and thereby reduce chemical pesticide residual, was a hard sell. Along a similar line of mixed messages, Markosyan et al. conducted surveys of consumers in grocery stores in October 2006 in the Pacific Northwest to test their willingness to pay for “naturally enriched antioxidant coatings” embedded in the wax on retail fresh apples.41 In general, consumers were willing to pay a little, 4%–8% more, for the antioxidant enhanced apples, but a number of consumers were quite negative about the technology, for example, “I don’t want to eat wax,” “it is unnatural,” “additives to fruit are not necessary,” “washing apples removes the wax,” “prefer foods without additives,” and “it is better to get nutrients naturally.” Hence, adding antioxidants to the wax of apples also raised the dormant issue that fresh commercial apples are waxed.

CONCLUSIONS AND PREDICTIONS FOR THE FUTURE More than a decade has passed since the first GM foods appeared in U.S. grocery stores. Early attempts to market fresh horticultural products, in particular, the FlavrSavr tomato and the Russet Burbank Newleaf potato failed after very brief appearances in the market. Input traits developed by transgenetic methods applied to field crops have been much more successful, but consumers continue to express some resistance to them because of environmental, biodiversity, or human health concerns. Recent developments of new intragenic GM methods are exciting because they permit scientists to use diverse genes and attributes identified in the genomes of particular horticultural plants to quickly enhance quality attributes of commercial varieties of crops like potato and tomato. This is very important in potato breeding, where it is impossible for scientists to dramatically enhance conventional levels of vitamin C and antioxidants using a range of conventional non-GM breeding methods. The new intragenic GM potato varieties enhanced with antioxidants and vitamin C promise to be the first successful commercial GM product with consumer traits on the market. Economists’ research has shown that U.S. consumer acceptance and willingness to pay for GM food crops is not only conditioned by the nature of the new trait and the method of DNA transfer, but also by the content of food labels, prior beliefs of consumers, and content of diverse information injected into the food market about GM technologies and food products. During the era of input-trait dominated GM foods, consumers have revealed that GM food products are weakly inferior to conventional products, which means that marketers of GM food products will not label voluntarily. With the commercialization of new intragenic GM products with enhanced consumer attributes, these new GM food products promise to command a premium relative to conventional food products. Hence, the private sector’s incentive to voluntarily label GM products will change dramatically. This promises to be a positive development in the commercialization of GM food products.

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During the era of input traits, consumers’ informed prior beliefs were somewhat negative about agricultural biotechnology and GM foods. These prior beliefs, however, have the potential to become more favorable toward GM horticultural crops as new products with intragenic GM-enhanced consumer attributes become generally available in the food market. Also, strong evidence exists that consumers are positively influenced by biotech industry or pro-biotech information, and negatively affected by environmental group or anti-biotech information. Moreover, third-party verifiable information has been shown to be a moderating influence on consumer interpretation of anti-biotech and pro-biotech information and on willingness to pay for GM foods. Hence, a future role exists for public sector provision of thirdparty verifiable information about GM technologies and GM food products. This new information will affect prior beliefs of consumers about GM food products and be a useful input to objective assessments of new GM food products that enter the market, which could be important to the commercial successes of new GM horticultural crops.

APPENDIX A:â•…INFORMATION INJECTED INTO THE 2001 DES MOINES AND ST. PAUL EXPERIMENTS42 A. The following is a collection of statements and information on genetic modification from Greenpeace, a leading environmental group. General Information Genetic modification is one of the most dangerous things being done to your food sources today. There are many reasons that genetically modified foods should be banned, mainly because unknown adverse effects could be catastrophic! Inadequate safety testing of GM plants, animals, and food products has occurred, so humans are the ones testing whether or not GM foods are safe. Consumers should not have to test new food products to ensure that they are safe. Scientific Impact The process of genetic modification takes genes from one organism and puts them into another. This process is very risky. The biggest potential hazard of GM foods is unknown. This is a relatively new technique, and no one can guarantee that consumers will not be harmed. Recently, many governments in Europe assured consumers that there would be no harm to consumers over mad cow disease, but unfortunately, their claims were wrong. We do not want consumers to be harmed by GM food. Human Impact GM foods could pose major health problems. The potential exists for allergens to be transferred to a GM food product that no one would suspect. For example, if genes from a peanut were transferred into a tomato, and someone who is allergic to peanuts eats this new tomato, they could display a peanut allergy. Another problem with GM foods is a moral issue. These foods are taking genes from one living organism and transplanting them into another. Many people think it is morally wrong to mess around with life forms on such a fundamental level.

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Financial Impact GM foods are being pushed onto consumers by big businesses, which care only about their own profits and ignore possible negative side effects. These groups are actually patenting different life forms that they genetically modify, with plans to sell them in the future. Studies have also shown that GM crops may get lower yields than conventional crops. Environmental Impact GM foods could pose major environmental hazards. Sparse testing of GM plants for environmental impacts has occurred. One potential hazard could be the impact of GM crops on wildlife. One study showed that one type of GM plant killed Monarch butterflies. Another potential environmental hazard could come from pests that begin to resist GM plants that were engineered to reduce chemical pesticide application. The harmful insects and other pests that get exposed to these crops could quickly develop tolerance and wipe out many of the potential advantages of GM pest resistance. B. The following is a collection of statements and information on genetic modification provided by a group of leading biotechnology companies, including Monsanto and Syngenta. General Information GM plants and animals have the potential to be one of the greatest discoveries in the history of farming. Improvements in crops so far relate to improved insect and disease resistance and weed control. These improvements using bioengineering/ GM technology lead to reduced cost of food production. Future GM food products may have health benefits. Scientific Impact Genetic modification is a technique that has been used to produce food products that are approved by the FDA. Genetic engineering has brought new opportunities to farmers for pest control and in the future will provide consumers with nutrient-enhanced foods. GM plants and animals have the potential to be the single greatest discovery in the history of agriculture. We have just seen the tip of the iceberg of future potential. Human Impact The health benefits from genetic modification can be enormous. A special type of rice called “golden rice” has already been created which has higher levels of vitamin A. This could be very helpful because the disease vitamin A deficiency (VAD) is devastating in third-world countries. VAD causes irreversible blindness in over 500,000 children, and is also responsible for over 1 million deaths annually. Since rice is the staple food in the diets of millions of people in the third world, golden rice has the potential of improving millions of lives a year by reducing the cases of VAD. The FDA has approved GM food for human consumption, and Americans have been consuming GM foods for years. While every food product may pose risks, there has never been a documented case of a person getting sick from GM food.

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Financial Impact GM plants have reduced the cost of food production, which means lower food prices, and that can help feed the world. In the United States, lower food prices help decrease the number of hungry people and also let consumers save a little more money on food. Worldwide the number of hungry people has been declining, but increased crop production using GM technology can also help further reduce world hunger. Environmental Impact GM technology has produced new methods of insect control that reduce chemical insecticide application by 50% or more. This means less environmental damage. GM weed control is providing new methods to control weeds, which are a special problem in no-till farming. Genetic modification of plants has the potential to be one of the most environmentally helpful discoveries ever. C. The following is a statement on genetic modification approved by a third-party group, consisting of a variety of individuals knowledgeable about GM foods, including scientists, professionals, religious leaders, and academics. These parties have no financial stake in GM foods. General Information Bioengineering is a type of genetic modification where genes are transferred across plants or animals, a process that would not otherwise occur (in common usage, genetic modification means bioengineering). With bioengineered pest resistance in plants, the process is somewhat similar to the process of how a flu shot works in the human body. Flu shots work by injecting a virus into the body to help make a human body more resistant to the flu. Bioengineered plant-pest resistance causes a plant to enhance its own pest resistance. Scientific Impact The FDA standards for GM food products (chips, cereals, potatoes, etc.) are based on the principle that they have essentially the same ingredients, although they have been modified slightly from the original plant materials. Oils made from bioengineered oil crops have been refined, and this process removes essentially all the GM proteins, making them like non-GM oils. So even if GM crops were deemed to be harmful for human consumption, it is doubtful that vegetable oils would cause harm. Human Impact While many GM foods are in the process of being put on your grocers’ shelves, there are currently no foods available in the United States where genetic modification has increased nutrient content. All foods present a small risk of an allergic reaction to some people. No FDAapproved GM food poses any known unique human health risks. Financial Impact GM seeds and other organisms are produced by businesses that seek profits. For farmers to switch to GM crops, they must see benefits from the switch. However, genetic modification technology may lead to changes in the organization of the

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agri-business industry and farming. The introduction of GM foods has the potential to decrease the prices to consumers for groceries. Environmental Impact The effects of genetic modification on the environment are largely unknown. Bioengineered insect resistance has reduced farmers’ applications of environmentally hazardous insecticides. More studies are occurring to help assess the impact of bioengineered plants and organisms on the environment. A couple of studies reported harm to Monarch butterflies from GM crops, but other scientists were not able to recreate the results. The possibility of insects growing resistant to GM crops is a legitimate concern.

APPENDIX B:â•…INFORMATION INJECTED INTO THE 2007 DES MOINES AND HARRISBURG EXPERIMENTS A. The following is a collection of statements and information on genetic modification from Greenpeace, a leading environmental group. General Information Genetic modification takes genes from one organism and places them into another. The process lets scientists manipulate genes in an unnatural way. Inadequate safety testing of GM plants and food products has occurred. Humans and the Earth are being used as guinea pigs for testing whether “Frankenfoods” are safe. GM foods should be banned because their effect on consumers and the environment is unknown and potentially catastrophic! Genetic modification is one of the most risky things being done to your food sources today and should be stopped before more damage is done. Scientific Impact All genetic modifications of plants are risky. All GM techniques are relatively new and no one can guarantee that consumers or the environment will not be harmed. The biggest potential hazard of GM foods is unknown. Human Impact GM foods could pose serious risks to human health. Some foods contain allergens, and the potential exists for allergens to be transferred into a GM food product that no one would suspect. For example, if the genes from a peanut were transferred into a tomato, and someone who is allergic to peanuts eats this GM tomato, he could display a peanut allergy. Another problem with transgenic foods is a moral issue. Many GM techniques transfer genes across species. We believe it is morally wrong to alter life forms on such a fundamental level. Financial Impact GM foods are being pushed onto consumers by big businesses which only care about their own profits and ignore possible negative side effects. These groups are actually patenting new life forms they create with plans to sell for profits. Studies have shown that GM crops may even get lower yields than conventional crops.

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Environmental Impact GM foods could pose major environmental hazards. Little testing of GM plants for environmental impacts has occurred. One potential risk of GM crops is their impact on wildlife, including wild species of plants and insects. A study showed that one type of GM plant killed Monarch butterflies. Another potential environmental hazard could come from pests that become resistant to new naturally occurring toxic substances engineered into plants to kill pests— insects and worms—or to make a plant resistant to a particular herbicide application. The target pests that get exposed to these new GM crops could quickly develop tolerances and wipe out many of the potential advantages of GM pest resistance. B. The following is a collection of statements and information on genetic modification provided by a group of leading biotechnology companies, including Monsanto, Pioneer, and Syngenta. General Information GM plants have the potential to be one of the greatest discoveries in the history of farming. GM crops have lowered food production costs by improving insect and disease resistance and weed control in plants. New genetic engineering techniques could dramatically enhance consumer benefiting attributes of food such as vitamins, antioxidants, flavor, and shelf life. These improvements to plant quality can only be attained through GM, not conventional breeding. The process of genetic modification takes genes from one organism and places them into another. There are two distinct types of GM used by biotechnology companies. Transgenic GM transfers genes between two unrelated organisms, for example, from soil bacteria to corn. Intragenic GM involves transferring genes between two breeds of the same organism, for example, from wild species of corn to a commercial variety of corn. Scientific Impact Both transgenic and intragenic techniques are used to produce food products that are approved by the FDA. Intragenic modification is a genetic technique for significantly speeding up the conventional process of plant cross-breeding, which has been undertaken by farmers and plant breeders for thousands of years. Many industry groups believe intragenics should require minimal FDA testing because no foreign genes or proteins are added to the GM plant. We have only seen the tip of the iceberg of the future potential of GM for improving worldwide health and nutrition through enhanced plants. Human Impact The potential exists for GM to dramatically enhance traits that have direct value to consumers, such as increased vitamins and antioxidants, more flavor, longer shelf life, lower pesticide use, and reduced cost of production. Superior GM plants will help reduce worldwide malnutrition and improve the healthiness of foods. The FDA has approved GM food for human consumption, and Americans have been consuming GM foods for a decade. While every food (modified or not) poses some risks, there has never been a documented case of a person getting sick from GM food.

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Financial Impact With the introduction of enhanced nutrition, antioxidants, shelf life, flavors, and other consumer-desired attributes using GM technology, consumers will for the first time enjoy the direct benefits of genetic engineering. GM plants have reduced farmers’ costs, which mean lower food prices. Worldwide the number of hungry people is declining. GM technology is helping to feed the world and improve worldwide nutrition. Environmental Impact Genetic modification of plants has the potential to be one of the most environmentally helpful discoveries ever. GM technology has produced new methods of insect control that reduce chemical insecticide application by 50% or more. GM weed control is providing new methods to control weeds, which are a problem in no-till farming. This means greater crop yields and less environmental damage. C. The following is a statement on genetic modification approved by a third-party group consisting of a variety of individuals knowledgeable about GM foods, including scientists, professionals, religious leaders, and academics. These parties have no financial stake in GM foods. General Information The process of genetic modification takes genes from one organism and places them into another. There are two distinct types of GM used by biotechnology companies. Transgenic GM transfers genes between two unrelated organisms, for example, from soil bacteria to corn. Intragenic GM involves transferring genes between two breeds of the same organism, for example, from wild species of corn to a commercial variety of the crop. Hence, intragenic modification has much in common with conventional plant breeding. Scientific Impact The FDA standard for GM food products is based on the principle that they have essentially the same ingredients, although modified from the original plant. Almost all GM crops meet the FDA’s substantive equivalent requirement. Hence, they do not require special testing before commercial marketing can occur. Human Impact Many scientists see intragenics as having real potential for enhancing consumer attributes of plants such as dramatically increasing vitamin and antioxidant levels, extending shelf life, and reduced chemical pesticide application without concerns about gene transfer across species. These improvements to plants are only possible using genetic modification and not conventional breeding. All foods present a risk of an allergic reaction to a small fraction of the population. No FDA-approved GM food poses any known unique human health risks, but when genes are transferred across species, a new allergen is possible. This is more likely with transgenics than intragenics. While GM crops can result in higher yields and enhanced nutrition, there is no consensus whether GM foods have or will reduce worldwide hunger.

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Many people have moral or religious objections to GM. Some groups see intragenics as being more acceptable because genes are transferred between two breeds of the same species. Financial Impact GM seeds and other organisms are produced by businesses that seek profits. For farmers to switch to GM crops, they must see benefits from making a change. Consumers must also see benefits from consuming GM foods—lower price or enhanced consumer attributes. However, GM technology may lead to changes in the organization of the agri-business industry and farming. Environmental Impact The long-term effects of GM on the environment are largely unknown. Bioengineered insect resistance has reduced farmers’ applications of environmentally hazardous insecticides, but resistance to this bio-control system will increase over time. More studies are occurring to help assess the impact of bioengineered plants on the environment. Some studies reported harm to Monarch butterflies from GM crops, but other scientists were not able to recreate the results. Enhanced consumer attributes, such as vitamins, antioxidants, and longer shelf life due to intragenics pose no known environmental hazards.

REFERENCES

1. Alston, J.M., K. Bradford, and N. Kalaitzandonakes. The economics of horticultural biotechnology. Journal of Crop Improvement, 18, 413, 2006. 2. Bradford, K.J. and J.M. Alston. Diversity of horticultural biotech crops contributes to market hurdles. California Agriculture, 58, 84, 2004. 3. Alcamo, I.E. DNA Technology, 2nd edn. New York: Harcourt Academic Press. 1999. 4. Soil Association. Flavr-Savr tomato & GM tomato puree: The failure of the first GM foods. Briefing Paper, November 29, 2005. Available at: http://www.soilassociation.org/ web/sa/sweb.nsf/ (accessed April 11, 2007). 5. Fernandez-Cornejo, J. and M. Caswell. The first decade of genetically engineered crops in the United States. USDA-ERS, Economic Information Bulletin, No. 11, April 2006. 6. Zilberman, D. The economics of biotechnology regulation. In R.E. Just, J.M. Alston and D. Zilberman, eds., Regulating Agricultural Biotechnology: Economics and Policy. New York: Springer, 2006, pp. 243–261. 7. Gonsalves, D. and S. Ferreira. Transgenic papaya: A case of managing risks of papaya ringspot virus in Hawaii. Plan Health Progress, November 13, 2003 (6pp). 8. Gonsalves, D. Transgenic papaya in Hawaii and beyond. AgBioForum, 7, 36, 2004. 9. Zilberman, D. The economics of pesticide usage. Department of Agricultural and Resource Economics, University of California Berkeley, 2004. Available at: http:// are.berkeley.edu/%7Ezilber/ (accessed April 12, 2007). 10. Rommens, C.M., J.M. Humara, J. Ye, H. Yan, C. Richael, L. Zhang, R. Perry, and K. Swords. Crop improvement through modification of the plant’s own genome. Plant Physiology,135, 421, 2004. 11. Rommens, C.M., O. Bougri, H. Yan, J.M. Humara, J. Owen, K. Swords, and J. Ye. Plantderived transfer DNAs. Plant Physiology, 139, 1338, 2005.

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12. Rousu, M., W.E. Huffman, J.F. Shogren, and A. Tegene. Effects and value of verifiable information in a controversial market: Evidence from lab auctions of genetically modified food. Economic Inquiry, 45, 409, 2007. 13. Falck-Zepeda, J.B., G. Traxler, and R.G. Nelson. Surplus distribution from the introduction of a biotechnology innovation. American Journal of Agricultural Economics, 82, 360, 2000. 14. Moschini, G., H. Lapan, and A. Sobolevsky. Roundup ready soybeans and welfare effects in the soybean complex. Agribusiness, 16, 33, 2000. 15. Huffman, W.E. and M. Rousu. Consumer attitudes and market resistance to biotech products. In R.E. Just, Julian Alston, and D. Zilberman, eds., Regulating Agricultural Biotechnology: Economics and Policy. New York: Springer, 2006, pp. 201–226. 16. Moschini, G. and H. Lapan. Labeling regulations and segregation of first- and secondgeneration GM products: Innovation incentives and welfare effects. In R.E. Just, J. Alston, and D. Zilberman, eds., Regulating Agricultural Biotechnology: Economics and Policy. New York: Springer, 2006, pp. 263–282. 17. Balasubramanian, S.K. and C. Cole. Consumers’ search and use of nutrition information: The challenge and promise of the Nutrition Labeling and Education Act. Journal of Marketing, 66, 113, 2002. 18. Silverglade, B. The Nutrient Labeling and Education Act: Progress to date and challenges for the future. Journal of Public Policy and Marketing, 15, 148, 1996. 19. Tversky, A. and D. Kahneman. The framing of decisions and the psychology of choice. Science, 211, 453, 1981. 20. Just, R.E., D. Zilberman, and J.M. Alston. Regulating agricultural biotechnology: Introduction and overview. In R.E. Just, J. Alston, and D. Zilberman, eds., Regulating Agricultural Biotechnology: Economics and Policy. New York: Springer, 2006, pp. 3–17. 21. Wilson, W.W. and B.L. Dahl. Costs and risks of testing and segregating genetically modified wheat. Review of Agricultural Economics, 27, 212, 2005. 22. Roe, B. and I. Sheldon. Credence good labeling: The efficiency and distributional implications of several policy approaches. American Journal of Agricultural Economics, 89, 1020, 2007. 23. Greenpeace International. We want natural food! 2001. Available at http://www.greenpeace. org/∼geneng/ (accessed March 15, 2004). 24. Akerlof, G. The market for lemons: Quality uncertainty and the market mechanism. Quarterly Journal of Economics, 84, 488, 1970. 25. Molho, I. The Economics of Information: Lying and Cheating in Markets and Organizations. Oxford, U.K.: Blackwell Publishing Ltd. 1997. 26. Huffman, W.E. and R.E. Evenson. Science for Agriculture: A Long-Term Perspective. Ames, IA: Blackwell Publishing, 2006. 27. Rousu, M., W.E. Huffman, J.F. Shogren, and A. Tegene. Estimating the public value of conflicting information: The case of genetically modified foods. Land Economics, 80, 125, 2004. 28. Milgrom, P. and J. Roberts. Relying on the information of interested parties. Rand Journal of Economics, 17, 18, Spring 1986. 29. Huffman, W.E. and A. Tegene. Public acceptance of and benefits from agricultural biotechnology: A key role for verifiable information. In V. Santaniello, R.E. Evenson and D. Zilberman, eds., Market Development for Genetically Modified Foods. Wallingford, U.K.: CAB International, 2002, pp. 179–189. 30. Smith, V. L. Experimental economics: Induced value theory. American Economic Review, Papers and Proceedings, 66, 274, 1976. 31. Huffman, W.E., M. Rousu, J.F. Shogren, and A. Tegene. The public good value of information from agribusinesses on genetically modified foods. American Journal of Agricultural Economics, 85, 1309, Dec 2003.

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32. Haneman, M.W. Welfare evaluations in contingent valuation experiments with discrete responses. American Journal of Agricultural Economics, 66, 332, 1984. 33. Mendenhall, C.A. and R.E. Evenson. Estimates of willingness to pay a premium for nonGM foods: A survey. In V. Santaniello, R.E. Evenson, and D. Zilberman, eds., Market Development for Genetically Modified Foods. Wallingford, U.K.: CAB International, 2002, pp. 55–62. 34. Chern, W.D. and K. Rickertsen. A comparative analysis of consumer acceptance of GM foods in Norway and in the USA. In R.E. Evenson and V. Santaniello, eds., Consumer Acceptance of Genetically Modified Foods. Cambridge, MA: CABI 2004, p. 95 35. Shogren, J.F., M. Margolis, C. Koo, and J.A. List. A random nth-price auction. Journal of Economic Behavior and Organization, 46, 409, 2001. 36. McFadden, D. The human side of mechanism design: A tribute to Leo Hurwicz and JeanJacque Laffont. Working Paper, Department of Economics, University of California, Berkeley, April 2007. Available at: http://emlab.berkeley.edu/econ/faculty/mcfadden_d. shtml (accessed April 30, 2007). 37. Huffman, W.E. Consumer acceptance of genetically modified foods: Traits, labels and diverse information. Iowa State University, Department of Economics, Working Paper No. 10029, August 2010. Available at: http://www.econ.iastate.edu/research/workingpapers/p11835 (accessed August 30, 2010). 38. Zhoa, X., E. Chambers IV, Z. Matta, T.M. Loughin, and E.E. Carey. Consumer sensory analysis of organically and conventionally grown vegetables. Journal of Food Science, 72, S87, 2007. 39. Huffman, W.E., M. Rousu, J.F. Shogren, and A. Tegene. The effects of prior beliefs and learning on consumers’ acceptance of genetically modified foods. Journal of Economic Behavior and Organization, 63, 193, 2007. 40. Rousu, M., W.E. Huffman, J.F. Shogren, and A. Tegene. Are US consumers tolerant of GM foods? Review of Agricultural Economics, 26, 19, Spring/Summer 2004. 41. Markosyan, A., J.J. McCluskey, and T.I. Wahl. Functional foods in the marketplace: Willingness to pay for apples enriched with antioxidants. School of Economics, Washington State University, July 2007. 42. Huffman, W.E., J.F. Shogren, M. Rousu, and A Tegene. Consumer willingness to pay for genetically modified food labels in a market with diverse information: Evidence from experimental auctions. Journal of Agricultural and Resource Economics, 28, 481, December 2003. 43. Fernandez-Cornejo, J., and W.D. McBride. The Adoption of Bioengineered Crops. U.S. Department of Agriculture, Economic Research Service, Agricultural Economic Report No. 810. May 2002.

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Intellectual Property and Development of Transgenic Horticultural Crops Cecilia L. Chi-Ham and Alan B. Bennett

CONTENTS Intellectual Property in Horticultural Biotechnology............................................. 219 Types of Intellectual Property to Protect Horticultural Inventions......................... 220 Freedom to Operate.................................................................................................224 Strategies for IP Access.......................................................................................... 228 References............................................................................................................... 230

INTELLECTUAL PROPERTY IN HORTICULTURAL BIOTECHNOLOGY For centuries, horticulturists have applied science and art to genetically improve edible and ornamental plant varieties. Horticultural industries have produced new flowers, fruit and perennial trees, landscape plants, and vegetables. Also, the industry has provided technological advances to improve plant cultivation and postharvest practices. The time and economic investment by plant-breeding programs is significant because it may take decades for the development of a new plant variety or improved seed. Historically, breeders’ inventions have been freely distributed, diminishing the incentive to invest and the ability to capture value. In order to reconcile societal needs for new crops, recognize breeders’ creativity, and provide a means that ensures financial returns for the economic investment, international and national legal intellectual property protections systems have been developed. As a consequence, there is an increasing need for horticultural scientists to complement their technical knowledge with intellectual property information and awareness. This chapter is intended to provide horticulturists with an understanding of intellectual property issues affecting the development and deployment of genetically engineered horticultural crops. The chapter is divided into three sections. First, we describe the different forms of intellectual and tangible property rights and their application in the development of horticultural crops, particularly transgenic crops. The second part of the chapter addresses scientists’ increasing needs to complement their technical knowledge with intellectual property information and awareness. 219

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We specifically define freedom-to-operate, a systematic way of considering legal implications and navigating through IP landscape and issues in research and commercial development. Finally, the third section describes strategies of accessing intellectual property rights (IPRs) to enhance broad access and dissemination of new transgenic horticultural crops. We present PIPRA as a model of a public sector initiative that provides expertise to understand and to facilitate access to IP for the development and deployment of horticultural crops.

TYPES OF INTELLECTUAL PROPERTY TO PROTECT HORTICULTURAL INVENTIONS In the case of horticultural crops, the legal systems protect human-made improvements, such as a new plant variety, with IPRs and the physical materials, such as seeds, with tangible property rights (TPRs). These forms of IPRs and TPRs can be used by themselves or compounded to protect and manage innovations in horticultural biotechnology. The main types of intellectual property protection applicable to horticultural inventions include patents, plant variety rights, trade secrets, trademarks, and copyrights (Table 10.1).1,2 Tangible property rights are also important and are often managed through material transfer agreements (MTAs) or bailments and can be used to limit the use of physical materials. These forms of IPRs and TPRs are the cornerstone of the innovation process in the United States and Europe, and these rights are increasingly expanding into other countries in the world. Below, we describe the different forms of IPRs and TPRs and how these are related to the development of horticultural crops. There are significant differences between intellectual and tangible property rights, particularly in terms of geographic and time limitations. IPRs, like plant-variety protection (PVP) or plant patents, are granted by national offices for a specific country-jurisdiction and for a specified time period. In contrast, tangible property rights are not limited by geography or time and the terms governing their use are left to the discretion of the tangible property owner. The terms governing tangible property use are usually specified in MTAs or bailments. Thus, the tangible property rights may extend beyond the protection offered by IPRs. Of the many forms of IPRs, patents provide the legal rights to exclude others from using your invention that must be novel, useful, and nonobvious to one skilled in the art of horticultural breeding. Patent rights are conferred by national governments for a specified period of time, usually 20 years, and are limited to the country in which the patent is granted. In exchange for exclusivity, the inventor makes a public disclosure of the invention. In the United States, the two forms of patents most pertinent to the horticultural industry are plant patents and utility patents. Plant patents are specific to the U.S. legal system and are granted only for asexually propagated plants, such as strawberries and fruit trees, and exclude tuberpropagated and uncultivated species. At the time the U.S. Plant Patent Act was enacted in the 1930s, sexually produced plants were believed to be unstable and nonuniform and, as a result, were excluded from plant patent protection. Utility patents offer one of the most robust forms of IP protection. In horticultural biotechnology, utility patents can be used to protect the genetically modified seeds or plants and

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TABLE 10.1 Overview of IP Protection Systems Means of Protection

What Can Be Protected

How to Protect

Term of Protection

Rights of the Owner

If filing date is after June 8, 1995, 20 years from filling date; if filing date is before June 8, 1995, 17 years from issue If filing date is after June 8, 1995, 20 years from filling date; if filing date is before June 8, 1995, 17 years from issue date 20 years most crops; 25 years trees, vines

Right to exclude others from making, using, manufacturing, selling, and offering to sell Right to exclude others from asexually reproducing, selling, or using claimed plant

Utility patent

Any useful, novel, and nonobvious invention

USPTO

U.S. plant patent

Asexually produced plants (excludes uncultivated and tuber propagated plants)

USPTO

U.S. PVP

Sexually produced plants (excludes first generation hybrids, uncultivated plants)

USDA

UPOV91

All genera and species

UPOV

20 years most crops; 25 years trees, shrubs, and vines

Trademarks

Words, phrases, and logos that can distinguish the goods and services from those of others

Use or have a bona fide intent to use and apply for a federal registration

Unlimited duration as long as the mark is in use; the mark has to be renewed every 10th year

Right to exclude others from importing or selling, sexually or asexually reproducing, distributing without proper notice, producing a hybrid or new variety, and using the claimed patent Prevents others from producing or reproducing, conditioning for the purpose if propagation, offering for sale, selling or other marketing, importing, exporting, and stocking for any purposes Right to exclude others from using the mark and other marks so similar they cause confusion (continued)

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TABLE 10.1 (continued) Overview of IP Protection Systems Means of Protection

What Can Be Protected

How to Protect

Copyright

Literary works, software, dramatic works, music, pictures, sound recordings, architectural works, and movies Any technical or business information that is secret and that gives the holder an advantage over a competitor who does not have the information

Apply for federal registration

Life of the author plus 70 years

Right to prevent unauthorized copying or public performance

Keep secret; no registration available

Unlimited duration as long as the subject matter is secret

Right to prevent unlawful use

Trade secrets

Term of Protection

Rights of the Owner

Sources: Modified from Dodds, J. et al., Plants, germplasm, Genebanks, and intellectual property: Principles, options, and management, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices, MIHR (Oxford, U.K.) and PIPRA (Davis, CA), 2007; Dodds, J. and Krattiger, A., The statutory toolbox: An introduction, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices, MIHR (Oxford, U.K.) and PIPRA (Davis, CA), 2007.

the methods embodied in the invention such as plant transformation and selection methods. Utility patents may protect different aspects of the invention. For instance, Calgene Inc., now Monsanto Company, used a series of claims in the family of utility patents to protect the first horticultural biotechnology crop, the FlavrSavr™ tomato that was genetically engineered to suppress the expression of the tomato fruit ripening polygalacturonase enzyme. Using a portfolio of patents, the company protected the DNA sequence of the enzyme, a DNA construct to suppress the enzyme expression in plants, the transgenic tomato cell, and even, the overall method for antisense-based gene suppression of this or any other gene. In the United States, plant and utility patents are awarded by the United States Patent and Trademark Office (USPTO). A more widely employed international legal system to award rights to breeders was created in Europe through The International Union for the Protection of New Varieties of Plants, UPOV, commonly referred to as Plant Breeder’s Rights (PBRs).1,3 The original international convention that established UPOV was held in 1961, and

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the treaty has been revised in 1972, 1978, and more recently in 1991. UPOV provides the international standardized framework in which each national government can establish their own legal system. As a member of UPOV, the U.S. Plant Variety Protection Act offers protection of sexually propagated or tuber-propagated plant species. In the United States, PBRs are granted through Plant Variety Protection (PVP) Certificates issued through the U.S. Department of Agriculture. UPOV requires that a plant variety meet a set of criteria known as DUS requirements; distinct, uniform, and stable. UPOV provides a PVP-like system, which can offer robust protection worldwide. The horticultural industry can use a combination of plant patents, PVP, and PRBs to protect and market its varieties in most parts of the world. For example, the University of California relies on UPOV and U.S. plant patents to manage a successful Strawberry Licensing Program in domestic and international markets. The Strawberry Licensing Program has expanded its international markets in countries with strong IP protection and the UC strawberry cultivars now represent up to 60% of the worldwide production.4 Trade secrets are one of the oldest forms of IP protection and are used to protect confidential business information, processes, know-how, data, and materials.5 However, a trade secret does not protect against competitors reverse engineering a product. In the horticultural and seed industries, trade secrets have been widely used to protect parent inbred lines for hybrid seed production but are clearly not useful for the protection of a variety that can be directly propagated asexually or with open-pollinated seeds. Where appropriate, however, trade secrets can be maintained indefinitely so long as confidentiality is not breached. Another form of IP protection of limited utility in horticulture is a copyright. A copyright protects the expressed idea, plant variety description or diagrams, or other material that may be displayed in catalogs or seed packages. This form of IP protection is weaker than others because anyone is free to use the actual plant or depicted idea. Also, anyone is free to use the copyright information for the development of a product. Copyright use in plant sciences could apply to plant genome information in electronic databases and bioinformatics software and hardware. For example, the use of information in electronic databases can be limited or controlled by granting access to databases through a copyright license or an information access agreement. The International Center for Tropical Agriculture (CIAT), for example, limits the ability to claim intellectual property protection over any data or information in its databases through an information access agreement (http://isa.ciat.cgiar. org/urg/ita.do;jsessionid=9FEEC2C59FAB82D4EABF5052083238A3). Trademarks are another form of IP protection applicable to brand names of horticultural products; however, as with copyright, it does not protect the technology itself. In the United States, trademarks are registered at the USPTO. A searchable database of trademark registrations is available on the USPTO website at the Trademark Electronic Search System (http://tess2.uspto.gov). For horticultural crops, trademarks are often used to protect any word, phrase, symbol, or logo that represents brand varieties or products such as Dole® (a registered trademark of Dole Fruit Company), Sunkist™, and Monsanto Company’s Roundup Ready® products.

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Tangible property rights reside with the property owner. MTAs are typically used to transfer possession of materials but not ownership. The party who transfers the materials retains full ownership, and the transfer is governed by a contract, typically specifying the term of the transfer, how the materials may and may not be used, and other related issues, such as confidentiality. MTAs are legal contracts and are also referred to as bailments, which is the legal term for a contract that transfers physical possession but not ownership of personal property. In research, MTAs are often used for transfer of materials for research use only, allowing scientist to perform academic research. However, there is typically no guarantee or promise that the owner will give rights for commercial applications. Also, the time period and geographical limitations can be established by the property owner, and, thus, unlike patents, limitations on the use of tangible property typically have no  temporal or geographic limitations. Research-use only MTAs are typically easier to obtain, especially between public or nonprofit research institutions, but could represent a considerable obstacle downstream if the materials cannot be used for commercial applications.

FREEDOM TO OPERATE Navigating the complex IP landscape of a research project in horticultural biotechnology requires some analytical tools and specialized analytical capabilities.6 The analysis requires both legal and scientific knowledge as well as access to both patent and literature databases and typically takes the form of what is known as a freedom to operate (FTO) opinion. The FTO opinion is a legal assessment about whether a research project or the development of a new product can proceed with a low, or tolerable, likelihood that it will not infringe existing patents or other types of IP rights. It is important to note that the FTO determination is not absolute but reflects an evaluation of risk since there is typically some uncertainty around the interpretation of patent claims as well as uncertainty as to whether new IP may issue or be discovered at a later date. The FTO opinion may lead to a range of options: identifying in-licensing targets, considering the substitution of technologies or processes, deciding to ignore the potential infringement, investing in work-around technologies, or perhaps deciding to abandon the project all together. Although, private firms are more likely to engage in FTO analysis because any infringement risk may directly affect their ability to develop new products, and their ultimate profitability, public, and not-for-profit private institutions are becoming increasingly aware of the need for better FTO information. This is particularly true for research projects undertaken by universities or not-for-profit research centers for the specific purpose of producing new crops for developing countries. In these cases where deployment is anticipated, it is critical that IP considerations be taken into account early in the research process to avoid the need for re-engineering due to IP constraints. Similarly, horticulturists should address IP needs to ensure that commercialization is not halted due to a lack of IP clearance. While patents are the most common type of IP right encountered, a thorough FTO analysis needs to assess all types of existing property rights for the likelihood that the research project or the product being commercialized infringes terms of an MTA.

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Of particular concern are tangible property rights, such as cell lines, transgenic plants, germplasm, and plasmids. Because, as described above, the transfer of tangible property often occurs under the terms of an MTA that has no geographic or temporal limitation. These terms can be particularly problematic and directly impact FTO. Enabling technologies for plant transformation or transformation vectors combine several components such as promoters, selectable markers, marker removal systems, and more. Because of the fundamental role that these technologies play, they have been extensively patented. In addition, the FTO surrounding enabling technologies related to plant transformation is further complicated because these technologies are not used individually but are combined with a suite of related enabling technologies, specific trait technologies, and deployed in many different plant species. We can look at a relatively simple example of a single component of a transformation vector to illustrate the elements of an FTO analysis. The target technology for this case study was a fruit-specific promoter from the tomato E8 gene. The E8 promoter has been used to improve fruit quality, extend fruit shelf life, and to express edible human vaccines specifically in ripening tomato fruit.7,8 The first step in an FTO investigation is to clearly define the target technology. In this hypothetical case, the fruit-specific promoter will be used exactly as described in the initial publications.9,10 The promoters in these publications are virtually identical and consist of about 2100 nucleotides upstream of the E8 structural gene. Further promoter characterization identifying the location and sequence of functional elements within the promoter and upstream nucleotide sequence was reported in Deikman et al.9 These publications draw the technical boundaries surrounding the target promoter technology and provide important prior art to subsequently filed patents. To establish the relationship of publications and patents that describe or claim the E8 promoter, a patent landscape must first be established. The patent landscape should include patents and patent applications closely related to the technology. Keywords and authors of key publications are used to search for patents or patent applications. A separate search should then be conducted to identify patents or patent applications, which referenced the scientific publications describing the technology. Additionally, in the E8 case, patented DNA and protein sequence databanks were searched using the promoter’s DNA sequence as a query. The patent landscape will reveal “family” relationships among different patents and published patent applications. Patent families include later patent applications that claim the benefit of an earlier, related, application, or later patent applications that arise from foreign filings of the parent application. Figure 10.1 illustrates a patent family arising from a 1989 patent application related to the E8 promoter filed by Agritope, an agricultural biotechnology company. An informative way of analyzing the FTO search results is to construct a timeline of scientific literature, patent applications, and issued patents on the specific technology and on potentially overlapping subject matter. Ordering the patents and published applications according to their priority dates (also known as effective filing dates) reveals important relationships. For example, it reveals what publications or patents are prior art against newer patents. Since patents may only be granted if the claims are both novel and nonobvious over the prior art, this analysis reveals the relative dominance of earlier, broader patents over later, narrower patents.

U.S. patent USSN 5,750,864 Published 05/12/1998

U.S. patent 6,054,635 Filed 04/25/2000

U.S. application USSN 777,147 Filed 12/27/1996

CIP

U.S. application USSN 331,355 Filed 10/27/1994

CIP

CIP

U.S. application USSN 046,583 Filed 04/09/1993

CIP

CIP

U.S. patent 5,589,623 Filed 12/31/1996

U.S. application USSN 360,974 Filed 12/20/1994

U.S. application USSN 255,833 Filed 06/08/1994

U.S. patent 5,416,250 Published 05/06/1995

FIGURE 10.1â•… The epitope/agritope patent family6 displays the numerous United States (U.S.) and World (WO) patent filings stemming from a 1989 U.S. patent application. The patent family includes a series of continuation in part (CIP) applications filed in the United States that claim new aspects of the invention while retaining the parent application filing date.

U.S. patent 5,859,330 Published 01/12/1999

WO application 95/35387 Filed 10/27/1994

U.S. application USSN 261,677 Filed 06/17/1994

U.S. patent 5,723,746 Published 03/03/1998

WO application 94/24294 Filed 04/08/1994

U.S. application USSN 613,858 Filed 12/12/1990

CIP

U.S. application USSN 448,095 Filed 12/12/1989

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Literature timeline Deikman and Fischer (1988) Deikman et al. (1992) Giovannoni et al. (1989) 1985

1990

Agritope 448,095 12/12/1989 Patent and patent application timeline

Agritope 613,858 12/12/1990 Monsanto 632,440 12/26/1990

1995 Epitope 046,583 04/09/1993 U.S. 5,723,746 WO 94/24294 Agritope 255,833 06/08/1994 U.S. 5,416,250

Agritope 360,974 12/20/1994 U.S. 5,589,623 Epitope 10/27/1994 U.S. 5,859,330 Epitope 261,677 06/17/1994 U.S. 5,750,864

FIGURE 10.2â•… Timeline of tomato E8 promoter scientific publications and United States (U.S.) and World (WO) patent filing. (From Fenton, G. et al., eds., Freedom to operate: the law firms approach and role, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices, eds. A. Krattiger et al. 2007, available online at www.ipHandbook.org)

Figure 10.2 illustrates the IP priority timeline for the E8 promoter. A thorough FTO analysis may require direct contact with the researchers, and, in this analysis, it was learned from the authors of the Deikman and Fischer publication10 that they did not apply for patent protection prior to their publication. This information was also confirmed by searching patent databases. Based on this information, it was presumed that the basic E8 promoter technology was in the public domain based on the publications, which constituted a public disclosure. However, this conclusion required thorough review and documentation of the published literature or “prior art” relative to the subject matter of subsequent patents. As shown in the priority timeline, the Deikman and Fischer10 and Giovannoni et  al.11 publications initially describe the E8 promoter technology. This precluded the novelty of any subsequent patent claims on the E8 promoter per se (for example, applications filed by Agritope and Epitope). Counsel concluded that the tomato E8 promoter constructs per se and as described in the scientific publications can be reasonably considered to be in the public domain. However, patent issues subsequently may limit the use of the E8 promoter to drive specific heterologous genes or to use in combination with other transcription regulatory elements (see Figure 10.2). For example, Epitope’s patent U.S. 5,859,330 contains very narrow claims to the use of the E8 promoter with a specific gene, S-adenosylmethionine hydrolase. Therefore, this specific E8 promoter-gene combination would fall under the scope of the patent.

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Similarly, other patents may limit the use of the E8 promoter in chimeric constructs that contain other promoter elements. For example, another of Agritope’s patent, U.S. 6,118,049, claims the use of the tomato E8 promoter fused to a portion of the tomato E4 promoter. These examples illustrate the complexities in analyzing the FTO of particular DNA elements. We exemplify that FTO may differ when you consider a DNA element by itself (i.e., promoters) or in combination with other elements (i.e., trait genes). This example provides an overview of the data and information that should be considered in an FTO analysis. It is not difficult to imagine how the complexity of an FTO analysis would grow dramatically with the inclusion of multiple enabling technologies, one or more trait technologies and proprietary germplasm. This is one of the challenges of understanding IP constraints and developing FTO strategies in plant biotechnology where multiple complementary technologies are necessarily integrated to develop new crop varieties.

STRATEGIES FOR IP ACCESS During the 1990s, it became clear that universities and nonprofit agricultural research institutions were constrained in their historical role of providing many new agricultural innovations—particularly biotechnology-derived innovations—directly to the agricultural industry or to farmers. This was especially apparent for specialty or horticultural crops that do not individually occupy the large acreage of agronomic crops but as a group represent a high-value agricultural sector. While there are many contributing factors, access to the suite of proprietary technologies required to produce a genetically modified crop has been frequently cited as a barrier to the commercialization of public sector agricultural research.12 In Europe, a similar picture emerged in 2000, when a Swiss scientist developed “Golden Rice,” genetically modified rice with elevated provitamin A,13 which triggered an intellectual property audit. The audit revealed that 70 proprietary technologies had been infringed in the development of Golden Rice and illustrated the complex patent thicket that surrounded biotechnology innovations for crop improvement.14 The complex IP environment surrounding agricultural biotechnology research and development, exemplified by the Golden Rice case or even the relatively simple FTO opinion described above, has spawned some new strategies and new organizations committed to lower the IP barriers to new crop developments and to provide more open access to patented technologies. These issues are critical for small private companies attempting to enter this sector but can also be important for public or notfor-profit research institutions. Most plant biotechnology laboratories routinely use patented technologies in their research without specific legal permissions. Although patent owners have rarely been concerned about academic research infringement in agriculture, there are many examples where fundamental biomedical research has been challenged because of IP issues.15 In addition, projects carried out in public or not-for-profit institutions that are targeted toward the development of crops for developing country farmers must consider the IP inputs to the project. Increasingly, the sponsors of such research are requiring the full analysis and disclosure of strategies to ensure global access to all IP embodied in a project.

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To develop a strategy for IP access, it is important to consider both the IP and TP rights. Most researchers are still relatively unfamiliar with how to find, understand, and utilize IP information, including published patents and patent applications. Therefore, it is important for scientists to seek legal expertise to understand the FTO and IPRs of the components and processes used to develop agricultural biotech products. In addition, management of other TP agreements (like MTAs) is crucial. Keeping track of contractual obligations specified in sponsor research agreements, employment contracts, and MTAs are important to address the development of a project because they may be critically important in the downstream deployment of a biotechnology product. For example, agreements may define who holds patent rights, whether a technology can be commercialized or transferred to third parties, or if the license is for “research-use only.” Therefore, understanding these complex IP and TP issues are critical in order to develop strategies for IP access. Scientists worldwide are realizing the increasing need to address IP and TP issues in order to deploy their biotechnology discoveries. In the private sector, the corporate culture prioritizes preemptive mitigation of IP and TP issues, and, as a consequence, biotechnology companies have robust legal resources. In contrast, public sector scientists usually do not have access to the legal information necessary to address these issues. In 2003, several public sector and not-for-profit agricultural research institutions recognized the need to develop an organization to address the relative inaccessibility of IP information and to provide a framework to ensure that IP does not block applications of agricultural biotechnology and, in particular, to facilitate projects that can have broad humanitarian benefits.16 Numerous universities, including the University of California, the Donald Danforth Plant Science Center, North Carolina State University, Ohio State University, Boyce Thompson Institute for Plant Research, Michigan State University, Cornell University, University of WisconsinMadison, University of Florida, the United States Department of Agriculture, Rutgers University, Texas A&M University, and Purdue University developed the Public Sector Intellectual Property Resource for Agriculture (PIPRA; www.pipra. org). These institutions made a public commitment to participate and promote strategies to collectively manage public-sector intellectual property in support of both U.S. and developing country agriculture. This initial founding group of PIPRA members has grown to over 53 institutional members in 15 countries, illustrating the widespread concern and interest in working collectively to remove and avoid IP barriers that might slow development of new crops. PIPRA operates on several levels to effectively support the broad application of agricultural technologies developed in public-non/profit research institutions. Its primary strategy has been to adopt a highly collaborative program built on consensus views of its members—all of whom fundamentally believe that intellectual property protection is an important tool to support innovation. PIPRA is working within the context of its members to support both commercial and humanitarian applications of technologies and to develop strategies and mechanisms to stimulate even more innovation globally. A number of strategies have been implemented to enhance FTO using publicsector IP for agricultural biotechnology projects. For example, informed decisions regarding dissemination of new knowledge via open publication or protecting it with

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a patent are clearly important and FTO can be improved if public-sector institutions systematically consider how, when, and if to use the patent system to support broad innovation.17 Even when using the patent system, PIPRA encourages its members to reserve rights to use their newest and best technologies for humanitarian purposes, particularly when they issue exclusive commercial licenses.18 For U.S. agriculture, it is also important to use licensing strategies that promote commercialization of technologies for both large and small acreage crops. Thus, for technologies with potential applications in many crops, instead of granting an exclusive license for all fields and in all crops, it may be prudent to license for a specific field-of-use or specific crop and retain rights to use the technology, or to issue additional licenses, for the development of other crops that may not be within the commercial interests of large companies. The anticipated benefits of a collective IP management regime are to enable an effective assessment of FTO issues to overcome the fragmentation of public-sector IPR and re-establish the necessary FTO in agricultural biotechnology for the public good and to enhance private sector interactions by more efficiently identifying collective commercial licensing opportunities. Among PIPRA’s core activities is working with scientists and funding agencies on a project-specific basis to understand the underlying FTO, IP, and TP necessary to develop and deploy agricultural biotechnology projects. Based on this information, PIPRA works with scientists and technology managers to design and implement strategies to access and negotiate third party rights. PIPRA offers services to develop patent landscapes in particular technology spaces, for example, gene suppression in plant biotechnology.19 Delmer et al. described other initiatives designed to play roles in enabling access to IPRs for the development of crops, including horticultural crops, the African Agricultural Technology Foundation, and CAMBIA.20 The African Agricultural Technology Foundation focuses on negotiating access to proprietary technologies for the benefit of smallholder farmers in Sub-Saharan Africa (www.aatf-africa.org).21 CAMBIA offers a number of resources and initiatives to promote innovation (www.cambia.org). In particular, foundations with an interest in supporting broad innovation as well as corporations and corporate foundations whose future depends on the broad acceptance of biotechnology in both agronomic and horticultural crops are likely to be important sponsors of activities to enable access to IPRs.

REFERENCES



1. Kesan, J.P., The statutory toolbox: Plants, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices 2007, MIHR (Oxford, U.K.) and PIPRA (Davis, CA). 2. Dodds, J., A. Krattiger, and S.P. Kowalski, Plants, germplasm, Genebanks, and intellectual property: Principles, options, and management, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices 2007, MIHR (Oxford, U.K.) and PIPRA (Davis, CA). 3. Lesser, W.H., Plant breeders’ rights: An introduction, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices 2007, MIHR (Oxford, U.K.), and PIPRA (Davis, CA).

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4. Bennett, A.B. and M. Carriere, The University of California’s strawberry licensing program, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices 2007, MIHR (Oxford, U.K.) and PIPRA (Davis, CA). 5. Dodds, J. and A. Krattiger, The statutory toolbox: An introduction, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices 2007, MIHR (Oxford, U.K.) and PIPRA (Davis, CA). 6. Fenton, G., C. Chi-Ham, and S. Boettiger, eds. Freedom to operate: The law firms approach and role, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices, eds. A. Krattiger et al., 2007, available online at www.ipHandbook.org (accessed January 28, 2011). 7. Sandhu, J.S. et al., Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Transgenic Res., 9(2), 127, 2000. 8. Sheehy, R.E., M. Kramer, and W.R. Hiatt, Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc. Natl. Acad. Sci. USA, 85(23), 8805, 1988. 9. Deikman, J., R. Kline, and R.L. Fischer, Organization of ripening and ethylene regulatory regions in a fruit-specific promoter from tomato (Lycopersicon esculentum). Plant Physiol., 100, 2013, 1992. 10. Deikman, J. and R.L. Fischer, Interaction of a DNA binding factor with the 5′-flanking region of an ethylene-responsive fruit ripening gene from tomato. EMBO J., 7(11), 3315, 1988. 11. Giovannoni, J.J. et al., Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell, 1(1), 53, 1989. 12. Graff, G.D. et al., Accessing intellectual property for biotechnological development of horticultural crops. Calif. Agric., 58, 122, 2003. 13. Ye, X. et al., Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science, 287(5451), 303, 2000. 14. Kryder, R.D., S.P. Kowalski, and A.F. Krattiger, The intellectual and technical property components of pro-vitamin a rice (GoldenRice™): A preliminary freedom-to-operate review. ISAAA Briefs, 20, pp. 1–56, 2002. 15. Marshall, E., Intellectual property. DuPont ups ante on use of Harvard’s OncoMouse. Science, 296(5571), 1212, 2002. 16. Atkinson, R.C. et al., Intellectual property rights. Public sector collaboration for agricultural IP management. Science, 301(5630), 174, 2003. 17. Boettiger, S. and C. Chi-Ham, eds. Defensive publishing and the public domain, in Intellectual Property Management in Health and Agricultural Innovation: A  Handbook of Best Practices, eds. A. Krattiger et al., 2007, available online at www.ipHandbook.org (accessed January 28, 2011). 18. Bennett, A.B., Reservation of rights for humanitarian use, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices 2007, MIHR (Oxford, U.K.) and PIPRA (Davis, CA). 19. Chi-Ham, C.L., K.L. Clark, and A.B. Bennett, The intellectual property landscape for gene suppression technologies in plants. Nat. Biotechnol., 28(1), 32, 2010. 20. Delmer, D.P. et al., Intellectual property resources for international development in agriculture. Plant Physiol., 133(4), 1666, 2003. 21. Boadi, R. and M. Bokanga, eds. The African agricultural technology foundation approach to IP management, in Intellectual Property Management in Health and Agricultural Innovation: A Handbook of Best Practices, eds. A. Krattiger et al., 2007, available online at www.ipHandbook.org (accessed January 28, 2011).

11

Structuring University– Private Partnerships for Developing and Commercializing Transgenic Horticultural Crops Gordon Rausser and Reid Stevens

CONTENTS Introduction............................................................................................................. 233 Operational Framework.......................................................................................... 235 Stage 1: Setting the Bargaining Space and Negotiating the Contract................ 235 Stage 2: Decision Making through Bargaining.................................................. 238 Stage 3: Is There a Shock?................................................................................. 238 General Guidelines.................................................................................................. 239 PPPs and Public Good Research........................................................................ 239 Two Leading University–Private R&D Partnerships......................................... 241 PPPs in Horticultural Research...............................................................................244 References...............................................................................................................246

INTRODUCTION Horticultural research is conducted primarily in the public sector, with private research institutions playing a relatively minor role. As a result, research gaps emerge between the basic research generated by public institutions and the applied research needs of industry. This gap can be reduced by forming public–private partnerships (PPPs) that allow academic researchers access to commercialization technologies (such as gene expression profiles and genome maps) and give private firms access to new research and innovation. However, significant obstacles hinder the formation of successful research partnerships. Both parties in a partnership face substantial risks. These risks are rooted in the conflict between a university’s academic objectives and the private 233

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firm’s corporate incentives. One critical risk is the potential co-opting of the academic research agenda by private interests. University researchers risk the loss of academic freedom and integrity while industry risks the loss of investment capital, privacy, and proprietary information. Differences between the university’s educational objectives and the private sector’s goals, as well as differences in the cultures, institutional incentives, and time frames, can lead to a clash of values. Intellectual property (IP) rights issues are also a frequent source of contention. Given these risks, partnerships must be based on carefully structured contracts to protect the interests of the private and public partners. Though there has been much discussion of public–private research partnerships in both the popular press and academic community, there is no consensus on the optimal contract structure for these partnerships. We have developed a three-stage framework for evaluating PPP contracts. This framework is unique because it takes into account the type of economic good the partnership produces as well as the incomplete nature of contracts that deal with sponsored research. Partnerships in horticultural research deal with the management and provision of impure public goods.* The optimal structure of a PPP depends on the degree of impurity, and, if a PPP produces a good that is impure to some extent, its contract should differ from that of a PPP that produces a purely private good. The contracts that govern these partnerships and assign ownership of the impure goods they produce are incomplete in the sense that there is a set of events that can influence the partnership, which cannot be enumerated in the contract. These events could be unanticipated research opportunities that arise during the research process or could be an unexpected discovery that falls outside of the contract’s scope. An optimal contract will take into account the uncertainty inherent in the research process and assign control in the case of unanticipated events in a manner that does not pervert the partner’s incentives. Contracts for PPPs in horticultural research and their associated control and property rights come in many forms, ranging from large, multiproject, multiyear alliances to small-scale projects. Our three-stage framework to analyze these contracts is based on the contractual assignment of control in the case of unanticipated events. In Stage 1, the public and private partners negotiate to determine the allocation of the front-end control rights and the back-end property rights.† The front-end control rights determine the nature and scope of the research activities that the partnership will undertake as well as decision-making authority over those activities while back-end property rights determine how any benefits generated by the research will be distributed among the partners. The partners also make relationship-specific investments according to the contract in Stage 1. In Stage 2, the partners bargain over management decisions with bargaining power determined by the contract rights * Impure public goods are goods that are either nonrival or nonexcludable. A nonrival good can be consumed by one person without preventing simultaneous consumption by others (e.g., intellectual property). A good is nonexcludable if it is not possible to prevent anyone from consuming the good (e.g., a lighthouse). † In this chapter, we will use control rights to refer to the authority to make decisions during the research process (the “front-end”) and property rights to refer to ownership of the research produced by the partnership (the “back-end”).

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and investments made in Stage 1. The outcome of this bargaining is a decision that lies between the noncooperative bargaining solution (where each partner unilaterally exercises their control rights) and the cooperative bargaining solution (where the partners maximize their joint benefit). In Stage 3, there may be an unanticipated “shock.” For our purposes, a shock is an event that affects the partnership over which there are no explicit contract provisions. Such a shock may cause the players to revisit their control and property rights. Depending on the nature of the shock, the partners may engage in renegotiation that reassigns the control and property rights (and begin again at Stage 1) or they may conclude the partnership. The remainder of the chapter is organized as follows: in the “Operational framework” section, we present the three-stage framework for evaluating public–private research contracts; in the “General guidelines” section, general guidelines for university–private partnerships in research and development (R&D) are provided; and in the “Public– private partnerships in horticultural research” section, we visit the implications of our framework for PPPs in horticultural research and more specifically for the development and commercialization of transgenic horticultural crops.

OPERATIONAL FRAMEWORK There is a considerable body of literature related to PPP theory, and the reader interested in developing a greater depth of knowledge in this area is directed to these selected citations.1–14 In this section, we describe an operational framework that can be used as a conceptual lens to analyze PPP contracts in horticultural research that includes the terms and conditions used to assign front-end control rights and back-end property rights. The following three-stage framework provides a lens that will allow us to evaluate PPP contracts.

Stage 1: Setting the Bargaining Space and Negotiating the Contract In Stage 1, the partners negotiate a contract and make investments. The public institution should begin this process with a self-assessment to identify their primary objectives in seeking out private partners, their strengths and assets, and the desired complementarities. This assessment is essential to form an efficient partnership. Though the order in which partnership negotiations proceed is not of much consequence, it is vital for the public institution to be deliberately early in the process, when seemingly innocuous decisions ex-ante may severely limit its control or flexibility at crucial junctures ex-post. At each point in a relationship, it is important for the public institution to consider the long-term consequences of all relationshiprelated decisions. Consider the importance of the public institution’s method for finding and selecting a partner. Often firms seek government contracts and make specific offers, leaving public institutions the passive role of waiting to be approached and evaluating one partnership at a time. Alternatively, the public institution can take a proactive role. Given the results of the self-assessment, the public institution can seek out wellmatched partners that complement their strengths. Although deliberately seeking out

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partners, rather than waiting to be approached with a proposal, requires more effort up front, it gives the public institution the greatest degree of control over the selection of partners, which implicitly define the control that the public institution will have over the remainder of the structuring process. In addition, by actively approaching potential partners in the private sector, a public institution can form a consortium with a group of specialized partners if that better suits the public institution’s objective. This active approach can substantially broaden the public institution’s choice set. Likewise, a proactive approach on the part of a private firm can increase their control in the bargaining process. Once a partner is selected, the public and private institutions engage in negotiations that result in a contract that allocates to each of the partners a share of the front-end control rights and back-end property rights. The front-end control rights enumerate the resources committed by both partners and give the partners decisionmaking power over the partnership’s R&D processes, and the back-end property rights specify the manner in which the partnership’s assets will be distributed at the conclusion of the partnership. At the end of this stage, the partners invest in the partnership according to the contract. In this negotiation, each partner works to minimize its share of input while making sure the combined resources will be sufficient for a successful joint effort. The commitment of resources in the front-end is fairly transparent; however, the implications of choosing particular governing structures for the partnership are less transparent. Given the diversity of assets, it is difficult for potential partners to balance their respective asset contributions. These assets can be tangible, as with financial assets or equipment, or intangible “knowledge” assets.15 Unlike tangible assets, the value of intangible assets is not easily defined as it relies on many factors such as the nature of the assets and the degree of complementarity. Knowledge assets may be tacit (e.g., know-how) or codifiable. Tacit knowledge draws on skills and techniques and is transferred by demonstration, apprenticeships, personal instruction, and provision of expert services. Codifiable knowledge can be reduced to messages and is easily transferred. Knowledge assets can assume the form of a nonrivaled or inexhaustible good. In other words, sharing this information will not reduce the amount available to others. Although nonrivaled, these assets are not necessarily public goods since it may be possible to exclude access by others. This is certainly true of patents, copyrights, and trade secrets. To be sure, exclusive control over a specified set of knowledge assets creates rents for its holders. A further, important distinction is between generic and specialized assets.16,17 Generic assets are useful for most research, such as basic scientific knowledge concerning biotechnology research. In contrast, specialized assets are suited to a narrow set of specific applications that can be more easily commercialized. In the area of plant R&D, these assets can be further characterized as input(e.g., herbicide-resistant transgenic seed) or output-trait assets (e.g., high-nutrient content seeds). Identifying these aspects of the partner’s assets is important to create complementarities among the different assets held by the public and private partners, and when negotiating over the contributions each partner will make to the relationship. Private institutions are likely to have more access to funding, state-of-the-art scientific tools,

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experience in commercialization, and marketing expertise. Public institutions, in return, offer preferential access to academic resources and assistance in navigating bureaucracies. The objective of the contract is to combine each partner’s assets in the most productive combinations. The governance structure of the partnership must be determined in the first stage. Fundamentally, it defines each partner’s front-end control rights and back-end property rights. This assignment of control will determine how the partners will interact, make decisions, resolve conflicts, and terminate the agreement if necessary. The most important consequence of the governance structure is that it determines how the project will be evaluated, and the governance structure specifies how the agreement will be administered and if the scope will be changed (i.e., the agreement extended or terminated). At the conclusion of the partnership, the back-end options in the agreement determine how benefits are disseminated and the process for establishing ownership. Each of these issues is crucial in determining how both the pecuniary and nonpecuniary benefits of the project are shared by the partners and by the public. In the case of partnerships that involve research, licensing options are important when the PPP generates a new, marketable product. Currently, it is common for the industry partner to be given a first-to-negotiate licensing option for some subset of the innovations generated under the partnership. Generally, these options must be exercised within a specified time period, or else the option is extended to third parties. In response to public outcry over poorly structured agreements and concern about blocking patents, right-of-first-refusal options evolved into right-tonegotiate options. If the industry partner is granted the more limited option of rightto-negotiate, a public institution should have greater control over licensing rights and can prevent blocking patents from being awarded. Other aspects of licensing agreements receive less attention but are also critical. One such aspect is the percentage of the total innovation for which the industry partner holds an option to negotiate an exclusive license or access option. For example, in some partnerships, the private partner can exercise this option for an “allowable percentage” of patents, equal to the percentage of the funding that came from private partner. Under other agreements, the industry partner holds this option for all patented discoveries generated by the agreement. Third-party options are also a critical aspect of licenses options. These options are the rights that parties outside the agreement have to innovation generated by the agreement. In some agreements, third parties hold open options on patents not included in the allowable percentage and on patented innovations either covered by nonexclusive license, or for which the first-to-negotiate option has expired for private partner. In this case, the private partner has no recourse once their licensing option has expired, and the public partner is free to enter licensing negotiations with third parties. In contrast, other agreements give third parties only a conditional option. The private partner has right-of-first refusal on any licensing arrangement between the public institution and third parties, even if their original licensing option has expired. Thus, private partner is guaranteed an option of first refusal on any third party offers made to the public partner. This severely limits the options available to third parties.

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Stage 2: Decision Making through Bargaining In Stage 2, the partners jointly manage the partnership by making decisions based on a two-person, two-phase bargaining game.* In the first phase, the public partner and the private partner decide what threats to invoke if no agreement is reached, where the threat strategies are chosen to maximize their pay-off while minimizing effort and are based on the control rights and property rights assigned in Stage 1. These threat strategies determine the disagreement point and are taken as given in the second phase. These strategies need not actually be carried out and may not even be explicit. All that is required is the potential of threat. In this stage, the partners will achieve an efficient outcome, in which the partner with control rights implements a decision, and a pay-off is exchanged between the partners. The partner holding the control rights is aware of the noncontrolling partner’s reaction function and unilaterally selects an action that maximizes the controlling partner’s objective function given the noncontrolling partner’s reaction pattern.13 The reaction function allows the noncontrolling partner to influence controlling partner’s decision. Beyond their choice of threat strategy, each partner exercises control over the resources dedicated to the relationship through its choices in Stage 1. For example, if a public institution is approached by a single firm and considers only their offer, it has a very limited choice set and is likely to have little leverage over that firm’s resources. In contrast, if a public institution considers multiple offers from partners with varied assets, its choice set is broader.

Stage 3: Is There a Shock? In the final stage of the agreement (Stage 3), the partners deal with unanticipated shocks. When there is a shock, the partners have two options: (1) they can conclude the partnership and exercise their back-end property rights over the partnership’s property, or (2) they can renegotiate the control rights and property rights assigned and begin again at Stage 1. If the partners choose to renegotiate, the allocation of bargaining power in renegotiation may be different from the allocation in the previous stages. By this stage, a partner might find themselves in a more vulnerable position due to the nature of the shock or relationship-specific investments. This potential for changes in relative bargaining power could lead to a reassignment of control rights. After Stage 3, the partners assess the outcome of their partnership and consider whether to renew the agreement. Although many public institutions have developed policies to evaluate partnerships, there is no generally accepted method for formal review of partnerships with private institutions. These methods rely mainly on anecdotal feedback from involved personnel to measure the merits of specified projects and to monitor unintended consequences. The informal reviews and vague impressions of both partners are coupled with more tangible outcomes, such as the project’s revenue, in assessing whether a partnership was successful or not. * See Rausser et al.18 for an extension of this analysis to a multi-person bargaining game.

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A key policy issue is developing concrete measures of PPP productivity. Much of the literature on PPPs focuses on developing proper incentives for behavior covered within the scope of an individual agreement, but little consideration is given to incentives that fall outside a specific agreement. Because many of these agreements are up for renewal once completed, there are incentives for the public institution to make sure that the private partner is satisfied with the outcome of the agreement, and, under increasing financial pressure, this may affect behavior within a current agreement. In other words, these agreements are not necessarily one-shot games, but might be a single round of a repeated game. As such, there are incentives for the public institution to develop a certain reputation so that the private partner will support a renewed relationship. This speaks to one of the primary concerns with these agreements that public institutions will fail to look for funding from other sources, will become dependent on renewing these agreements, and will therefore lose their ability to walk away from negotiations, and, as a result, will sacrifice much of their bargaining power. If recognized, these issues may be addressed by choosing a partner with which there is strong incentive alignment as well as safeguards in the agreement.

GENERAL GUIDELINES In this section, we will apply our operational framework to structure general guidelines and evaluate the control rights for two leading university–private partnerships in R&D. These case studies provide insights into structuring horticulture university– private partnerships.

PPPs and Public Good Research As public funding of scientific research has declined, and knowledge inputs have played an increasingly important role in industrial processes, universities and other public research institutions have looked to private sources to increase their research budgets. Many lessons have been learned as public criticism and scrutiny of these research partnerships have evolved.19 Issues such as conflict of academic and industry interests, ownership of, and access to, IP (e.g., issues of hold-up and blocking patents), and publication delays have fueled the current debate and often present insurmountable obstacles to forming research partnerships. A host of external forces have shaped the current environment in which public researchers are seeking to actively engage with private firms. Among these are diminishing federal and state funds for public good research and increased state funding incentives for private–public research. In addition, legislation (e.g., the Bayh–Dole Act), the restructuring of many large life sciences firms, and an alignment of private and public research incentives have contributed to this trend.20 Moreover, the traditional research paradigm, which presumes that there is a oneway flow from basic science conducted in public institutions to applied research and commercialization undertaken by private industry, has begun to be replaced by a chaotic R&D feedback loop paradigm.13,20 Increasingly, public universities and

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private companies are engaging in joint research, establishing relationships with exchange and collaboration in all stages of research. The potential benefits from university–industry partnerships are clear. CompleÂ� mentarities between scientific and practical knowledge have the capacity to generate rapid and far-reaching innovation. Under the best of circumstances, each partner is seeking attributes and assets in prospective partners that complement their own abilities and resources. Industry is interested in combining its knowledge of markets with information on new research and innovation in order to identify those developments that are likely to lead to commercial applications. This motivation may be obvious, but industry is also interested in more subtle assets such as access to academic expertise, networks, and first-hand information about up-and-coming scientists (current graduate students). And while universities are very clearly interested in financial capital, they are also seeking intellectual capital, cutting-edge research technologies,21 proprietary research tools (e.g., databases), and in many instances enabling IP.22 Access to these research assets enhances a university’s ability to provide first-rate education to its graduate students. Although the potential benefits of research partnerships are reasonably transparent, the potential risks to both parties are opaque. These risks pose serious obstacles to the successful formation of public–private research partnerships. In addition to the uncertainty inherent in any research process, the differences between university educational objectives and corporate goals are an important source of risk in these relationships. Recent data show that almost 70% of research in universities has been categorized by the National Science Foundation as basic, while the proportion is reversed in industry. In 2000, while universities accounted for only 14% of total R&D funding in the United States, they performed about 50% of the total basic research.23 With private financing comes the concern that the traditional orientation of the academic research agenda toward basic, public goods research will be directed toward more applied, appropriable research that serves the objectives of the private partner, and that this, in turn, will result in a loss of academic integrity. Not only research direction but research results from sponsored studies might be biased toward sponsors’ interests. Bekelman et al., for instance, show that in biomedical research there is a statistically significant association between industry sponsorship and proindustry conclusions.24 Industrial sponsors may also impose constraints on communication between grantees and other colleagues, which, in turn, may hinder research progress and increase research costs.23 Planning horizons tend to differ; university researchers focus on long-term research while companies often seek quick turn-around projects. In addition, the cultures and values of research partners may simply clash, creating insurmountable blocks to a continuing relationship. Furthermore, the incentives to secure a renewal or extension of an existing contractual agreement may adversely influence university scientist behavior under a current collaboration. Rights to IP are especially contentious. Hold-up and background rights are of primary concern to an industry partner interested in commercializing the products of a research partnership. Researchers at universities and other public institutions often use proprietary or enabling IP research tools in their research without obtaining rights. They are sometimes blocked, however, for any research discoveries that have

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commercial value. Generally, one researcher in a university institution may freely access another researcher’s patented research tool for academic study. This opportunity does not typically extend to private researchers unless a formal agreement is forged. Thus, a private company looking to partner with a particular researcher, for example, may experience hold-up at the commercialization stage because the public research partner did not obtain formal rights to all research inputs (i.e., background rights) from some other private company. Note also that if numerous university researchers and graduate students are involved in a research project, industry risks loss of privacy, and protection for proprietary information. The interests of parties outside a research agreement (i.e., third parties) are also at risk under public–private research agreements. If an agreement is not effectively structured with regard to patenting and licensing rights, a third-party interest in having access to research products and innovations may not be adequately represented. In fact, blocking patents can and do arise.22 In summary, the cooperation between universities and industry opens a series of questions. Does the profit-driven sponsor shift the university’s mission away from basic research? Does industry’s desire to exploit IP rights interfere with communication within and between universities to an extent harmful to open science?23 These conflicts are an inevitable consequence of a fundamental clash between a public system that encourages openness in science and an industrial system that gives financial rewards based on secrecy. In the end, this all boils down to one question: can a university–industry partnership be socially beneficial or, more precisely, Pareto improving?* Scotchmer23 argues that a public–private venture is justifiable for big science projects. For certain large projects, the public sector may face the problem of choosing the right investments (those with high probability of success) and making sure the funds are used as intended; the private sector, on the other hand, has the expertise needed to screen likely successful projects but sometimes cannot reap unappropriable social benefits, thus is unable to recover the cost of research. In this situation, a PPP can help solve the duality problem. If the partners do not have access to the same information, however, industry may manipulate the public sector into subsidizing its’ privately profitable projects.

Two Leading University–Private R&D Partnerships The University of California formed a partnership with the Novartis Agricultural Discovery Institute, Inc. (NADI) in 1998 that allowed Berkeley to retain control of an open research agenda. Initially, an open call is put out to participating faculty for research proposals with neither the University nor NADI defining the type of project proposals that would be considered. Further, the committee that allocates funding to each project (all proposed projects receive some amount of funding) is made up of three Berkeley University faculty members and two members representing NADI. In addition, the criteria used for ranking project include the quality and intellectual * A partnership is Pareto improving if at least one partner is made better off by joining and no partner is made worse off by joining.

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merit of the proposed research, potential advancement of discovery, and the past and present productivity of the research. As a result, the direct value of any potential projects to NADI is not considered. An alternative structure governs a biological research agreement between Washington University (WU) in St. Louis and the agricultural biotechnology company, Monsanto. An advisory committee solicits proposals that conform to research areas specified by the committee. In this case, the committee is equally split with three university members and three representatives from Monsanto. Therefore, the interests of Monsanto have more weight, both in defining the choice set of research proposals that will be considered by the committee and in selecting which of those proposals are funded. Moreover, the agreement specifically directs the committee to identify and fund projects that not only have exceptional academic merit but also serve the research interests of Monsanto. Depending on the mission of the university and its role in the community, both of these alternative governance structures have merits. Because the research interests of the private partner carry more weight, the WU/Monsanto agreement may be more likely to generate innovations that result in commercial applications, meeting the objective of serving the community with successful technology transfers. On the other hand, the Berkeley/NADI agreement more adequately protects the academic freedom of participating faculty. What is important is that the public institutions make conscious decisions about where they are comfortable on this spectrum of control over the research agenda and that they are fully aware of the implicit trade-offs contained in the related contract language. The primary interest of universities is to share their research results with colleagues as rapidly as possible, through publications and presentations at conferences, with the hope that scientific knowledge and research will be advanced. This academic mission conflicts with the interest of the private partner to appropriate innovation and technological advancements, thus requiring, for a certain amount of time, that research results are kept from competing interests until the private partner establishes rights to the innovation. As such, the publication delay provisions of research agreements usually come under considerable scrutiny. In fact, guidelines issued by the National Institutes of Health recommend a delay of 30–60 days.25 A more relevant question concerns control of the option for terminating the delay period rather than the specified maximum length of this period. For example, under the Berkeley/NADI agreement, NADI has an initial 30 day delay during which they must decide whether an innovation has the potential to be patented. If they decide that the parties should proceed with a patent application, publications can be delayed only up until the time the patent application is filed or 90 days—whichever is shorter. And Berkeley has right to file the patent application at any time. The filing process can be expedited, with an initial application filed in a day or so. Therefore, under this agreement, although the maximum publication delay is 120 days, Berkeley has complete control to end the delay (past the initial 30 day period). In contrast, in the WU/Monsanto agreement, important issues concerning patenting of innovations that are generated from the partnership include who is responsible for filing for the patents, the right not to file for a patent, control of patent litigation, the right to “know-how” transfer, and ownership of the core technology. Who holds

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the option to file a patent application is important not only because of control over the publication delay period but also because the content of the application defines who is responsible for generating the innovation as well as the nature of the innovation. Both of these parameters can be defined broadly or more conservatively; this will have significant implications for who controls the licensing rights and how strong these rights turn out to be. In some instances, the university partner is responsible for filing the patent applications, but the degree to which the private partner provides guidance and is involved in patent litigation is varied. A more subtle issue is whether a university is obligated to file for a patent if requested to do so by the industry partner, or whether it has some discretion. A university partner may wish to avoid expending the effort required to patent innovations if it does not foresee that it will be applied commercially. For example, under the Berkeley/NADI agreement, Berkeley can elect not to file for a patent that NADI does not intend to commercialize. In other words, Berkeley can make sure that innovation, or know-how, that would not otherwise be commercialized remain freely available to the public and that limited administrative resources are not diverted to pursue meaningless patents. Of vital importance for industry–university research agreements is the nature of the licensing options. Currently, it is common for the industry partner to be given a first-to-negotiate licensing option for some subset of the innovations generated under the research agreement. Generally, these options must be exercised within a specified time period, or else the option is extended to third parties. In response to public outcry concerning previous, poorly structured agreements, such as the Sandoz/Scripps agreement,* and concern about blocking patents, right-of-first-refusal options evolved into right-to-negotiate options. In theory, if the industry partner is granted the more limited option of right-to-negotiate, a university has greater control over licensing rights and can prevent blocking patents from being awarded. Other aspects of licensing agreements receive less attention but are also critical. One such aspect is the percentage of the total innovation for which the industry partner holds an option to negotiate an exclusive license or access option. For example, under the Berkeley/ NADI agreement, NADI can exercise this option for an “allowable percentage” of patents, equal to the percentage of the research funding that came from NADI; therefore, NADI has limited access options. Under other agreements, the industry partner holds this option for all patented discoveries generated by the agreement. Third-party options are also a critical aspect of licenses options. These options are the rights that parties outside the agreement have to innovation generated by the agreement. Under the Berkeley/NADI agreement, third parties hold open options on patents not included in the allowable percentage, and on patented innovations either covered by nonexclusive license or for which the first-to-negotiate option has expired for NADI. NADI has no recourse once their licensing option has expired, and Berkeley is free to * In 1993, the publicly funded Scripps Research Institute agreed to form a research partnership with Sandoz Pharmaceuticals. Sandoz agreed to provide $300 million in funding for research over 10 years in return for a worldwide license for all of discoveries made by researchers at Scripps. This controversial agreement, which would have given Sandoz licensing rights to nearly $1 billion of research funded by the federal government, was restructured after the government threatened to cut off funding for the institute.

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enter licensing negotiations with third parties. In contrast, under the WU/Monsanto agreement, third parties hold only a conditional option. Monsanto has right-of-firstrefusal on any licensing arrangement between the university and third parties, even if Monsanto’s original licensing option has expired. Thus, Monsanto is guaranteed an option of first refusal on any third party offers made to WU. This severely limits the options available to third parties and ill serves the interests of the university.

PPPs IN HORTICULTURAL RESEARCH The horticultural research industry is composed primarily of small- to medium-sized enterprises with small markets for individual products.26 Even though these firms are relatively small, they have demonstrated a capacity to apply new discoveries for possible commercialization. However, when it comes to genetically engineered crops, these small firms generally do not have the assets to develop new products. As a result, research funds in horticulture are sourced primarily from the public sector.26,27 The reluctance of major biotechnology R&D companies to dedicate funds to horticultural research is, in part, because technological advances in horticulture are not viewed as “low-hanging fruit.” The commercial value is not nearly as attractive as that of annual agronomic crops grown on large acreages. In addition, consumer acceptance of genetically modified foods is considered a major obstacle to the adoption and commercialization of agricultural biotechnology. These constraints on the application of modern biology to the market for horticultural crops are the most important impediment to the formation of public–private research partnerships. Hence, public institutions might be best served by forming partnerships with private associations or consortia (i.e., Western Fruit Growers’ Association) rather than single firms. Such partnerships are more likely to “crowd-in” in contrast to “crowd-out” public good research at land-grant universities.13 Groups of horticultural firms have expressed interest in funding research for crop and seed improvement in response to the recent global food shortages.27 These firms, attracted by the research expertise of university faculty, have begun working with universities to develop effective new technologies. Some firms have indicated that partnerships with universities have the added advantage of improving the image of genetically modified crops by allowing for increased public exposure to the benefits of their work. Though many firms have expressed interest and some partnerships have been formed, there is potential for many more research partnerships. Public research institutions have much to gain by forming partnerships with consortia composed of small private firms, nurseries, or grower’s associations. Currently, these groups sponsor relatively little horticultural research in the United States.28 A model similar to Australia’s check-off funding organizations* could be used to increase private sector sponsorship of public agricultural research institutions. One check-off organization, Horticulture Australia Limited (HAL), invests over $80 million annually in more than 1200 research partnerships. All HAL members are required to donate a percentage, determined by an external council, of their revenue * A check-off program levies a small tax on the producers of a horticultural product and uses these funds to sponsor research at public institutions.

Structuring University–Private Partnerships for Commercialization Firms

Universities

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Public institutions

Commerical discoveries Nurseries Grower adoption Packaging and processing Wholesale Food service

Retail

Consumers

FIGURE 11.1â•… Sources of discovery and commercialization.

to HAL, which is distributed to a broad range of research programs in response to the horticulture industry’s current needs. The establishment of similar organizations in the U.S. horticulture industry (see Figure 11.1), with mandatory contributions, would dramatically increase funding for public good research. Domestic and international public–private research partnerships in horticulture are considered especially important for developing economies.29 Dixon26 notes that successful entrepreneurs in horticulture maintain a continuous dialogue with scientists; partnerships are one approach for guaranteeing this dialogue. Dixon also notes that linkages between research and industry (public and private relationships) have improved “where levy funding systems have been established to support scientific endeavors.” In other words, more formal financial arrangements between partners are likely to yield a superior exchange. The most relevant partnership model for the horticulture industry is that of less formal, single, or multiple-project partnerships (sponsored project and informal arrangements). Private horticulture institutions should seek to align research incentives and form consortia of small and medium firms with parallel research interests to concentrate intellectual and financial resources. These consortia are organized by crop or pest type (or other research interests) to facilitate networking, identify key researchers at public institutions, and propose specific research projects. A proportional-contribution burden sharing scheme between consortia members is likely to be the most effective self-governing approach given the public nature of research leads and outcomes. The university should accept or reject these proposals based on the research synergy and embedded options. Although all universities share a common set of core principles that guide their decisions, different institutions emphasize different

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objectives; the private partner should consider the university’s research culture when considering research partners. Given the nature of research objectives at universities, the horticulture industry partner should propose research projects that are more basic, have longer time frames, and are not adequately addressed by current private research efforts. These partnerships are more likely to be successfully negotiated if the industry partner understands, ex ante, the need for academic freedom. On the back-end, university guidelines and policy usually constrain its researchers to specific conditions for patenting research and licensing and disseminating results (publication delays). Although there is some variation, these constraints are fairly common among research universities. Both partners should establish links, so that industry can effectively utilize public research, and universities can secure access to research funding and complimentary enabling technologies. These collaborations can serve as stepping stones to more formal, long-term agreements. Alternatively, once initial consortiauniversity research partnerships are established, more enterprising members of the consortia can capitalize on the research contacts and pursue firm-specific, appliedresearch partnerships. The primary obstacle to forming these research partnerships is high transaction costs. The process of identifying appropriate researchers as potential partners can involve significant search costs. And once the potential partners have been selected, the time and effort involved in negotiating a research agreement, especially given the differing objectives of public versus private institutions, can be substantial. The consortium approach is a strategy for sharing these costs. If the consortia are not well structured, however, reduced external transaction costs may face higher internal costs of organizing and maintaining the consortia. Inequitable benefits sharing within a consortium may also be a source of conflict. And although this approach is intended to serve the needs of medium-tosmaller-sized firms, the smallest enterprises may still be excluded (especially in subsequent partnerships). Dramatic improvement in the development and commercialization of transgenic horticultural crops can come from increased involvement from the private sector. Though the potential benefits from university–industry partnerships in the field of agricultural biotechnology are obvious, the private sector will not increase their investment in horticultural R&D without active solicitation by the public sector. As soon as the public sector is able to form horticultural PPPs, whether they are composed of a single firm or a consortia, formal or informal, or in developing or industrialized nations, and demonstrate their value to the private sector, there will be fewer obstacles to forming PPPs in the future, and those partnerships are likely to have more favorable terms for the public sector.

REFERENCES

1. Aghion, P. and Bolton, P. An incomplete contracts approach to financial contracting, Rev. Econ. Stud., 59, 473, 1992. 2. Bennett, C. and Iossa, E. Delegation of contracting in the private provision of public services, Rev. Ind. Organ., 29, 75, 2006. 3. Besley, T. and Ghatak, M. Government versus private ownership of public goods, Q. J. Econ., 116, 1343, 2001.

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4. Engel, E., Fischer, R., and Galetovic, A. The basic public finance of public–private partnerships, Working Paper, 13284, NBER, 2007. 5. Francesconi, M. and Muthoo, A. Control rights in public–private partnerships, Discussion Paper, 2143, IZA, 2006. 6. Grossman, S. and Hart, O. The costs and benefits of ownership: A theory of vertical and lateral integration, J. Polit. Econ., 94, 691, 1986. 7. Hart, O. Firms, Contracts, and Financial Structure, Oxford, U.K.: Oxford University Press, 1995. 8. Hart, O. Incomplete contracts and public ownership: Remarks, and an application to public–private partnerships, Econ. J., 113, C69, 2003. 9. Hart, O. and Moore, J. Incomplete contracts and renegotiation, Econometrica, 56, 755, 1988. 10. Hart, O and Moore, J. Property rights and the nature of the firm, J. Polit. Econ., 98, 1119, 1990. 11. Hart, O., Shleifer, A., and Vishny, R. The proper scope of government: Theory and an application to prisons, Q. J. Econ., 112, 1126, 1997. 12. Martimort, D. and Pouyet, J. “Build it or not”: Normative and positive theories of public–private partnerships, Discussion Paper, 5610, CEPR, 2006. 13. Rausser, G., Simon, L., and Stevens, R. Public vs. private good research at land-grant universities, J. Agric. Food Ind. Organ. 6, artic. 4, 2008. 14. Wang, C. Public investment policy and industry incentives in life science research, PhD thesis, Oregon State University, Corvallis, OR, 2007. 15. Rausser, G., Ameden, H., and Simon, L. Public–private alliances in biotechnology: Can they narrow the knowledge gaps between rich and poor? Food Policy, 25, 499, 2000. 16. Teece, D. Profiting from technological innovation: Implications for integration, collaboration, licensing, and public policy, Res. Policy, 15, 285, 1986. 17. Vonortas, N. Cooperative Research in R&D-Intensive Industries, Aldershot, U.K.: Avebury Academy, 1991. 18. Rausser, G., Swinnen, J., and Zusman, P. 2010. Political Power and Endogenous Policy Formation, Cambridge, U.K.: Cambridge University Press (in press). 19. Press, E. and Washburn, J. The kept university, Atl. Mon., 285, 39, 2000. 20. Rausser, G. Private/public research: Knowledge assets and future scenarios, Am. J. Agric. Econ., 81, 1011, 1999. 21. Blumenstyk, G. Temple University shuts down for-profit distance-education company, Chron. High. Educ., 47, 29, 2001. 22. Heller, M. and Eisenberg, R. Can patents deter innovation? The anticommons in biomedical research, Science, 280, 698, 1998. 23. Scotchmer, S. Innovation and Incentives, Cambridge, MA: MIT Press, 2004. 24. Bekelman, J., Gross, C., and Li, Y. Scope and impact of financial conflicts of interest in biomedical research, JAMA, 289, 454, 2003. 25. National Institutes of Health. NIH Guidelines for Research Involving Recombinant DNA Molecules, Washington, DC: Department of Health Human Service, 1994. 26. Dixon, G. Market-led horticultural research: Does this provide what the industry needs? World Conference on Horticultural Research, Rome, Italy, 1998. http://www.agrsci. unibo.it/wchr/wc3/dixon.html (accessed July 9, 2010). 27. American Seed Research Foundation. Strategic research, education, and policy goals for seed and crop improvement, American Seed Research Summit, 2008. 28. Alston, J. and Pardey, P. Making science pay: The economics of agricultural R&D policy. AEI Studies in Agricultural Policy, Washington, DC: The AEI Press, 1996. 29. Robitaille, H. Needs and expectations of the horticulture-related industry. World Conference on Horticultural Research, Rome, Italy, 1998. http://www.agrsci.unibo.it/ wchr/wc3/robitail.html (accessed July 9, 2010).

12

Why Are Regulatory Requirements a Major Impediment to Genetic Engineering of Horticultural Crops? Steven H. Strauss

CONTENTS Need for Biotechnology Scientists to Be Informed and to Inform Regulations..... 250 Consequences of Failure to Comply with Regulations Are Large.......................... 252 A Multitude of Regulations Exist at National and International Levels................. 253 Regulatory Compliance Is Especially Problematic for Horticultural Crops........... 253 Deregulated Horticultural Varieties Do Not Provide General Models.................... 254 Causes of Our Stringent Regulatory System.......................................................... 254 Presumption of Harm from Transgenic Methods............................................... 255 Environmental Concerns Prompt Strong Regulations....................................... 256 Familiar Genes Meet Same Regulatory Review................................................ 256 Environmental Studies Are Seriously Compromised by Regulations.................... 258 Presumption That Stasis Is Desirable..................................................................... 258 Consequences of Simple Definitions of Clean and Green...................................... 259 Conclusions.............................................................................................................260 References...............................................................................................................260 Regulations in the United States and in most other countries treat all plants produced using recombinant DNA methods (genetic engineering or genetic modification) as illegal for use in the environment or in commercial products until their safety and acceptability has been specifically authorized. The costs of complying with regulations and the legal risks of not complying place severe constraints on the use of recombinant DNA breeding methods at both research and commercial phases. In particular, the limitations to gene release in the environment pose severe constraints for required field research, development, and commercial applications for most horticultural crops, a problem that is exacerbated in many cases by their incomplete

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domestication and wild or feral relatives. This chapter explores the direct and indirect causes for the stringent regulatory system in place, discusses the opportunity costs they impose, and proposes some alternative regulatory concepts. I maintain that until regulatory systems incorporate a tier that provides, at the outset of field research, exemptions or workable tolerances for adventitious presence, the ability to use transgenic approaches for horticultural breeding will be severely limited, thus foreclosing a number of important options for improving pest management, stress tolerance, and product quality. It is common to see lay discussions of the social controversies and the potential of genetic engineering (GE)* virtually ignore the federal regulatory gauntlet that GE products must get through. Those who tend to be in favor of GE crop solutions often assume that regulations are well-crafted and essential to protect the public safety; their efficacy, cost, and what products might have been discouraged even before they are created are rarely considered. In contrast, those against GE argue that regulations are not strong enough, as evidenced by the very existence of GE products with the absence of full scientific certainty about their effects. Because of the esoteric nature of regulations, it often seems to be only the practitioners of GE who really understand the implications of regulations in practice. Who else would know what it costs in time and labor to conduct a regulated field trial apart from those conducting the trials? Or of what it costs to bring a product to market, other than public sector institutions or companies that have sought to do so? The goal of this chapter is to discuss the costs and impediments to research and development of transgenic horticultural crops from the perspective of a public sector biotechnologist who works on ornamental and forest trees. In addition to my own experience, this article is motivated by the apparent absence of any new horticultural transgenic crops in the public sector pipeline (an observation based on discussions with many colleagues) in spite of a rather large number of field trials that have been conducted during the past two decades (see http://www.isb.vt.edu/cfdocs/fieldtests1.cfm for listings). This suggests that regulatory costs and obstacles, in combination with market risks, are severely impeding transgenic variety development.

NEED FOR BIOTECHNOLOGY SCIENTISTS TO BE INFORMED AND TO INFORM REGULATIONS Few scientists or students who are drawn to plant science or to its practical applications such as horticulture and forestry like the idea of studying government regulations. It sounds about as exciting as reading the United States Internal Revenue tax code, and about as enticing as a trip to the dentist to have your teeth drilled. As a scientist, I fully share these sentiments, but my work over the years with field trials of genetically modified trees1,2 (Figure 12.1), and the small part I have played in writing * Throughout this chapter, which specifically addresses the products of genetic engineering or genetic modification, I use the terms “biotechnology” or “GE” or “GM” as shorthand. I am referring to crops produced using methods where plants are modified by asexually induced, specific genetic modification and regeneration of the modified cells into plants.

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FIGURE 12.1â•… (See color insert.) USDA-APHIS authorized field trial of transgenic poplars in Oregon (United States) during its first (top) and second (bottom) growing seasons. The population, part of a gene discovery program using a method called “activation tagging” (where genes are randomly upregulated by insertion of a gene expression enhancer), was being screened for novel morphologies under field conditions. The trees had to be removed prior to the desired long-term nature of this experiment because of regulatory costs associated with long-term containment, monitoring, reporting, and removal costs for large trees. There is no obvious scientific basis for intensively regulating such trees while interspecies hybrid poplar trees, and those produced through non-transgenic forms of mutagenesis, are essentially unregulated throughout the world.

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about regulatory reforms3,4 and taking part in national and international workshops designed to inform or influence regulations,5 have shown me how important they are. I now often argue that every plant scientist who works in and understands the potential benefits of transgenic plant biotechnology needs to understand regulations, and play a role in improving them. The goal is to craft regulations that more effectively target and limit very high risk applications, while minimizing encumbrances to field research on safe and highly valuable applications. At least for now, regulations and their implementation are still evolving, providing an opportunity for influence from scientists.6 In addition, all applications for permits and petitions for deregulation (USDA) and registration of GE pest-tolerant plants (EPA) have required periods of open public comment; the high quality science-based or data-based input that is often received from biotechnology scientists is valued by regulatory agencies.

CONSEQUENCES OF FAILURE TO COMPLY WITH REGULATIONS ARE LARGE Regulations are informed by science, but mainly they embody the overall “attitude” of a society about a technology.7 Emotions, perceptions, economics, and politics generally dwarf the influence of science in developing regulatory policies. Regulations can be written with a tone of aversion and extreme caution when society senses risk and harm rather than direct benefit—as we see today with plant biotechnology. Or, they can be written with a sense of optimism and encouragement, as we tend to see today with respect to wind power and related technologies. Because regulations have the force of law behind them, even minor violations can have significant penalties including heavy fines and even imprisonment. Thus, they have a power and gravity very different from research procedures or recommendations, such as those followed in molecular biology laboratories in the United States under the National Institute of Health (NIH) recombinant DNA research guidelines. The risks and costs of complying with regulations—or being unable to comply—often determine, not just inform, scientific and business strategies. As seen with the StarLink GE maize debacle8 and with the ongoing multimillion dollar lawsuits over accidental infusion of USDA-approved GE rice that harmed U.S. exports,9 the consequences of getting the regulations about gene dispersal (often called adventitious presence [AP] or low-level presence [LLP]) wrong, even in small detail, can be enormous for companies and for the entire agricultural sector. In addition, recent successful lawsuits over USDA decisions on herbicide resistant sugar beets, alfalfa, and creeping bentgrass have set new precedents for use of the National Environmental Protection Act. Its requirement for Environmental Impact Statements in regulatory decisions on crop biotechnologies has brought the courts into the regulation of crop biotechnology in a major way10 that requires far more work and legal detail to the process in order to increase the likelihood that Animal and Plant Health Inspection Service (APHIS) decisions can withstand legal challenges. In addition, given the broad interpretation of what National Environmental Policy Act (NEPA) covers in these cases—which include economic damages to organic and conventional farmers from AP—it is unclear whether the preparation of Environmental Impact Statement (EISs) will improve the quality of scientific analysis of the underlying biological issues.

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A MULTITUDE OF REGULATIONS EXIST AT NATIONAL AND INTERNATIONAL LEVELS The problems with AP and consequent trade disruptions described above also remind us that we have not one, but a multitude of national regulatory regimes that can vary widely by country, as well as an overarching international regulatory policy in the Cartagena Protocol of the Convention on Biological Diversity (CBD).11 The CBD’s provisions must be addressed if living agricultural products such as seeds are traded, or if pollen, seed or vegetative propagules can move across country boundaries. Such rules are critical for trade in many horticultural crops, where the products are often living (e.g., nuts, fruits, horticultural varieties), and where wind and insect vectors often can move pollen, fruits, and sometimes small seeds many kilometers. This network of regulations means that making changes to regulations is truly a glacial process; it involves seeking coordinated changes in the attitudes of highly diverse societies, as well as through fractious and highly political bureaucracies such as the United Nations. Given the negative attitude inherent in most regulatory regimes concerning crop biotechnology, it is not hyperbole to state that the regulatory challenges facing horticultural biotechnology are both global and monumental.

REGULATORY COMPLIANCE IS ESPECIALLY PROBLEMATIC FOR HORTICULTURAL CROPS For most horticultural crops,* the implications of the stringent regulatory system are even more grave than for field crops. This is because the high regulatory costs12 per gene insertion event tend to be spread over a smaller variety base, with a smaller economic return, and with a longer time for the return to be manifest. This results because these crops are far more diverse in their genetics and geography, transferring approved biotech traits into new varieties through breeding is slow due to a longer generation time and biological limits to inbreeding, and because valuable genotypes tend to be cloned rather than sexually propagated. Thus, it is expected that individual transgenic events from elite clones, not progeny from deregulated or registered events, will each require separate regulatory dossiers and decisions.3 Moreover, these crops as a category tend to be less domesticated and thus can more readily mate with wild or feral relatives, and spread directly via seed or vegetative propagation in wild or feral environments. Because of their large size and potential for wide pollen or seed dispersal by wind, insect, or animal vectors, containment when plants are old enough to flower and are producing fruit can be very difficult, costly, and often impossible to assure. This creates a situation where gathering needed regulatory data on environmental effects, under the very strong confinement mandated by regulations, poses a kind of “Catch-22” situation (i.e., where the required information, at a high level of scientific rigor and ecological relevance, is nearly impossible to obtain while assuring full containment). Even if the data could be obtained, the required depth of analyses (e.g., of nontarget * Throughout this chapter my focus is on woody fruit, shade, and ornamental horticulture species.

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effects, fitness, potential for spread, effect on endangered species) is very costly and by their nature imprecise, requiring, for reasonable estimates, large experiments and years of study over many environments. For pest-tolerant crops (i.e., those with plant incorporated protectants, [PIPs]), the required analyses by EPA are expected to be even more costly and complex. Rarely are the paybacks to developers from improved horticultural crops sufficient to cover all of these large up-front costs.

DEREGULATED HORTICULTURAL VARIETIES DO NOT PROVIDE GENERAL MODELS The very few woody horticultural crops that appear to have successfully navigated the regulatory maze have special characteristics, and thus provide few general lessons. They are trees that have genes that protect against a major viral pest and make no actual novel pest-toxic compound (papaya and plum: they invoke the natural RNA interference mechanism), and also cannot spread in the wild to any significant degree. The GE cold-tolerant and male-sterile eucalypt, now in extensive field trials and part of a petition for deregulation, is also dependent for its approval to allow flowering and commercial planting on its presumed sterility or inability to spread.13 It is as yet unclear if, in a practical and affordable way, normally fertile horticultural varieties that have wild or feral relatives can comply with regulations and obtain regulatory approval for commercialization.

CAUSES OF OUR STRINGENT REGULATORY SYSTEM How have we, in the Unites States, produced a regulatory environment that appears so hostile to transgenic innovation in horticultural crops? The political and legal history of our regulatory framework is well known,6,14 and there are a number of very significant political issues15 that appear to have played a major role in shaping the negative, or at least highly divided, public view of crop biotechnology. Major sources of controversy include:





1. The relatively new and major roles for strong patents in crop breeding, which provide no breeder’s rights to the use of genetic material and no limits on ownership of genes and transgenic plants when they move in the environment. This appears to be considered an overstep or an outright ethical transgression to many. 2. The growing role of multinational corporations in biotechnology. This is in no small part due to the costly intellectual patent and regulatory landscapes discussed above. Negative attitudes toward these corporations and their dominance in the development of commercial biotech crops are also a result of the legacy of the production and marketing of pesticides, and of divisive products such as recombinant bovine somatotropin (rBST), by these companies or their predecessors. 3. The lack of direct benefits to consumers and food production/service companies, in the face of perceived risks to people or retail chains, from use of herbicides and pesticidal molecules in the current major transgenic varieties.

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4. Divided scientific advice on the risks versus benefits of GE in relation to the stringency of regulations. The large majority of ecologists I have met with, including during services on National Research Council panels, show a strong negative attitude toward GE of crops, whereas most breeders, agronomists, and biotechnologists seem to view them positively. The concern expressed by ecologists is prompted in no small part by the commonly made analogy between transgenic and invasive exotic species. Moreover, many serious invasive plant species are the result of intentional introductions from the horticulture industry. Thus, although there is only limited biological homology between a novel invasive organism and introduction or modification of one or a few genes in a familiar organism, the legacy of exotic species problems creates a climate that dictates extreme precaution and concern. 5. The growing popularity of organically certified forms of agriculture and its strong direct and indirect campaigns against transgenic breeding methods and varieties as dangerous and “unnatural.” 6. Waning trust in government and government organized science panels to make wise judgments about the safety of novel genes in foods and environment. 7. The strong political and legal pressures for stringent regulations from wellfunded nongovernmental organizations that are opposed to, or highly concerned about, GE crops.

All of these are clearly major problems for any efforts to produce what GE crop developers would view as more balanced science-based regulations. However, I will discuss what I see as deeper, more foundational issues that I believe have contributed to making the regulatory system such a difficult barrier to progress in horticultural biotechnology.

Presumption of Harm from Transgenic Methods Thomas Jefferson is widely quoted as having said that “the greatest service which can be rendered any country is to add a useful plant to its culture.”* Clearly, something has changed since the era of transgenic biotechnology began. Whereas all products of traditional breeding are considered generally regarded as safe (GRAS), all varieties produced using transgenic methods are in effect considered the opposite, that is, hazardous until “proven” safe.† This is despite the common scientific knowledge, and FDA rulings, that the transgenic method per se is not more risky than conventional breeding methods such as inbreeding, wide hybridization, and mutagenesis. Moreover, the established legacy of plant breeding includes importations of exotic plants that can spread widely; enabling agriculture and humans—arguably the * Thomas Jefferson, “A Memorandum of Services to My Country,” September 2, 1800 (PTJ, 32:124). Polygraph copy at the Library of Congress. http://wiki.monticello.org/mediawiki/index.php/ Useful_plant_%28Quotation%29 † It is not in fact possible to prove the absence of any risk.

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most environmentally destructive forces on the planet—to migrate around the globe as plants have been and are bred for adaptation to new regions. Clearly, the distinct regulatory treatments, which impose such a striking double standard of strong regulation versus the absence of regulation, are a legacy of history. It is fair to say that if conventional breeding were forced to undergo the same scrutiny as does GE, much of it would not be legally permissible today. At a minimum, conventional breeding would all be subject to much higher costs and long delays, with inestimable penalties for yield and product quality improvement. It is also very likely that environmental impacts of agriculture would be far greater, as the amount of output per unit area of land, water, and fertilizer would certainly be far lower in the absence of vigorous plant breeding programs.

Environmental Concerns Prompt Strong Regulations The pressing environmental problems facing society are another motivation for strong regulations of this new agricultural technology. Whether one considers climate change, non-point-source pollution, soil erosion, or water quality, there is clearly a pressing need to reduce the environmental footprint of agriculture. However, is the intensive regulation of all forms of transgenic biotechnology, and only transgenic biotechnology among breeding methods, a sensible means for doing this? Such a practice seems especially specious in that the environmental benefits of transgenic crops have, on the whole, been strongly positive to date (primarily in the form of tillage and pesticide ecotoxicity reductions),16 yet many crops with similar expected benefits have not made it to market at all.12 Some of the most notable examples of transgenic crops that, though developed and field proven, have not made it to market, are horticultural crops. These include virus-resistant berries, disease-resistant apples, and disease- and insect-resistant potatoes. All of these would have reduced pesticide applications. Although business and market factors also contributed, sometimes substantially, to decisions not to commercialize such varieties, the overarching hostile regulatory environment made the business proposition marginal at the outset, especially for public sector breeders and smaller companies. It is not difficult to argue that the stringent regulation of plant biotechnology has had the opposite environmental consequence of what was intended.

Familiar Genes Meet Same Regulatory Review Unfortunately, the “guilty until proven innocent” framework applies not just to biotechnologies that impart novel properties, such as new kinds of pest resistance proteins or metabolites, but it applies to all cases where a transgenic method is used. Thus, it is the method, not the actual biological novelty of the new gene that triggers the regulatory system. As a result, we scrutinize all changes from the method, not just the novel property imparted, presuming all changes are hazardous until “proven” otherwise. This means that mutagenesis due to the gene insertion process is intensely scrutinized—though mutagenesis in various forms has been long applied in conventional breeding. The nature of the insertion site and any changes in general plant chemistry are studied in detail, not just transgene expression and

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its associated phenotypes. Regulatory agencies apply similar scrutiny even where genes from sexually compatible or closely related species are transferred, or normally expressed genes are attenuated, shut down, or mutated (often called cisgenics or intragenics). Incremental changes to existing phenotypes such as cold hardiness, reduced rate of ripening, and pest resistance—even when due to modified expression of native genes—are treated as ecologically novel traits if GE is involved. Canada has attempted to put in place a method-neutral regulatory system that covers GE as well as conventional breeding, called the “plants with novel traits” system (http:// www.inspection.gc.ca/english/sci/biotech/gen/terexpe.shtml). In practice, however, it appears to regulate all forms of GE crops similarly to method-based systems in the United States, while upsetting conventional breeders when their new varieties come under regulation for the first time. The intense scrutiny compelled by the GE method creates serious legal and epistemic problems. How can we prove safety when the variances for the system we regard as GRAS are so extraordinarily wide? Food is known to contain “toxins” and contaminants whose concentrations vary widely and can cause adverse effects in high dosage tests, and breeders often make crosses with wild relatives that have not been widely consumed for food and may even be poisonous. For example, if a modified crop has chemical components whose levels are elevated but are still within the enormous range of variation seen among conventional varieties, hybrids, and environments (e.g., of a natural alkaloid or terpenoid), such changes might not be considered safe or desirable from a toxicological viewpoint (i.e., in light of the known biochemical actions of those compounds). How such cases would fare under legal scrutiny in the EU where the Precautionary Principle prevails, or under legal challenge in the United States where FDA could declare such changes as adulteration if supported by toxicological science, is unclear. In addition, because it is logically impossible to prove the absence of a risk, it is very difficult to scientifically declare safety for the whole organism, especially for crops or where gene products that do not fit the standard toxicology model (i.e., where they have complex phenotypic changes, and thus simple dose–response tests performed in the laboratory are not meaningful). This has led to continued political debate over how safe is safe enough, including over whether “substantial equivalence” is a satisfactory regulatory attribute. This indiscriminant system also means that gene transfers from related species, such as the transfer of a pest resistance gene from a wild relative, faces the same regulatory system. Why should a gene introduced through hybridization from a wild relative, with its usual linkage drag, be considered less risky than the same gene isolated and introduced using GE methods and accompanied by a well-studied vector and associated sequences? In other words, why are they regulated at all, when the same or a similar result can be produced with conventional breeding, though with less precision? Although it seems likely that regulators will require less data for low novelty transfers compared to wide phylogenetic transfers or newly synthesized genes, just by entering the highly politicized regulatory arena—where agencies simply respond to each case as they come in the door—the costs, delays, and outcomes are unpredictable, and thus can result in costly delays or roadblocks. The unpredictability of the regulatory process is a very serious problem for companies, investors,

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and grant agencies choosing among research and technology transfer options. They have little idea what the cost and time delays will really be, and agencies provide no guarantees up front.

ENVIRONMENTAL STUDIES ARE SERIOUSLY COMPROMISED BY REGULATIONS From an environmental viewpoint, the presumption of harm creates even larger problems and regulatory obstacles. As discussed above, it is very difficult to predict ecological impact from small studies that are performed under containment. Although simple extrapolations are possible when the toxicology model applies (as with a pest toxin whose effects on wild species can be roughly estimated in the greenhouse or short-term field study), even this simple case is fraught with difficulty. Such studies say little about the effect of such genes under varying abiotic and biotic environments in the field, and they cannot predict in any meaningful way what might happen in a future dominated by climate change, nor can assess how biological communities will adapt and evolve in response to the new gene product and phenotype. In other words, under current regulatory constraints we are unable to adequately answer any of the big questions about transgene impacts. For example, how will the myriad species that might be exposed to a naturalized transgene-expressing plant be affected over time? Can the novel gene/toxin have so strong an effect as to drive an herbivorous species to extinction, or will most species, or other ecosystem adjustments, attenuate such effects over evolutionary time? Do the perturbations matter given the very large effects of agriculture, breeding, climate-induced variation, anthropogenic change, and exotic species in general? How often will genes of value in the management of simple agricultural systems, or as a result of crop domestication for human tastes in food and fiber, be ecologically powerful in diverse wild or feral systems? The point is that while the goal of regulations is to force informed and wise decisions, the reality is that the process imposed, with its high costs and legal risks, appears to do more harm than good by impeding most forms of transgenic research and development with horticultural crops. A  more efficient option might be to exempt the transgenic method and small or contained field trials from regulation, but require substantially novel gene products—such as phylogenetically novel and broadly effective toxins, or pharmacologically active molecules that result from synthetic biology or long distance phylogenetic transfers—to undergo regulatory review prior to large scale, uncontained field research or commercial use. We have provided more specific recommendations for regulatory reform elsewhere.3,4,14

PRESUMPTION THAT STASIS IS DESIRABLE The USDA regulations for transgenic biotechnology treat all transgenic innovations as risks. The benefits of transgenic plants are not formally considered. This framework is not surprising given the evolution of the current regulatory scheme from a plant pest oriented system.17 The framework therefore implicitly assumes that crop species and their wild relatives that might receive transgenes via gene flow

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are superior in their present form to what they would be with the modified genes— unless a strong case can be made otherwise. This presumption (and the underlying conservation-oriented value that supports it) seems reasonable, until one considers the very strong barrier it also poses to the transgenic use of pest or stress resistance genes to promote the health of horticultural woody plants in cases where they have wild or feral relatives (as nearly all do). If the gene disperses, the genetic diversity and fitness of wild relatives might be increased to some degree. This might in fact be beneficial because woody horticultural and forest species are often foundational members of terrestrial ecosystems, providing much of the structural habitat and primary productivity. Thus, some increased vigor and adaptability would generally be expected to be ecologically advantageous, not disadvantageous. In addition, many woody species are under serious threat from climate change and the emergence or invasion of newly epidemic and/or exotic pests,18 and thus could benefit from genes that increased their resilience or pest/stress tolerance. Of course, in cases where a wild relative is already a problematic exotic species that is having a strong negative environmental impact, such improvements of vigor would not be considered desirable. Such cases could be specially identified and disallowed (e.g., by presence on a noxious weed list), rather than imposing a blanket preclusion to gene flow to wild relatives as a result of the transgenic method. The core regulatory and ecological problem is the extreme difficulty in predicting the outcome of transgene introductions in terms of their ultimate ecological impact in advance, without actual field releases and monitoring over many years and sites. This, however, is very costly, especially where strong containment must be imposed during these trials. As stated above, this is a reasonable requirement for species with high risk relatives such as a Johnson grass or a scotch broom, but unfortunately under the current operational “presumption of harm” such precaution is applied to all transgenes and species. This makes commercialization of each transgenic product a multidecade and multimillion dollar undertaking, even when pest resistance genes from related plant species are used, and appears to make transgenic solutions prohibitive except in special cases (e.g., American Chestnut, a dominant tree that was driven near to extinction and has strong private and public foundation support for the use of biotechnology for its restoration).19 Given the growing pace of such serious threats to wild and cultivated trees, it would appear that new, expedited regulatory options—such as exemptions for species in crisis and/or genes from related species—are critically needed.

CONSEQUENCES OF SIMPLE DEFINITIONS OF CLEAN AND GREEN Finally, a major impediment to the use of transgenic methods appears to reside in the blanket manner with which society seems to categorize technologies as good or bad. Organically certified food is currently considered by much of the public to be greener, safer, and thus superior to conventionally produced food. Yet when scrutinized it has not shown any consistent advantages for food safety or nutrition, and its net environmental benefits are also questionable (e.g., when full life cycle studies of nitrogen, land use, runoff, soil erosion, transport, and energy consumption— and even pesticide ecotoxicity in some cases—are considered).20–22 Nonetheless,

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perceived economic harms to marketing organic products as a result of “contamination” of organic food by GE, even when at very low levels, have prompted successful lawsuits. The courts have viewed organic agriculture as an environmental good, whose possible harm thus requires careful consideration via an environmental impact statement. Such a ruling for alfalfa has resulted in its withdrawal from the marketplace,23 and a similar case is pending for sugar beet. These legal precedents and the high costs they impose are likely to continue to slow, and in many cases will prevent development of GE crops. Unfortunately, these legal decisions appear to be informed by a popular, rather than a scientific, view of the relative environmental value of GE versus organically certified food. Simple green labels that presume GE is bad and any GE “contamination” of “green” products is bad—when uncritically accepted by courts and a large section of the public—pose considerable challenges to revision of the current regulatory system.

CONCLUSIONS Regulatory change that would decriminalize the GE process is needed to move forward. But how can that happen? It could be motivated by growing urgency for improved food production, as expanded uses of crops for bioenergy, and climate change-induced crop losses, continue to drive up food prices. Change may also be motivated by the many humanitarian GE projects underway for the developing world, of which Golden Rice is the best known. A single major, highly publicized success could shift public opinion substantially. Change may also be motivated by informed, popular, and powerful thought and environmental leaders, such as Stewart Brand and Michael Specter, who have embraced the benefits and debunked the myths surrounding GE crops and other environmental and scientific technologies.22,24 However, as discussed above, due to the many layers of national and global regulations, and the strong political influences on them, the timescale of change may be on the order of decades or more. For change to ultimately occur scientists must play a key role. By educating decision makers and the public in understandable, contextually relevant, and generationally appropriate forms, and by taking an active part in providing public input to regulatory decisions, biotechnologists can help to craft a new era of intelligent, discriminating, science-based regulations. Transgenic biotechnology is too powerful a tool to surrender. Our precarious world, the billions of needy people, and threatened nonhuman species need it to become a potent and central part of the crop technology toolkit.

REFERENCES

1. Li, J. et al., Stability of herbicide resistance over 8 years of coppice in field-grown, genetically engineered poplars. Western Journal of Applied Forestry, 23, 89, 2008. 2. Strauss, S.H. et al., Ten lessons from 15 years of transgenic Populus research. Forestry, 77, 455, 2004. 3. Bradford, K.J. et al., Regulating transgenic crops sensibly: Lessons from plant breeding, biotechnology and genomics. Nature Biotechnology, 23(4), 439, 2005.

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4. Strauss, S.H., Genetic technologies: Genomics, genetic engineering, and domestication of crops. Science, 300(5616), 61, 2003. 5. Strauss, S.H. et al., Strangled at birth? Forest biotech and the convention on biological diversity. Nature Biotechnology, 27, 519, 2009. 6. APHIS, Programmatic Environmental Impact Statement (for new proposed regulations). http://www.aphis.usda.gov/publications/biotechnology/content/printable_version/ fs_programmatic_eis.pdf, 2007. (Accessed January 27, 2011). 7. Devos, Y. et al., The interplay between societal concerns and the regulatory frame on GM crops in the European Union. Environmental Biosafety Research, 5(3), 127, 2006. 8. Bucchini, L. and L.R. Goldman, Starlink corn: A risk analysis. Environmental Health Perspectives, 110(1), 5, 2002. 9. Vinluan, F., Genetically modified rice leads to ruling against Bayer CropScience. Triangle Business Journal, December 7, 2009. http://www.bizjournals.com/triangle/ stories/2009/11/30/daily70.html (Accessed January 27, 2011). 10. Bryson, N., APHIS programmatic EIS–Implications for crop biotechnology regulation. In Farm Foundation Conference, Washington, DC, January 16–17, 2008. 11. Kinderlerer, J., The Cartagena protocol on biosafety. Collection Biosafety Reviews, 4, 12, 2008. 12. Kalaitzandonakes, N., J.M. Alston, and K.J. Bradford, Compliance costs for regulatory approval of new biotech crops. Nature Biotechnology, 25(5), 509, 2007. 13. APHIS, ArborGen, LLC; Availability of an environmental assessment for controlled release of a genetically engineered Eucalyptus Hybrid. 74 Federal Register, 74, 26648– 26719, June 3, 2009. 14. Strauss, S.H. et al., Far-reaching deleterious impacts of regulations on research and environmental studies of recombinant DNA-modified perennial biofuel crops in the USA. BioScience, 60(9), 729, 2010. 15. Herring, R.J., Opposition to transgenic technologies: Ideology, interests and collective action frames. Nature Reviews Genetics, 9(6), 458, 2008. (Accessed January 27, 2011). 16. NRC, Impact of genetically engineered crops on farm sustainability in the United States. http://www.nap.edu/catalog/12804.html, 2010, p. 240. (Accessed April 20, 2011). 17. Medley, T.L. and S.L. McCammon, Strategic regulations for safe development of transgenic plants. In Biotechnology, H.-J. Rehm and G. Reed, eds. Weinheim: Federal Republic of Germany, 1995. 18. Chornesky, E.A. et al., Science priorities for reducing the threat of invasive species to sustainable forestry. BioScience, 55(4), 335, 2005. 19. Merkle, S. et al., Restoration of threatened species: A noble cause for transgenic trees. Tree Genetics & Genomes, 3(2), 111, 2007. 20. DiGregori, T.R., Bountiful Harvest: Technology, Food Safety, and the Environment. Washington, DC: Cato Institute, 2002, p. 262. 21. McWilliams, J.E., Just Food: Where Locavores Get It Wrong and How We Can Truly Eat Responsibly. New York: Little, Brown & Company, 2009. 22. Specter, M. Denialism: How Irrational Scientific Thinking Hinders Scientific Progress, Harms the Planet, and Threatens Our Lives. New York: Penguin Press, 2009. 23. Peck, A.E., Plant biotechnology law after Geertson Seed Farms: Potential impacts on regulation, liability, and coexistence measures. National AgLaw Center, 2008. http://www.nationalaglawcenter.org/assets/articles/peck_aftergeertson.pdf (Accessed January 27, 2011). 24. Brand, S., Whole Earth Discipline: An Ecopgramatist Manifesto. New York: Viking, 2009.

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Virus-Resistant Transgenic Horticultural Crops: Safety Issues and Lessons from Risk Assessment Studies Jonathan E. Oliver, Paula F. Tennant, and Marc Fuchs

CONTENTS Introduction.............................................................................................................264 Pathogen-Derived Resistance and Engineered Resistance to Viruses in Plants.... 265 RNA Silencing and Engineered Resistance to Viruses in Plants....................... 265 Safety Issues and Risks...........................................................................................266 Transencapsidation.............................................................................................266 Recombination................................................................................................... 267 Gene Flow to Free-Living Relatives.................................................................. 268 Effects on Nontarget Organisms........................................................................268 Allergenicity and Human Health Effects........................................................... 269 Durability and Specificity of Engineered Resistance to Viruses in Plants......... 270 Breakdown of Engineered Virus Resistance................................................. 270 Specificity of Engineered Virus Resistance................................................... 271 Examination of Risks Associated with Commercialized Transgenic Horticultural Crops................................................................................................. 271 Squash Resistant to Cucumber Mosaic Virus, Zucchini Yellow Mosaic Virus, and Watermelon Mosaic Virus................................................................. 272 Background................................................................................................... 272 Risk Studies................................................................................................... 272 Papaya Resistant to Papaya Ringspot Virus....................................................... 273 Background................................................................................................... 273 Risk Studies................................................................................................... 274 Tomato and Sweet Pepper Resistant to Cucumber Mosaic Virus...................... 275 Background................................................................................................... 275 Risk Studies................................................................................................... 276

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Examination of Risks Associated with Transgenic Plum Which Is under Consideration for Deregulation................................................................. 276 Background........................................................................................................ 276 Risk Studies........................................................................................................ 277 Overview of Risk Lessons of Transgenic Horticultural Crops............................... 278 Perspectives............................................................................................................. 279 Introduction........................................................................................................ 279 Future Trends.....................................................................................................280 Conclusions............................................................................................................. 281 References............................................................................................................... 281

INTRODUCTION Horticultural crops were the first transgenic crops commercialized in the United States in the mid-1990s. Among the first of these were virus-resistant transgenic summer squash (Cucurbita pepo ssp. ovifera var. ovifera L.). The first transgenic squash cultivars, deregulated in 1994, were resistant to Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV).1–3 Other transgenic summer squash cultivars resistant to ZYMV, WMV, and Cucumber mosaic virus (CMV) were released in 19962–4 followed by papaya (Carica papaya L.) genetically modified for resistance to Papaya ringspot virus (PRSV) in 1998.2,5,6 To date, virus-resistant summer squash and papaya are the only transgenic horticultural crops that are commercially released in the United States, along with Bt sweet corn (Zea mays L.).7 Potato (Solanum tuberosum L.) resistant to Potato virus Y (PVY), Potato leafroll virus (PLRV) and the Colorado potato beetle were released in 1998, but were withdrawn from the market almost immediately thereafter due to anti-biotechnology campaigns and international trade barriers.8 In the People’s Republic of China, transgenic tomato (Solanum esculentum L.) and sweet pepper (Capsicum annuum L.) resistant to CMV and papaya resistant to PRSV have also been released.9 The virus-resistant transgenic horticultural crops commercially available have been developed by applying the concept of pathogen-derived resistance.10 Pathogenderived resistance refers to the use of a pathogen’s own genes to confer resistance in a host to that pathogen. In the case of the horticultural crops released to date in the United States, engineered virus resistance has been achieved through expression of viral coat protein genes.3,6 The development and release of transgenic horticultural crops, in particular those engineered for virus resistance, have raised potential safety issues regarding their impact on the environment and human health.2,11–13 Similarly, concerns have been expressed over the release of horticultural crops expressing cry toxin genes from the bacterium, Bacillus thuringiensis (Bt).14,15 Over the years, a significant amount of research has been done to address safety issues and examine potential risks. In this chapter, we provide a synopsis of transgenic horticultural crops, in particular virusresistant transgenic crops with the major emphasis on those that are currently available in commerce. We also examine the mechanisms underlying engineered virus resistance and discuss potential safety issues with this technology. We then examine risk assessment research by focusing on commercial crops for which data from realistic

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field studies are available, thereby using each released crop as a case study in our examination of the safety issues associated with transgenic horticultural crops. Finally, we summarize lessons from risk assessment research and evaluate whether safety issues account for the limited number of horticultural transgenic crops released to date.

Pathogen-Derived Resistance and Engineered Resistance to Viruses in Plants The first report on engineered resistance to viruses in plants was published in 1986.16 These researchers noted that tobacco plants expressing the coat protein gene of  Tobacco mosaic virus (TMV) exhibited delayed infection following mechanical inoculation with TMV. This observation conformed to the concept of pathogen-derived resistance that had been postulated earlier.10 Following the initial breakthrough by Abel et al.,16 viral coat protein genes from various viruses were introduced into numerous economically important crop species in hopes of achieving resistance.17 It was initially believed that resistance was provided by the viral protein itself via a mechanism involving excess plant-expressed coat protein that interfered with the uncoating step in viral replication.18 However, it soon became apparent that resistance could be achieved in transgenic plants producing low or undetectable levels of coat protein.19 Further observations indicated that the mechanism involved degradation of the transgene-derived messenger ribonucleic acid (mRNA) into small fragments in a sequence-specific manner.20–23

RNA Silencing and Engineered Resistance to Viruses in Plants Extensive research has shown that engineered virus resistance in transgenic plants works primarily through the antiviral pathways of the mechanism known as RNA silencing. This mechanism regulates the expression of genes at the RNA level following RNA transcription by the host plant. RNA silencing is triggered by the replication of genomic viral RNA within the host cell.18,21,24 Following entry into the cell, most viruses form double-stranded RNA (dsRNA) intermediates during their replication. These dsRNA are recognized by host RNase III Dicer-like enzymes and cleaved into short fragments called small interfering (si) RNA (∼21 nts). These fragments then associate with the RNA-induced silencing complex (RISC), which proceeds to target homologous RNA sequences within the cell. Once identified by the RNA silencing machinery, these target RNA sequences are similarly cleaved.18,21,23,24 In this way, the host cell severely limits or entirely prevents viral replication, resulting in resistance. Though originally identified in plant–virus interaction studies, RNA silencing has been shown to be present in a wide range of organisms including humans, plants, nematodes, and fruit flies and is believed to function as a sort of primitive immune system.25 The activation of these cellular viral defenses within host plants is believed to explain the long-recognized phenomenon of recovery from viral symptoms seen in the upper leaves of some infected plants, as it has been shown that a silencing signal can move systemically within the plant, activating silencing ahead of the viral replication advance.26

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Lending further support to the importance of the RNA silencing system in plants is the discovery that most virus families encode one or more proteins that suppress silencing.21,24,27–29 Viral suppressors have been shown to interact in multiple ways with the RNA silencing machinery to prevent effective control of the expression of the viral genome within the host. One such example is the HC-Pro protein from PVY. This protein binds the siRNAs produced by the cell’s RNA silencing machinery, thereby squelching the signal, which effectively suppresses RNA silencing within the host cell.30 This not only can allow for continued PVY replication, but has also been shown to be primarily responsible for the observed phenomenon of viral synergism where two unrelated viruses, when co-infecting the host plant, can exhibit more severe symptoms than either virus on their own. Experiments with PVY and Potato virus X (PVX) have shown that HC-Pro of PVY can suppress host defenses and allow for PVX, which very weakly suppresses silencing on its own, to replicate and produce much more severe symptoms than either PVX or PVY cause on their own.13 The fact that RNA silencing is a nucleotide sequence-based resistance mechanism has unique implications for risk assessment and the development of new virusresistant transgenic plants. This feature is discussed in the next section.

SAFETY ISSUES AND RISKS Due to the expression of viral gene constructs to provide resistance to viruses in transgenic plants, there are unique safety issues associated with this technology2,11–13 versus many of the other commercialized transgenic crop plants. Among these are the risks of viral recombination and transencapsidation. However, not all of the potential risks associated with this technology are unique to virus-resistant transgenic plants. Risks also associated with other transgenic plant technology including gene flow to free-living relatives, allergenicity and other human health effects, breakdown of resistance, and effects on nontarget organisms also need to be assessed with regard to engineered resistance against viruses. In the following section, we examine each of these areas of concern and assess their scientific merits.

Transencapsidation One of the concerns associated with virus-resistant transgenic crops is the potential that viral capsid proteins, when produced in a transgenic host plant, may transencapsidate the genome of a challenge virus.2,11–13 Since viral capsid proteins play roles in such diverse processes as movement within the host, replication, suppression of gene silencing, and vector transmission specificity,31 it is plausible that a transencapsidated virus may have altered properties. If the coat protein of a virus vectored by aphids, for example, were transgenically produced within a host plant, it is conceivable that an aphid non-transmissible virus could be encapsidated within the coat protein derived from the transgene. This transencapsidated virus (with the coat protein from the aphid-borne virus and the genome of the aphid non-transmissible virus) might then acquire the characteristics of an aphid-borne virus, potentially allowing it to move more expeditiously into new host plants—possibly even to plant species

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that the parent viruses heretofore had not come into contact with. Transencapsidation has been shown experimentally.11 However, many of the concerns with respect to transencapsidation have been alleviated by our current understanding of RNA silencing. This is true because when RNA silencing is active (as is expected if the transgenic plants exhibit viral resistance), the expression of viral-derived proteins is regulated posttranscriptionally, with little to no detectable capsid protein produced. In the case of untranslatable coat protein transgenes, this concern is alleviated altogether. Also, since the interactions between viral proteins or between capsid proteins and viral genomes is often very specific,31 it seems unlikely that interactions necessary for a viral coat protein to aid in the replication or movement of its corresponding viral genome would take place in coordination with genomic material or proteins from a heterologous virus. Finally, it is unclear how these risks are substantially different from the risks already present when a host plant is infected with two distinct viruses—which has been shown to lead to transencapsidation11—and how transencapsidation would be any more likely to occur in the transgenic case than in the non-transgenic multiple infection scenario.2,11,12 Even if transencapsidation did occur, it is questionable how it could result in viruses with permanently altered properties, since it is not conceivable how the movement of a transencapsidated virus to a new host would be anything other than a so-called dead end, since (due to the lack of a coat protein from the heterologous virus in the new host) all new viruses produced in the new host plant would be encapsidated within their own capsid protein.2,11–13 An exception would be if the new host were transgenic and expressing the coat protein gene of a closely related heterologous virus. In any event, if problems with transencapsidation did arise, the problem could be eliminated by ending the cultivation of the transgenic crop in question.25

Recombination Another potential risk of virus-resistant transgenic crop plants expressing viral genes is that of recombination between viral-derived transgene mRNA and the genomic RNA of an infecting virus.2,11–13 It is thought that this type of recombination could potentially give rise to a new virus strain or new virus species possessing characteristics different from those of the parent viruses. Recombination may involve, for example, an incoming virus containing a viral coat protein gene whose product is defective in its ability to be vectored, and transcripts of a homologous viral transgene sequence possessing a functional copy of the coat protein gene. Recombination between these sequences may then restore vector transmissibility. Unlike the risk of transencapsidation, recombination cannot be as easily dismissed as an evolutionary dead end, since viral progeny identical to the recombined strain could be produced in a new host.2,12,13 Recombination between transcripts of a viral gene construct in a transgenic plant and an incoming virus has been shown by several groups.32 If resistance were effective through RNA silencing, the occurrence of recombination is less likely unless the incoming virus were a related, but divergent, isolate of the virus from which the transgene is derived. In that case, it is conceivable that the incoming virus would replicate to the same extent as in a susceptible plant, providing opportunities for recombination.

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Nevertheless, it is unclear how recombination would be more likely to occur between a viral transgene mRNA and an infecting virus than between two viruses that are coinfecting the same host (a common situation).2,33–35 It is likewise unclear how recombination is any more likely to occur in the transgenic case than in the case of crossprotection, an accepted method used in controlling viral diseases. Cross-protection relies on the use of mild virus strains to protect plants from economic damage caused by closely related severe virus strains.36,37 Therefore, RNA molecules of distinct viral strains have ample opportunities to recombine in cross-protected plants. Though not shown to have emerged in cross-protected plants, recombinant viral strains resulting from recombination between Arabis mosaic virus (ArMV) and Grapevine fanleaf virus (GFLV) have been observed.38 The use of recombinant mild strains of ArMV and GFLV to cross-protect against GFLV is an accepted experimental control method for GFLV39 despite their deliberate dissemination in the environment.

Gene Flow to Free-Living Relatives Another concern, not unique to virus-resistant transgenic crops, is the risk of transgene flow.2,12,13 Transgene movement from a transgenic crop species to a free-living relative, through pollen flow, can be a significant concern. If transgenes provide a selective advantage, it is conceivable that hybrids between transgenic and free-living compatible species might acquire a fitness benefit and eventually a competitive edge over free-living plants.40 In an extreme scenario, a transgenic hybrid may outcompete free-living plants and thereby lead to an elimination of entire species, land races, or varieties of non-transgenic plants. In the case of virus-resistant transgenic plants, it is postulated that a hybrid possessing a transgene conferring virus resistance might outcompete compatible free-living plants and become established in the natural environment. Though this is unlikely to be due to an increase in weediness potential of the transgenic crop itself, such a scenario may have far-reaching environmental consequences in the case of free-living species.2 The likelihood of outcrossing between transgenic crop plants and free-living relatives depends on numerous factors, including pollen phenology, pollen compatibility, and spatial proximity.40 Each of these factors could vary significantly between any two given transgenic crop species and environments. Therefore, evaluations of the likelihood of gene flow (and its effects) must be carried out on a case-by-case basis. Even if gene flow from a virus-resistant transgenic crop to free-living relatives could readily occur, it is not obvious what effect this would have. Gene flow from domesticated crop species developed through traditional breeding practices has also been shown to occur,40–44 but in the case of traditional breeding, problems arising from gene flow to free-living relatives have not been seen, and it is unclear how the effect of transgenic virus resistance genes would be substantially different from those of resistance genes derived from traditional breeding.

Effects on Nontarget Organisms An additional concern regarding transgenic horticultural crops is their potential to have negative effects on nontarget organisms,2,45 for example, organisms that are not intentionally targeted by the disease or pest management strategy. Effects on

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nontarget organisms can be difficult to evaluate, and even when an effect is seen, it can be difficult to assess the significance of this observation. With respect to virus-resistant transgenic crops, it is not easy to identify a mechanism which might result in an effect on nontargets, because the titer of the transgene protein product is likely manyfold lower than the amount of the corresponding viral protein in a non-transgenic virus-infected plant.46,47 In addition, given the fact that the resistance mechanism is likely to be RNA silencing, in many cases little to no protein is likely produced at all, especially in the case of untranslatable transgenes. Furthermore, crop plants derived through traditional breeding practices have been shown to produce a wide range of allergens and toxins with clear effects on animals, plants, insects, and nematodes that may come into contact with these plants during their lifetime.48

Allergenicity and Human Health Effects Another area of concern regarding transgenic horticultural crops is the possibility of the introduction of allergenic proteins into the food supply and the introduction or increase in the production of toxic compounds.48–52 Although this concern also applies to crop varieties developed using conventional breeding methods, transgenic products have received strict scrutiny presumably because of the nature of the transgene proteins. The underlying concept of safety evaluations of genetically modified foods was proposed in the early 1990s53 and is based on comparative analyses of the transgenic crop with the conventionally bred parent that has a history of safe use (i.e., substantial equivalence).54 Additionally, safety testing of whole foods in animals is used to determine toxicity and allergenicity of genetically modified foods as well as toxicity testing of individual proteins. The latter tests, in combination with nutritional analysis, are regarded as more sensitive and accurate.55,56 While postmarket monitoring of transgenic food crops provides data on patterns of human nutritional exposure and may be useful in confirming premarket risk assessment and the detection of rare unintended effects on health, the evaluation is not regarded as a component of risk assessment and is not a substitute for thorough premarket risk assessment.57 Based on this approach, a number of transgenic crops expressing protein products, such as those derived from Bt-derived toxins (cry) and marker transgenes (nptII, uidA), have been found to present little to no risk to food or feed safety.2,58–62 Further, analysis of potential pleiotropic effects on inherent plant toxins and antinutrients of transgenic plants and their progenitor cultivars (e.g., maize, rape, tomato, potato, and soybean) has shown minor to perceptible variations, albeit within the ranges documented in literature, in the contents of these compounds.63 Natural biological variation, including nutrient variation, of individual plants grown under the same conditions is expected, given the influences of differences in plant development, metabolism, and biotic factors.63,64 However, the differences between the transgenic and the progenitor cultivar can also be attributed to somaclonal variation, given that the transformation of many crops, including papaya, involves an adventitious regeneration protocol, and in some cases, 2,4-dichlorophenoxyacetic acid,65–68 a plant growth regulator known to introduce genetic mutations, is used. Nonetheless, backcrossing to the original parental variety (and selecting progeny with appropriate traits) effectively eliminates composition alterations caused by tissue

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culture methods.69 It is important to also note that variation in composition is not limited to transgenic crops generated by recombinant DNA technologies and tissue culture methods. Nutritional variation has been reported for a number of plant products derived from conventionally bred varieties, and ranges for most of the compositional variables are available in the literature.70 Variation in conventional crops is attributed to genetics as well as preharvest conditions, maturity at harvest, harvesting methods, postharvest handling, and storage conditions. Although numerous animal studies have been conducted with transgenic crops carrying cry insecticidal, cowpea trypsin inhibitor, phytase, and snowdrop lectin genes,58 there are only a few published studies on the safety assessment of whole foods derived from transgenic crop plants transformed with viral coat protein genes. Presumably viral coat proteins are not regarded as potential allergens or toxins given the physicochemical and structural properties of the proteins and the low exposure levels due to low or undetectable transgene protein expression (because of RNA silencing). For transgenic viral proteins expressed in commercialized horticultural crops, sequence relatedness of 35% (or higher) or a continuous stretch of eight amino acids is not shared with known allergens.71 Moreover, resistance to digestion under acidic conditions has not been demonstrated.2,72,73 It is important to bear in mind that many of the crop plants available in commerce contain natural toxins and allergens.48 Peanuts, tomatoes, soybeans, kiwi, and potatoes are a few examples.

Durability and Specificity of Engineered Resistance to Viruses in Plants The issues of broad-spectrum, durable resistance with regard to virus-resistant transgenic horticultural crops do not conceivably have any impact on the environment and human health. If the engineered resistance to viruses were to show limitations in terms of durability and specificity, it would likely only create an agronomic problem and affect growers.2 However, these issues should be considered in light of an effective management of the technology. Breakdown of Engineered Virus Resistance The risk that virus resistance may break down or not prove durable is not unique to virus-resistant transgenic crops. It is a risk shared by conventional crops and other transgenic technologies including the pest resistance of Bt crops. The durability of resistance refers to the ability of a gene conferring resistance to hold up over time after being widely deployed. In the case of virus resistance, however, the potential mechanisms for resistance breakdown are different. As alluded to previously, one potential breakdown of resistance could occur if virus isolates that are genetically divergent enough, at the nucleotide sequence level, from the transgene are not recognized by the RNA silencing machinery and are subsequently capable of infecting the genetically modified host plant. Another potential mechanism of resistance breakdown centers on the virus’ ability to mutate in such a way as to overcome the resistance triggered by the transgene. A third potential mechanism for resistance breakdown involves infection of the resistant host plant with a heterologous virus74,75 encoding a strong suppressor of gene silencing. This heterologous virus

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might suppress the host resistance provided by the host transgene, thereby allowing the host to be infected by the original virus toward which their resistance had been targeted. Though each of these mechanisms could lead to a potential breakdown in host resistance, it is important to note that the risk of resistance breakdown is not unique to virus-resistant transgenic crops nor is it unique to transgenic crops. Resistance breakdown is also an issue associated with the deployment of resistance genes derived via traditional breeding. Pathogens capable of overcoming deployed resistance genes have been extensively documented;76 likewise, the deployment of new resistance genes has been shown to alter pathogen populations to overcome that resistance.76 Initial infection by a virus not targeted by a traditional breeding-derived resistance gene has also been shown to lead to a breakdown of the resistance to the virus targeted by the resistance gene77—analogous to the breakdown of resistance due to a co-suppressor of RNA silencing. Therefore, it is unclear how the risks of breakdown (with transgenic virus resistance) are substantially different from those associated with the use of resistance genes in traditional breeding. Specificity of Engineered Virus Resistance Another concern unique to virus-resistant transgenic technology is the so-called specificity of resistance provided by the transgene. As has been shown previously,78 the resistance provided by the transgene might only be specific to the virus isolate from which it was derived and a few closely related isolates. Though previously not well understood, the current understanding of RNA silencing suggests that this may be due to the sequence specificity of the resistance mechanism itself. Since the RNA silencing mechanism relies on the alignment of cleaved fragments of the target RNA in the search for invading RNA sequences, divergence at the sequence level (over ∼10%) can lead to an apparent breakdown in resistance.79 However, this has not been shown to be the case with all of the virus-resistant transgenic plants,80 as a single transgene is able to confer resistance to challenge from numerous isolates of the same virus.81 Also, it is important to note that similar specificity has been shown with resistance genes derived from traditional breeding,82 and it is unclear how the resistance provided in the transgenic case is substantially more specific than the resistance provided by the resistance genes derived by traditional breeding.

EXAMINATION OF RISKS ASSOCIATED WITH COMMERCIALIZED TRANSGENIC HORTICULTURAL CROPS Once safety issues associated with transgenic horticultural crops are identified, how are risks assessed? How does one examine the significance of risk assessment data? When is there enough evidence to start drawing conclusions on the safety of transgenic horticultural crops? From risk assessment conclusions, are there any safety issues that need to be examined further? Or, are there any that can be put to rest, so to speak? In the following section, we address these questions with regard to virusresistant transgenic horticultural crops.

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Given the tremendous amount of transgenes that have been engineered to provide virus resistance and the numerous crops that these genes have been introduced into, there is a staggering amount of scientific literature on resistance to viruses in transgenic crop plants.17 To focus our efforts on findings that we believe to be the most relevant, we have chosen to examine risk assessment of virus-resistant transgenic horticultural crops already commercialized (e.g., papaya, summer squash, tomato, and sweet pepper) or awaiting deregulation (e.g., plum). In our examinations, we will rely primarily on realistic field studies, which may provide the most accurate reflection of risks, and to a lesser extent on laboratory and greenhouse studies. We will look at the conclusions that can be made about the safety of virus-resistant transgenic horticultural crops by analyzing the significance of risk assessment studies, as well as at identifying gaps in knowledge where further experimental evidence may be needed before conclusions can be drawn with respect to the safety of these crops.

Squash Resistant to Cucumber Mosaic Virus, Zucchini Yellow Mosaic Virus, and Watermelon Mosaic Virus Background The first disease-resistant transgenic crop to be commercialized in the United States was transgenic summer squash. This squash, which possesses resistance to ZYMV and WMV, was deregulated in 1994.2,3 Another summer squash cultivar resistant to CMV, ZYMV, and WMV was later released in 1996.2–4 Virus-resistant transgenic squash possess the coat protein genes from each virus. Early testing indicated that they provide high resistance to viral infection1,3,4 and prevent viral epidemics by reducing secondary plant-to-plant spread.83 No similar resistance to multiple viruses is available in traditionally bred commercial summer squash.2 Squash is unique among commercial transgenic plants in that it is monoecious and readily outcrosses,44 emphasizing the significance of gene flow issues for this crop. Since the center of origin for many squash species is in the southern United States and Mexico,42 the commercialization of virus-resistant transgenic squash marks the first transgenic crop to be released within its center of origin. Risk Studies As summer squash was the first virus-resistant crop with a coat protein transgene to be commercialized, the potential allergenicity and impacts on human health were considered extensively.46 No significant difference in protein, total fat, dietary fiber, carbohydrate, calories, vitamins A (and its precursor, β-carotene) and C, calcium, iron, sodium, ash, moisture, and sugar profiles (fructose, glucose, sucrose, maltose, and lactose) was found between transgenic and non-transgenic squash.46 Also, an examination into human consumption of virus-infected non-transgenic summer squash fruits led to the conclusion that there was likely to be no significant negative impact on human health beyond those of virus-infected traditionally bred squash cultivars with which consumers have a long history of exposure to without any clear hazards arising.46

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Field trials relating to gene flow from virus-resistant transgenic summer squash to a free-living relative (C. pepo ssp. ovifera var. texana) and the persistence of transgenes among hybrids of transgenic and free-living C. pepo have been carried out. Gene flow occurred with sympatric populations (populations of related species existing in the same geographic area) under conditions of low disease pressure.84 Hybrid plants containing transgenes were likely to produce more fruit, seed, and be more vigorous than free-living C. pepo and non-transgenic hybrids under conditions of high disease pressure.85 This was not true under conditions of low virus pressure, where free-living plants outperformed the transgenic hybrids.85 These results clearly indicated the advantage that the transgenes might provide under conditions of high disease pressure, although it is not clear whether this poses a significant risk in terms of population dynamics. Surveys of free-living C. pepo for viruses in areas where transgenic summer squash had not yet been released showed an extremely low incidence of viruses, including CMV, ZYMV, and WMV.86 These results suggested that viruses have a limited effect on the dynamics of free-living C. pepo populations.86 Also, the studies on gene flow and its consequence in squash do not necessarily indicate any risk of engineered virus resistance beyond that of conventionally bred resistance genes, as far as free-living populations are concerned.2 An additional study by Fuchs et al.4 investigated the likelihood of transencapsidation in transgenic squash, tomato, and melon, so as to allow the transmission of an aphid non-transmissible strain of CMV by aphids through interaction with the coat protein transgene derived from an aphid transmissible strain of CMV. The results of this study failed to demonstrate that transencapsidation could occur over two consecutive growing seasons.4 However, transencapsidation of an aphid nontransmissible strain of ZYMV likely occurred in transgenic squash expressing the coat protein gene of an aphid transmissible strain of WMV at a very low rate and without triggering an epidemic.87 Similarly, virus-resistant transgenic summer squash had no effect on the genetic diversity of CMV strains, suggesting that these plants did not facilitate the emergence of recombinant viruses.88

Papaya Resistant to Papaya Ringspot Virus Background An early success story in the development and commercialization of virus-resistant transgenic fruit crop plants is the case of papaya resistant to PRSV.6 This virus causes one of the most devastating viral diseases of papaya. There is no practical resistance known to PRSV in Carica germplasm; therefore, control of this virus has relied on exclusion, movement to new growing regions where PRSV is not found (often involving the destruction of native rainforest habitats), and the use of mild isolates of PRSV in attempts to control the disease via cross-protection.6 PRSV is an aphid-borne potyvirus and can readily spread over long distances by its vectors. Papaya is grown in tropical and semitropical regions and Hawaii is by far the largest producer of papaya in the United States. The production center for Hawaiian papaya is in the Puna district of the island of Hawaii. Production from this district comprises

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over 95% of the total Hawaiian papaya production. In 1992, PRSV was discovered in Hawaii’s Puna district5 and within 3 years nearly all of the plants in Puna were severely affected. By 1997, papaya production had declined by 36% from 21,800 tons (at the start of the outbreak) down to 14,000 tons.6 Transgenic papaya cultivars, ‘SunUp’ and ‘Rainbow,’ resistant to PRSV were released in 1998 and widely planted in Hawaii providing effective virus control and allowing for increased papaya production (from the low in 1998).2,6 Since the release of the PRSV-resistant transgenic papaya in Hawaii, additional papaya cultivars resistant to PRSV containing other PRSV coat protein transgenes have also been developed in Australia, Florida, Brazil, Taiwan, Jamaica, the Philippines, Thailand, and Venezuela.68,81,89–94 The transgenic varieties are at various stages of development and evaluation. Other transgenic papaya purportedly resistant to PRSV have also been developed utilizing the viral replicase gene67 but the major focus here will be on PRSV-resistant papaya involving the coat protein gene. Risk Studies Since the development and commercial release of transgenic papaya expressing the coat protein gene of PRSV, numerous experiments and field trials have probed the potential risks associated with this transgenic technology. As one of the first widely commercialized fruit crops, there has been much interest in the effects of the coat protein transgene on human health and potential allergenicity as well as investigations into compositional changes in fruit with respect to nutrient and antinutrient content. Descriptions of transgenic papayas developed in Hawaii report on percent soluble solids above the minimum required for commercial fruit and yields of almost three times those of industry averages.5 Comparable values for vitamin C and minerals (potassium, phosphorus, calcium, magnesium, sodium, iron, copper, zinc, and boron) for the transgenic and non-transgenic cultivars have been published.95,96 Also, no evidence of ill effects has been linked to the consumption of transgenic papaya in the United States and Canada.2 Another study on transgenic papaya from Thailand reported on comparable nutrient composition with the non-transgenic counterpart.93 Recently, the levels of nutrients (protein, fat, carbohydrate, minerals) and antinutrients (oxalates, hydrocyanic acid, and benzyl isothiocyanate) in three transgenic papaya lines expressing a PRSV coat protein gene construct, which are not currently available on the market, were compared to those of the commercial papaya cultivar ‘Sunrise solo’ grown under the same conditions in an experimental plot in Jamaica.73 Since papaya is a climacteric fruit, three stages of maturity were considered to facilitate an evaluation of the changes in various parameters that accompany the ripening process after harvest. With the exception of one transgenic line, no significant differences were observed in selected nutrients and antinutrients between the control and test samples at three stages of maturity, although a few random variations were noted.73 Overall, the compositional changes over the three maturities were as expected and comparable to those reported.97–99 Sugars, vitamin C, and carotenoids followed a general upward trend, whereas slight decreases in moisture, ash, and fat at the final stage of ripening were observed. Some variability in the concentrations of the three antinutrients tested was observed, but the values were within the range of concentrations reported for

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the parental variety.100 A general trend of decreasing antinutrient levels was noted during ripening in transgenic and control fruits. Similar nonsignificant variations in selected horticultural traits (water, lipid, nitrogen, protein, reducing sugar, vitamin A (and its precursor), vitamin C) were obtained with transgenic papaya expressing the replicase gene of a PRSV isolate from the People’s Republic of China.67,101 Recently, Powell et al.102 evaluated the safety of transgenic papaya in a subchronic feeding study. A diet formulated with 10% transgenic papaya, the equivalent of twice the average daily human consumption of fresh papayas based on food consumption data from the Caribbean and Latin America,103,104 was administered to rats for 90 consecutive days. For comparison, reference non-transgenic papaya ‘Sunrise solo’, from which the transgenic papaya was derived, and a control laboratory rodent diet formulation were also evaluated. Markers of general health, including body weight, food intake, and activities of plasma, liver, and kidney function enzymes (acid and alkaline phosphatases and alanine and aspartate transaminases) were comparable for the test, reference, and control groups. No significant effects were observed in organ weights or histopathology.102 Changes in the liver and kidney, the sites of biotransformation and detoxification, and of excretion of metabolic waste products, respectively, were not observed.102 Overall, the plasma cholesterol levels, which are markers of cardiovascular risk, were similar to the control as were triglycerides, which are biomarkers for hepatotoxicity. Although not statistically relevant, variations in the values of the parameters monitored with the control, reference, and test groups were observed.102 Based on literature ranges, the variations were attributed to natural biological fluctuations and were not regarded as reflecting a toxicologically meaningful effect. In addition to looking at the effect of transgenes on papaya fruit, other researchers have focused on the effects of transgenic papaya on nontarget organisms including soil microbial organisms. Hsieh and Pan,105 looking at populations of fungi, bacteria, and actinomycetes present in the soil from field plantings of both transgenic and nontransgenic fields, found highly similar (>80%) populations in both soil environments (transgenic vs. non-transgenic) as well as in upper and lower soils within the environments. These authors concluded that the planting of transgenic papaya’s effect on the soil microorganisms is limited.105 Minor effects on nontargets have been observed,106 but these effects are varied and not consistent across all virus-resistant transgenic crops. Another study, also examining the effects of transgenic papaya on the soil, relied upon polymerase chain reaction to determine the persistence and availability of transgenic genes that may be released by transgenic papaya during growth.107 Though transgenic DNA was detected at low levels, no gene transfer events from soil DNA extracts to Acinetobacter (a bacterium well known for its ability to uptake foreign DNA) were observed.107

Tomato and Sweet Pepper Resistant to Cucumber Mosaic Virus Background With regard to transgenic tomato and sweet pepper engineered for CMV resistance, the body of publications is much smaller than for the aforementioned transgenic crops. Transgenic tomato and sweet pepper containing the coat protein gene from

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a local isolate of CMV were released in the People’s Republic of China.108 CMV is a cucumovirus that is transmitted in a nonpersistent manner by several aphid species.109 It has the widest host range of any known plant virus with 1300 species in more than 500 genera of over 100 families. Control of CMV can be achieved by planting resistant crops but resistance in many crops species is often not available to a broad range of CMV strains.109 Risk Studies A study on gene flow was performed with transgenic sweet pepper and tomato by Ming et al.110 Seeds and pollen of many different plants at varying distances from fields of transgenic plants were examined to determine whether gene flow had occurred. Selection of seedlings on antibiotic-containing medium and polymerase chain reaction were used to monitor transgene movement, but transfer of transgenes was not detected.110 For assurance of food safety, transgenic sweet peppers and tomatoes expressing the coat protein gene of CMV were evaluated in animal feeding studies.108 The animals received about 12,600 and 7100 times the average daily human consumption of sweet peppers and tomatoes, respectively. Comparable performance of rats fed transgenic and non-transgenic diets was demonstrated.108 Significant differences were not reported in mean weekly body weights, body weight gains, or food consumption of rats fed transgenic or non-transgenic sweet pepper and tomato diets. Similarly, significant differences were not observed with the hematological and blood biochemical parameters monitored (including cholesterol and triglyceride), although fluctuations in the values were observed.108 In another study, Cai et al.111 conducted 30-day acute toxicity (LD50) experiments with male and female rats and mice using gavage administration of a series of doses ranging from 1 to 10â•›g /kg body weight of dry pepper fruit containing the coat protein gene of TMV and CMV. Abnormalities in body weights, organ weights, histopathology, and hematology were not observed.111

EXAMINATION OF RISKS ASSOCIATED WITH TRANSGENIC PLUM WHICH IS UNDER CONSIDERATION FOR DEREGULATION Background Though not yet commercially available, the transgenic plum cultivar ‘Honey sweet’ (aka C5) resistant to Plum pox virus (PPV) has been deregulated by the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service (USDAAPHIS) and registered by the Environmental Protection Agency (EPA) in the United States and is being widely tested in Europe under varying growing conditions. PPV is a potyvirus that is considered the most important pathogen in Prunus by the U.S. and E.C. agencies.112 Conventional breeding has not been able to produce any trees of commercially acceptable varieties with high resistance to PPV. Therefore, control of PPV has relied on prevention via certified planting material, quarantine measures, and eradication.112 PPV is spread by multiple aphid species, but control of the vector is not feasible for both efficacy and environmental impact reasons.113

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Transgenic plum trees containing multiple copies of the PPV coat protein gene were developed114 and line C5 was shown to be highly resistant to PPV infection.113 The involvement of the posttranscriptional RNA silencing mechanism in the resistant line C5 was confirmed.115,116 Clone C5 was tested extensively in the field in the Czech Republic, Poland, Romania, and Spain to demonstrate the effectiveness of the engineered resistance. Transgenic C5 trees all remained free from PPV infection even 6117,118 and 8 years after transfer to the field,119 while 100% of the control, non-transgenic trees were infected with PPV after these periods. Though these data indicate the durability of the engineered resistance to PPV, trees inoculated with PPV via chip budding exhibited very mild symptoms after several years, though these symptoms did not progress to severe.118,119

Risk Studies Fruit compositional analyses indicated that PPV-resistant transgenic C5 is typical for P. domestica plums in terms of protein, total fat, antioxidant capacity, phenolics, starch, dietary fiber, ash, moisture, acidity, carbohydrates, sugar profiles (glucose, sucrose, lactose, maltose, and fructose), calcium, magnesium, sodium, potassium, iron, and vitamins A, B1, B2, B3, and C.120 The potential for viral recombination between transgene transcripts and incoming PPV RNA was examined in transgenic plums as well as the effects of PPV-resistant transgenic plums on aphid vector populations.121 Utilizing transgenic European plum lines as well as non-transgenic plums from an experimental orchard and Japanese plums from an external control plot, 85 PPV isolates were collected from these three populations of trees and their genetic diversity was compared. Looking at variable regions of the PPV genome including the coat protein gene, no significant differences in genetic variability were found among isolates from the three populations, indicating that the PPV populations were not being selectively altered in the transgenic trees.121 Subsequent analysis of 12 PPV isolates showed no detectable recombinant virus.121 Of note in this experiment, C5 could not be used as a source of virus isolates for testing since it remained free from infection 8 years after natural exposure to PPV populations. Recombination in C5 trees therefore would have been impossible due to lack of virus infection. For assessing the impact of transgenic plums on nontarget organisms, the diversity of aphid populations visiting transgenic and non-transgenic plums was investigated.121 Aphids were captured, identified, counted, and their viruliferous potential was subsequently characterized. These comparisons found no significant differences between the aphid populations from either transgenic or control plums in terms of total aphid numbers, aphid species distributions, and viruliferous potential over the 2 year period of the study.121 To test the effect of heterologous viruses on the stability of RNA silencing in transgenic plum line C5, trees were graft-inoculated with different combinations of PPV and either Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), or Apple chlorotic leafspot virus (ACLSV).75 PNRSV, PDV, and ACLSV are common in Prunus sp. The engineered resistance to PPV was stable and was not suppressed by the presence of heterologous viruses during a 3 year field trial in Romania and Spain.75

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OVERVIEW OF RISK LESSONS OF TRANSGENIC HORTICULTURAL CROPS Based on the existing body of research into the potential risks posed by virus-resistant genetically modified horticultural crops, there appears to be a significant amount of evidence that these crops have little to no detrimental impact on the environment and human health beyond those of conventional horticultural crops. The work on summer squash supports the aforementioned claim that transencapsidation is not a significant environmental risk beyond that already posed by multiple infected conventionally bred plants.4,87 This is also true for the risks of gene flow to free-living relatives84–86 and human health effects, specifically on allergenicity.46 Furthermore, there is a documented safe release of this transgenic crop over the past 12 years in the United States.2 In the case of papaya, the results from the allergenicity and nutritional experiments seem to very strongly refute risks to human health.73,93,102,111 The threat of negative nontarget effects appears to be lessened though the papers published on this topic are very limited in scope.105–107 With respect to the other areas of risk, direct experimental evidence does not appear to exist in sufficient quantities to make any firm conclusions on transencapsidation, recombination, and gene flow. However, the safe commercial use of transgenic papaya over a decade and evidence from other transgenic crops may speak to some of these issues enough to be extrapolated to the PRSV papaya case.2 The work on plums supports the fact that recombination is unlikely to facilitate the emergence of virus species with altered or new biological properties beyond the occurrence in conventional plums subjected to mixed virus infection.121 This also applies to the risks on nontarget organisms.121 It is doubtful that transencapsidation could occur to a meaningful level in transgenic plum line C5, because of undetectable levels of PPV coat protein.115–117 This also casts doubt on whether the coat protein is expressed at a meaningful level to have human health impacts differing significantly from those seen in PPV-infected non-transgenic plums. Based on the information from the transgenic tomato and sweet pepper, there is further evidence to support the claim that gene flow is not a major concern in this case.109 Most significantly, the animal feeding studies give further support to the safety of virus-resistant transgenic crops expressing viral coat protein genes.108,111 Similar conclusions on the risk assessment of other transgenic horticultural crops have been reported. The only Bt horticultural crop commercially available in the United Stated is Bt sweet corn, although other Bt transgenic vegetables (i.e., cauliflower, cabbage, and eggplant) are being considered for commercialization in India.62 Studies have shown that Bt sweet corn provides consistently excellent control of lepidopteran pests. This technology also substantially reduces insecticide applications and better preserves predators of the European corn borer than commonly used broad-spectrum insecticides.62 Bt sweet corn, like virus-resistant summer squash and papaya, are consumed in the United States with no ill effects reported. In summary, transgenic horticultural crops have become important components of disease and pest management programs in the United States and the People’s Republic of China. Their adoption rate is constantly increasing since their first release

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in the mid-1990s. Safety issues have been expressed during their development and release, but most of these risks are the same or similar to those posed by traditionally bred plants with host resistance. Since the commercialization of virus-resistant transgenic squash in 1996, considerable data have been gathered from many parts of the world on the effects on the environment and human health of virus-resistant and Bt horticultural crops. From these studies, some general trends have emerged. Commercialized summer squash, tomato, sweet pepper, and papaya expressing viral coat protein genes, and Bt sweet corn have effectively controlled viruses and species of Lepidoptera, respectively. Also, extensive research has been published showing that these transgenic crops have little to no impact on the environment and human health beyond those of virus-infected plants in natural settings, in traditional agriculture, and exposed to conventional pest management strategies.2,14,45,62,121,122

PERSPECTIVES Introduction Since the initial discovery of engineered resistance to viruses via expression of the TMV coat protein gene in plants,16 pathogen-derived resistance10 and coat proteinmediated resistance have proven to be effective tools to control viruses in horticultural crops.17 As discussed, PRSV, ZYMV, CMV, and WMV have been effectively controlled in commercial settings of transgenic papaya, summer squash, tomato, and sweet pepper with the use of coat protein genes. In addition, in 2007 alone, more than 25 field trial permits for resistance against other viruses were granted by the USDA-APHIS.123 Target viruses included Tomato spotted wilt virus, Beet necrotic yellow vein virus, Sorghum mosaic virus, Grapevine fanleaf virus, Grapevine leafroll-associated virus 2, Grapevine leafroll-associated virus 3, Citrus tristeza virus, Cassava mosaic virus, Sugarcane mosaic virus, Sugarcane yellow leaf virus, Papaya leaf distortion mosaic virus, PPV, and PVY.123 In recent years, new knowledge of the mechanism behind engineered virus resistance has been gained. This has greatly expanded the potential for utilizing the antiviral pathways of RNA silencing to control plant viral diseases. Though all the currently available commercial virus-resistant transgenic crops utilize the coat protein-mediated resistance, recent publications, patents, and field trial data reveal that there are many alternative approaches currently in the pipeline to engineer virus resistance in plants. Transgenes incorporating short fused sequences derived from different viral strains have been engineered successfully to provide resistance to several virus species.124 Other methods which, unlike the RNA silencing-based approaches, do seem to depend on protein production have also been shown to have some promise, including the use of defective viral movement protein and replicase genes.25 The risks that these gene constructs pose are not yet fully determined and are likely different from those risks already discussed with regard to the viral coat protein gene. Though the risk assessment of the coat protein technology has, as discussed, alleviated most of the concerns of this technology, questions remain about the new technologies that are becoming available. Do they alleviate any of the problems of the existing technology? Do they raise new safety issues that may pose their

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own potential risks? Do they alleviate a previous concern that was no longer seen as a problem only to raise new issues that are potentially more difficult to assess? In the following section, we will describe some of the latest approaches toward virus resistance in transgenic plants and discuss if and how these technologies alleviate some of the existing concerns.

Future Trends Given the discovery and elucidation of the antiviral pathways of RNA silencing, many new approaches have been used to develop transgenes more likely to stimulate RNA silencing via the design of sophisticated transgenes. Since RNA silencing theoretically is stimulated in a plant cell by the presence of dsRNA, many of these transgene constructs attempt to transcribe RNA molecules that are more likely to form dsRNA structures. One strategy for accomplishing this is the use of inverted repeats, which involves the creation of a transgene containing two copies of the viral target complementary DNA (cDNA) sequence separated by a spacer DNA of some length.25,125 After transcription, it is thought that the resulting RNA will form a hairpin with the inverted RNA forming a double-stranded structure. The use of introns is a similar approach in that two inverted regions of viral-derived cDNA are separated by an intron.126 Once the intron is spliced by host machinery following transcription, a dsRNA structure is formed. Another approach consists of producing two complimentary pieces of RNA which can then form a dsRNA from bidirectional promoters.127 These new strategies have been shown to produce a significantly higher proportion of virus-resistant transgenic plants than the use of full-length coat protein transgenes.25,125,126 In addition, they hold a seeming advantage over a full-length coat protein gene in the sense orientation as they are generally unable to produce a functional protein, alleviating concerns arising from the presence of the coat protein in plant material. Coat protein expression can also be prevented by using transgenic approaches involving a transgene that produces an RNA product which is untranslatable, either because it lacks the necessary translation start codon for ribosomal processing or because it is oriented in antisense directions. It should be noted that the use of introns, often derived from plants themselves, potentially poses the risk of silencing host genes from where the intron was derived, if the RNA silencing machinery incorrectly processes the transgene RNA. A similar concern applies to the siRNA technology overall.128 Another approach utilizing the knowledge of viral silencing is to produce resistance by using modified plant microRNA (miRNA) cistrons to produce a range of artificial antiviral miRNAs.129,130 The durability of this approach compared to the use of longer dsRNA approaches has not been demonstrated.131 Some nonviral sources of virus resistance have also been investigated. These would theoretically alleviate concerns about synergism, recombination, and transencapsidation. These include the transfer of host resistance genes against viruses into other hosts via genetic engineering, or the silencing of host genes that are necessary for viral replication.25 The use of plant-generated antibodies against viruses, which failed to progress for many years though initially perceived as promising, has recently been shown to be effective in controlling viruses in plants expressing the transgenes

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for the production of these antibodies.132 The potential risks of these technologies remain largely undiscussed and untested.

CONCLUSIONS To date only a handful of horticultural transgenic crops, including vegetable crops (summer squash, sweet pepper, tomato, and sweet corn) and one fruit crop (papaya) are available commercially. Most of these crops have been engineered for virus resistance (summer squash, sweet pepper, tomato, and papaya), while sweet corn has been developed for insect tolerance. Noteworthy, the majority of virus-resistant transgenic crops were released over a decade ago. Why have not more transgenic horticultural crops been released? Though a virus-resistant transgenic plum has been deregulated in the United States, why have transgenic horticultural crops not been released recently? Can safety issues be held accountable for the limited number of transgenic horticultural crops released commercially? As discussed in this chapter, extensive research on risk assessment of transgenic horticultural crops has been carried out in various environments and varied conditions of disease and pest pressure.2,14,45,62,133 This wealth of information implies that safety issues should not hinder the release of new horticultural crops that are engineered based on the identical or similar technologies to those used for the development of the transgenic horticultural crops already released. Factors other than safety issues (e.g., institutional, policy, and economical factors) are apparently playing more important roles worldwide in stymieing the adoption of transgenic horticultural crops.134–136

REFERENCES



1. Fuchs, M. and Gonsalves, D., Resistance of transgenic squash Pavo ZW-20 expressing the coat protein genes of zucchini yellow mosaic virus and watermelon mosaic virus 2 to mixed infections by both potyviruses, Biotechnology, 13, 1466, 1995. 2. Fuchs, M. and Gonsalves, D., Safety of virus resistant transgenic plants two decades after their introductions: Lessons from realistic field risk assessment studies, Annu. Rev. Phytopathol., 45, 173, 2007. 3. Tricoli, D.M. et al., Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to Cucumber mosaic virus, Watermelon mosaic virus 2, and Zucchini yellow mosaic virus, Biotechnology, 13, 1458, 1995. 4. Fuchs, M. et al., Transgenic melon and squash expressing coat protein genes of aphidborne viruses do not assist the spread of an aphid non-transmissible strain of Cucumber mosaic virus in the field, Trans. Res., 7, 449, 1998. 5. Ferreira, S.A. et al., Virus coat protein transgenic papaya provides practical control of Papaya ringspot virus in Hawaii, Plant Dis., 86, 101, 2002. 6. Gonsalves, D., Control of Papaya ringspot virus in papaya: A case study, Annu. Rev. Phytopathol., 36, 415, 1998. 7. James, C., Global status of commercialized biotech/GM crops: 2007. ISAAA, Brief no. 37, International Service for the Acquisition of Agri-Biotech Applications, Ithaca, NY, 2008. http://www.isaaa.org/resources/Publications/briefs/37/default.html (accessed December 1, 2008). 8. Kaniewski, W.K. and Thomas, P.E., The potato story, Agric. Biol. Forum, 7, 41, 2004. 9. Stone, R., China plans $3.5 billion GM crops initiative, Science, 321, 1279, 2008.

282

Transgenic Horticultural Crops: Challenges and Opportunities

10. Sanford, J.C. and Johnston, S.A., The concept of parasite-derived resistance-deriving resistance genes from the parasite’s own genome, J. Theor. Biol., 113, 395, 1985. 11. Hammond, J., Lecoq, H., and Raccah, B., Epidemiological risks from mixed virus infections and transgenic plants expressing viral genes, Adv. Virus Res., 54, 189, 1999. 12. Robinson, D.J., Environmental risk assessment of releases of transgenic plants containing virus-derived inserts, Trans. Res., 5, 359, 1996. 13. Tepfer, M., Risk assessment of virus-resistant transgenic plants, Annu. Rev. Phytopathol., 40, 467, 2002. 14. Shelton, A.M., Zhao, J.Z., and Roush, R.T., Economic, ecological, food safety and social consequences of the deployment of Bt transgenic plants, Annu. Rev. Entomol., 47, 845, 2002. 15. Romeis, J. et al., Assessment of risk of insect-resistant transgenic crops to nontarget arthropods, Nat. Biotechnol., 26, 203, 2008. 16. Abel, P.P. et al., Delay of disease development in transgenic plants that express the Tobacco mosaic virus coat protein gene, Science, 232, 738, 1986. 17. Fuchs, M., Plant resistance to viruses: Engineered resistance. In Encyclopedia of Virology, 3rd edn., Mahy, B.W.J. and Van Regenmortel, M.V.H., eds., Elsevier, San Diego, 2008, vol. 4, pp. 156–164. 18. Lindbo, J.A. and Dougherty, W.G., Plant pathology and RNAi: A brief history, Annu. Rev. Phytopathol., 43, 191, 2005. 19. Lindbo, J.A. et al., Induction of a highly specific antiviral state in transgenic plants: Implications for regulation of gene expression and virus resistance, Plant Cell, 5, 1749, 1993. 20. Dietzen, R.G. and Mitter, N., Transgenic gene silencing strategies for virus control, Austral. Plant Pathol., 35, 605, 2006. 21. Eamens, A. et al., RNA silencing in plants: Yesterday, today and tomorrow, Plant Physiol., 147, 456, 2008. 22. Lin, S.-S. et al., Strategies and mechanisms of plant virus resistance, Plant Biotechnol. Rep., 1, 125, 2007. 23. Voinnet, O., Induction and suppression of RNA silencing: Insights from viral infections, Nat. Rev. Gen., 6, 206, 2005. 24. Voinnet, O., Post-transcriptional RNA silencing in plant–microbe interactions: A touch of robustness and versatility, Curr. Opin. Plant Biol., 11, 464, 2008. 25. Prins, M. et al., Strategies for antiviral resistance in transgenic plants, Mol. Plant Pathol., 9, 73, 2008. 26. Kehr, J. and Buhtz, A., Long distance transport and movement of RNA through the phloem, J. Exp. Bot., 59, 85, 2008. 27. Díaz-Pendón, J.A. and Ding, S.-W., Direct and indirect roles of viral suppressors of RNA silencing and pathogenesis, Annu. Rev. Phytopathol., 46, 303, 2008. 28. Ding, S.-W. and Voinnet, O., Antiviral immunity directed by small RNAs, Cell, 130, 413, 2007. 29. Moissiard, G. and Voinnet, O., Viral suppression of RNA silencing in plants, Mol. Plant Pathol., 5, 71, 2004. 30. Lakatatos, L. et al., Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors, EMBO J., 25, 2768, 2006. 31. Callaway, A. et al., The multifunctional capsid proteins of plant RNA viruses, Annu. Rev. Phytopathol., 39, 419, 2001. 32. Fuchs, M., Potential for recombination and creation of new viruses in transgenic plants expressing viral genes: Real or perceived risk? In Biotechnology and Plant Disease Management, Punja, Z.K., DeBoer, S.H., and Sanfaçon, H., eds., CABI Press, Wallingford, U.K., 2007, pp. 416–435. 33. Falk, B.W. and Bruening, G., Will transgenic crops generate new viruses and new diseases? Science, 163, 1395, 1994.

Virus-Resistant Transgenic Horticultural Crops

283

34. Rubio, T. et al., Recombination with host transgenes and effects on virus evolution: An overview and opinion, Mol. Plant–Microbe Interact., 12, 87, 1999. 35. Turturo, C. et al., Evaluation of potential risks associated with recombination in transgenic plants expressing viral sequences, J. Gen. Virol., 89, 327, 2008. 36. Fuchs, M., Ferreira, S., and Gonsalves, D., Management of virus diseases by classical and engineered protection, Mol. Plant Pathol. On-line http://www.bspp.org.uk/mppol /1997/0116fuchs, 1997 (accessed April 20, 2011). 37. Lecoq, H., Control of plant virus diseases by cross-protection. In Plant Virus Disease Control, Hadidi, A., Khetarpal, R.K., and Koganezawa, H., eds., APS Press, St. Paul, MN, 1998, pp. 33–40. 38. Vigne, E., Marmonier, A., and Fuchs, M., Multiple interspecies recombination events within RNA2 of Grapevine fanleaf virus and Arabis mosaic virus, Arch. Virol., 153, 1771, 2008. 39. Komar, V. et al., Cross-protection as control strategy against Grapevine fanleaf virus in naturally infected vineyards, Plant Dis., 92, 1689, 2008. 40. Ellstrand, N.C., Prentice, H.C., and Hancock, J.F., Gene flow and introgression from domesticated plants into their wild relatives, Annu. Rev. Ecol. Syst., 30, 539, 1999. 41. Hoc, P.S. et al., Hybridization between wild and domesticated types of Phaseolus vulgaris L. (Fabaceae) in Argentina, Gen. Res. Crop Evol., 53, 331, 2006. 42. Kirkpatrick, D. and Wilson, H., Interspecific gene flow in Cucurbita: C. texana and C. pepo, Am. J. Bot., 75, 519, 1988. 43. Martínez-Castillo, J. et al., Gene flow and genetic structure in the wild-weedy-domesticated complex of Phaseolus lunatus L. in its Mesoamerica center of domestication and diversity, Crop Sci., 47, 58, 2007. 44. Wilson, H.D., Gene flow in squash species, Bioscience, 40, 449, 1990. 45. Keese, P., Risks from GMOs due to horizontal gene transfer, Environ. Biosafety Res., 7, 123, 2008. 46. Tricoli, D., Petition for determination of nonregulated status: Squash containing the coat protein genes from Cucumber mosaic virus (CMV), Watermelon mosaic virus 2 (WMV 2) and Zucchini yellow mosaic virus (ZYMV), 1995, http://www.aphis.usda.gov/brs/ aphisdocs/95_35201p.pdf (accessed April 20, 2011). 47. Vigne, E., Komar, V., and Fuchs, M., Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus, Transgenic Res., 13, 165, 2004. 48. Lemaux, P.G., Genetically engineered plants and foods: A scientist’s analysis of the issues (Part I), Annu. Rev. Plant Biol., 59, 771, 2008. 49. Atherton, K.T., Safety assessment of genetically modified crops, Toxicology, 181–182, 421, 2002. 50. Lack, G., Clinical risk assessment of GM foods, Toxicol. Lett., 127, 337, 2002. 51. Kuiper, H.A. and Kleter, G.A., The scientific basis for risk assessment and regulation of genetically modified foods, Trends Food Sci. Tech., 14, 277, 2003. 52. Mills, E.N.C. et al., Food allergens of plant origin—Their molecular and evolutionary relationships, Trends Food Sci. Tech., 14, 145, 2003. 53. Organization for Economic Cooperation and Development (OECD), Safety considerations of foods derived by modified biotechnology: Concepts and principles. OECD, Paris, France, 1993. http://www.oecd.org/pdf/M00007000/M00007573.pdf (accessed December 1, 2008). 54. Constable, A. et al., History of safe use as applied to the safety assessment of novel foods and foods derived from genetically modified organisms, Food Chem. Toxicol., 45, 2513, 2007. 55. Chassy, B. et al., Nutritional and safety assessments of foods and feeds nutritionally improved through biotechnology: An executive summary, Compr. Rev. Food Sci. Food Saf., 3, 25, 2004.

284

Transgenic Horticultural Crops: Challenges and Opportunities

56. Deng, P. et al., Edible safety requirements and assessment standards for agricultural genetically modified organisms, Food Chem. Toxicol., 46, 1414, 2008. 57. European Food Safety Agency, Report of the GMO Panel Working Group on animal feeding trials, safety and nutritional assessment of GM plants and derived food and feed: The role of animal feeding trials, Food Chem. Toxicol., 46, S2, 2008. 58. Craig, W. et al., An overview of general features of risk assessments of genetically modified crops, Euphytica, 64, 853, 2008. 59. Fuchs, R.L. et al., Safety assessment of the neomycine phosphotransferase II (NPTII) protein, Biotechnology, 11, 1543, 1993. 60. Gilissen, L.J.W. et al., Biosafety of E. coli β-glucuronidase (GUS) in plants, Transgenic Res., 7, 157, 1998. 61. Ramessar, K. et al., Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: A case of the science not supporting the politics, Transgenic Res., 16, 261, 2007. 62. Shelton, A.M., Fuchs, M., and Shotkoski, F.A., Transgenic vegetables and fruits for control of insects and insect-vectored pathogens. In Integration of Insect-Resistant Genetically Modified Crops within IMP Programs, Romeis, J., Shelton, A.M., and Kennedy, G.G., eds., Springer, Dordrecht, the Netherlands, 2008, pp. 249–272. 63. Novak, W.K. and Haslberger, A.G., Substantial equivalence of antinutrients and inherent plant toxins in genetically modified foods, Food Chem. Toxicol., 38, 473, 2000. 64. Koenig, A. et al., Assessment of the safety of foods derived from genetically modified (GM) crops, Food Chem. Toxicol., 42, 1047, 2004. 65. Fitch, M.M.M. et al., Stable transformation of papaya via microprojectile bombardment, Plant Cell Rep., 9, 189, 1990. 66. Cai, W.Q. et al., A protocol for efficient transformation and regeneration of Carica papaya L., In Vitro Cell. Dev. Biol. Plant, 35, 61, 1999. 67. Chen, G. et al., Cloning of the Papaya ringspot virus (PRSV) replicase gene and generation of PRSV resistant papayas through the introduction of the PRSV replicase gene, Plant Cell Rep., 20, 272, 2001. 68. Tennant, P., Ahmad, M.H., and Gonsalves, D., Transformation of Carica papaya L. with virus coat protein genes for studies on resistance to Papaya ringspot virus from Jamaica, Trop. Agric., 79, 105, 2002. 69. Dahleen, L.S. and Manoharan, M., Recent advances in barley transformation, In Vitro Cell. Dev. Biol., 43, 493, 2007. 70. Shewfelt, R.L., Sources of variation in the nutrient content of agricultural commodities from the farm to the consumer, J. Food Qual., 13, 37, 1990. 71. Hileman, R.E. et al., Bioinformatic methods for allergenicity assessment using a comprehensive allergen database, Int. Arch. Allergy Immunol., 128, 280, 2002. 72. Herman, R.A., Storer, N.P., and Gao, Y., Digestion assays in allergenicity assessment of transgenic proteins, Environ. Health Persp., 114, 1154, 2006. 73. Roberts, M. et al., Assessment of compositional changes during ripening of transgenic papaya modified for protection against papaya ringspot virus, J. Sci. Food Agri., 88, 1911, 2008. 74. Bau, H.-J. et al., Potential threat of a new pathotype of Payapa leaf distortion mosaic virus infecting transgenic papaya resistant to Papaya ringspot virus, Phytopathology, 98, 848, 2008. 75. Zagrai, I. et al., Plum pox virus silencing of C5 transgenic plums is stable under challenge inoculation with heterologous viruses, J. Plant Pathol., 90, S1.63, 2008. 76. McDonald, B.A. and Linde, C., The population genetics of plant pathogens and breeding strategies for durable resistance, Euphytica, 124, 163, 2002.

Virus-Resistant Transgenic Horticultural Crops

285

77. García-Cano, E. et al., Synergistic interaction between Tomato chlorosis virus and Tomato spotted wilt virus results in breakdown of resistance in tomato, Phytopathology, 96, 1263, 2006. 78. Tennant, P. et al., Papaya ringspot virus resistance of transgenic Rainbow and SunUp is affected by gene dosage, plant development, and coat protein homology, Eur. J. Plant Pathol., 107, 645, 2001. 79. De Haan, P. et al., Characterization of RNA-mediated resistance to Tomato spotted wilt virus in transgenic tobacco plants, Biotechnology, 10, 1133, 1992. 80. Tripathi, S. et al., The ability of Papaya ringspot virus strains overcoming the transgenic resistance of papaya conferred by the coat protein gene is not correlated with higher degrees of sequence divergence from the transgene, Eur. J. Plant Pathol., 110, 871, 2004. 81. Bau, H.-J. et al., Broad-spectrum resistance to different geographic strains of Papaya ringspot virus in coat protein gene transgenic papaya, Phytopathology, 93,112, 2003. 82. Thakur, R.P., Host plant resistance to diseases: Potential and limitations, Ind. J. Plant Protect., 35, 17, 2007. 83. Klas, F.E., Fuchs, M., and Gonsalves, D., Comparative spatial spread overtime of Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV) in fields of transgenic squash expressing the coat protein genes of ZYMV and WMV, and in fields of nontransgenic squash, Transgenic Res., 15, 527, 2006. 84. Fuchs, M., Chirco, E.M., and Gonsalves, D., Movement of coat protein genes from a commercial virus-resistant transgenic squash into a wild relative, Environ. Biosafety Res., 3, 5, 2004. 85. Fuchs, M. et al., Comparative fitness of a wild squash species and three generations of hybrids between wild x virus resistant transgenic squash, Environ. Biosafety Res., 3, 17, 2004. 86. Quemada, H. et al., Population size and incidence of virus infection in free-living populations of Cucurbita pepo, Environ. Biosafety Res., 7, 185, 2008. 87. Fuchs, M., Gal-On, A., Raccah, B., and Gonsalves, D., Epidemiology of an aphid nontransmissible potyvirus in fields of nontransgenic and coat protein transgenic squash, Transgenic Res., 99, 429, 1999. 88. Lin, H.X. et al., Genetic diversity and biological variation among California isolates of Cucumber mosaic virus, J. Gen. Virol., 84, 249, 2001. 89. Davis, M.J. and Ying, Z.T., Development of papaya breeding lines with transgenic resistance to Papaya ringspot virus, Plant Dis., 88, 353, 2004. 90. Fermin, G. et al., Engineered resistance against PRSV in Venezuelan transgenic papayas, Plant Dis., 88, 516, 2004. 91. Hautea, R., Chan, Y.K., Attathom, S., and Krattiger, A., The papaya biotechnology network of Southeast Asia: Biosafety considerations and papaya background information. ISAAA, Brief no. 11, International Service for the Acquisition of Agri-Biotech Applications, Ithaca, NY, 1999. 92. Lines, R.E. et al., Genetically engineered immunity to Papaya ringspot virus in Australia papaya cultivars, Mol. Breeding, 10, 119, 2002. 93. Sakuanrungsirikul, S. et al., Update on the development of virus-resistant papaya: Virus-resistant transgenic papaya for people in rural communities of Thailand, Food Nutr. Bull., 26, 422, 2005. 94. Souza, M.T. Jr., Nickel, O., and Gonsalves, D., Development of virus resistant transgenic papayas expressing the coat protein gene form a Brazilian isolate of Papaya ringspot virus, Fitopathol. Bras., 30, 357, 2005. 95. Manshardt, R.M., ‘UHRainbow’ papaya, in University of Hawaii College of Tropical Agriculture and Human Resources Germplasm. P G1-2, 1998.

286

Transgenic Horticultural Crops: Challenges and Opportunities

96. Mutsuga, M. et al., Comparison of carotenoid components between GM and non-GM papaya, J. Food Hyg. Soc. Jpn., 42, 367, 2001. 97. Chan, J.T. et al., Sugar composition of papayas during fruit development, Hortic. Sci., 14, 140, 1979. 98. Bari, L. et al., Nutritional analysis of two local varieties of papaya (Carica papaya L.) at different maturation stages, Pak. J. Biol. Sci., 9, 137, 2006. 99. Yamamoto, H.Y., Comparison of the carotenoids in yellow- and red-fleshed Carica papaya, Nature, 201, 1049, 1964. 100. Umoh, I.B., Commonly used fruits in Nigeria. In Nutritional Quality of Plant Foods, Osagie, A.U. and Eka, O.U., eds., Post Harvest Research Unit, Dept. of Biochemistry, University of Benin, Benin City, Nigeria, 1998, p. 134. 101. Xiangdong, W. et al., Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya, Front Biol. China, 2, 284, 2007. 102. Powell, M. et al., Effects of subchronic exposure to transgenic papayas (Carica papaya L.) on liver and kidney enzymes and lipid parameters in rats, J. Sci. Food Agr., 88, 2638, 2008. 103. Caribbean Food and Nutrition Institute (CFNI), Food composition tables for use in the English speaking Caribbean supplement. Pan American Health Organization. J7, 5, 2000. 104. World Health Organization/Environmental Monitoring System/Food Contamination Monitoring and Assessment Programme (WHOGEMS)/Food, Regional diets, Regional per capita consumption of raw and semi-processed agricultural commodities/Global Environment Monitoring System/Food Contamination Monitoring and Assessment Programme. World Health Organization, Geneva, Switzerland, 2003, pp. 1–27. 105. Hsieh, Y.-T. and Pan, T.-M., Influence of planting Papaya ringspot virus resistant transgenic papaya on soil microbial biodiversity, J. Agric. Food Chem., 54, 130, 2006. 106. Wei, X.D. et al., Field released papaya affects microbial communities and enzyme activities in soil, Plant Soil, 285, 347, 2006. 107. Lo, C.-C., Chen, S.-C., and Yang, J.-Z., Use of real-time polymerase chain reaction (PCR) and transformation assay to monitor the persistence and bioavailability of transgenic genes released from genetically modified papaya expressing nptII and PRSV genes in the soil, J. Agric. Food Chem., 55, 7534, 2007. 108. Chen, Z.-L. et al., Safety assessment for genetically modified sweet pepper and tomato, Toxicology, 188, 297, 2003. 109. Garcia-Arenal, F. and Palukaitis, P., Cucumber mosaic virus. In Encyclopedia of Virology, 3rd edn., Mahy, B.W.J. and Van Regenmortel, M.V.H, eds., Elsevier, San Diego, 2008, vol. 1, pp. 614–619. 110. Ming, X.T. et al., Field release and biosafety assessment of transgenic plants, JIRCASInt. Symp. Ser., 5, 279, 1997. 111. Cai, W.Q. et al., Development of CMV- and TMV-resistant chili pepper: Field performance and biosafety assessment, Mol. Breeding, 11, 25, 2003. 112. Capote, N. et al., A review of Plum pox virus, Bulletin OEPP/EPPO, 36, 201, 2006. 113. Ravelonandro, M. et al., The use of transgenic fruit trees as a resistance strategy for virus epidemics: The plum pox (sharka) model, Virus Res., 71, 63, 2000. 114. Scorza, R. et al., Transgenic plums (Prunus domestica L.) express the Plum pox virus coat protein gene, Plant Cell Rep., 14, 18, 1994. 115. Scorza, R. et al., Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene, Transgenic Res., 10, 201, 2001. 116. Kundu, J.K. et al., Role of the 25–26 nt siRNA in the resistance of transgenic Prunus domestica graft inoculated with Plum pox virus, Virus Genes, 36, 215, 2008.

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117. Hily, J.-M. et al., Stability of gene silencing-based resistance to Plum pox virus in transgenic plum (Prunus domestica L.) under field conditions, Transgenic. Res., 13, 427, 2004. 118. Polák, J. et al., Behaviour of transgenic Plum pox virus-resistant Prunus domestica L. clone C5 grown in the open field under a high and permanent infection pressure of the PPV-Rec strain, J. Plant Pathol., 90, S1.33–S1.36, 2008. 119. Malinowski, T. et al., Field trials of plum clones and transformed with the Plum pox virus coat protein (PPV-CP) gene, Plant Dis., 90, 1012, 2006. 120. Scorza, R., Application for determination of non-regulated status for C5 (‘HoneySweet’) resistant to Plum pox virus, 2004, http://www.aphis.usda.gov/brs/aphisdocs/04_2641p.pdf (accessed December 1, 2008). 121. Capote, N. et al., Assessment of the diversity and dynamics of Plum pox virus and aphid populations in transgenic European plums under Mediterranean conditions, Transgenic Res., 17, 367, 2008. 122. Widmer, F., Assessing effects of transgenic crops on soil microbial communities, Adv. Biochem. Engin./Biotechnol., 107, 207, 2007. 123. Information Systems for Biotechnology (ISB). Field test release application in the USA, National resource in agbiotech information, 2008, http://www.isb.vt.edu/ (accessed December 1, 2008). 124. Bucher, E.C. et al., Multiple virus resistance at a high frequency using a single transgene construct, J. Gen. Virol., 87, 3697, 2006. 125. Waterhouse, P.M., Graham, M.W., and Wang, M.-B., Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA, Proc. Natl. Acad. Sci. USA, 95, 13959, 1998. 126. Smith, N.A. et al., Total silencing by intron-spliced hairpin RNAs. Nature, 407, 319, 2000. 127. Li, Z.T., Jayasankar, S., and Gray, D.J., Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco, Transgenic Res, 13, 143, 2004. 128. Snove, J. and Holen, T., Many commonly used siRNAs risk off-target activity, Biochem. Biophys. Res. Commun., 319, 256, 2004. 129. Niu, Q.-W. et al., Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance, Nat. Biotechnol., 24, 1420, 2006. 130. Qu, J., Ye, J., and Fang, R.X., Artificial microRNA-mediated virus resistance in plants, J. Virol., 81, 6690, 2007. 131. Garcia, J.A. and Simón-Mateo, C., A micropunch against plant viruses: Artificial microRNAs show promise for combating viral infections in plants, Nat. Biotechnol., 24, 1358, 2006. 132. Nölke, G., Fischer, R., and Shillberg, S. Antibody-based pathogen resistance in plants, J. Plant Pathol., 86, 5, 2004. 133. Fuchs, M. et al., Safety assessment of transgenic plums and grapevines expressing viral coat protein genes: New insights into real environmental impact of perennial plants engineered for virus resistance, J. Plant Pathol., 89, 5, 2007. 134. Davidson, S.N., Forbidden fruit: Transgenic papaya in Thailand, Plant Physiol., 147, 487, 2008. 135. Fermin, G. et al., Comparative development and impact of transgenic papayas in Hawaii, Jamaica and Venezuela. In Methods in Molecular Biology, 286, Transgenic Plants: Methods and Protocols, Peña, L., ed., Humana Press, Totowa, NJ, 2004, pp. 399–430. 136. Gonsalves, C., Lee, D.R., and Gonsalves, D., The adoption of genetically modified papaya in Hawaii and its implications for developing countries, J. Dev. Stud., 43, 177, 2007.

14

Molecular Approaches for Transgene Containment and Their Potential Applications in Horticultural Crops Yi Li and Hui Duan

CONTENTS Challenges to Transgenic Horticultural Crops........................................................ 289 Candidate Molecular Strategies Addressing Concerns over Transgenic Horticultural Crops............................................................................... 290 Male and Female Sterility..................................................................................290 Genesafe Technologies....................................................................................... 293 Parthenocarpy Technology................................................................................. 294 Chloroplast Transformation............................................................................... 295 Gene Deletor Technology................................................................................... 296 Prospectives............................................................................................................ 299 Acknowledgments................................................................................................... 299 References............................................................................................................... 299

CHALLENGES TO TRANSGENIC HORTICULTURAL CROPS Horticultural crops were the first commercialized transgenic plants in the United States. From 1994 to 1995, Flavr Savr tomato, Endless Summer tomato, and virus-resistant squash were marketed.1,2 A tomato paste (puree) produced from a transgenic processing tomato was the best-selling paste in 1999 and 2000 in the United Kingdom. Despite these early commercial successes, the number of transgenic horticultural crops currently marketed in the United States is very small, limited to papayas, sweetcorn, squash, and carnations.1,2 Also, the economic impact of transgenic horticultural crops is minimal. Except papaya, transgenic horticultural crops have had very small market shares.3,4 The well-known estimate that 70% of food products in U.S. supermarkets contain transgenic crop ingredients is due to the widespread use of transgenic corn, canola, and soybean products in virtually all processed foods.1 289

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There are a number of challenges for commercialization of transgenic horticultural crops. The biggest obstacle is probably high costs associated with food and environmental safety testings required by federal agencies. While traditionally bred crop plants are not subjected to any testing or regulations, testing required for a deregulation approval of a transgenic crop is extensive, and can cost $10–20 million per novel gene in a crop variety.5 Because each horticultural plant species has many varieties and each variety represents a small market value, recovering high costs associated with the required testing can be a challenge. As a consequence, commercial horticultural companies are not enthusiastic to pursue transgenic horticultural crops.1 Another hurdle for commercialization of transgenic horticultural crops is environmental and food safety concerns.4 One example is transgenic glyphosate-resistant creeping bentgrass. The herbicide-resistant bentgrass should make weed control more effective and also enhance the uniformity, quality, aesthetics, and playability of golf course turf.6 Studies by the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) suggest that (1) there appear no major unintended effects resulting from the introduction of the glyphosate resistance gene into the creeping bentgrass genome, (2) the transgenic bentgrass is not sexually compatible with any federally acknowledged threatened or endangered species or with any species on the federal noxious weed list, and (3) the transgenic bentgrass does not differ in pest and pathogen susceptibility or resistance from its parent.7 However, Watrud8 reported that pollen of the herbicide-resistant transgenic creeping bentgrasses traveled up to 21â•›km and the pollen could fertilize wild bentgrasses and a close relative, redtop (Agrostis gigantean). Reichman9 detected nine herbicide-resistant creeping bentgrass plants that were found up to 3.8â•›km beyond the control area, and they therefore concluded that there were both seed dispersal and pollen-mediated crossing with wild creeping bentgrass. Although the herbicide-resistant creeping bentgrass displays no increase in vegetative spread or relative fitness,10 the public opposition to deregulation of the herbicide-resistant bentgrass is strong. It appears that the transgenic herbicide-resistant bentgrass has a long way to go before it is marketed.6

CANDIDATE MOLECULAR STRATEGIES ADDRESSING CONCERNS OVER TRANSGENIC HORTICULTURAL CROPS Gene-based technologies that can reduce environmental and food safety concerns over transgenic plants could facilitate commercialization of transgenic horticultural crops. A number of transgene containment technologies that may reduce gene flow have been reported.11–15 Some of these technologies should also be useful to address food safety concerns on transgenic horticultural crops. In this chapter, we will provide a brief review on some of these molecular technologies and discuss their potential applications and limitations in horticultural crops.

Male and Female Sterility Male sterility is defined as the inability of pollen to fertilize the ovum, and female sterility is defined as the inability of the female organ to be pollinated or to produce seeds upon pollination. In both cases, sterility can be caused by inadequacy in

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structure or function of the genital organs. To create sterility in higher plants, male and female organs have been targeted for manipulation. Several types of male and female sterility technologies have been developed.14,16,17 The first group of sterility genes is cytotoxin genes. Cytotoxin genes used for creating sterility include ribonuclease genes such as the bacterial barnase gene cloned from a soil bacterium, Bacillus amyloliquefaciens,16,18–24 diptheria toxin A chain (DTA; ADP-ribosyl-transferase),25,26 and the ribosome inactivating protein (RIP).27 Mariani et al.23 used a tobacco tapetum-specific gene promoter (TA29) to control the expression of the bacterial barnase gene in tobacco and oilseed plants and induced male sterility. Expression of the barnase gene destroyed the tapetal cells and successfully prevented pollen formation, thereby producing male-sterile plants. Because the expression of the barnase gene was restricted to tapetum tissues, vegetative growth and floral development of the transgenic plants were normal. Similarly, female sterility has been achieved using the same barnase gene. Goldman28 used a stigmatic secretory zone-specific gene promoter sequence, the 5′ untranslated region of the STIG1 gene cloned from tobacco plants, to drive the expression of the barnase gene. The pistils of transgenic plants that expressed the STIG1::barnase gene underwent normal development but lacked the stigmatic secretory zone and therefore became female sterile. The pollen grains of the transgenic plants could germinate on the ablated stigmatic surface although they failed to penetrate the transmitting tissue of the style, suggesting that pollen was normal. Using a developing seed-specific gene promoter, the FBP7 gene promoter cloned from petunia, to control the expression of the barnase gene in tobacco plants, Colombo et al.29 were able to generate transgenic plants that produced no ovules or seeds. The FBP7 promoter was specifically active in the coat of developing seeds and is completely silent in the gametophytically derived tissues. Because normal seeds were produced if wild-type plants were pollinated with transgenic plants, the pollen of the FBP7::barnase plants was fertile. A large body of experimental evidence has demonstrated that the bacterial barnase gene is highly effective to produce sterile plants when expressed in reproductive organs. However, barnase expression sometimes also results in deleterious effects on vegetative growth of transgenic plants if its expression is leaky and affects nontarget organs.30 The bacterial barstar protein, a potent inhibitor of barnase, has been used successfully to restore fertility of barnase-mediated sterility23 although the efficacy of the barstar gene can vary.30,31 Also, there are concerns that the bacterial barnase protein may be toxic to humans and animals because barnase can be a poison for human and animal cells.32–34 Even though it is unclear whether minute amounts of the bacterial barnase protein present in transgenic crops can be of harm to humans and animals, the use of a barnase gene in edible horticultural crops should be avoided. For nonedible horticultural crops in which seeds or fruits are not used for human and animal consumption, the barnase gene would provide an outstanding tool for inducing sterility. A second group of genes that can be used to produce sterility is one involved in specific metabolic pathways in higher plants. For example, inhibition of pyruvate dehydrogenase production in mitochondria resulted in male sterility.35 Tissue-specific repression of an extracellular invertase, Nin88, caused male sterility.21 Disruption of

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flavonoid biosynthesis through manipulation of chalcone synthase gene expression could lead to male sterility.36,37 Overexpression of β-ketothiolase via integration of the transgenes into the chloroplast genome of tobacco plants produced male sterile phenotype.38 Expression of a bacterial enoyl-CoA hydratase/lyase enzyme, which led to re-routing of the phenylpropanoid pathway, and an unedited atp9 gene, a protein involved in the proton channel of ATP synthase, also caused male sterility.39–43 Compared to the use of the barnase gene, food safety concerns over transgenic plants can be minimized if these metabolic genes are used to create sterility. However, the effectiveness of using metabolic genes to create sterility is much lower than that of using the barnase gene. Also, the efficacy of the metabolic genes can be plant species dependent. Pilot studies to test the effectiveness of a chosen metabolic gene to produce sterility in a plant species of interest should always be carried out before it is used as a sterile gene tool in that particular crop. The third group of useful genes to create sterility involves hormone biosynthesis, catabolism or signaling pathways in higher plants. Plant hormones play an important role in the reproductive organ development and altering expression of genes involved in hormone levels or response in reproductive organs often can lead to sterility. For instance, an inhibition of expression of an ethylene-forming enzyme, l-aminocyclopropane-l carboxylate oxidase, in pistils was effective in disrupting ovule development and therefore caused female sterility.44 Transgene-mediated reduction in jasmonic acid concentration in anthers led to male sterility due to defects in anther and pollen development.45,46 Expression of the rolC gene, cloned from Agrobacterium rhizogenes, resulted in male sterility and reduced female fertility, presumably due to the RolC-mediated changes in hormone sensitivity or concentration in reproductive organs.47 Tissue-specific overexpression of a cytokinin oxidase (CKX1) gene involved in cytokinin degradation in transgenic maize (Zea mays) also produced male-sterile plants.48 Similarly, expression of a mutant version of a gibberellins receptor gene (gai) in anthers and pollen of tobacco and Arabidopsis caused the abortion of these reproductive organs.48 Male infertility or abnormal male organ development as a result of expression of the CKX1 could be restored by applications of exogenous cytokinins. Similar to the Group 2 genes discussed earlier, using hormone-related genes should be more acceptable by the public regarding food safety issues. However, the effectiveness of these hormone genes can vary from one plant species to another, and may be further influenced by environmental factors. A fourth group of genes that can be used to produce sterility is homeotic genes, important for floral organ development. Genetic and molecular studies demonstrate that homeotic genes act alone and together to specify the fate of floral organ primordia in higher plants (see reviews by Ferrandiz,49 Lohmann and Weigel,50,51 Ma,52 Weigel and Meyerowitz,53 and Zik and Irish.54). Sterility can be achieved by ectopic expression of homeotic genes. For example, overexpression of the Brassica napus AGAMOUS gene, which regulates the development of stamens and pistils, leads to the conversion of stamens into carpel-like structures in transgenic tobacco plants.55 Expression of a tobacco AGAMOUS homolog, TAG1, resulted in the replacement of stamens with petaloid organs and the conversion of pistils to nonreproductive organs.56 In both cases, the transgenic plants produced were male sterile. On the other hand, Mitsuda et al.57 used a Chimeric REpressor Gene-Silencing Technology

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(CRES-T) to repress expression of four transcription factor genes, namely APETALA3, AGAMOUS, LEAFY, and AtMYB26 that are involved in the control of floral meristem and organ identities, and the regulation of anther dehiscence, respectively. Transgenic plants expressing each chimeric silencer gene construct were sterile, and resembled the loss-of-function phenotype of each corresponding gene. One advantage of using these floral organ genes to create sterility is that the products of floral organ development genes have no toxic effects on human and animal health and therefore there should be few concerns if used in edible horticultural crops. However, ectopic expression of floral organ development genes frequently leads to alterations in floral morphology. For horticultural crops, alteration in floral organ morphology can be desirable or undesirable depending on plant species and applications.

Genesafe Technologies Genesafe technologies, also called genetic use restriction technologies (GURTs), involve the use of genetic switch mechanisms to control expression of a toxin gene in seeds (see review by Kausch et al.14). It is an extension of sterility technologies discussed in the “Male and female sterility” section of this chapter. The best example of the genesafe technologies is the one invented by Oliver et al.,58 many called it the “terminator” seed technology. The strategy uses a late embryogenesis specific gene promoter to control the expression of a lethal gene, the bacterial barnase gene. However, the barnase gene is interrupted by a short blocking DNA sequence so that it is inactive. The blocking DNA sequence is flanked by two loxP sequences in the same orientation. LoxP is the recognition sequence of the Cre recombinase from the bacteriophage P1 loxP/Cre DNA recombination system.59,60 The interrupted lethal gene is introduced into one parental plant line. The Cre gene that is under the control of a chemically induced gene promoter such as the tetracycline inducible promoter is introduced into another parental line. Both of these plant lines can produce viable seeds if self-pollinated. If the lethal/barnase line and the Cre line are hybridized, their progeny should contain both the chemically inducible Cre gene and the interrupted barnase gene that is under the control of a late-embryogenesis active gene promoter. If hybrid seeds produced from the cross of these two plant lines are treated with the chemical inducer, the Cre enzyme will be produced in seeds and the interrupting DNA sequence should be deleted. As a consequence, excision of the interrupting DNA sequence should make the barnase gene functional in mature embryos during the seed development. If these resulting seeds are used for planting, when the plants derived reach the reproductive stage, the lethal gene should be expressed specifically in mature embryos. The barnase gene expression in mature embryo will cause the death of embryo and thus prevent seed germination. Similar to the “terminator” seed technology, Kuvshinov et al.61 described a “recoverable block of function” (RBF) technology. A lethal gene, also the barnase gene, was driven by a germination-specific promoter, and the barstar (an inhibitor of barnase) gene was placed under the control of a heat-shock promoter. Under natural conditions, the barnase gene will be expressed during seed germination but the barstar gene will be inactive because the heat-shock promoter is not induced. The expression of the barnase gene in germinating seeds will prevent seed germination.

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However, when needed, seed germination and seedling growth can be restored through a heat shock treatment of germinating seeds. Thus, the RBF system could be a useful tool to reduce seed-mediated escape of transgenes. As represented by the “terminator” seed and RBF systems, genesafe technologies are conditional seed sterility technologies. For the “terminator” seed technology, one important requirement is high efficiencies of the chemical-induced removal of the blocking DNA sequence from the barnase gene. On the other hand, high efficiency of the barstar gene in the RBF system is not critical. Regarding the food safety concerns, it should be noted that the barnase gene used in the two genesafe systems discussed in “Male and female sterility” section may be replaced with a gene whose product is nontoxic to humans or animals.

Parthenocarpy Technology Parthenocarpy (seedlessness) is the production of normal-sized fruits without fertilization and the fruit produced contains no embryo and endosperm. Fruit development in higher plants normally requires pollination, fertilization, and seed development that stimulate cell division of specific floral and fruit tissues. Traditionally, parthenocarpic fruits are produced from mutants, triploid plants, or flowers treated with exogenous growth regulators.62 Transgenic parthenocarpy was reported in auxinoverproducing transgenic petunia63 and in tobacco plants64 using the iaaM gene cloned from Agrobacterium tumefaciens.63 With ovary- or fruit-specific gene promoters to direct expression of the iaaM gene or RolB gene, seedless fruits were produced from eggplant, tobacco, tomato, and watermelon.65–69 Rotino69 reported that transgenic tobacco and eggplants that expressed the coding region of the iaaM gene from Pseudomonas syringae pv. savastanoi, driven by an ovule-specific DefH9 gene promoter from Antirrhinum majus, produced parthenocarpic fruits. If flowers were emasculated, seedless fruits were produced from these transgenic plants. If pollinated, the plants produced fruits containing seeds. In the case of eggplant, the expression of the iaaM transgene enabled fruit set and growth under environmental conditions where no fruit setting was observed in the wildtype control plants. Under normal growth conditions, the transgenic plants produced marketable fruits from pollinated or unpollinated transgenic flowers, but the control plants produced fruits of marketable size only if flowers were fertilized.69 We have used two types of gene promoters to control the expression of the Agrobacterium iaaM gene. One is the auxin inducible, ovary active GH3 promoter cloned from soybean70,71 and another is the ovary specific AGL5 promoter.72 Using these two iaaM fusion genes, we produced transgenic Arabidopsis, tobacco, tomato, and watermelon plants.68 Transgenic plants that expressed the GH3 promoter::iaaM or AGL5 promoter-iaaM gene were capable of producing seedless fruits. Also, because overproduction of auxin in the transgenic plants was restricted to the ovary and developing fruit, no obvious side effects were evident. The seedless tomato and watermelon fruits produced from the transgenic plants contained similar levels of acids and sugars, vitamins, and other nutrients relative to the seeded fruits produced from control plants. Furthermore, the seedless tomatoes and watermelons were normal in size or sometimes were significantly larger than those produced from the

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wild-type control plants grown under identical conditions, although fruit productivity per plant was not significantly altered. Seeds can be produced from many naturally or transgenically produced seedless fruit plants if pollination is enforced. To have a total sterility of seedless fruit plants, we have also constructed a number of gene cassettes that can lead to both male and female sterility and also production of normal sized fruits with no seeds.67 We used reproductive organ-specific gene promoters to control the expression of a cytotoxin gene, the bacterial barnase gene for the initial testing, to achieve both male and female sterility. We used an ovary-specific gene promoter to control overproduction of auxin for seedless fruit production.68 With a combination of the male- and female-sterility gene and the seedless fruit gene, we have demonstrated that transgenic tobacco and tomato plants are totally sterile (both male and female sterile) and also capable of producing normal sized fruit with no seeds.67 Nonhormonal genes have also been shown to be capable of producing seedless fruits. For example, Yao et al.73 have shown that the loss of function mutation in the MdPI MADS-box transcription factor, a homolog of Arabidopsis mutant pistillata, confers parthenocarpic fruit development in apple plants. Ampomah-Dwamena74 reported that repression of a tomato MADS box gene, TM29, using either cosuppression or antisense techniques, resulted in infertile stamens and ovaries and subsequently led to the production of parthenocarpic fruits. However, these transgenic tomato plants also produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Ito and Meyerowitz75 reported that overexpression of the cytochrome P450 gene could lead to production of parthenocarpic fruits. Wild-type Arabidopsis fruits developed to normal size only if the ovules were fertilized. When expression of the cytochrome P450 gene was hyperactivated, fruits grew without fertilization and reached nearly normal size. When wild-type pollen was used to pollinate the cytochrome P450 overexpressing plants, the pollinated fruits became more than 10% longer and 40% wider than wild-type fruits but they produced very few seeds. Seedless fruit technologies can be used to reduce seed-mediated transgene escape from horticultural crops if seeds of these plant species are of little value. Since seeds can be produced from seedless fruit plants if pollination is enforced, seedless fruit technologies can be used in both vegetatively and sexually propagated crops. Further, the use of a seedless fruit technology should lead to improvement of fruit quality and productivity.68,69,76

Chloroplast Transformation Chloroplasts of higher plants are semiautonomous organelles with a small, highly polyploid genomes and their own transcription–translation machinery.77 In most flowering plants, chloroplasts are inherited from their female parents although for some plant species such as pines their chloroplasts are inherited from pollen.78,79 For the plant species whose chloroplasts are inherited only from the female, if transgenes are intergrated into the chloroplast genome, pollen produced from the resulting transgenic plants should be transgene free. In the case of tobacco plants with transgenes inserted into chloroplast gemone, for instance, only three out of 1,000,000 pollen grains contain transgenes.78

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Chloroplast transformation offers several advantages.77,80–82 First, each plant cell contains a large number of chloroplasts, with some having up to 100 chloroplasts per cell.83 Also, each chloroplast contains 50–100 or more copies of its own genome84. As a consequence, if a trait gene is incorporated into the chloroplast genome, the gene can be enriched up to 100,000 copies per each cell. High copy number of transgenes per cell leads to high expression levels. For example, 47% leaf total proteins were the Bt cry2Aa2 protein if the Bt cry2Aa2 gene was introduced into the choloroplast genome of tobacco.85 Second, transgene expression via chloroplast transformation is more stable because gene silencing that can affect expression of nuclear transgenes appears to have little effect on transgenes that are introduced into the chloroplast genome.86 Third, for plant species in which chloroplasts are inherited exclusively through the female line, chloroplast transformation can provide an effective means of preventing transgenes from transferring to non-transgenic crops or wild relatives through pollen. In spite of the technical challenges in transforming chloroplasts, stable incorporation of trait genes into chloroplast genomes has been reported in Arabidopsis thaliana, cotton, poplar, rice, and soybean.87,88 Also, chloroplast transformation has been described in a number of horticultural crops such as cabbage,89 cauliflower,90 carrot,91 egg plant,92 lettuce,93,94 bladderpod,95 petunia,96 tobacco,97–100 potato,101 and tomato.102–104 Genetic transformation of chloroplasts should provide an effective tool to reduce pollen-mediated transgene flow in these horticultural species whose chloroplasts are inherited from female parents. On the other hand, problems associated with the seed-mediated transgene escape will not be addressed with a chloroplast transformation strategy.

Gene Deletor Technology Gene deletor technology refers to techniques that can eliminate all transgenes in a particular organ or in the entire plant when transgene functions are completed or the transgene presence is of concern.105–108 The principle of the gene deletor technology is illustrated in Figure 14.1. All transgenes, such as trait and selection marker genes, are placed within the two loxP-FRT hybrid sites. In addition, the FLP or Cre recombinase gene under the control of a conditionally active (e.g., organ- or tissuespecific, or developmental stage specific) gene promoter sequence is inserted into the two loxP-FRT sites. If a pollen and seed-specific gene promoter such as PAB5 from Arabidopsis109 is used to control the expression of the FLP gene, the FLP protein will be produced only in pollen and seed. We have shown that transgenic plants have transgenes present and functional in all organs during the plant life cycle except in seed and pollen. Because of pollen- and seed-specific FLP expression, all transgenes except an 86-bp nonprotein encoding loxP-FRT fusion sequence are excised from pollen and seed. The excised DNA sequence, consisting of one loxP-FRT site, the trait gene, the selection marker gene, and the FLP gene should be destroyed by nonspecific nucleases in the cell.110,111 Site-specific recombination systems (e.g., bacterial phage Cre/loxP and SacchaÂ� romyces cerevisiae FLP/FRT) function through interactions of a recombinase with its specific recognition sites.112–115 Recombinase-mediated excision between directly oriented recognition sites results in removal of the intervening DNA leaving one

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Host plant genome

Non-transgenic plant Transformation using trait genes and a gene deletor cassette

Integration of gene deletor system and trait genes into host plant genome

Host plant genome

Transgenic plant hosting trait gene and a gene deletor system All transgenes including the gene deletor system are deleted from pollen and seed if FLP is expressed in pollen and seed specifically

All transgenes including the gene deletor system are deleted from the entire plant if FLP is conditionally expressed in all cells

Host plant genome

Organ-specific or conditionally inducible expression of FLP

loxP-FRT

loxP-FRT

Deletion of all transgenes in cells where FLP is expressed Excised transgenes destroyed in the cell

Pollen and seed reverted back to non-transgenic

Entire plant reverted back to non-transgenic

Host plant genome with transgenes deleted in FLP expressed cells

FIGURE 14.1â•… (See color insert.) Schematic illustration of the principle of the gene deletor technology. On the left, an application of the gene deletor technology to generate non-transgenic pollen, seed, or plant from a transgenic plant is illustrated. The schematics presented on the right show an FLP-mediated transgene deletion at the DNA level. Any transgenes, such as trait genes, selection marker gene, and FLP or Cre recombinase gene that have been inserted into the two loxP-FRT sites (86 bp in length), will be deleted from any cell, in which the FLP recombinase is expressed. When a pollen- and seed-specific gene promoter is used to control recombinase expression, all functional transgenes are deleted from these specific organs. If a conditionally inducible gene promoter, such as chemically or high-temperature inducible, is used to control recombinase expression, the system will delete all transgenes throughout the plant upon induction. (Modified from Luo, K. et al., Plant Biotechnol. J., 5, 263, 2007; Moon, H.S. et al., Trends Biotechnol., 28, 3, 2010.)

recognition site intact;116–120 FLP/FRT and Cre/loxP function through interactions of a recombinase with its specific recognition sites.116–120 Traditionally, site-specific DNA recombination systems, such as bacterial phage Cre/loxP and Saccharomyces cerevisiae FLP/FRT systems, have been used to excise marker genes or short spacer sequences in higher plants.114,121 Recombinase recognizes these flanking recognition sites and excises any intervening DNA. Further, in addition to DNA recombination systems, other site-specific DNA excision systems can also be used in the gene deletor technology. With a combined use of loxP and FRT sequences as the flanking sites for FLP or Cre recombinase, we have observed high efficiency for deleting all functional

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transgenes from pollen and/or seed of transgenic tobacco plants.105 Based on analysis of more than 25,000 progeny for each representative transgenic event, efficiencies for automatic deletion of all transgenes from pollen and/or seed are as high as 100% under greenhouse conditions. Also, we have demonstrated that the high excision efficiency trait remained stable in vegetatively propagated progeny plants. The current version of the gene deletor technology is readily applicable to address the pollenand seed-mediated transgene flow problems of vegetatively propagated plants that are important to the agricultural, forestry, bioenergy, ornamental, and paper/pulping industries. This technology can also be used directly in sexually propagated crops if artificial seeds (i.e., no pollination or fertilization is needed) are used for the propagation of these crops. Artificial seeds have been produced from many major agricultural crops.122 A modified version of the current gene deletor technology could be used in sexually propagated horticultural crops such as tomato, lettuce, eggplants, etc. One possible strategy is to introduce a chemically inducible RNAi-FLP gene cassette into the gene deletor system. Application of a chemical inducer at the correct time would prevent the deletion of transgenes in pollen and seeds. For instance, the ethanol inducible ALCR/alcA (alc) two-component system123 could be used to control RNAi-FLP gene expression. With the ALCR/alcA (alc) system, application of ethanol during pollen/seed development would activate the RNAi-FLP gene. RNAi-FLP represses the pollen/seed-specific expression of FLP recombinase. As a result, all transgenes will remain in the pollen or seeds during that generation. However, if the inducer is not applied in a subsequent generation, FLP will be expressed. Consequently, FLP expression will lead to deletion of all functional transgenes in pollen and seeds. The gene deletor technology may be extended to generate non-transgenic edible parts from transgenic plants to mitigate consumer concerns over the presence of transgenes in food products. For instance, in the case of the transgenic processing tomatoes grown in California, the final products, tomato paste, ended up being exported to many countries around the world.2 Approvals prior to exporting the tomato paste to these countries had to be obtained from each of these countries. If the processed product contains viable seeds, environmental studies and approvals are then required in the importing country, even if the importation is intended only for food consumption. A fruit-specific gene deletor system to eliminate all transgenes from fruit tissues might help to simplify some of these procedures and thus reduce associated costs. With desirable organ- and tissue-specific gene promoters or developmental stage specifically or conditionally inducible gene promoters to control the FLP gene expression, it is possible to obtain non-transgenic organs from transgenic plants or to revert the transgenic plants back to “non-transgenic” plants when needed. Volunteer crops are plants growing from seeds that are left in the field from a previous planting. Volunteer transgenic plants are a source of perceived or actual environmental risks and have resulted in lawsuits from neighboring farms or seed companies. Because the gene deletor technology can remove all functional transgenes from pollen and seeds, volunteer plants in subsequent seasons will then be non-transgenic. Deleting all transgenes from pollen, seeds or other organs once their functions are completed probably is one of the best transgene containment concepts, but efficiency

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of the gene deletor systems under field environments and in agricultural crops remains to be demonstrated. While the gene deletor technology should be readily used in vegetatively propagated crops, its application in sexually propagated plants requires further modifications and refinements.

PROSPECTIVES Transgenic technology will undoubtedly play an important role in genetic improvement of horticultural crops in the future. As discussed earlier even though transgenic horticultural crops are among the first group of commercialized transgenic crops, the market values of transgenic horticultural crops is insignificant.1,2 In recent years, the potential utilities of many trait genes have been well demonstrated in plants including in some horticultural crops (see other chapters of this book); their applications in horticultural plants remain uncertain because of high costs associated with deregulation approvals and environmental and food safety concerns. If the safety concerns over transgenic horticultural crops can be reduced and if the costs associated with the regulatory approval for deregulation of a transgenic horticultural crop can also be reduced, commercialization of transgenic horticultural crops should be accelerated. Thus, it is crucial to develop and use highly reliable gene-based technologies to help minimize transgene flow or food safety concerns over transgenic horticultural crops and to help reduce costs associated with their deregulation approvals. Finally, we would like to end this chapter with quotation from a report by The National Research Council124 on biological confinement of genetically engineered organisms: “Making biosafety a primary goal from the start of any project will be a more effective and efficient way to prevent safety failures and it will increase commercial investment ratings and reduce financial risks posed by possible liability claims and loss of consumer confidence.” “Each (gene containment) method has its strengths and weaknesses, and all vary in efficacy depending on circumstances. No one method will achieve 100% confinement in the real world.” “Therefore, it is sometimes necessary to employ more than a single method. In many technological applications, the principle of redundancy reduces the occurrence of predictable hazards while achieving the benefits of technological application.”

ACKNOWLEDGMENTS The work described in the Yi Li Laboratory at the University of Connecticut has been supported by grants from USDA, DOE, New England Invasive Plant Center, and the Storrs-Connecticut Agriculture Experiment Station. The authors would like to thank K. S. Cao for his help in editing references cited in this chapter.

REFERENCES

1. Clark, D., Klee, H., and Dandekar, A. Despite benefits, commercialization of transgenic horticultural crops lags. Calif. Agric., 58, 89, 2004. 2. Redenbaugh, K. and McHughen, A. Regulatory challenges reduce opportunities for horticultural biotechnology. Calif. Agric., 58, 106, 2004.

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3. Alston, J., Bradford, K., and Kalaitzandonakes, N. The economics of horticultural biotechnology. J. Crop Improv., 18(1/2), 413, 2006. 4. Bradford, K. J. et al. Challenges and opportunities for horticultural biotechnology. Calif. Agric., 58(2), 68, 2004. 5. Bradford, K. J. et al. Regulating transgenic crops sensibly: Lessons from plant breeding, biotechnology and genomics. Nat. Biotechnol., 23, 439, 2005. 6. Jones, P. B. C. Approval for genetically engineered bentgrass creeps through agency turfs. ISB News Report, http://isb.vt.edu/articles/jan0504.htm, 2005. (Accessed January 31, 2011). 7. Meilan, R. Challenges to commercial use of transgenic plants. J. Crop Improv., 18(1/2), 433, 2006. 8. Watrud, L. S. et al. Evidence for landscape-level, pollen-mediated gene flow from genetically modified creeping bentgrass with CP4 EPSPS as a marker. Proc. Natl. Acad. Sci. USA, 101, 14533, 2004. 9. Reichman, J. R. et al. Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats. Mol. Ecol., 15, 4243, 2006. 10. Gardner, D. S., Danneberger, T. K., and Nelson, E. K. Lateral spread of glyphosateresistant transgenic creeping bentgrass (Agrostis stolonifera) lines in established turfgrass swards. Weed Technol., 18, 773, 2004. 11. Daniell, H. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol., 20, 581, 2002. 12. Gressel, J. and Al-Ahmad., H. Molecular containment of genes within crops, prevention of gene establishment in volunteer offspring and feral strains. In Gressel, J., ed. Crop Ferality and Volunteerism. CRC Press, Boca-Raton, FL, pp. 371, 2005. 13. Hills, M. J. et al. Genetic use restriction technologies (GURTs): Strategies to impede transgene movement. Trends Plant Sci., 12, 177, 2007. 14. Kausch, A. P. et al. Transgenic perennial biofuel feedstocks and strategies for bioconfinement. Biofuels, 1(1), 163, 2010. 15. Stewart, C. N. Jr. Biofuels and biocontainment. Nat. Biotechnol., 25, 283, 2007. 16. Goldberg, R. B., Beals, T. P., and Sanders, P. M. Anther development: Basic principles and practical applications. Plant Cell, 5, 1217, 1993. 17. Schnable, P. S. and Wise, R. P. The molecular basis of cytoplasmic male sterility. Trends Plant Sci., 3,175, 1998. 18. Hartley, R. W. Barnase and Barstar: Expression of its cloned inhibitor permits expression of a cloned ribonuclease. J. Mol. Biol., 202(4), 913, 1988. 19. De Block, M. and Debrouwer, D. RNAN–RNA in situ hybridization using digoxigeninlabeled probes: The use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. Anal. Biochem., 215, 86, 1993. 20. Denis, M. et al. Expression of engineered nuclear male sterility in Brassica napus L. genetics, morphology, cytology and sensitivity to temperature. Plant Physiol., 101, 1295, 1993. 21. Goetz, M. et al. Induction of male sterility in plants by metabolic engineering of the carbohydrate supply. Proc. Natl. Acad. Sci. USA, 98, 6522, 2001. 22. Lemmetyinen, J. et al. Prevention of flower formation in dicotyledons. Mol. Breeding, 7, 341, 2001. 23. Mariani, C. et al. Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature, 347, 737, 1990. 24. Zhan, X. Y., Wu, H. M., and Cheung, A. Y. Nuclear male sterility induced by pollenspecific expression of a ribonuclease. Sex. Plant Reprod., 9, 35, 1996. 25. Kandasamy, M. K. et al. Ablation of papillar cell function in Brassica flowers results in the loss of stigma receptivity to pollination. Plant Cell, 5, 263, 1993. 26. Thorsness, M. K. et al. A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana. Dev. Biol., 143, 173, 1991.

Molecular Approaches for Transgene Containment

301

27. Cho, H. J. et al. Production of transgenic male sterile tobacco plants with the cDNA encoding a ribosome inactivating protein in Dianthus sinensis L. Mol. Cells, 11, 326, 2001. 28. Goldman, M. H., Goldberg, R. B., and Mariani, C. Female sterile tobacco plants are produced by stigma-specific cell ablation. EMBO J., 13, 2976, 1994. 29. Colombo, L. et al. Downregulation of ovule-specific MADS box genes from petunia results in maternally controlled defects in seed development. Plant Cell, 9, 703, 1997. 30. Mourgues, F., Brisset, M. N., and Cheveau, E. Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends Biotechnol., 16, 203, 1998. 31. Wei, H. et al. Transgenic sterility in populus: Expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S promoters in vegetative tissues. Tree Physiol., 26, 401, 2006. 32. Ilinskaya, O. N. and Vamvakas, S. Nephrotoxic effects of bacterial ribonucleases in the isolated perfused rat kidney. Toxicology, 120, 55, 1997. 33. Leuchtenberger, S. et al. Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells. Nucleic Acids Res., 29, E76, 2001. 34. Prior, T. I., Kunwar, S., and Pastan, I. Studies on the activity of barnase toxins. Bioconjug. Chem., 7, 23, 1996. 35. Yui, R. et al. Antisense inhibition of mitochondrial pyruvate dehydrogenase E1a subunit in anther tapetum causes male sterility. Plant J., 34, 57, 2003. 36. van Tunen, A. J. et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: Coordinate, light-regulated and differential expression of flavonoid genes. EMBO J., 7, 1257, 1988. 37. van der Meer, I. M. et al. Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell, 4, 253, 1992. 38. Ruiz, O. N. and Daniell, H. Engineering cytoplasmic male sterility via the chloroplast genome by expression of {beta}-ketothiolase. Plant Physiol., 138, 1232, 2005. 39. Hernould, M. et al. Male-sterility induction in transgenic tobacco plants with an unedited atp9 mitochondrial gene from wheat. Proc. Natl. Acad. Sci. USA, 90(6), 2370, 1993. 40. Hernould, M. et al. Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants. Plant Mol. Biol., 36, 499, 1998. 41. Mayer, J. The golden rice controversy: Useless science or unfounded criticism? Bioscience, 55, 726, 2005. 42. Tsuchiya, T. et al. Tapetum-specific expression of the gene for an endo-beta-1,3-glucanase causes male sterility in transgenic tobacco. Plant Cell Physiol., 36, 487, 1995. 43. Worrall, D. et al. Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Plant Cell, 4, 759, 1992. 44. De Martinis, D. and Mariani, C. Silencing gene expression of the ethyleneforming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants. Plant Cell, 11, 1061, 1999. 45. Park, J. H. et al. A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis. Plant J., 31, 1, 2002. 46. Sanders, P. M. et al. The Arabidopsis DELAYED DEHISCENCE 1 gene encodes an enzyme in the jasmonic acid synthesis pathway. Plant Cell, 12, 1041, 2000. 47. Schmulling, T. et al. Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants. Mol. Gen. Genet., 237, 385, 1993. 48. Huang, C. Y. et al. Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature, 422, 72, 2003. 49. Ferrandiz, C., Pelaz, S., and Yanofsky, M. F. Control of carpel and fruit development in Arabidopsis. Annu. Rev. Biochem., 68, 321, 1999. 50. Lohmann, J. U. and Weigel, D. Building beauty: The genetic control of floral patterning. Dev. Cell, 2, 135, 2002.

302

Transgenic Horticultural Crops: Challenges and Opportunities

51. Lohmann, J. U. and Weigel, D. From tough nuts to touch-me-nots. Cell, 116, 763, 2004. 52. Ma, H. Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants. Annu. Rev. Plant Biol., 56, 393, 2005. 53. Weigel, D. and Meyerowitz, E. M. The ABCs of floral homeotic genes. Cell, 78, 203, 1994. 54. Zik, M. and Irish, V. F. Flower development: Initiation, differentiation, and diversification. Annu. Rev. Cell Dev. Biol., 19, 119, 2003. 55. Mandel, M. A. et al. Manipulation of flower structure in transgenic tobacco. Cell, 71, 133, 1992. 56. Pnueli, L. et al. Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. Plant Cell, 6, 163, 1994. 57. Mitsuda, N. et al. Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice. Plant Biotechnol. J., 4, 325, 2006. 58. Oliver, M. J. et al. Control of plant gene expression. U.S. Patent, 5, 723, 1998. 59. Austin, S., Ziese, M., and Sternberg, N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell, 25, 729, 1981. 60. Sauer, B. Identification of cryptic lox sites in the yeast genome by selection for Cremediated chromosome translocations that confer multiple drug resistance. J. Mol. Biol., 223, 911, 1992. 61. Kuvshinov, V. V. et al. Molecular control of transgene escape from genetically modified plants. Plant Sci., 160, 517, 2001. 62. Varoquaux, F. et al. Less is better: New approaches for seedless fruit production. Trends Biotechnol., 18, 233, 2002. 63. Klee, H. J. et al. The effects of overproduction of two Agrobacterium tumefaciens TDNA auxin biosynthetic gene products in transgenic petunia plants. Genes Dev., 1, 86, 1987. 64. Guilfoyle, T. J. et al. Auxin-regulation transcription. Aust. J. Plant Physiol., 20, 489, 1993. 65. Acciarri, N. et al. Genetically modified parthenocarpic eggplants: Improved fruit productivity under both greenhouse and open field cultivation. BMC Biotechnol., 2, 4, 2002. 66. Carmi, N. et al. Induction of parthenocarpy in tomato via specific expression of the rolB gene in the ovary. Planta, 217, 726, 2003. 67. Chen, Y. et al. Biotech approach to neutralize the invasiveness of burning bush (Euonymus alatus), a progress report on development of its genetic transformation system and functional analysis of sterile genes. Acta Hort., 769, 10, 2008. 68. Li, Y. Transgenic seedless fruit and methods. U.S. Patent, 6, 268, 552, 1998. 69. Rotino, G. L. et al. Genetic engineering of parthenocarpic plants. Nat. Biotechnol., 15, 1398, 1997. 70. Hagen, G. et al. Auxin-induced expression of soybean GH3 promoter in transgenic tobacco plants. Plant Mol. Biol., 17, 567, 1991. 71. Li, Y. et al. Expression of the GH3/GUS gene as a molecular marker for auxin physiology. Plant Cell Physiol., 40, 675, 1999. 72. Sieburth, L. E. and Meyerowitz, E. M. Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell, 9, 355, 1997. 73. Yao, J., Dong, Y., and Morris, B. A. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. Proc. Natl. Acad. Sci. USA, 98, 1306, 2001. 74. Ampomah-Dwamena, C. et al. Down-regulation of TM29, a tomato SEPALLATA homolog, causes parthenocarpic fruit development and floral reversion. Plant Physiol., 130, 605, 2002. 75. Ito, T. and Meyerowitz, E. M. Overexpression of a cytochrome P450, CYP78A9, induces large and seedless fruit in Arabidopsis. Plant Cell, 12, 1541, 2000. 76. Pandolfini, T., Molesini, B., and Spena, A. Molecular dissection of the role of auxin in fruit initiation. Trends Plant Sci., 12, 327, 2007.

Molecular Approaches for Transgene Containment

303

77. Maliga, P. Plastid transformation in higher plants. Annu. Rev. Plant Biol., 55, 289, 2004. 78. Ruf, S., Karcher, D., and Bock, R. Determining the transgene containment level provided by chloroplast transformation. Proc. Natl. Acad. Sci. USA, 104 (17), 6998, 2007. 79. Stegemann, S., Hartmann, S., Ruf, S., and Bock, R. High-frequency gene transfer from the chloroplast genome to the nucleus. Proc. Natl. Acad. Sci. USA, 100, 8828, 2003. 80. Bock, R. Transgenic plastids in basic research and plant biotechnology. J. Mol. Biol., 312, 425, 2001. 81. Grevich, J. J. and Daniell, H. Chloroplast genetic engineering: Recent advances and future perspectives. Crit. Rev. Plant Sci., 24, 83, 2005. 82. Sharma, K. K., Bhatnagar, P., and Thorpe, T. A. Genetic transformation technology: Status and problems. In Vitro Cell Dev. Biol. Plant, 41, 102, 2005. 83. Martin, W. and Borst, P. Secondary loss of chloroplasts in trypanosomes. Proc. Natl. Acad. Sci. USA, 100, 765, 2003. 84. Franklin, S. E. and Mayfield, S. P. Prospects for molecular farming in the green alga Chlamydomonas. Curr. Opin. Plant Biol., 7, 159, 2004. 85. De Cosa, B. et al. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol., 19, 71, 2001. 86. van Bel, A. J. E. et al. Novel approach in plastid transformation. Curr. Opin. Biotechnol., 12, 144, 2001. 87. Koop, H. U. et al. The genetic transformation of plastids. Top. Curr. Genet., 19, 457, 2007. 88. Wang, H. H., Yin, W. B., and Hu, Z. M. Advances in chloroplast engineering. J. Genet. Genomics, 36, 387, 2009. 89. Liu, C. W. et al. Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep., 26, 1733, 2007. 90. Nugent, G. D. et al. Nuclear and plastid transformation of Brassica oleracea var. Botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts. Plant Sci., 170, 135, 2006. 91. Kumar, S., Dhingra, A., and Daniell, H. Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiol., 136, 2843, 2004b. 92. Singh, S., Verma, S., and Bansal, K. C. Plastid transformation in eggplant (Solanum melongena L.). J. Transgenic Res., 19, 113, 2010. 93. Lelivelt, C. et al. Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol. Biol., 58, 763, 2005. 94. Kanamoto, H. et al. Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res., 15, 205, 2006. 95. Skarjinskaia, M., Svab, Z., and Maliga, P. Plastid transformation in esquerella Fendleri, an oilseed brassicacea. Transgenic Res., 12, 115, 2003. 96. Zubko, M. K. et al. Stable transformation of petunia plastids. Transgenic Res., 13, 523, 2004. 97. Koop, H. U. et al. Integration of foreign sequences into the tobacco plastome via polyethylene glycol-mediated protoplast transformation. Plants, 199, 193, 1996. 98. O’Neillt, C. et al. Chloroplast transformation in plants: Polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems. Plant J., 3, 729, 1993. 99. Svab, Z., Hajdukiewicz, P., and Maliga, P. Stable transformation of plastids in higher plants. Proc. Natl. Acad. Sci. USA, 87, 8526, 1990. 100. Svab, Z. and Maliga, P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA, 90, 913, 1993. 101. Sidorov, V. A. et al. Technical advance: Stable chloroplast transformation in potato: Use of green fluorescent protein as a plastid marker. Plant J., 19, 209, 1999. 102. Ruf, S. et al. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol., 19, 870, 2001.

304

Transgenic Horticultural Crops: Challenges and Opportunities

103. Nugent, G. D. et al. Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep., 24, 341, 2005. 104. Wurbs, D., Ruf, S., and Bock, R. Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J., 49, 276, 2007. 105. Luo, K. et al. GM-gene-deletor: Fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol. J., 5, 263, 2007. 106. Li, Y., Duan, H., and Smith, W. Gene-deletor: A new tool to address concerns over GE crops. USDA Information Systems for Biotechnology News Report, June 2007. 107. Mlynarova, L., Conner, A. J., and Nap, J. P. Directed microspecific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes. Plant Biotechnol. J., 4, 445, 2006. 108. Moon, H. S., Li, Y., and Stewart, C. N. Keeping the genie in the bottle: Transgene biocontainment by excision in pollen. Trends Biotechnol., 28, 3, 2010. 109. Belostotsky, D. A. and Meagher, R. B. A pollen-, ovule-, and early embryo-specific poly (A) binding protein from Arabidopsis complements essential functions in yeast. Plant Cell, 8, 1261, 1996. 110. Wilson, C. M. Plant nucleases. Annu. Rev. Plant Physiol., 26, 187, 1975. 111. Srivastava, V. and Ow, D. W. Rare instances of Cre-mediated deletion product maintained in transgenic wheat. Plant Mol. Biol., 52, 661, 2003. 112. Chen, Y. and Rice, P. A. New insight into site-specific recombination from Flp recombinase-DNA structures. Annu. Rev. Biophys. Biomol. Struct., 32, 135, 2003. 113. Lyznik, L. A., Gordon-Kammm, W. J., and Taom, Y. Site-specific recombination for genetic engineering in plants. Plant Cell Rep., 21, 925, 2003. 114. Ow, D. W. Recombinase-directed plant transformation for the post-genomic era. Plant Mol. Biol., 48, 183, 2002. 115. van Duyne, G. D., A structural view of cre-loxP site-specific recombination. Annu. Rev. Biophys. Biomol. Struct., 30, 87, 2001. 116. Bayley, C. C. et al. Exchange of gene activity in transgenic plants catalysed by the Crelox site specific recombination system. Plant Mol. Biol., 18, 353, 1992. 117. Dale, E. C. and Ow, D. W. Gene transfer with the subsequent removal of the selection gene from the host genome. Proc. Natl. Acad. Sci. USA, 88, 10558, 1991. 118. Gilbertson, L. Cre-lox recombination: Cre-active tools for plant biotechnology. Trends Biotechnol., 21, 550, 2003. 119. Odell, J. et al. Site-directed recombination in the genome of transgenic tobacco. Mol. Gen. Genet., 223, 369, 1990. 120. Russell, S. H., Hoopes, J. L., and Odell, J. T. Directed excision of a transgene from the plant genome. Mol. Gen. Genet., 234, 49, 1992. 121. Ow, D. W. GM maize from site-specific recombination technology, what next? Curr. Opin. Biotechnol., 18, 115, 2007. 122. Fujii, J. A. et al. Artificial seeds for plant propagation. Trends Biotechnol., 5, 335, 1987. 123. Caddick. M. X. et al. An ethanol-inducible gene switch for plants used to manipulate carbon metabolism. Nat. Biotechnol., 16, 177, 1998. 124. National Research Council, Biological Confinement of Genetically Engineered Crops. Washington, DC: The National Academies Press, 2004.

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Prospects for the Commercialization of Transgenic Ornamentals Michael S. Dobres

CONTENTS Introduction............................................................................................................. 305 Patents, Plants, and Costs........................................................................................307 Field Testing and Deregulation...............................................................................309 Global Distribution and Deregulation..................................................................... 311 Consumer Acceptance............................................................................................. 313 References............................................................................................................... 314

INTRODUCTION Over the past 20 years, there has been an explosion in the range of technologies available to breeders of commodity crops. This has included the use of molecular markers1 and transgenic technologies2 to bring about significant increases in crop yields. Despite this availability and a significant amount of academic and industrial experimentation, these technologies have not been effectively applied to the development and commercialization of ornamental plants. This essay seeks to explain the commercial and business reasons behind this apparent missed opportunity. In the United States, the entire wholesale value of the ornamental market exceeds $8 billion annually.3 The market can be divided into several major product sectors including cut flowers, potted plants, as well as all categories of garden and landscape plants (trees, shrubs and herbaceous annuals, and perennials). In the nursery and landscape industry, there is significant consumer and grower demand for lowmaintenance plants. This includes plants with improved disease resistance, drought tolerance, and cold and heat tolerance as well as plants with an extended seasonal flowering period. Disease and insect resistance traits have the potential to reduce the environmental and human health impact of gardening and landscaping in terms of reduced pesticide and chemical input, as has been reported for the use of transgenic commodity crops.4,5 Traits such as compact habit and improved branching could reduce the time and labor needed to maintain landscaped areas. Compared to the achievements of conventional breeders, genetic engineers have a tough act to follow. For centuries, breeders of ornamental plants have pursued genetics that confer improved disease resistance, improved habit, more flowers, extended 305

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flowering time range, increased branching, and better shelf life. However, once created, these improvements are almost always restricted to the genus, sometimes restricted to the species, and occasionally restricted to the variety in which they are bred. Commercial examples of shrubs and herbaceous perennials displaying some of the above listed traits have been and continue to be developed by conventional breeding. These include The Knock Out® Rose (U.S. patent PP11836), Endless Summer Hydrangea (U.S. plant patent 15298), and Salvia ‘May Night.’ The Knock Out Rose is highly resistant to black spot, is drought tolerant, and flowers from early summer to fall. Endless Summer Hydrangea blooms throughout the summer into late fall, and Salvia ‘May Night’ is repeat blooming and highly drought tolerant. The market success of these examples demonstrates that there is indeed a strong demand market for these traits. At the same time, it emphasizes that the technology used to develop new varieties needs to be comparable in cost to conventional approaches or needs to confer traits unobtainable by conventional breeding. Transgenic technology is unique among other breeding tools in its ability to precisely move single traits over large genetic distances. Although crossing the species and the genus barrier has been achieved by conventional breeding on numerous occasions,6,7 genetic engineering differs in its ability to move specific genes across greater distances: between families, between divisions, and between kingdoms. Transferring genes for color, fragrance, and other desirable traits to species devoid of such traits represents a prime target for genetic engineering.8 To date, only Suntory has commercialized a transgenic ornamental in the form of cut carnations in the United States, Europe, and Japan9 and more recently lavendercolored “blue” cut roses in Japan.10 Given the dearth of commercial exploitation in ornamentals, it is ironic that one of the first published examples of a transgenic plant involved petunia.11 Since then, there has been considerable excitement about the prospect for improving ornamental crops and other specialty food and nonfood crops through genetic engineering. Several companies were founded either partially or solely on the concept of developing and commercializing genetically engineered ornamental plants. Companies included NovaFlora Inc., Sanford Scientific, Calgene Pacific, and Florigene NV. Many established companies formed their own genetic engineering groups. These included Scotts, Ball Seeds, Syngenta, Suntory, and Kirin. Corporate and university-based activity in genetic engineering can be tracked by examining the online United States Department of Agriculture-Animal and Plant Health Inspection ServiceBiotechnology Regulatory Service (USDA-APHIS-BRS) movement and release permit database (www.aphis.usda.gov/biotechnology). More than 91 movement permits and 73 release permits (field trials) for ornamental genera have been issued. Permits were issued equally to both corporations and university and government institutions. The targets cover more than 10 genera of bedding plants, foliage plants, and shrubs. In the history of technology, this spurt of corporate and entrepreneurial interest in a new technology is fairly common. Similar entrepreneurial bursts have been seen in the advent of electrical, automotive, photographic, computer, and medical technology industries. As with other new technologies, the growth of commercial interest was matched with skepticism and sometimes fear. This has been true for the automotive industry, the computer industry as well as biotechnology and genetic engineering. Some of the above-mentioned “ornamental biotech” companies are still in existence.

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Others have long since gone out of business or have been absorbed into larger corporations or have curtailed or terminated their in-house programs. The main reason were cost of patents and increased regulatory oversight.12 Although there are significant costs in the development and introduction of conventionally bred ornamentals, these do not carry with them the same administrative and legalistic burden associated with the testing and government approval of transgenic ornamental crops. These issues are described in more detail in the following paragraphs. Despite the decrease in corporate expenditures, university and government funds are still used at a significant level for research centered around the development of genetically engineered ornamentals. This includes research on fragrance, flower form, and flower color.13,14

PATENTS, PLANTS, AND COSTS For genetic engineering, like any new and innovative technology, its success and persistence in the marketplace centers around the patent protection of new inventions and discoveries. The ornamental industry is no stranger to the legal protection of new inventions. Since 1930, new varieties of trees, shrubs, and herbaceous plants generated as spontaneous or induced mutations, or by cross-hybridization have been protected by Plant Patents.15 The Plant Patent Act provides patent protection for such clonally propagated varieties. This includes varieties propagated by grafting of scions onto rootstock, the direct rooting of cuttings, or the division of plants or bulbs. For recalcitrant varieties that are difficult or slow to propagate, this can also be �performed by tissue culture micropropagation. Others can breed with these varieties but cannot propagate them without a license from the patent owner. For the past 90 years, this method of protection has provided a simple, affordable, and enforceable means of protecting the inventive endeavors of hobby breeders, university researchers, and corporations alike. Ornamental varieties that are produced and/or propagated by seed can potentially be protected under the Plant Variety Protection (PVP) Act.16 In practice, however, novel ornamental F1 hybrids are not protected by their inventors. Instead the F1 parents are held by the inventors as a trade secret. Since a novel F1 variety will not breed true, the breeder has little to fear from a competitor or consumer who attempts to produce the new variety through seed. Most of the varieties protected as PVPs are pure breeding open pollinated varieties of grasses and vegetables (http://www.ars-grin.gov/cgi-bin/ npgs/html/pvplist.pl). The developer of F1 hybrid seed avoids the cost of patent protection, but in turn invests more in the development of the genetics required to develop true breeding parent lines needed for large scale F1 seed production. Production of hybrid seed is also more technically demanding and less predictable than asexually propagated varieties. In general, the ornamental industry has shifted away from F1 hybrid seed production and more and more towards vegetative propagation. Outside of the United States, new plant varieties can be protected under the International Convention for the Protection of New Varieties of Plants (UPOV) in any country that is a signatory of the 1991 convention.17 There are currently 67 member states, including countries in Europe, Asia, Africa, and the Americas. UPOV provides a codified system, whereby a developer can seek protection in parallel in multiple member states. This greatly simplifies the protection for a developer seeking to produce, distribute, and market new varieties in multiple member states.

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At first sight transgenic technology represents a rapid and simple way of modifying plant traits for the development of novel patentable varieties. At its simplest level, it allows the modification of traits by transfer of specific trait genes to a targeted variety. If the gene brings about a change in a protected variety, for example petal color, a plant patent in the United States could potentially protect that variety. However, in UPOV countries, the new variety would be viewed as essentially the same as the parent variety, or “essentially derived.” The developer of the transgenic variety would require a license from the owner of the original variety before commercialization. In practice, however, transgenic technology has proven to be both technically demanding and expensive, especially when the full range of intellectual property issues are considered. Unlike other breeding technologies, the transgenic approach requires use of multiple novel technologies, genes, and vectors each of which can be covered by a utility patent restricting the use of that technology in one or more territories. These patent-related issues have been discussed in detail in a previous review.12 In short, the breadth and strength of a patent can vary greatly. For example, engineering novel petal color might involve the use of a coding sequence for an enzyme involved in the formation of a novel pigment. In a hypothetical case, a broad patent covering all uses of the coding sequence to modify pigment formation may cover this coding sequence. In other cases the breadth of the patent might be restricted to a narrower subset of uses. For example, claims may only be granted for use of the coding sequence for its use in color modification of petals of a specific species. The developer and his/her patent counsel need to carefully read the patent, its claims, and the prosecution history to fully understand the breadth of a patent. If the developer’s use of the coding sequence falls outside the granted claims of the patent, the developer’s legal counsel may determine that no license is needed. Conversely, the patent may be sufficiently broad to make commercialization without a license from the patent holder impossible. In such cases, the developer needs to negotiate with the patent holder for a license. Licenses may also be required for the use of promoter and other regulatory sequences, as well as for selectable marker cassettes and vectors used in the plant transformation process. In addition to those covering trait gene technology, patents exist for all of the commonly used plant transformation methods. Although many of the major patents have recently expired, or are close to expiring, the existence of even a few patents for which licenses are required can add significantly to the cost of developing and commercializing a transgenic ornamental. Even if the dominant patent for a given transformation method has expired, there may be genus specific transformation patents covering the use of a specific transformation method in a particular genus. The situation is further complicated by complexities of patent law and the complex way in which a patent is issued and defined or claimed. In some cases a patent is so narrowly claimed that it is possible to easily circumvent the patent. However, to do this often requires engaging services of a skilled patent lawyer familiar with the technology field in hand. A full legal opinion can add tens of thousands of dollars to the cost of product development. Future prospects are, however, not quite so gloomy. Thus, within the next decade or so, most of the key enabling patents should expire and fall into the public domain and the prospect for developing transgenic ornamentals should improve. In addition, university and non-profit efforts such as the

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Public Sector Intellectual Property Resource (PIPRA) (www.pipra.org) and Cambia (www.cambia.org) should help facilitate access to the technology needed to develop transgenic ornamentals and other specialty crops.

FIELD TESTING AND DEREGULATION In addition to the complex intellectual property issues discussed above, an ornamental breeder needs to consider the increased burden associated with field-testing a transgenic variety. For conventionally bred varieties, the focus during fieldtesting involves evaluation of such characteristics as: phenotypic stability, hardiness, flowering time and length, diseases resistance, and in some cases invasiveness. The breeder needs to ensure that the variety will perform as expected in the market place. Ornamentals released in the United States have to perform well in a wide variety of conditions that range from the hot humid summers and cold damp winters of the East Coast to the year-round arid conditions of the Southwest. For this reason, developers often test in multiple locations. In some cases, this is organized through grower organizations such as the All America Rose Selections (AARS, http://www.rose.org/). The AARS organizes multi-site testing for new varieties developed by its members. New varieties are ranked based on disease resistance, ease of care, and beauty. The best varieties are awarded the prestigious AARS winners certificate. The developer uses the award to promote the variety to the trade and consumers. Before entering an AARS trial, developers perform their own evaluations for several years. Such field tests for conventionally bred ornamentals are often conducted under tight security to ensure the public and competitors cannot gain access to commercially valuable varieties before they are released or undergo industry trials. Transgenic landscape plants would, in addition to the private and public field evaluations, need to undergo studies on trans-gene flow and impact on insect populations. These studies would significantly add to the size and complexity of the field evaluations. Such studies are aimed at understanding the ecological benefits and/or risks of introducing the transgenic crop. For large acreage commodity crops, a great deal of scientific effort and literature has been devoted to the subject of gene flow and impact on nontarget organisms.18 Such data is used in the compilation and review of regulatory packages. The garden and landscape industry represents a special case. Plants are produced on a much smaller scale than commodity crops. For a popular perennial or shrub, this could be in the range of 100,000 to a million plants covering 0.5–10â•›ha. The final end use in the landscape might range from a single specimen plant to a few hundreds in a large municipal landscape planting. Hybridization and gene flow between native species is a naturally occurring event. Spontaneous interspecific hybrids are well documented in numerous ornamental genera.19 The exact level and frequency of outcrossing from a cultivated variety (whether transgenic or not) will, however, likely vary from species to species, and variety to variety. A 2005 study in Germany documented the level of outcrossing from non-transgenic hybrid roses to wild roses.20 The study made use of microsatellite markers to follow gene flow. The authors concluded that the frequency of outcrossing from cultivated to wild roses was rare. A developing body of similar literature is helping to define the level of regulatory oversight needed for transgenic ornamentals.21

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Studies on gene flow and impact on flora and fauna for a new transgenic crop are compiled and submitted as a petition for deregulation by the USDA. In addition to the detailed molecular and ecological studies required by the USDA, the Environmental Protection Agency (EPA) requires registration of what it defines as Plant Incorporated Protectants (PIP).12 A PIP is a “pesticidal substance that is produced in a living plant and the genetic material necessary for the production of the substance, where the substance is intended for use in the living plant.” This definition does not include genes moved into a variety through conventional breeding or cell culture techniques, but only covers genes transferred to a variety by genetic engineering. It also does not cover pesticidal genes transferred from closely related species. To conduct the necessary field experiment prior to filing for EPA approval and registration of a PIP containing variety, a developer must obtain an Experimental Use Permit (EUP) in addition to the USDA-APHIS permit. Final registration requires a commitment to post-registration monitoring and stewardship. For commodity crops registered to-date, this has included preparation of an Insect Resistance Management Plan (IRM) including the use of non-transgenic refuges to reduce the potential for insects resistant to the PIP. There are currently no examples of EPA approved PIPs in ornamental crops, and it is unclear what refuge and stewardship requirements would be required for transgenic ornamentals. Natural refuges would exist in all other nontransgenic plants in the surrounding landscape. Furthermore, EPA PIP registration for commodity crops is valid for about 3–5 years. This would not be practical for plants used in a garden or landscape environment. Would the consumer be expected to discard his/her aphid-resistant rose bush after 5 years? Perhaps one of the most significant differences in design of transgenic and nontransgenic field tests is that conventional field tests and trials are conducted in parallel on hundreds of candidate varieties. Commercial field-tests typically contain several hundred distinct varieties. Each variety may differ in one or more traits. It is fairly common for a developer to include lines selected from a broad array of parents that exhibit wide variation in color range, habit, and disease resistance traits in a single field-test location. In contrast, for transgenic varieties, gene flow studies and insect impact population studies are typically conducted on one variety at a time. In some cases, multiple lines each carrying a distinct gene insertion event may be characterized in parallel. This greatly increases the cost per variety for transgenic varieties compared to non-transgenic varieties. Many of the genes proposed for use in ornamentals are derived from plants. These include genes involved in pigment biosynthesis and genes conferring altered plant habit.8 The consequences of gene flow between related species of ornamentals will probably depend on the exact transgene in question. In a hypothetical case, would a gene derived from Arabidopsis and conferring dwarf phenotype22 confer any selective advantage if transferred from a transgenic hybrid rose to a multiflora rose? Would it make a difference if the gene were derived from rose instead of Arabidopsis? Indeed in 2007, the USDA proposed a tiered regulatory structure in which certain low-risk transgenics are regulated less stringently than higher risk transgenics.23 In this context, technical refinements may help lower the regulatory cost and burden. The development of native selectable markers,24 positive selection systems,25,26 marker removal systems,27 and marker-free systems is one approach.

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The development of cisgenic technology, in which all genes used in the gene transfer process are derived from the same genus as the target genus, is another approach.28,29 Other approaches that could be adopted by a breeder to mitigate risk and reduce costs include the careful selection of parental breeding lines. One approach would be to introduce the transgene into male and/or female sterile lines. Many varieties or ornamentals have been bred by conventional means to have full flowers with multiple whorls of petals but no male or female reproductive organs. Commercial examples include Impatiens ‘Cameo Pink Surprise’ (U.S. patent 13, 308), Petunia ‘Condowhite’ (U.S. patent 14, 509), and Begonia ‘Double White’ (U.S. patent 12, 674). Such transgenic versions of these sterile ornamentals would be unable to hybridize with wild or other related cultivated species, and would be unable to spread by seed dispersal. This approach, however, might be expensive from a regulatory approach, since every new variety would need to be separately deregulated. Thus, the U.S. system regulates transformation “events.” By first introducing the transgene into a good parent or breeding line, a high-expressing stable transgenic line can be characterized and deregulated and used as a parent line for subsequent breeding and development of new varieties in accordance with conventional breeding and evaluation practices. The two approaches could be potentially combined if the final variety created by the breeder were rendered sterile through use of male sterile lines or parents of different ploidy levels. Even if issues related to gene-flow could be addressed, there would still be costs related to monitoring and post-deregulation stewardship that could add significant costs to field trials and commercial release of transgenic varieties.

GLOBAL DISTRIBUTION AND DEREGULATION Difficulties also arise in the way that different regions and countries regulate transgenic crops. Thus, although many countries are signatories of the Cartagena Protocol on Biosafety,30 significant differences exist in the exact way in which regulatory policy is practiced and enforced. For example, in the European Economic Community (EEC), transgenic crops are not regulated based on the insertion events, but based on the variety.31,32 Thus, the EEC must separately approve two or more varieties derived from a single transformation event. Such differences in regional and national regulatory practices add a further level of cost and complexity to the development and commercialization of transgenic ornamentals and can impact the prospects for international distribution. This is discussed in more detail below. Varieties of bedding plants, perennials, shrubs, and cut flowers are produced, distributed, and sold to consumers in many different regions and countries. The supply chain for a new bedding plant variety typically starts in a low-cost production region such as Africa or Central America. From there, cuttings are shipped to rooting stations in Europe, North America, or Asia. These rooting stations, in turn, sell plant plugs to growers within that country or region. The grower then sells finished plants to garden centers and large chain retailers. Similarly, the supply chain for cut flowers starts in South America or North Africa for the United States or Europe, respectively. Cut flowers are harvested and shipped by overnight air-freight to major distribution centers in the United States and Europe. At this point commercial brokers are

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instrumental in distributing products to large regional wholesalers. The wholesalers distribute products to local florists and supermarkets. There are some exceptions to these general patterns. For example, a few internet-direct importers of cut flowers allow florists to ship directly from farms in South America to destinations in Europe and the United States. Some of the large chain retailers control their own distribution chain directly from Africa or South America to the United States or Europe. Certain locations, such as California or Holland, have either favorable climate or favorable infrastructure that allow for the production of cut flowers in the Northern Hemisphere. For international trade of conventionally bred varieties, growers routinely deal with the phytosanitary regulations required for the export and import of plant material. These are, for the most part, fairly similar from country to country. They are mainly written and enforced to ensure that plant material is pest and disease free as it travels from one country to another. For a transgenic variety, however, a developer would need to apply for government approval and permission to import and distribute in each member state or region of the supply chain. A considerable amount of time and effort would be required to identify the appropriate government agency, understand the authorization process, liaise with the appropriate government officials, and prepare the appropriate application documents required for government approval. For example, in a hypothetical case, starting in Costa Rica, a developer of a transgenic petunia would need to gain approval for import and growth of stock plants from the Costa Rican government. Costa Rica is one of the largest producers of un-rooted cuttings for the North American bedding plant industry. In Costa Rica, the National Technical Biosafety Commission (NTBC) regulates the production of transgenic crops.33 The NTBC has approved the production of transgenic cotton and soybean seeds and would presumably be amenable to the production of transgenic cuttings of ornamentals. Imports of such cuttings into the United States would require that the developer files a petition for deregulation with USDA-APHIS-BRS and the EPA, if the variety contained a PIP. If the developer wanted to ship transgenic petunia cuttings to Europe, he would be required to file and obtain approval for release of the variety throughout the EEC.31,32 In theory, a developer can file for approval in a single member state. If the variety is approved in that member state (e.g., Holland) and no other member state objects, the variety can be released and commercialized in all member states. Even if one member state objects, an opinion is sought from the European Food Safety Authority (EFSA) and used by the European Commission to approve or reject the application. For cut flower production and import into the United States, Columbia and Ecuador are the two main sites of production. The regulatory system in Columbia is more accommodating for the production of transgenic crops than Ecuador’s.34 A 2006 law in Ecuador prohibits the trade, use, and handling of transgenic products for human consumption. Though limited quantities of transgenic carnations have been produced in Ecuador, transgenic cotton is widely planted in Columbia, and the government and agricultural industry appear to be generally supportive of biotechnology.35 Similar technology driven import barriers exist for countries such as Japan36 and China.37 Thus import of cutting material for growth and sale in Japan would require

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approval by the Plant Safety Division of the Food Safety and Consumer Affairs Bureau of the Japanese Ministry of Agriculture, Forestry, and Fisheries (MAFF). Due to consumer and industry resistance, no transgenic crops have been commercially cultivated in Japan. This is despite the approval for import of more than 90 transgenic crop-derived products. In China, the application process appears to be more complicated than in most other countries. There are only two application deadlines per year. Before applying for approval in China, the transgenic variety must first be approved in the country of origin. Thereafter, it must be retested for the Chinese application. Overall a company seeking to commercialize a transgenic crop in one or more of the major markets of the developed world has to deal with the regulatory agencies not only in the country where the crop will be sold but also in any country in which the crop is produced. This presents a significant challenge in terms of the resources needed to compile the regulatory packages and communicate with the regulatory agencies in each of these countries. In some respects, the regulatory process is just as much about managing the perception of risk and the perception of benefits as it is about managing the risks and benefits themselves. In this regard effective communication between industry, the regulatory agencies, and the public is important. Furthermore, the effectiveness of the regulatory process will be aided by the growing body of literature on the ecological consequences and extent of gene flow for transgenic and non-transgenic crops. Additional funding to support basic research, and the dispersal of this research to industry and the public could substantially increase the prospects for the development of transgenic ornamentals for garden and landscape use. In the attempt to reduce the impact of the regulatory process on the development of ornamentals and other specialty crops the Specialty Crop Regulatory Assistance consortium (SCRA) was formed in 2005. SCRA is a public–private effort that is modeled somewhat on the Federal Drug Administration’s successful Orphan Drug Program. It plans to work alongside the existing regulatory agencies and their existing regulatory framework to facilitate passage of transgenic specialty crops through the regulatory process. This is expected to include clear delineation of regulatory requirements for specific crop-trait combinations, assistance in compiling regulatory packages, and general guidance for navigating the regulatory process (http://www. specialtycropassistance.org). The effort is currently seeking industry and government funding to run and maintain the necessary support programs. If successful, this effort could significantly increase the prospects for the development and commercialization of transgenic ornamentals and other specialty crops.

CONSUMER ACCEPTANCE Genetically engineered carnations, sold as cut flowers, have been marketed in Europe, the United States, and Japan for many years.9 There has been little adverse public reaction to these varieties. This bodes well for the marketing of transgenic landscape plants. Indeed, a 2004 survey of 602 Master Gardeners in Tennessee revealed that 73% of respondents reported an interest in buying transgenic ornamentals, if and when they become available.38 Overall respondents perceived slight environmental

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and human health benefits due to transgenic ornamentals. Respondents were particularly interested in modifications that confer increased disease resistance, pest resistance, plant longevity, and increased flowering time. When asked, respondents indicated a strong preference for the labeling of transgenic ornamentals. The authors of the study suggest that the results indicate that developers should emphasize the benefits gained, not the technology used to develop new varieties. Consumer and grower acceptance will also be influenced by any added cost related to the regulatory and patent hurdles discussed above. Will there be sufficient added value to justify the increased cost and can the developer pass the cost on to the grower and/or the consumer? If so, how will this be done? In commodity crops, such as herbicide-tolerant soybeans, this has been successfully accomplished by use of a technology fee. The farmer buys the seed and pays an additional fee in consideration of the additional cost and benefits of the herbicide tolerance gene technology contained within the seed.39 It is unlikely that such a fee-based system would be necessary or be practical for transgenic landscape plants. However, if the value added can be clearly demonstrated to the grower and consumer, it is possible that transgenic landscape plants could be marketed at a premium relative to existing varieties. Indeed, in the ornamental sector, it is not uncommon for new improved varieties (developed by conventional breeding) to carry a higher wholesale, royalty, and retail price than older established varieties. The changing structure of the industry itself may eventually favor the development of transgenic landscape plants. The past few years have seen tremendous consolidation in the ornamental industry.40 As a result of these consolidations, the number of breeding companies has decreased while their size has increased. This does not necessarily change the likelihood that the remaining large companies will adopt transgenic technologies. Most of the major ornamental companies have experimented with genetic engineering and most have shelved their efforts in transgenic ornamentals. Only Suntory has commercialized a transgenic product in the form of cut carnations in the United States, Europe, and Japan9 and more recently lavender-colored “blue” cut roses in Japan.10 In other words, the size of a company does not necessarily change the fundamental economics of commercializing transgenic ornamentals. Currently, the main factors influencing the prospects for the development of transgenic ornamentals are those related to regulatory oversight and those related to freedom to operate. Additional streamlining of the regulatory process and additional research aimed at a further assessment of the risks and benefits associated with transgenic ornamentals will help facilitate the commercialization and public acceptance of transgenic ornamentals.21,41 At the same time, the expiration of key enabling patents and the activities of organizations such as Cambia and PIPRA should help facilitate access by breeders to the necessary technology.

REFERENCES

1. Bernardo, R., Molecular markers and selection for complex traits in plants: Learning from the last 20 years, Crop Sci. 48(5), 1649, 2008. 2. Castle, L. A. and McElroy, D., Agricultural input traits: Past, present and future, Curr. Opin. Biotechnol. 17, 105, 2006.

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3. Jerado, A., Floriculture and nursery crops outlook, Report No. FLO-05, 2006. United States Department of Agriculture, Washington, DC. 4. Cattaneo, M. G. et al., Farm-scale evaluation of the impacts of transgenic cotton on biodiversity, pesticide use, and yield, Proc. Nat. Acad. Sci. USA 103(20), 7571, 2006. 5. Matin, Q. and Greg, T., Roundup ready soybeans in Argentina: Farm level and aggregate welfare effects, Agric. Econ. 32(1), 73, 2005. 6. Adams, R. P., Rushforth, K., and Trimble, S. N., The origins of Leyland’s cypreses (XCupressocyparis leyandii) based on DNA data, Phytologia 88(1), 1, 2006. 7. Furutaa, H. et al., Production of intergeneric somatic hybrids of chrysanthemum [Dendranthema × grandiflorum (Ramat.) Kitamura] and wormwood (Artemisia sieversiana J. F. Ehrh. ex. Willd) with rust (Puccinia horiana Henning) resistance by electrofusion of protoplasts, Plant Sci. 166(3), 695, 2004. 8. Tanaka, Y. et al., Genetic engineering in floriculture, Plant Cell Tissue Organ Cult. 80, 1, 2005. 9. Chandler, S., Genetic modifications in floral crops: Research to marketplace, Acta Hort. 738, 37, 2007. 10. Westcott, K., My love is like a blue, blue rose. Online at: http://news.bbc.co.uk/2/hi/ asia-pacific/8318511.stm, 2009 (accessed January 21, 2011). 11. Horsch, R. B. et al., A simple and general method for transferring genes into plants, Science 227, 1229, 1985. 12. Dobres, M., Barriers to genetically engineered ornamentals: An industry perspective, in Floriculture, Ornamental and Plant Biotechnology, Teixera da Silva, J. (ed.) Global Science Books, London, U.K., 2008, pp. 1–14. 13. Muñoz-Bertomeu, J., Ros, R., Arrillaga, I., and Segura, J., Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves, Metab. Eng. 10(3–4), 166, 2008. 14. Verdonk, J. C. et al., Flower-specific expression of the Agrobacterium tumefaciens isopentenyltransferase gene results in radial expansion of floral organs in Petunia hybrida, Plant Biotechnol. J. 6(7), 694, 2008. 15. Plant Patent Act, in USC Title 35, Chapter 15, 1930. Online at: http://uscode.house.gov/ download/pls/35C15.txt (accessed January 21, 2011). 16. The Plant Variety Protection Act, in USC Title 7, Chapter 57, 1970. Online at: http:// uscode.house.gov/download/pls/07C57.txt (accessed January 21, 2011). 17. Act of the international convention for the protection of new varieties of plants. International union for the protection of new varieties of plants. Online: http://www.upov. int/en/publications/conventions/1991/act1991.htm, 2009 (accessed January 21, 2011). 18. Chandler, S. and Dunwell, J., Gene flow, risk assessment and the environmental release of transgenic plants, Crit. Rev. Plant Sci. 27(1), 25, 2008. 19. Auer, C., Ecological risk assessment and regulation for genetically modified ornamental plants, Crit. Rev. Plant Sci. 27, 255, 2008. 20. Debener, T., The probability of outcrosses between cultivated and wild roses, in GMO Safety Federal Ministry of Education and Research, Germany, 2005. Online at: http://www.gmo-safety.eu/database/900.probability-outcrosses-between-Â�cultivatedwild-roses.html, 2009 (accessed January 21, 2011). 21. Snow, A. A. et al., Genetically engineered organisms and the environment: Current status and recommendations, Ecol. Appl. 15(2), 377, 2005. 22. Peng, J. et al., ‘Green revolution’ genes encode mutant giberellin response modulators, Nature 400, 256, 1999. 23. Introduction of genetically engineered organisms. Draft environmental impact Statement— July 2007, Report, 2007. USDA-APHIS Marketing and Regulatory Programs. 24. Hsiao, P. et al., Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation, Planta 225(4), 897, 2007.

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25. Chen, I. C. et al., Lysine racemase: A novel non-antibiotic selectable marker for plant transformation, Plant Mol. Biol. 72(1–2), 153, 2010. 26. Haldrup, A., Petersen, S. G., and Okkels, F. T., Positive selection: A plant selection principle based on xylose isomerase, an enzyme used in the food industry, Plant Cell Rep. 18(1), 76, 1998. 27. Yoder, J. and Lassner, M., Biologically safe plant transformation system using a Ds transposon. U.S. Patent 5225341, 1993. 28. Rommens, C. M. et al., The intragenic approach as a new extension to traditional plant breeding, Trends Plant Sci. 12(9), 397, 2007. 29. Schouten, H. J., Krens, F. A., and Jacobsen, E., Cisgenic plants are similar to traditionally bred plants, EMBO Reports 7(8), 750, 2006. 30. Cartagena protocol on biosafety to the convention on biological diversity: Text and annexes. Secretariat of the Convention on Biological Diversity, Montreal, Quebec, Canada, 2000. 31. Directive 2001/18/EC of the European Parliament and of the council off 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing council directive 90/220/EEC, Off. J. Eur. Union L 106, 1, 2001. 32. Regulation (EC) No 1829/2003 of the European Parliament and of the council of 22 September 2003 on genetically modified food and feed, Off. J. Eur. Union L 268, 1, 2003. 33. Gonzalez, V., Costa Rica, Biotechnology, annual report, GAIN report CS7011, USDA Foreign Agricultural Service, Washington, DC, 2007. 34. Alarcon, R. A., Ecuador, Biotechnology, Ecuador standing biotechnology report, 2006, GAIN report EC6007, USDA Foreign Agricultural Service, Washington, DC, 2006. 35. Uribe, A. and Restrep, A., Colombia, biotechnology, agricultural biotechnology, 2006, GAIN report CO7014, USDA Foreign Agricultural Service, Washington, DC, 2006. 36. Hamamoto, T., Japan, Biotechnology, agricultural biotechnology report, GAIN report JA5038, 2005, USDA Foreign Agricultural Service, Washington, DC, 2006. 37. Cino, A., Latner, K., and Bugang, W., China, Peoples Republic of, biotechnology, agricultural biotechnology report, 2005, GAIN report CH5069, USDA Foreign Agricultural Service, Washington, DC, 2005. 38. Klingeman, W., Babbit, B., and Hall, C., Master gardener perception of genetically modified ornamental plants provides strategies for promoting research products through outreach and marketing, HortScience 41(5), 1263, 2006. 39. Lence, S. H. and Hayes, J. D., Technology fees versus GURTs in the presence of spillovers: World welfare impacts, AgbioForum 8(2&3), 172–186. Online at: http://www. agbioforum.org/v8n23/v8n23a14-lence.pdf, 2005 (accessed January 21, 2011). 40. Onofrey, D., Big get bigger in breeding, Greenhouse Grower (Mid-September), 8, 2009. 41. Romeis, J., Lawo, N. C., and Raybould, A., Making effective use of existing data for case-by-case risk assessments of genetically engineered crops, J. Appl. Entomol. 133(8), 571, 2009.

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Genetic Engineering of Grapevine and Progress toward Commercial Deployment Dennis J. Gray, Sadanand A. Dhekney, Zhijian T. Li, and John M. Cordts

CONTENTS Introduction............................................................................................................. 318 Grape Genetic Transformation................................................................................ 318 Culture Systems................................................................................................. 319 Methods of Gene Insertion................................................................................. 319 Marker Genes..................................................................................................... 319 Functional Genes................................................................................................ 319 Field Tests.......................................................................................................... 320 Issues Related to Commercialization of Transgenic Grapevines............................ 322 Native Species and Gene Flow........................................................................... 322 Grape Speciation and Range......................................................................... 322 Potential for Environmental Risk.................................................................. 322 Potential Impacts of Transgene Flow in Grapevine........................................... 323 Potential for Transgene Pollen Flow into Non-GE Vineyards....................... 323 Potential for Contamination of Commercial Vineyards via Seed.................. 324 Potential for Transgene Movement into Native Vitis Populations................. 324 Mitigating Environmental Risks via Cisgenics Engineering............................. 324 Endogenous Genes........................................................................................ 324 Marker-Free Plants........................................................................................ 326 Rootstocks..................................................................................................... 327 Conclusion.............................................................................................................. 327 References............................................................................................................... 327

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INTRODUCTION Grapevine is the world’s most valuable fruit crop due to its multipurpose uses for fresh fruit, wine, and other processed products. While many species of grapevine exist, as described below, the predominant species used in commerce is Vitis vinifera L. This species originated in the Middle East and is adapted to a Mediterranean environment. It has diversified into hundreds of varieties since antiquity. Major production areas in the world tend to be those possessing Mediterranean climatic conditions. Despite widespread consumption of grape products, attempts to grow V. vinifera in climates that are too cold or, especially, too humid have met with failure. It would be advantageous to have V. vinifera varieties with more resistance to diseases caused by bacteria, fungi, and viruses in order to foster more widespread production and thus address local market needs and opportunities. While V. vinifera has been hybridized with native species to create land races for suboptimal regions, this approach has met with limited success due to the consumer’s strict preference for the exact phenotypic characteristics of well-known varieties. This is because grapevine is genetically self-incompatible, making it impossible to create inbred lines needed to introgress desirable traits from resistant native species into existing elite varieties. Although clonal selection of randomly occurring mutations has been successful in creating new varieties, it is a haphazard process and cannot produce grapevines with desirable resistance traits.1 Genetic transformation offers an alternate method for adding specific traits into otherwise desirable varieties. Presumably, varieties altered only by the addition of genes for resistance (or other traits) would retain their elite characteristics.2 Only recently has genetic transformation technology matured for grapevine, such that it is now commonplace in certain laboratories to insert genes and produce transgenic versions of popular varieties.3–6 It now remains to study grapevines containing various transgenes for their performance, which includes expression of new desirable traits as well as whether the high level of clonal fidelity demanded of grape is maintained in transgenic vines. This chapter will review the progress in genetic transformation of grapevine, including discussion of the most recent methods and procedures that have led to successful transformation. Status of field tests and issues related to environmental release of transgenic grapevines will be presented.

GRAPE GENETIC TRANSFORMATION Significant progress in grapevine transformation has been made.1–3,5,6 Genetic transformation has become relatively routine for ‘Chardonnay’, ‘Merlot’, ‘Superior Seedless’, and ‘Thompson Seedless’ (=‘Sultana’, ‘Sultanina’), among others. However, several fundamental technologies were required before transformation of grapevine could be studied. These included regenerative cell culture systems, methods for gene delivery, and then advances in molecular genetic techniques to identify useful genes and place them into functional DNA cassettes that could be tested.

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Culture Systems Somatic embryogenesis is well documented for grapevine.7–9 Most successful examples of transformation have utilized such embryogenic culture systems because embryogenic cells are capable of being genetically transformed and can be induced to regenerate into plants.1,2 Thus, once a cell is stably transformed, a transgenic plant can be produced. A drawback to use of embryogenic cultures is that they have been difficult to obtain for many varieties. However, this obstacle is slowly being overcome through experimentation and refinement of culture techniques.7,10 Cells of the shoot apical meristem obtained through in vitro micropropagation were considered as a potential alternative to embryogenic cells for transformation. The ease of producing and maintaining in vitro micropropagation cultures from a large number of cultivars11,12 makes development of a shoot tip-based transformation system attractive. However, use of shoot apical meristems for transformation has met with limited success in grapevine.13 Problems that inhibit their use likely include a relatively low number of totipotent cells in the shoot apex to be transformed, when compared to embryogenic cultures, and the propensity for chimeric plants to be recovered.

Methods of Gene Insertion As reviewed by Gray et al.,1 the two commonly used methods of gene insertion, biolisticmediated and Agrobacterium-mediated transformation, have been successfully utilized for grape. Biolistic bombardment resulted in intense transient beta-glucuronidase (GUS) expression in somatic embryos of V. vinifera ‘Thompson Seedless’14 and has been used to produce transgenic plants from embryogenic suspension cultures of ‘Chardonnay’, ‘Merlot’, and ‘Chancellor’.15,16 However, Agrobacterium-mediated transformation has been utilized more frequently and currently appears to be the transformation system of choice for grape (see Dhekney et al.3–5 and Li et al.6 for recent examples).

Marker Genes Development of transformation protocols for grape has been greatly facilitated by refinement of genetic vectors that efficiently express various reporter genes. Visible reporter genes inserted in grape include GUS,14 green fluorescent protein (GFP),17 and luciferase.18 The selectable marker genes, phosphinothricin acetyl transferase (PAT, or BAR for Bialaphos resistance) gene,19 hygromycin B phosphotransferase (HPH) gene,19 and neomycin phosphotransferase (NPTII) gene,17 have been used, with the latter being most common.

Functional Genes Establishment of reproducible transformation protocols has enabled the insertion of transgenes for quality trait improvement including fungal, bacterial, and viral resistance in elite grapevine varieties. Genes encoding pathogenesis-related (PR) proteins have been widely tested to enhance grapevine fungal resistance. ‘Thompson Seedless’ grapevines transformed with an endogenous thaumatin like protein (VVTL-1) gene exhibited significant resistance to powdery mildew (Uncinula necator) and black rot (Guignardia bidwellii) in greenhouse and field tests.20,21 Berries of ‘Shiraz’ grapevines transformed

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with an endogenous proprietary Vitis gene (EG2) exhibited significantly lower development of powdery mildew (Uncinula necator) compared to control grapevines.22–24 Resistance to fungal diseases in grapevines transformed with other PR proteins has also been reported.25,26 Low molecular weight chain antimicrobial peptides, also known as lytic peptides, have been widely tested to impart bacterial resistance in grapevines. Transgenic ‘Chardonnay’, carrying an antimicrobial peptide magainin, exhibited significant reduction in crown gall symptoms caused by Agrobacterium vitis.27 ‘Merlot’, ‘Shiraz’, and ‘Thompson Seedless’ grapevines transformed with proprietary hybrid lytic peptides, LIMA-A and LIMA-B, exhibited enhanced resistance to Pierce’s disease (Xylella fastidiosa) in greenhouse tests.28 Field tests are currently underway to test resistance of a number of transgenic Vitis varieties and rootstocks to Pierce’s disease.29,30 Virus resistant transgenic grapevines have also been developed using virus-derived genetic elements. Grapevine varieties and rootstocks carrying the coat protein for grape fan leaf virus (GFLV) and grape leaf roll virus (GLRV) have been produced31–34 and tests are under way to study the response of vines to virus infection under field conditions.

Field Tests Field trials of GE grapevines (Vitis vinifera and other Vitis species) have been conducted worldwide since the mid-1990s (Table 16.1). Early trials in Colmar, France, consisted of gene constructs for resistance to GFLV. Other trials in the United States (several locations), Canada, and Germany were designed to evaluate resistance to fungal diseases (powdery mildew and botrytis, primarily). Those trials included a number of different genes including chitinases/endochitinases, glucanases, a ribosome inactivating protein, a polygalacturonase inhibiting protein, lytic peptides, antimicrobial peptides, a lignin biosynthesis protein, and endogenous grape genes. Field trials that have ended include work in Canada by Chateau des Charmes to develop both fungal resistance and improved cold tolerance in vines (Cordts, personal communication); work by AgriVitis/GenApps in the United States to develop resistance to GFLV, GLRV, and crown gall (http://www. isb.vt.edu/cfdocs/fieldtests1.cfm); research in Italy to improve berry number and size (http://www.gmo-compass.org/eng/database/plants/73.grape_vine.html)35; research by SUNY/Geneseo to develop mildew resistant V. labrusca (http://www. isb.vt.edu/cfdocs/fieldtests1.cfm); and trials by the University of California and Anton Caratan & Son for fungal resistance and improved product quality (http:// www.isb.vt.edu/cfdocs/fieldtests1.cfm). Current field trials of Vitis species worldwide are being conducted by Cornell University, the University of Florida, and by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) (http://www.isb.vt.edu/cfdocs/fieldtests1. cfm, http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/ir-1). The Cornell vines have been developed to evaluate resistance to GFLV, GLRV, crown gall disease, fungal pathogens, and Xylella fastidiosa (http://www.isb.vt.edu/cfdocs/ fieldtests1.cfm). The University of Florida vines are being evaluated for resistance to Xylella and fungal diseases. CSIRO is evaluating vines for a number of traits including alterations to tannins, anthocyanins, sugars, browning, and various flowering/ fruiting parameters (Table 16.1).

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TABLE 16.1 Field Tests of Transgenic Grapevine Country Australia CSIRO (1999–2009), http://www.ogtr. gov.au/internet/ogtr/publishing.nsf/ Content/ir-1

Traits Tested

V. vinifera

Expression of modified color, sugar composition, flowering and fruit development/improved product quality (ppo, sh4, ufgt, dfr, inv, ARMGs, GFP, GUS) browning, flowers, anthocyanins, tannins, sugars

V. vinifera

Fungal tolerance, cold tolerance

V. vinifera

GFLV resistance using viral coat proteins (VCPs)

V. vinifera

Improved product quality (fungal resistance—chitinase, glucanase, ribosome inactivating protein)

V. vinifera

Fecundity (Increased auxin, increased tryptophan-2-mono-oxygenase) Defâ•›H9-iaaM designed to increase fruit size, number35

University of Florida (2006–2009)

Vitis rootstock species V. vinifera

Cornell (2000–2009)

V. vinifera

SUNY/Geneseo (1999–2007)

V. labrusca

University of California/Davis (2004+)

V. vinifera

GFLV, GVLR resistance based on coat protein gene insertions Xylella resistant and powdery mildew resistant (lytic peptides and endogenous grape genes) Crown gall resistant, Xylella resistant, Botrytis resistant, powdery mildew resistant (antimicrobial peptides, chitinases, endochitinases) Powdery mildew resistant (lignin biosynthesis protein, glucanase) Fungalâ•›/â•›bacterial resistance (polygalacturonase inhibiting protein)

Canada Chateau des Charmes, Ontario, Canada (ended 2001) France INRA, Colmar, France (1994, 1999, 2004) (DeFrancesco36; http://www. gmo-compass.org/eng/database/ plants/73.grape_vine.html) Germany Bundesanstalt fur Zuchtungsforschung, Germany (2000) (DeFrancesco36; http://www.gmo-compass.org/eng/ database/plants/73.grape_vine.html) Italy Universita degli Studenti Ancona, Italy (1999) (DeFrancesco36; http://www. gmo-compass.org/eng/database/ plants/73.grape_vine.html) United States of America Cornell (2007–2009)



Species

(continued)

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TABLE 16.1 (continued) Field Tests of Transgenic Grapevine Country

Species

Traits Tested

AgriVitis/GenApps (1997–2000)

Rootstocks

Anton Caratan & Son (2000+)

V. vinifera

GLRV, GFLV, crown gall resistant (based on VCPs and other) Improved product quality

V. vinifera

Traits to be tested unclear

South Africa University of Stellenbosch, South Africa

No submissions for regulatory approvals for unconfined release of GE grapevines have been noted from current sources.

ISSUES RELATED TO COMMERCIALIZATION OF TRANSGENIC GRAPEVINES Native Species and Gene Flow Because several species of grapevine are endemic to temperate and tropical areas of the world, genetic modification to adapt V. vinifera into such regions should be studied for environmental impact on native flora. Grape Speciation and Range Vitis is divided into two subgenera, Euvitis Planch, the bunch grape species that all contain 38 somatic chromosomes, and Muscadinia Planch, the muscadine grapes with 40 somatic chromosomes.37,38 Species in each subgenus are interfertile but are only partially fertile between subgenera. Estimates of the number of Euvitis species range from 28 to 43. These are separated into an American group of 18–28 species, an Asian group of 10–15 species, and a European or central Asian group of one species (V. vinifera).37 Muscadinia contains three species; V. munsoniana and V. rotundifolia are native to the southeastern United States and V. popenoeii Fennell is native to Central America. With the exception of muscadine cultivars, most cultivated grapes are either pure strains or hybrids of V. vinifera. Two wild subspecies of V. vinifera, ssp. sylvestris Gmel. and ssp. caucasis Vav., and one cultivated subspecies, ssp. sativa D.C., are recognized.37 Potential for Environmental Risk Transgenic grapevines that contain genes to make them resistant to diseases, much like native species, could pose the possibility of environmental risk if they became invasive. It is important to consider whether there is potential for adverse environmental impacts from field release of genetically modified disease-resistant grapevines. Research should be accomplished to determine the characteristics, rates, and methods of gene transfer that may occur between genetically engineered non-native grapevines and cross-fertile wild and agricultural species currently in the environment to determine if the GE vines or progeny produced by cross-fertilization could

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become invasive pests. Because transgenes are dispersed from genetically engineered vines by pollination and/or seed production, certain aspects of grape reproductive biology should be evaluated. Molecular methods using marker genes for detection of transgenes and transproteins in grape seed/fruit tissues to monitor movement of such genes through sexually produced progeny are readily available. In addition to gene movement, the relative vigor and adaptation, including survivability of transgenic progeny, must be determined to assess their potential to become invasive pests. Gene Flow via Pollen In varieties with perfect flowers, the calyptrate flower structure and arrangement of many flowers on a panicle favor self-pollination.37 The extent to which natural cross-pollination occurs in grape is controversial. Natural hybridization among native species has been detected.39 Grape has been considered to be wind-pollinated, with pollen flow occurring no further than 20 ft. from the source.40 However, cross-pollination has been suggested to be enhanced by insect pollinators in certain instances. For example, in muscadine grape vineyards composed of female (self-sterile) vines that require pollinator vines, fruit set has been enhanced by incorporation of honeybee hives (reviewed by McGregor41). However, in a controlled study, vines isolated from wind-blown pollen by cages did not produce appreciatively more fruit when bee hives were added.42 Airborne pollen yields have been accurately measured in order to predict seasonal fruit yield.43,44 The latter studies described airborne pollen loads, but did not address hybridization/gene flow. Thus, the maximum distance that viable pollen moves from one grapevine to another and/or the frequency of hybridization resulting from such movement have not been adequately documented. This issue has taken on new importance with the advent of genetically GE grapevine. Gene Flow via Seed The possibility for gene flow via seed dispersal from transgenic vines would be low if seeds result from selfs, as mentioned above, and because grape has a high level of inbreeding depression.45 Therefore, the resulting selfed plants lack vigor and tend not to survive. For example, of plants grown from over one thousand selfed seeds, none were able to survive to flowering when planted in a research vineyard at Leesburg, Florida (Gray, personal observation). Further, when seedless varieties are used, there is no opportunity for seed dispersal. Thus, frequency of natural self versus crossfertilization should be investigated.

Potential Impacts of Transgene Flow in Grapevine Potential for Transgene Pollen Flow into Non-GE Vineyards Transgene movement into non-GE vineyards as a consequence of pollen flow should have minimal impact. This is because only the embryo and endosperm, which are encased in a non-transgenic hard, durable seed coat, can become transformed by pollen from a transgenic vine. Our current research using the GFP marker system confirms this. Grape seed is discarded whether fruit is used fresh or for processed products, with the exception of use for oil extraction.46 Thus, if seed is properly disposed, there is little-to-no risk of transgene movement.

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Furthermore, seedless varieties used for fruit, raisins, or other processing contain abortive ovules without embryos.47,48 Potential for Contamination of Commercial Vineyards via Seed Seeds could be distributed from transgenic vines into non-transgenic vineyards via animals or unintentionally by workers. However, as discussed above, the selfed seeds that typically would result lack vigor and plants from them do not mature to flowering. Regardless, even if selfed or hybrid seeds capable of flowering were produced, the plants would tend to be out of place and discarded in the normal practices of a commercial vineyard. Potential for Transgene Movement into Native Vitis Populations There are native species and feral vines of Euvitis grape that are cross fertile with transgenic varieties. The possibility that transgenes may be transferred to native populations of Vitis centers both on the frequency of selfing versus out crossing that naturally occurs and the dynamics of pollen flow. If only selfed seeds are produced, inbred progeny transferred from transgenic plants into the wild will tend to lack sufficient vigor to survive. How frequently hybridization occurs, how far viable pollen moves, and whether hybrid progeny containing transgenes for disease resistance have a competitive advantage in the ecosystem are issues that should be considered. For example, if out crossing occurs to produce hybrid seed among transgenic vines, or if transgenes are detected in seeds of outlying vines, or progeny are more vigorous in nature than their parents, more stringent management schemes might be required than if transgene flow is found to be restricted or its consequences benign.

Mitigating Environmental Risks via Cisgenics Engineering The advancements in molecular genetics and its application now allow a genetic engineering approach that utilizes only grape-derived genes (cisgenes) and genetic elements.23 Such a cisgenic approach to genetic engineering solves issues related to the release of foreign genes into the environment and should overcome reluctance to GM foods, which normally contain foreign genes and gene products.36 There are a number of grape-derived (endogenous) genes already available for testing. Furthermore, the common use of grafting in viticulture may allow transgenic rootstocks to be utilized to which non-transgenic scions could be grafted. Endogenous Genes Certain concerns about implementation of GE technology might be allayed if endogenous genes from the grape genome itself were used for genetic improvement through overexpression strategies. Recent advances in plant molecular biology including whole genome sequencing, rapid cloning, and efficient gene expression techniques have greatly facilitated efforts to exploit plant genes associated with agronomic traits based on manipulation of native endogenous genes already existing in a plant’s genome.49 Manipulation of endogenous genes includes removal of introns and codon optimization to increase efficiency. Endogenous genes may be placed in a construct with a promoter chosen to modify expression characteristics and then

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reinserted into the plant. Such genes often modify metabolism in a manner similar to natural or induced mutations without causing genetic contamination.49 The improvements achieved via use of such endogenous genes should be qualitatively similar to those obtained via traditional breeding approaches, but significantly more precise, due to the ability to overexpress desired proteins, both temporally and spatially. In addition, endogenous/homologous transgenes can be defined and regulated differentially as compared to use of foreign transgenes for crop genetic engineering.50 It is possible that transgenic modification of native genes tends to inherently balance the selection process and maintain natural variance.49 PR Proteins A number of genes, commonly known as PR (pathogenesis related) protein genes, have been cloned from and expressed in the plant species from which they were cloned. They have been grouped (PR 1–PR 5) based on their structure and mode of action.51 A number of PR proteins exhibit antifungal properties, which variously cause inhibition of fungal cell wall synthesis and/or a disruption in cell wall structure leading to cell lysis.52 PR proteins are classified into different groups including cysteine rich antimicrobial peptides,53–57 glucanases and chitinases,52 chitin binding proteins53,58 and thaumatin like (TL) proteins.52 The PR 5, or TL proteins, share significant amino acid homology to thaumatin.52 TL proteins inhibit a wide range of plant pathogens in vitro52 and have been cloned from several plant species.59–62 TL proteins are known to be differentially expressed in reproductive tissues such as pistils and ripening fruits.63–66 The University of Florida/Institute of Food and Agricultural Sciences (UF/IFAS) grape biotechnology laboratory cloned V. vinifera thaumatin-like protein (VVTL-1) gene from grapevines derived from embryogenic cultures that were subjected to in vitro selection with the culture filtrate of Elsinoe ampelina, the causal agent of grapevine anthracnose.67 Protein produced from VVTL-1 significantly inhibited E. ampelina spore germination and hyphal growth in vitro. Plants regenerated from in vitro-selected cultures similarly inhibited fungal growth in leaf assays. Similar results have been obtained with VVTL-2 cloned from grape, which was expressed in leaves and ripening berries in response to powdery mildew infection68,69 and exhibited antifungal properties.70 Seed Proteins Seeds contain a wide array of antimicrobial compounds that can be expressed in a constitutive or inducible manner.71 Although 2S albumins are generally considered as storage proteins, they also inhibit growth of pathogenic fungi in part by permeabilization of hyphal plasmalemma.72,73 2S albumin genes have been cloned from sunflower,74 Brazil nut,75 grape,76 and other crop plants. In grape, we have cloned the entire 2S albumin gene along with the coding sequence and promoter elements76 and we are evaluating its expression (Li et al., unpublished data). Stilbene Synthase Phytoalexins are low molecular weight antimicrobial compounds that are synthesized by and accumulate in plants in response to biotic and abiotic stresses. Stilbenes and their derivatives have been regarded as phytoalexins that contribute to defense

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against fungal infection.77 Stilbene synthase is the key enzyme that catalyzes the formation of resveratrol via phenolic substrates in the shikimic acid pathway. Stilbene biosynthesis only specifically requires the presence of stilbene synthase. The family of stilbene synthase genes have been cloned and characterized from grape.78 Studies with transgenic apple,79 papaya,80 rice,81 tobacco,77 wheat, and barley82 overexpressing the STS 1 gene from grape reported enhanced resistance to various fungal diseases like blast, fruit, root, and stem rot. Polyphenol Oxidase Polyphenol oxidases (PPOs) are nuclear-encoded enzymes of almost ubiquitous distribution in plants and are known to catalyze the oxygen dependent oxidation of phenols to quinones.83 Systemic induction of PPO in response to wounding and pathogen attack may protect plants against further attack by pathogens and insects.84 PPOs have been cloned from a number of plant species including grape,85 potato,86 tomato,87 and Vicia species.88 Transgenic plants overexpressing PPO were found to exhibit enhanced resistance to bacterial diseases caused by Pseudomonas synringae.89,90 Additionally, an antisense downregulation of polyphenol oxidase in transgenic tomato plants resulted in increased disease susceptibility as compared to non-transgenic controls.90 Although only bacterial resistance has been specifically demonstrated, PPOs are considered to have broad antimicrobial activity. Marker-Free Plants Genetic transformation requires a method to separate transformed cells from nontransformed cells and typically is achieved by expression of a marker gene that provides a growth advantage to transformed cells in a selection medium (reviewed by Dutt et al.91). In the absence of a selectable marker gene, transformed cells tend to be at a competitive disadvantage compared to nontransformed cells and die out. The marker gene is needed only for selection of transgenic cells and typically is linked to a gene of interest. Thus, selection for ability to proliferate in the presence of a selective agent results in isolation of transgenic cells containing both the gene of interest and the marker gene. However, once a desired plant is selected from transgenic cells, the marker gene is no longer needed. The presence of marker genes may complicate future commercialization due to concerns regarding their effects on ecosystems and/or human health.91 For example, one concern is that selectable marker genes could become transferred to other organisms, leading to the creation of antibiotic resistant bacterial strains (from antibiotic resistance markers) or new, aggressive weedy plant species (from herbicide resistance markers). Development of transgenic plants without marker genes alleviates such concerns. Dutt et al.91 used a cotransformation treatment to demonstrate production of transgenic grapevines free of the selectable marker gene(s) that are typically required only for initial identification and selection of transgenic cells. Transgenic V. vinifera ‘Thompson Seedless’ plants containing only a single stably integrated gene of interest and not a selective marker gene were produced. The system provided the possibility of producing plants that contain only a gene of practical use while preventing the formation of genetic chimeras that might occur through meristem transformation. The technique can be

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used readily to produce grapevines with a range of other nonselectable genes and may be adapted for use in other vegetatively propagated crops as well. Rootstocks Grafting of scion to a rootstock is commonly used for grapevine in most production areas. The possibility of using a transgenic rootstock that resists soil stresses and/or transmits a useful substance into the scion is intriguing for several reasons. For example, only the non-transgenic scion would reproduce pollen or seed, so that there would be no possibility for transgene flow to occur, and virtually any scion could be grafted onto a transgenic rootstock, thus reducing the number of transgenic varieties to be developed and tested. Rootstocks that produce lytic peptides to inhibit the xylem-limited bacterium that caused Pierce’s disease of grapevine are in field tests.92 The direct vascular connection of rootstock to scion allows lytic peptide produced and accumulated in rootstock xylem to be transported into the scion.93

CONCLUSION Genetic transformation of grape is no longer an obstacle to progress in development of improved transgenic grapevines. However, it is difficult to predict when a transgenic grape product will become commercially available. While it is now possible to insert virtually any gene of interest into an increasingly wide range of elite grape varieties, field testing has been insufficient to prove the expected efficacy of new traits; one problem being the time required to evaluate such a perennial fruit crop. Also, needs to conduct risk assessment studies and develop consumer acceptance of any transgenic crop have proven to be costly and difficult. One alternate approach to reducing the concerns associated with transgenic plants is to adopt the aforementioned cisgenics approach to crop development wherein only genes and other genetic elements taken directly from grapevine are used. Such a green approach is more akin to “precision breeding” inasmuch as it disrupts the plant genome even less, and is more predictable, than conventional breeding. Similarly, the implementation of transgenic rootstocks to combat diseases in the scion could have a similar impact. We believe that grapevines developed in this manner to have improved disease resistance and quality attributes will be desirable to growers and be accepted by consumers.

REFERENCES

1. Gray, D.J., Jayasankar, S., and Li, Z., Vitaceae (Grape Vitis spp.), in Biotechnology of Fruit and Nut Crops, Biotechnology in Agriculture Series, No. 29, Litz, R.E., ed., CAB International, Wallingford, U.K., 2005, Chap. 22, pp. 672–706. 2. Gray, D.J. et al., Transgenic Grapevines, in Transgenic Plants, Khachatourians, G.G. et al., eds., Marcel Dekker, New York, 2002, Chap. 27, pp. 397–405. 3. Dhekney, S.A. et al., Factors influencing genetic transformation and plant regeneration of Vitis, Am. J. Enol. Vitic., 60, 285, 2009. 4. Dhekney, S. A. et al., Agrobacterium-mediated transformation of embryogenic cultures and regeneration of transgenic plants in Vitis rotundifolia Michx. (muscadine grape), Plant Cell Rep., 77, 865, 2008.

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Transgenic Horticultural Crops: Challenges and Opportunities

5. Dhekney, S.A. et al., Overcoming obstacles to genetic transformation in Vitis, In Vitro Cell. Dev. Biol., 44, S40, 2008. 6. Li, Z.T. et al., Optimizing Agrobacterium-mediated transformation of grapevine, In Vitro Cell. Dev. Biol. Plant., 42, 220, 2006. 7. Dhekney, S.A. et al., Optimizing initiation and maintenance of Vitis embryogenic cultures, HortScience, 44, 1400, 2009. 8. Li, Z.T. et al., An improved protocol for Agrobacterium-mediated transformation of grapevine, Plant Cell Tiss. Organ Cult., 93, 311, 2008. 9. Gray, D.J., Grape, in Somatic Embryogenesis in Woody Plants, Jain, S.M., Gupta, P.K., and Newton, R.J., eds., Kluwer Academic Publishers, Dordrecht, the Netherlands, 1995, Vol. 2, p. 191. 10. Jayasankar, S. et al., Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid and liquid culture systems, Am. J. Bot., 90, 973, 2003. 11. Gray, D.J. and Benton, C.M., In vitro micropropagation and plant establishment of muscadine grape varieties, Plant Cell Tiss. Org. Cult., 27, 7, 1991. 12. Gray, D. J. and Fisher, L.C., In vitro shoot propagation of grape species, hybrids and cultivars, Proc. Fla. State Hort. Soc., 98, 172, 1985. 13. Dutt, M. et al., Transgenic plants from shoot apical meristems of Vitis vinifera Thompson Seedless via Agrobacterium-mediated transformation, Plant Cell Rep., 26, 2101, 2007. 14. Gray, D.J., Songstad, D.D., and Compton, M.E., Expression of GUS in bombarded grape somatic embryos and cells, In Vitro Cell. Dev. Biol., 65, 29A, 1993. 15. Kikkert, J.R. et al., Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions, Plant Cell Rep., 15, 311, 1996. 16. Kikkert, J.R. et al., Expression of a fungal chitinase in Vitis vinifera L. ‘Merlot’ and ‘Chardonnay’ plants produced by biolistic transformation, in Proceedings of the VIIth International Symposium on Grapevine Genetics and Breeding (Acta Hort.), Bouquet, A. and Boursiquot, J.M., eds., International Society for Horticultural Sciences, Montpellier, France, 2000, pp. 528, 297. 17. Li, Z., Jayasankar, S., and Gray, D.J., Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera), Plant Sci., 160, 877, 2001. 18. Hanson, B. et al., A simple method to enrich an Agrobacterium-transformed population of plants containing only T-DNA sequences, Plant J., 19, 727, 1999. 19. Perl, A. et al., Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): The role of antioxidants during grape-Agrobacterium interactions, Nat. Biotechnol., 14, 624, 1996. 20. Dhekney, S.A. et al., Disease resistant transgenic grapevine constitutively expresses Vitis vinifera thaumatin-like protein, In Vitro Cell. Dev. Biol., 42, 3021, 2006. 21. Dhekney, S. A. et al., Greenhouse screening and field testing of transgenic grapevine for fungal resistance, In Vitro Cell. Dev. Biol., 43, S40, 2007. 22. Dhekney, S.A. et al., Using endogenous genes for producing disease resistant transgenic grapevines, In Vitro Cell. Dev. Biol., 45, S38, 2009. 23. Gray, D. J. et al., Green genetic engineering technology: The use of endogenous genes to create fungal disease-resistant grapevines, in Proceedings of the Caribbean Food Crops Society 44th Annual Meeting, Miami, FL 2008, Caribbean Food Crops Society, 2009, p. 44. 24. Gray, D.J. et al., Green genetic engineering technology: Rearrangement of endogenous functional genetic elements to create improved grapevines, In Vitro Cell. Dev. Biol., 45:S39, 2009. 25. Yamamato, T. et al., Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens, Plant Cell Rep., 19, 639, 2000. 26. Augero, C.B. et al., Evaluation of Pierce’s disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene, Mol. Plant Pathol., 6, 43, 2005.

Genetic Engineering of Grapevine

329

27. Vidal, J.R. et al., Evaluation of transgenic ‘Chardonnay’ (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew, Transgenic Res., 15, 69, 2006. 28. Gray, D.J. et al., Transgenic grapevines resistant to Pierce’s disease, HortScience, 40,1104, 2005. 29. Gray, D.J. et al., Field testing of transgenic grapevines for bacterial and fungal resistance, HortScience, 42, 858, 2007. 30. Gray, D.J. et al., Field testing of transgenic grapevines for disease resistance, In Vitro Cell. Dev. Biol., 44, S40, 2008. 31. Krastanova, S. et al., Transformation of grapevine rootstocks with the coat protein of grapevine fanleaf nepovirus, Plant Cell Rep., 14, 550, 1995. 32. Krastanova, S. et al., Development of transgenic grapevine rootstocks with genes from grapevine fanleaf virus and grapevine leafroll associated closterovirus 2 and 3, in Proceedings of the VIIth International Symposium on Grapevine Genetics and Breeding (Acta Hort.), Bouquet, A. and Boursiquot, J.M., eds., International Society for Horticultural Sciences, Montpellier, France, 2000, pp. 528, 367. 33. Xue, B. et al., Transformation of five grape rootstocks with plant virus genes and a vir— E2 gene from Agrobacterium tumefaciens, In Vitro Cell. Dev. Biol. Plant, 35, 226, 1999. 34. Gambino, G. et al., Molecular characterization of grapevine plants transformed with GFLV resistance genes, Plant Cell Rep., 24, 655, 2005. 35. Mezzetti, B., et al., Genetic transformation of Vitis vinifera via organogenesis. BMC Biotechnol., 2, 18, 2002. 36. DeFrancesco, L., Vintage genetic engineering, Nat. Biotechnol., 26, 261, 2008. 37. Einset, J. and Pratt, C., Grapes, in Advances in Fruit Breeding, Janick, J. and Moore, J.N., eds., Purdue University Press, West Lafayette, IN, 1975, p. 130. 38. Winkler, A.J. et al., General Viticulture, revised edition, University of California Press, Berkeley, CA, 1974. 39. Olmo, H.P. and Koyama, A., Natural hybridization of indigenous Vitis californica and V. girdiana in California, in Proceedings of the 3rd International Symposium on Grape Breeding, UCD Press, Davis, CA., 32, 1980. 40. Reisch, B., Grape Breeding Procedures, 2001. http://www.nysaes.cornell.edu/hort/ faculty/reisch/breeding/crossing1.html, 2001. (Accessed January 28, 2011). 41. McGregor, S.E., Insect Pollination of Cultivated Crop Plants, Virtual online version updated continuously. http://afrsweb.usda.gov/SP2UserFiles/Place/53420300/OnlinePollination Handbook.pdf, 1976. (Accessed April 20, 2011). 42. Sharples, G.C. et al., The importance of insects in the pollination and fertilization of the cardinal grape, Proc. Am. Soc. Hort. Sci., 86, 321, 1965. 43. Cristofolini, F. and Gottardini, E., Concentration of airborne pollen of Vitis vinifera L. and yield forecast: A case study at S. Michele all Adige, Trento, Italy, Aerobiologia, 16, 125, 2000. 44. Ribeiro, H.I. et al., Aeropalynological study of Vitis vinifera in the Braga region (1999–2003), Aerobiologia, 21, 131, 2005. 45. Alleweldt, G. and Possingham, J.V., Progress in grapevine breeding, Theor. Appl. Genet., 75, 669, 1988. 46. Gray, D.J. et al., Tracking pollen-mediated gene flow in transgenic grapevine, HortScience, 43, 1156, 2008. 47. Gray, D.J. et al., Comparison of methodologies for in ovulo embryo rescue of seedless grape cultivars, HortScience, 22, 1334, 1987. 48. Gray, D.J., et al., Ovule culture to obtain progeny from hybrid seedless grapes, J. Am. Soc. Hort. Sci., 115, 1019, 1990. 49. Strauss, S.H., Genomics, genetic engineering and domestication of crops, Science, 300, 61, 2003.

330

Transgenic Horticultural Crops: Challenges and Opportunities

50. Strauss, S.H., Application of biotechnology to study of trees and forests, in Proceedings of the Symposium on Ecological and Societal Aspects of Transgenic Plantations, Strauss, S.H. and Bradshaw, H.D., eds., July 22nd–24th, Stevenson, WA, 2001, p. 7. 51. Punja, Z., Genetic engineering of plants to enhance resistance to fungal pathogens— A review of progress and future prospects, Can. J. Plant Pathol., 23, 216, 2001. 52. Seliterennikoff, C.P., Antifungal proteins, Appl. Environ. Microbiol., 67, 2883, 2001. 53. Broekaert, W.F. et al., Plant defensins: Novel antimicrobial peptides as components of host defense systems, Plant Physiol., 108, 1353, 1992. 54. Cammue, B.P. et al., A potent antimicrobial protein from onion seeds shows sequence homology to plant lipid transfer proteins, Plant Physiol., 109, 445, 1995. 55. Epple, P., Apel, K., and Bohlmann, H., Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporium, Plant Cell, 9, 509, 1997. 56. Thevissen, K. et al., Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity, Mol. Plant Microbe. Interact., 13, 54, 2000. 57. Nielsen, K.K. et al., Characterization of a new antifungal chitin-binding peptide from sugar beet leaves, Plant Physiol., 113, 83, 1997. 58. Van Damme, E.J. et al., A gene encoding a heavein like protein from elderberry fruits is homologous to PR-4 and class V chitinase genes, Plant Physiol., 119, 1547, 1999. 59. Huynh, Q.K., Borgmeyer, J.R., and Zobel, J.F., Isolation and characterization of a 22â•›kDa protein with antifungal properties from maize seeds, Biochem. Biophys. Res. Commun., 182:1, 1992. 60. Hu, X. and Reddy, A.S., Cloning and expression of a PR-5 like protein from Arabidopsis, Plant Physiol., 107, 305, 1997. 61. Cheong, N.E. et al., Purification and characterization of an antifungal PR 5 protein from pumpkin leaves, Mol. Cells, 7:214, 1997. 62. Koiwa, H. et al., Crystal structure of tobacco PR 5d protein at 1.8A resolution reveals a conserved acidic cleft structure in antifungal thaumatic like proteins. J. Mol. Biol., 286, 1137, 1998. 63. Neale, A.D. et al., Chitinase, β-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation, Plant Cell, 2, 673, 1990. 64. Vu, L. and Huynh, Q.K., Isolation and characterization of a 27â•›kDa antifungal protein from the fruits of Diospyros texana, Biochem. Biophy. Res. Commun., 202, 666, 1994. 65. Fils-Lycaon, B.R. et al., A cherry protein and its gene, abundantly expressed in ripening fruit have been identified as thaumatin like protein, Plant Physiol., 111, 269, 1996. 66. Barre, A. et al., Purification and structural analysis of an abundant thaumatin like protein from ripe banana fruits, Planta, 211, 791, 2000. 67. Jayasankar, S., Li, Z., and Gray, D.J., Constitutive expression of Vitis vinifera thaumatin like protein after in vitro selection and its role in anthracnose resistance, Funct. Plant Biol., 30, 1105, 2003. 68. Jacobs, A.K. et al., Induction of different pathogenesis-related cDNAs in grapevine infected with powdery mildew and treatment with ethephon, Plant Pathol., 48, 325, 1999. 69. Tattersall, B.D. et al., Identification and characterization of a fruit-specific, thaumatinlike protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes, Plant Physiol., 114, 759, 1997. 70. Salzman, R.A. et al., Coordinate accumulation of antifungal proteins and hexoses constitutes a developmentally controlled defense response during fruit ripening in grape, Plant Physiol., 117, 465, 1998. 71. Cammue, B.P.A. et al., Isolation and characterization of a novel class of plant antimicrobial peptides from Mirabilis jalapa L. seeds, J. Biol. Chem., 267, 2228, 1992. 72. Terras, F.R. et al., A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species, FEBS Lett., 316, 233, 1993.

Genetic Engineering of Grapevine

331

73. Polya, G.M., Chandra, S., and Condron, R., Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase, Plant Physiol., 101, 545, 1997. 74. Allen, R.D. and Cohen, E.A., Sequence and expression of a gene encoding an albumin storage protein in sunflower, Mol. Gen. Genet., 210, 211, 1987. 75. Gander, E.S. et al., Isolation, characterization and expression of a gene coding for a 2S albumin from Bertholletia excelsa (Brazil nut), Plant Mol. Biol., 16, 437, 1991. 76. Li, Z., and Gray, D.J., Isolation by improved TAIL-PCR and characterization of a seed-specific 2S albumin gene and its promoter from grape (Vitis vinifera L.), Genome, 48, 312, 2005. 77. Hain, R. et al., Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol, Plant Mol. Biol., 15, 325, 1990. 78. Melchior, F. and Kind, H., Coordinate and elicitor-dependent expression of stilbene synthase and phenylalanine ammonia-lyase genes in Vitis cv. Optima, Arch. Biochem. Biophys., 288, 552, 1991. 79. Szankowski, I. et al., Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa), Plant Cell Rep., 22, 141, 2003. 80. Zhu, Y.J. et al., Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora, Planta, 220, 241, 2004. 81. Stark-Lorenzen, P.B. et al., Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.), Plant Cell Rep., 16, 668, 1997. 82. Leckband, G. and Lorz, H., Transformation and expression of a stilbene synthase gene from Vitis vinifera L. in barley and wheat for increased fungal resistance, Theor. Appl. Genet., 96, 1004, 1998. 83. Mayer, A.M., Polyphenol oxidases in plants—Recent progress, Phytochemistry, 26, 11, 1987. 84. Constabel, C.P., Bergey, D.R., and Ryan, C., Systemin activates synthesis of woundinducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway, Proc. Natl. Acad. Sci. USA, 92, 407, 1995. 85. Dry, I.B. and Robinson, H.B., Molecular cloning and characterization of grape berry polyphenol oxidase, Plant Mol. Biol., 26, 495, 1994. 86. Hunt, M.D. et al., cDNA cloning and expression of potato polyphenol oxidase, Plant Mol. Biol., 21, 59, 1993. 87. Thipyapong, P. and Steffens, J.C., Tomato polyphenol oxidase (differential response of the polyphenol oxidase F promoter to injuries and wound signals), Plant Physiol., 115, 409, 1997. 88. Cary, J.W., Lax, A.R., and Flurkey, W.H., Cloning and characterisation of cDNAs coding for Vicia faba polyphenol oxidase, Plant Mol. Biol., 20, 245, 1992. 89. Li, L. and Steffens, J., Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance, Planta, 215, 239, 2002. 90. Thipyapong, P., Hunt, M.D., and Steffen, J.C., Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility, Planta, 220, 105, 2004. 91. Dutt, M. et al., A co-transformation system to produce transgenic grapevines free of marker genes, Plant Sci., 175, 423, 2008. 92. Dhekney, S.A. et al., Transgenic grapevine rootstocks for Pierce’s disease resistance, HortScience, 43, 1155, 2008. 93. Dutt, M. et al., Transgenic rootstock protein transmission in grapevines, in Proceedings of the International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species (Acta Hort.), Litz, R.E. and Scorza, R., eds., International Society for Horticultural Sciences, Leuven, Belgium, 2007, pp. 738, 749.

Agriculture As the world debates the risks and benefits of plant biotechnology, the proportion of the global area of transgenic field crops has increased every year, and the safety and value continue to be demonstrated. Yet, despite the success of transgenic field crops, the commercialization of transgenic horticultural crops (vegetables, fruits, nuts, and ornamentals) has lagged far behind. Transgenic Horticultural crops: challenges and opportunities examines the challenges for the creation and commercialization of horticultural biotechnology and identifies opportunities, strategies, and priorities for future progress. A “must read” for anyone working in the fields of genetic engineering or plant breeding, for policy makers, educators, students, and anyone interested in the issues of genetic engineering of fruits, vegetables and ornamentals, this book covers: • Past achievements, newest developments, and current challenges in transgenic fruit, nut, vegetable, ornamental, and pharmaceutical crops • Reviews transgenic horticultural crops in the U.S., Europe, Africa, and Asia • Hurdles to the commercialization of transgenic technology in economics and the marketplace, consumer acceptance, intellectual property rights protection, public–private partnership, and regulation • Critical evaluation of the benefits and risks of genetically engineered horticultural crops, including risk assessment and transgene containment • Presents case studies and an industry perspective on transgenic horticultural crops The production and commercialization of transgenic horticultural crops are enormous tasks—their progress and realization require an informed research community, horticultural industry, government, and body of consumers. To aid in this effort, this book provides facts, analyses and insights by leading experts in this field to inform a wide audience of students, agricultural and genetic professionals, and the interested public. Part of the global conversation on the pros and cons of transgenic foods, Transgenic Horticultural crops aims to stimulate more interest and discussion on the subject and to promote the development of safe and sustainable genetically modified horticultural crop varieties.

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