Challenge of Plant and Agriculutral Sciences to the Crisis of Biosphere on the Earth in the 21st Century

ENVIRONMENTAL INTELLIGENCE UNIT

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Kazuo Watanabe • Atsushi Komamine

Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century

EIU

Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century

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WATANABE • KOMAMINE

INTELLIGENCE UNITS

ENVIRONMENTAL INTELLIGENCE UNIT

The Twelfth Toyota Conference

ENVIRONMENTAL INTELLIGENCE UNIT

Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century Kazuo Watanabe Faculty of Biology-Oriented Science and Technology Kinki University Uchita-Cho, Naga-gun Wakayama, Japan and

Atsushi Komamine Research Institute of Evolutionary Biology Kamiyoga, Setagaya Tokyo, Japan

LANDES BIOSCIENCE GEORGETOWN, TEXAS U.S.A.

EUREKAH.COM AUSTIN, TEXAS U.S.A.

Environmental Intelligence Unit

PROCEEDINGS OF THE 12TH TOYOTA CONFERENCE: CHALLENGE OF PLANT AND AGRICULTURAL SCIENCES TO THE CRISIS OF BIOSPHERE ON THE EARTH IN THE 21ST CENTURY EUREKAH.COM/LANDES BIOSCIENCE designed by KimMitchell Copyright ©2000 EUREKAH.COM All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: Eurekah.com/Landes Bioscience, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-58706-015-9

Library of Congress Cataloging-in-Publication Data

Toyota Conference (12th : 1998 : Shizuoka, Japan) Proceedings of the 12th Toyota Conference : Challenge of plant and agricultural sciences to the crisis of biosphere on the earth in the 21st century / Kazuo Watanabe, Atsushi Komamine p. cm. -- (Environmental intelligence unit) ISBN 1-58706-015-9 (alk. paper) 1. Crops -- Genetic engineering Congresses. 2. Plant biotechnology Congresses. I, Watanabe, Kazuo N. II. Komamine, Atsushi, 1929-. III. Title. IV. Title: Proceedings of plant and agricultural sciences to the crisis of biosphere on the earth in the 21st century. VI. Series SB123.57.T69 1998 631.5'233--dc21 99-34880 CIP

CONTENTS Section I Introduction to the Challenge: Guide for the Book .......................................... 1 A. Komamine, T. Fujimura and K. N. Watanabe

Section II Introduction—Prospects of Supply and Demand for Food: Research Strategies for the Coming Century ..................................................... 3 H. Ikehashi 1. Food Shortage in the 21st Century and Its Implications for Agricultural Research .......................................................................... 5 H. Tsujii Introduction ............................................................................................. 5 Factors Determining Food Shortage in the Early 21st Century ............ 7 Conclusion ............................................................................................. 23 2. Prospects for Grain Demand and Supply in the 21st Century ............. 29 H. Zhai Food Demand Analysis ......................................................................... 29 Grain Supply Potential .......................................................................... 32 Countermeasures Toward Making up the Balance Between Grain Supply and Demand ............................................................... 34 3. Agricultural Science In India—Shaping for the Future ........................ 39 M. Rai and A.K. Bawa Indian Scenario ...................................................................................... 39 National Agricultural Research System ................................................ 40 Challenges Ahead .................................................................................. 41 Opportunities and Strategies ................................................................ 42 Agricultural Extension .......................................................................... 48 Programs and Policies ........................................................................... 48 Human Resource Development ........................................................... 49 Conclusion ............................................................................................. 50 4. Setting Priorities for Agricultural Research: Theory and Experience ........................................................................... 51 D. Gollin Research Priority Setting: Theories and Models .................................. 52 Basic Principles ...................................................................................... 52 Congruence Rules .................................................................................. 53 Supply-Driven Resource Allocation ..................................................... 53 Economic Approaches to Priority Setting ............................................ 53 From Theory to Practice: A Case Study of the Rockefeller Foundation’s Decision to Prioritize Rice Biotechnology ................ 55

Priority-Setting and the Rockefeller Investments ................................ 56 Some Shortcomings of Existing Methods and Lessons for the Future ..................................................................................... 58 Managing a Portfolio of Research ........................................................ 58 Pursuing Comparative Advantage ........................................................ 58 Taking Advantage of Research Spillovers ............................................. 59 Realizing the Benefits of Delay .............................................................. 59 Conclusion ............................................................................................. 59 5. Sustainable Agriculture and Strategies in Rice Breeding ...................... 63 H. Ikehashi Rice Cultivation as a Model for Sustainable Agriculture .................... 63 Green Revolution .................................................................................. 64 Problems and Tasks in the Post Green Revolution Era ....................... 65 Strategies for Enhanced Yield Level ...................................................... 65 Application of Molecular Biology to Rice Breeding ............................ 67

Section III Introduction: The Present Situation of Biological Production and the Approach to the Sustainable Production in Arid Lands ................... 71 Satoshi Matsumoto 6. Drylands and Global Change: Rainfall Variability and Sustainable Rangeland Production ................................................. 73 J.F. Reynolds, R.J. Fernández and P.R. Kemp Introduction ........................................................................................... 73 Drylands and Global Change ................................................................ 73 Climate Variability ................................................................................ 76 Case Study .............................................................................................. 78 Integrating Ecological and Social Science Issues .................................. 82 Conclusion ............................................................................................. 85 7. Sustainable Water Management and Agriculture ................................. 87 W. Kinzelbach, D. McLaughlin and H. Kunstmann Introduction .......................................................................................... 87 The Global Water Situation ................................................................. 87 Important Water Resource Issues ......................................................... 89 Conclusions ........................................................................................... 96 8. Crop and Resource Management for Improved Productivity in Dryland Farming Systems .................................................................. 99 O. Ito and M. Kondo Introduction ........................................................................................... 99 Agriculture in the Semi-Arid Tropics ................................................. 100 Intercropping as One Cropping Option ............................................ 100

Interaction Among Water, Nutrients and Roots ............................... 102 Crop and Resource Management in Low Input Farming Systems ............................................................................................. 104 9. Sustainable Irrigated Agriculture in Arid Lands: Kazakstan Case Study ........................................................................... 107 T. Yano and S. Wang Introduction ......................................................................................... 107 Present Situation of Salt Accumulation in the Study Area ................ 107 Reclamation of Salt-Affected Soils ...................................................... 110 Permeability of Salt-Affected Soils ..................................................... 111 Conclusion ........................................................................................... 112 10. Distribution and Amelioration of Alkali Soils in Northeast China ... 113 S. Matsumoto Introduction ......................................................................................... 113 Alkali Soil Formation, Characterization and Its Distribution in China ........................................................................................... 114 Reclamation of Alkali Soils ................................................................. 115 Conclusion ........................................................................................... 119

Section IV Introduction:Conservation and Contribution of Plant Genetic Resources ........................................................................................... 123 Kazuo N. Watanabe 11. Integrated Plant Genetic Resources Management Systems for Sustainable Agriculture ................................................................... 125 M. Iwanaga, P. Eyzaguirre and J. Thompson Introduction ......................................................................................... 125 Biodiversity: A Foundation for Food Security, Poverty Elimination and Environmental Protection .................................. 125 Challenges of Agricultural Systems .................................................... 127 Plant Genetic Resources Systems View: Avenues for Meeting Agricultural Challenges ................................................................... 129 System View and Institutional Framework ........................................ 131 Conclusions ......................................................................................... 136 12. Genebank Management of Crop Genetic Resources ........................... 139 M.Nakagahra, S. Miyazaki and D.A. Vaughan Introduction ......................................................................................... 139 Features of Genebank Management in Japan .................................... 140 Success Stories in Management of Crop Genetic Resources ............. 145 Evolving Issues Related to the Management of Crop Genetic Resources ........................................................................... 148

13. Biodiversity Conservation Ex Situ and In Situ Conservation: A Case in Turkey ................................................................................... 151 A. Tan Introduction ......................................................................................... 151 Biodiversity Conservation Activities in Turkey ................................. 153 14. Plant Genetic Resources for Food and Agriculture: Status and Future Prospects ................................................................. 159 K.V. Raman and K.N. Watanabe Contribution and Value of PGRFA .................................................... 159 Utilization of PGRFA Including Genetic Vulnerability and Genetic Erosion ........................................................................ 161 Genetic Improvement and Use of Biotechnology Applications in PGRFA ......................................................................................... 162 Potato Late Blight as a Case Study ...................................................... 164 Conclusions ......................................................................................... 167

Section V Introduction: Improvements of Plant Function with Conventional Methods and Biotechnology ................................................................. 171 T. Fujimura 15. Engineering Carbohydrate Metabolism in Transgenic Plants ........... 173 A.G. Heyer Introduction ......................................................................................... 173 Source Capacity ................................................................................... 173 Sink Capacity ....................................................................................... 174 Transport Processes ............................................................................. 175 Altering Carbohydrate Composition: Starch ..................................... 176 Altering Carbohydrate Composition: Fructan ................................... 177 16. Super-RuBisCO for Improving Photosynthesis .................................. 183 A. Yokota, S. Okada, C. Miyake, H. Sugawara, T. Inoue and Y. Kai Introduction ......................................................................................... 183 Why is RuBisCO the Target? ............................................................... 183 Is Plant RuBisCO the Most Evolved Enzyme? ................................... 184 Structural Analysis ............................................................................... 185 Physiological Implications .................................................................. 189 17. Molecular Physiology of Nitrogen Recycling in Rice Plants .............. 191 T. Yamaya, S. Kojima, M. Obara, T. Hayakawa and T. Sato Introduction ......................................................................................... 191

Physiology and Biochemistry of Nitrogen Recycling in Rice Plants ................................................................................... 192 Export of Glutamine from Senescing Leaves ..................................... 192 Re-Utilization of the Transported Glutamine in Developing Organs .............................................................................................. 194 Primary Assimilation of Ammonium Ions in Rice Roots ................. 194 Variation in the Amounts of GS1 and NADH-GOGAT Protein in Rice Plants ...................................................................... 196 Conclusion ........................................................................................... 196 18. Agrobacterium-Mediated Cereal Transformation: Low Glutelin Rice Development ................................................................................. 199 T. Kubo, Y. Hiei, Y. Ishida, Y. Maruta, J. Ueki, N. Nitta and T. Komari Introduction ......................................................................................... 199 Agrobacterium-Mediated Genetic Transformation of Cereals .......................................................................................... 199 Development of Low Glutelin Rice .................................................... 201

Section VI Introduction: Environmental Adaptation and Generation of Resistant Plants ........................................................................................... 205 H. Uchimiya 19. Stress Tolerance in Crops—How Many and Which Genes? ............... 207 H.J. Bohnert, H.-X. Li and B. Shen Introduction ......................................................................................... 207 Osmotic Adjustment ........................................................................... 208 Functions of Compatible Solutes ........................................................ 210 Radical Oxygen Species are Unavoidable and Increase During Stress Episodes .................................................................... 210 Manipulation of Reducing Power ...................................................... 212 Controlled Ion and Water Uptake ...................................................... 212 How Many and Which Genes for Stress Tolerance? ......................... 216 20. Improving Drought, Salt and Freezing Stress Tolerance in Transgenic Plants .............................................................................. 223 K. Yamaguchi-Shinozaki, M. Kasuga, Q. Liu, Y. Sakuma, H. Abe, S. Miura and K. Shinozaki Introduction ......................................................................................... 223 Function of Water Stress-Inducible Genes ........................................ 224 Expression of Dehydration-Induced Genes in Response to Envitonmental Stresses and ABA ............................................... 224

Identification of Cis-Acting Element, DRE, Involved in Drought Responsive Expression .................................................................... 224 Important Roles of the DRE Binding Proteins During Drought and Cold Stresses ............................................................................. 225 Analysis of the In Vivo Roles of DREB1A and DREB2A by Using Transgenic Plants ............................................................. 227 Drought, Salt and Freezing Stress Tolerance in Transgenic Plants ................................................................................................ 227 21. Characterization of Salt Inducible Genes from Barley Plants ............ 231 T. Takabe, T. Nakamura, Y. Muramoto and S. Kishitani Introduction ......................................................................................... 231 Nuclease 12 .......................................................................................... 231 ATP-Dependent RNA Helicase .......................................................... 232 Betaine Aldehyde Dehydrogenase ...................................................... 233 Production of Transgenic Plants with Increased Salt Tolerance ......................................................................................... 234 22. Transgenic Rice: Development and Products for Environmentally Friendly Sustainable Agriculture ...................... 237 S.K. Datta Introduction ......................................................................................... 237 Case Study of Transgenic Rice ............................................................ 237 Environmentally Friendly Selectable Marker Genes ......................... 241 23. Plant Programmed Cell Death and Environmental Constraints—Adenylate Homeostasis and Aerenchyma Formation .............................................................................................. 247 H. Uchimiya, P. K. Samarajeewa and M. Kawai Introduction ......................................................................................... 247 Stimulation of Adenylate Kinase in Rice Seedlings Under Submergence Stress Introduction ....................................... 247 Sodium Chloride Stimulates Adenylate Kinase Level in Seedlings of Salt-Sensitive Rice Varieties ................................... 250 Dissection of Programmed Cell Death in Root Cortex in Rice ....... 251 Effects of NaCl on Cortical Cell Death ............................................... 253

Section VII Introduction: Biotechnology of Woody Plants ............................................. 257 S. Kitani Micropropagation ............................................................................... 257 Genetic Engineering ............................................................................ 257 Molecular Tools ................................................................................... 257

24. Molecular Tools for Capturing the Value of the Tropical Rain Forest ............................................................................................. 259 M. Van Montagu Introduction ......................................................................................... 259 The Model Plant Arabidopsis .............................................................. 259 The Genome Research ......................................................................... 260 Functional Genomics .......................................................................... 260 Biosynthetic Pathways for Secondary Metabolites ............................ 262 Tropical Diversity Studies ................................................................... 262 Forestry Research ................................................................................. 263 Stronger Plants Through Plant Engineering ...................................... 264 The Changing World of Industry ....................................................... 264 Conclusion ........................................................................................... 265 25. Improvement of a New Transformation Method: MAT Vector System .............................................................................. 267 H.Ebinuma Introduction ......................................................................................... 267 Principle of MAT Vectors ................................................................... 267 Transformation Procedure ................................................................. 267 Improvement of MAT Vectors ........................................................... 271 Plasmid Release .................................................................................... 274 26. Formation and Characterization of Transformed Woody Plants Inhibiting Lignin Biosynthesis .................................................. 275 Noriyuki Morohoshi Introduction ......................................................................................... 275 Development of the Technical Requisites to Inhibit Lignin Biosynthesis ..................................................................................... 276 Identification of a Peroxidase Enzyme Involved in Lignification ................................................................................ 276 Properties of the Poplar Controlled by the Peroxidase Gene ................................................................................................. 278 Conclusion ........................................................................................... 278 27. Tolerance of Acacia Mangium to Acid Soil ............................................................................................. 281 S. Kitani, N. Higuchi and I. Yasutani Introduction ......................................................................................... 281 Materials and Methods ........................................................................ 281 Results .................................................................................................. 283 Discussion ............................................................................................ 283 Introduction ......................................................................................... 289

28. Developing a Mass Propagation System for Woody Plants ................ 289 T. Kozai, C. Kubota, S. Zobayed QT Nguyen, F. Afreen-Zobayed and J. Heo Reasons for High Production Costs and Their Reduction by Photoautotrophic Micropropagation........................................ 290 Growth Promotion and Quality Improvement Using Small Culture Vessels ................................................................................ 290 Forced Ventilation Micropropagation Systems and Their Application ...................................................................................... 296 Scaled-Up Micropropagation System by Use of an Aseptic Culture Room25,26 ......................................................................... 300 Conclusion ........................................................................................... 300 29. Advances in Conifer Tree Improvement Through Somatic Embryogenesis ....................................................................................... 303 P. K. Gupta, R. Timmis, K. Timmis, J. Grob, W. Carlson, E. Welty and C. Carpentar Introduction ......................................................................................... 303 Culture Establishment ......................................................................... 303 Embryo Development, Maturation and Germination ...................... 304 Cryopreservation ................................................................................. 304 Field Performance ............................................................................... 305 Large Scale Production ........................................................................ 305 Embryo Sorting .................................................................................... 306 Manufactured Seed .............................................................................. 307 Clonal Field Tests ................................................................................ 307 Conclusion ........................................................................................... 308

EDITORS Kazuo Watanabe, Ph.D. Faculty of Biology-Oriented Science and Technology Kinki University Uchida-Cho, Naga-gun Wakayama, Japan

Atsushi Komamine, Dr. Sc. Research Institute of Evolutionary Biology Kamiyoga, Setagaya Tokyo, Japan

CONTRIBUTORS Hans J. Bohnert, Ph.D. Department of Biochemistry The University of Arizona Tucson, Arizona, U.S.A. Chapter 19

Pramod K. Gupta, Ph.D. Strategic Biology Research Weyerhaeuser Co. Tacoma, Washington, U.S.A. Chapter 29

Swapan K. Datta, Ph.D. Plant Breeding, Genetics and Biochemistry Division The International Rice Research Institute Manila, Philippines Chapter 22

Arnd G. Heyer, Ph.D. Max-Planck-Institute for Molecular Plantphysiology Golm, Germany Chapter 15

Hiroyasu Ebinuma, Dr. Agriculture Nippon Paper Industries Co., Ltd. Tokyo, Japan Chapter 25 Tatsuhito Fujimura, Dr. Sc. Institute of Agriculture and Forestry University of Tsukuba Ibaraki, Japan Introduction, Section V Introduction, Chapter 11 Douglas Gollin, Ph.D. Department of Economics Williams College Williamstown, Massachusetts, U.S.A. Chapter 4

Hiroshi Ikehashi, Dr. Agriculture Department of Agronomy Graduate School of Agriculture Kyoto, Japan Section II Introduction, Chapter 1 Osamu Ito, Ph.D. Plant Physiology and Agroecology Division International Rice Research Institute Manila, Philippines Chapter 8 Masaru Iwanaga, Ph.D. International Plant Genetic Resources Institute Rome, Italy Chapter 11

Wolfgang Kinzelbach, Dr. Ing. Institut für Hydromechanik and Wasserwirscchaft Eidgenössische Technische Hochschule Zurich, Switzerland Chapter 7 Shigekzu Kitani, Ms.Sc. Biology Research Lab Toyota Motor Corporation Toyota, Japan Section VII Introduction, Chapter 27 Toyoki Kozai, Ph.D. Department of Bioproduction Science Faculty of Horticulture Chiba University Matsudo, Japan Chapter 28 Tomoaki Kubo, Ph.D. Plant Breeding and Genetics Research Lab Japan Tabacco Inc. Shizuoka, Japan Chapter 18 Satoshi Matsumoto, Dr. Agriculture Graduate School of Agriculture University of Tokyo Tokyo, Japan Section III Introduction, Chapter 10 Noriyuki Morohoshi, Dr. Agriculture Graduate School of Bio-Application and Systems Engineering Tokyo University of Agriculture and Technology Fuchu, Japan Chapter 26 Masahiro Nakagahra, Dr. Agriculture National Agriculture Research Center MAFF Tsukuba, Japan Chapter 12

Mangala Rai, Ph.D. Crops Sciences Indian Council of Agricultural Research New Delhi, India Chapter 3 Kandukuri V. Raman, Ph.D. Department of Plant Breeding Cornell University Ithaca, NY, U.S.A. Chapter 14 James F. Reynolds, Ph.D. Department of Botany Duke University Durham, North Carolina, U.S.A. Chapter 6 Tetsuko Takabe Bioscience Research Center Nagoya University Nagoya, Japan Chapter 21 Hirofumi Uchimiya, Ph.D. Institute of Molecular and Cellular Biosciences The University of Tokyo Bunkyo-ku, Japan Section VI Introduction, Chapter 23 Ayfer Tan, Ph.D. Plant Genetic Resources Department Aegean Agricultural Research Institute Menemen Izmir, Turkey Chapter 13 Hiroshi Tsujii, Ph.D. Division of Natural Resource Economics Graduate School of Agriculture Kyoto University Kyoto, Japan Chapter 2

Marc Van Montagu, Ph.D. Flanders Interuniversity Institute for Biotechnology Gent University Gent, Belgium Chapter 24 Kazuko Yamaguchi-Shinozaki, Dr. Sc. Biological Resources Division Japan International Research Center for Agricultural Sciences MAFF Tsukuba, Japan Chapter 20 Tomoyuki Yamaya, Dr. Agriculture Department of Applied Plant Science Tohoku University Sendai, Japan Chapter 17

Tomohisa Yano, Dr. Agriculture Arid Land Research Center Tottori University Tottori, Japan Chapter 9 Akiho Yokota, Dr. Agriculture Department of Molecular Biology Nara Institue of Science and Technology Nara, Japan Chapter 16 Huqu Zhai, Ph.D. Nanjiing Agricultural University Nanjiing, China Chapter 2

FOREWORD For contribution to the growth and advancement of emerging fundamental science and technology, the first Toyota Conference was organized in 1987 in celebration of the 50th anniversary of the Toyota Motor Corporation. Since then, this converence has been held every year, dealing with a broad range of subjects in different fields. In this conference, dozens of Japanese and foreign experts spend four days and three nights living together, discussing a common theme. Participants have consistently praised this format, which encourages deeply involved discussions and fresh insights. Each Toyota Conference is planned and executed independently by a third-party organizing committee under the sponsorship of Toyota Motor Corporation, and its secretariat is placed in Toyota Central R&D Labs., Inc. One essential idea for the 21st century is “sustainable development.” I believe that agriculture has a vital role for this development. Many people are optimistic about plants’ ability to cleanse and protect the environment; for example, to help prevent global warming by fixing carbon monoxide with photosynthesis. Furthermore, experts forecast the global population topping 10 billion by the middle of the next century. If all these people are to enjoy meaningful lives, we must find environmentally friendly ways to produce sufficient supplies of safe foods. In other words, the world must adopt the principles and practices of sustainable agriculture. In view of this issue, the theme chosen for the 12th Toyota Conference was the “Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century.” I understand that 56 researchers from 16 countries have considered this theme, conducting lively discussions not only covering scientific subjects like biotechnology but also addressing social and economic issues. Today marks the release of the Toyota Conference record as part of the Environmental Intelligence Unit series published by Landes Bioscience. On this occasion, I would like to thank and congratulate all those who submitted papers, as well as those who helped edit the transcript of the conference proceedings. At the same time, I sincerely hope the proceedings of the 12th Toyota Conference will spur biological and scientific researchers around the world to take on this difficult challenge, while fostering greater cross-pollination efforts with other fields of study. And I also hope that these proceedings help mankind motivate to overcome the crises facing the earth’s biosphere in the 21st century. May 1999 Tatsuro Toyoda Chief Executive Officer Toyota Central R&D Labs, Inc.

ORGANIZING COMMITTEE OF THE 12TH TOYOTA CONFERENCE Chairperson:

Vice Chairperson:

Members:

Professor Atsushi Komamine Research Institute of Evolutionary Biology 2-4-28, Kamiyoga, Stegaya, Tokyo 158-0098, Japan Professor Tatsuhito Fujimura Institute of Agriculture and Forestry University of Tsukuba 1-1-1 Tennodai, Tsukuba. 305-8571, Japan Professor Hiroshi Ikehashi Department of Agronomy, Graduate School of Agriculture Kyoto University Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Mr. Shigekazu Kitani Toyota Motor Corporation 1 Toyota-cho, Toyota 471-8572, Japan

Professor Satoshi Matsumoto Department of Applied Biological Chemistry, Graduate School of Agriculture The University of Tokyo 1-1-1- Yayoi. Bunkyo-ku, Tokyo 113-8657, Japan Professor Hirofumi Uchimiya Institute of Molecular and Cellular Biosciences The University of Tokyo 1-1-1- Yayoi. Bunkyo-ku, Tokyo 113-8657, Japan Professor Kazuo N. Watanabe Faculty of Biology-Oriented Science and Technology Kinki University Uchita-cho, Naga-gun, Wakayama 649-6433, Japan Dr. Yukio Yamada Toyota Central Research and Development Laboratories, Inc. Nagakute, Aichi 480-1192, Japan Auditor:

Secretary General:

Dr. Atsushi Danno Toyota Central Research and Development Laboratories, Inc. Nagakute, Aichi 480-1192, Japan Ms. Sakoto Tanabe Toyota Central Research and Development Laboratories, Inc. Nagakute, Aichi 480-1192, Japan

TOYOTA CONFERENCES SPONSORED BY TOYOTA MOTOR CORPORATION The 1st TOYOTA Conference Molecular Conformation and Dynamics of Macromolecules in Condensed Systems September 28-October 1, 1987 Aichi, Japan organized by M. Nagasawa The 2nd TOYOTA Conference Organization of Engineering Knowledge for Product Modelling in Computer Integrated Manufacturing October 2-5, 1988 Aichi, Japan organized by T. Sata The 3rd TOYOTA Conference Integrated Micro Motion Systems—Micromachining, Control and Applications October 22-25, 1989 Aichi, Japan organized by F. Harashima The 4th TOYOTA Conference Automation in Biotechnology October 21-24, 1990 Aichi, Japan organized by I. Karube The 5th TOYOTA Conference Nonlinear Optical Materials October 6-9. 1991 Aichi, Japan organized by S. Miyata The 6th TOYOTA Conference Turbulence and Molecular Processes in Combustion October 11-14, 1992 Shizuoka, Japan organized by T. Takeno The 7th TOYOTA Conference Towards the Harnessing of Chaos October 31-November 3, 1993 Shizuoka, Japan organized by M. Yamaguti The 8th TOYOTA Conference Toward Global Planning of Sustainable Use of the Earth—Development of Global Eco-Engineering November 8-11, 1994 Shizuoka, Japan organized by S. Murai

The 9th TOYOTA Conference Brain and Mind—for Better Understanding of the Dynamic Function of Mind and Its Supporting Brain Mechanism December 5-8, 1995 Shizuoka, Japan organized by M. Ito The 10th TOYOTA Conference Atomic, Molecular and Electronic Dynamic Processes on Solid Surfaces November 5-8, 1996 Shizuoka, Japan organized by M. Aono The 11th TOYOTA Conference Nanostructured Materials in Biological and Artificial Systems November 5-8, 1997 Shizuoka, Japan organized by A. Yamagishi The 12th TOYOTA Conference Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century November 25-28, 1998 Shizuoka, Japan organized by A. Komamine The 13th TOYOTA Conference Affective Minds November 29-December 2, 1999 Shizuoka, Japan organized by G. Hatano

ACKNOWLEDGEMENTS

W

e take this opportunity to thank all participants in the Conference and the following members of the Organizing Committee of the Conference for their helpful cooperation.

Professor Tatsuhito Fujimura University of Tsukuba, Vice Chairperson Professor Hiroshi Ikehashi Kyoto University Mr. Shigekazu Kitani Toyota Motor Coporation Professor Satoshi Matsumoto The University of Tokyo Professor Hirofumi Uchimiya The University of Tokyo Dr. Yukio Yamada Toyota Central R&D Labs., Inc. Dr. Atsushi Danno Toyota Central R&D Labs., Inc.

We express our sincere thanks to Toyota Motor Corporation for sponsoring this Conference and to Ms. Satoko Tanabe, Secretary General, Mr. Kazufumi Morimoto and other members of Secretariat of Toyota Central Research and Development Labs., Inc. for their excellent organization of this fruitful conference and the Proceedings.

PREFACE

T

he most serious problem which we, as human beings, will face in the early 21st century is whether or not we can overcome the crisis in the biosphere that will occur on this planet. There are two reasons why we should anticipate a crisis in the biosphere. One is that we may be unable to achieve a sufficient level of agricultural production to support an explosively increasing population. If so, there will be rampant famines. The other is that all life on this small planet will be jeopardized by destruction of environments caused by pollution, global warming and desertification of lands. Such global disruptions would happen in the course of increased industrial production for raising the standard of living, and excessive use of fertilizers, herbicides and insecticides for promotion of crop production. Plants and agricultural sciences are playing a leading role in the rescue of human beings from the crisis in the biosphere; plant biotechnology may improve crop functions to rapidly promote food production. Plant and agricultural sciences also may produce plants tolerant to environmental stresses such as drought, salinity and coldness, and thus would expand land available for cultivation. It is also possible to produce crops resistant to diseases and arthropods using plant biotechnology, suppressing the excess usage of agricultural chemicals such as herbicides and pesticides. Woody plants will also play important roles in the suppression of CO2 increase in the atmosphere and in producing plant biomass. Plant biotechnology will improve the functions of woody plants and provide seedlings on a large scale to replace the destroyed tropical forests and to preserve environments. The aim of this international conference and the Proceedings was to discuss strategies for global crop production and environmental problems from the aspect of plant and agricultural sciences. With distinguished speakers and delegates from various areas of the world, we intended to discover research priorities toward overcoming the crisis in the biosphere in the coming century. The conference encompassed the following topics: 1. Prospect of supply and demand for food and research strategies for the coming century. 2. Improvements of plant functions with conventional methods and biotechnology. 3. Environmental adaptation and generation of resistant plants. 4. The present situation of biological production and an approach to sustainable production in arid lands. 5. Conservation and contribution of plant genetic resources. 6. Biotechnology of woody plants. It is my great pleasure to publish this book as the Proceedings of the 12th Toyota Conference, “Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century”, which contains 29 articles on the subjects mentioned. I trust that these proceedings may make a great contribution to rescue human beings from the biosphere crisis of the upcoming 21st century. Atsushi Komamine Chairperson of the Organizing Committee of the 12th Toyota Conference

SECTION I

Introduction to the Challenge: Guide for the Book A. Komamine, T. Fujimura and K. N. Watanabe

T

he most serious problem which we, as human beings, will face in the early 21st century is whether or not we can overcome the crisis in the biosphere that will occur on this planet. There are two reasons why we should anticipate a crisis in the biosphere. One is that we may be unable to achieve a sufficient level of agricultural production to support an explosively increasing population. If so, there will be rampant famines. The other is that all life on this small planet will be jeopardized by destruction of environments caused by pollution, global warming and desertification of lands. Such global disruptions would happen in the course of increased industrial production for raising the standard of living, and excessive use of fertilizers, herbicides and insecticides for promotion of crop production. Plants and agricultural sciences are playing a leading role in the rescue of human beings from the crisis in the biosphere; plant biotechnology may improve crop functions to

rapidly promote food production. Plant and agricultural sciences also may produce plants tolerant to environmental stresses such as drought, salinity and coldness, and thus would expand land available for cultivation. It is also possible to produce crops resistant to diseases and arthropods using plant biotechnology, suppressing the excess usage of agricultural chemicals such as herbicides and pesticides. Woody plants will also play important roles in the suppression of CO2 increase in the atmosphere and in producing plant biomass. Plant biotechnology will improve the functions of woody plants and provide seedlings on a large scale to replace the destroyed tropical forests, to preserve environments and biodiversity. The aim of this book is to discuss strategies for global food production and environmental problems associated with plant and agricultural sciences. With distinguished contributors from various disciplines around the world, we intend to discover research priorities for overcoming the crisis in the biosphere in the coming century.

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine. ©2000 Eurekah.com.

SECTION II INTRODUCTION

Prospects of Supply and Demand for Food: Research Strategies for the Coming Century H. Ikehashi

T

his session is organized as an interface between understanding of social or agricultural situations in the near future and evaluation of research strategies. Two papers from economists, two papers from leaders of national agricultural sciences and an additional one on genetic improvement of rice have been prepared for the discussion. An outlook for food supply and demand is delineated by Professor H. Tsujii, who predicts a substantial deficit of food in view of population growth and other factors. Following this general view, demand for food and agricultural sciences is discussed by one

leading agricultural scienticst from each of China and India, which support the largest and the second largest population, respectively, in the world. Each of the reports emphasizes the need for developing agricultural research. Then, policies to set priorities on each research area are discussed by Dr. D. Gollin, on the bases of past experience and economic principles. Lastly, a short note on the past achievements and expected potential in the genetic improvement of rice is given by the session organizer, who cites a set of available tools in the area.

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine. ©2000 Eurekah.com.

CHAPTER 1

Food Shortage in the 21st Century and Its Implications for Agricultural Research H. Tsujii

Introduction

T

he world grain stock/use ratios (ratio of stock volume against use volume) which are the criteria for global availability of grain have shown a tendency to fall since 1987. According to USDA data, except for 1997/98 the world average stock/use ratio for all grain has been below 17% since 1994/95; FAO considers this a dangerously low level. The ratio is at around the lowest level since the war, and is about the same as the level during the food crisis year of 1974. The stock/use ratios for rice and coarse grain have been lower than the average stock ratio since 1989/90, and they have been lower than 17% since 1993/94. The ratio for rice is projected to be at the dangerously low level of little more than 11% in 1998/ 99. These low stock/use ratios are caused by the following long term factors: transformation in agricultural policy during the late eighties and the nineties in both Europe and the United States, the stagnation in agricultural technology improvement, scarcity in and degradation of natural resources such as soil and water, yield constraint due to increased cropping intensity, the world population explosion and the rapid increase in demand for feed grain caused mainly by the high economic growth in Asia, most notably in China. In this paper, effects of these factors on the world food demand and supply in the past and in the near future are investigated and their implications to agricultural research are considered.

Since the last half of the eighties, the agricultural policies in Europe and the United States have changed from protectionist, surplus producing and dumping export policies to policies of reducing price support, subsidizing income on a decoupled basis, curtailment of surpluses, correction of interregional differences and environmental protection. Since these policy changes have been made under the influence of the Uruguay Round agricultural trade negotiations during 19861993 and under the WTO framework, these changes will continue into the 21st century and thus will keep the stock ratios for grain at a low level. The limitation in agricultural technology improvements and in natural resources is clearly represented by the sharp decline in the growth rates in grain yields across the globe during the post-Second World War years. According to the FAO data, the annual growth rate of the yield has declined continuously from about 3% during the 70s to about 1% during 1985 and 1996. Agricultural research expenditures in the international and national research institutions have been decreasing considerably. Yield decline or constraint for grain due to increase in cropping intensity has been reported in many parts of Asia. In order to cope with the exploding population, the grain yield must grow at 3% annually, and this seems very difficult to attain in the near future.

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine. ©2000 Eurekah.com.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Water shortage for agricultural production has been worsening, especially in fast growing Asia. Water demand for nonagricultural purposes has been growing at high speed in many developing countries. Annual stoppage of water flow in the Yellow River in China, which is a good indication of water shortage in northern China, has been rapidly worsening since 1990 in terms of number of days per year and distance of no water flow from the mouth of the river. In 1997 the number of days became more than 250 days and the distance became more than 700 km. In my interview with farmers and researchers in Punjab, India, which is the granary for the whole of the Indian people, in August this year, I heard that the underground water table in most of the Punjab is declining at about 50 cm per year because of too much pumping of water for agricultural production. This is also the case in many other places in India. If the decline continues at this speed, in the near future it will cause a severe reduction in Punjab grain production. Water supply suspension in most cities in India lasts very long hours every day, and rivers near large cities on the Deccan Plateau in India are extremely polluted. Arable land and planted areas of grain have been decreasing the last two decades over the globe. Soil degradation such as erosion, desertification and salinization has been spreading very fast on the globe. A very wide area of forest has been cut and burnt on the globe, and afterwards the area has been used for agricultural purposes, often by extensive soil mining techniques. The world population had increased by 2.5 billion over the past 4 million years. But, it increased by the same amount between 1950 and 1985. This population explosion started around 1960, and it will continue up to the year 2025.1 Annual growth of the world population is more than 70 million for the period of 1955/60 and 2020/25. It is more than 80 million during 1875/80 and 1995/ 2000. Population explosion is an important factor in increased food demand. Fast economic growth, especially in the developing countries, accelerates the increase in the world demand for food because fast income growth of the people causes shifts in the dietary

pattern of the people from more carbohydrate consumption to more animal protein consumption. This leads to a rapid increase in the demand for feed grain. Recent negative economic growth of the developing countries caused by monetary crises creates a temporary reversal in this tendency. Incorporating all the factors affecting world grain demand and supply discussed just above, using a simple projection model and assuming future values of exogenous variables of the model such as population, income, income elasticity and conversion ratio between feed and meat, I have projected the world demand and supply of grain in the year 2020. The projected world deficit of grain in 2020 is 417 million metric tons. The current world total grain trade is about 200 million tons, and the projected deficit is very large. Assuming world price elasticity of demand and supply of grain to be about 0.15, the world trade price of grain will increase by about 50% in the year 2020 compared to the base year of 1993. Lester Brown,2 Ministry of Agriculture, Forestry and Fishery of Japan, and FAO3 predict a shortage of grain in the early 21st century. International organizations such as IFPRI and the World Bank predict that grain prices will decrease by 10 to 30%, and thus think that we will face a surplus of grain in the world in the early 21st century. Reading the publications projecting the surplus, I think the assumptions for their projection are too optimistic, for instance, no limitation in arable land and water, positive price response in planted areas of grain, and considerable yield growth of grain in the future supported by technological improvements in agricultural production. A green revolution in rice and wheat that is based on intensification in modern inputs such as fertilizers, chemicals, agricultural machinery and irrigation water increased, on one hand, production of these grains in the world to reduce the huge number of world hungry. But, on the other hand, it destroyed natural environment and overused natural resources. Population explosion and income growth in the developing countries will cause an explosion of grain demand. This may lead to severe environmental destruction and exhaustion of natural resources if the demand is met by growth of grain production based

Food Shortage in the 21st Century and Its Implications for Agricultural Research on the conventional technology. The severe environmental destruction and exhaustion of natural resources will aggravate the constraints on the world grain supply, as has been shown by a large decline in the growth rate of grain yield during the last decade and a half. These factors will cause severe food shortage in the 21st century, and we will need further increases in food supply. I believe that developing countries should take more measures to slow down the population explosion, and high income countries should slow down economic growth, as was recommended by the Club of Rome in 1972.4 On the other hand, agricultural research that has pursued only yield increase in the past must emphasize technological improvements which not only increase yield but also conserve environment and natural resources at the same time. This means that so called socially optimal yield increase must be sought in agriculture. Researches for crops’ resistance to drought, pests, insects and salinity, etc. should be carried further, since improvement in these resistances will increase yield and at the same time reduce environmental destruction. Socioeconomic and engineering research may yield large supply increases of grain. Restructuring in the distribution of research funds is needed. Individual researchers would find it difficult to influence this restructuring because research is extremely sectionalized. There is a strong need for public research policy to direct and organize agricultural research in this direction.

Factors Determining Food Shortage in the Early 21st Century The world grain stock/use ratios (ratio of stock volume against use volume), which are the criteria for global availability of grain, have shown a tendency to fall since 1987 (Fig. 1.1). According to USDA data, except for 1997/98 the world average stock/use ratio for all grain has been below 17% since 1994/95; this is considered by the FAO to be a dangerously low stock level. The ratio is at around the lowest level since the war, and is about the same as the level during the food crisis year of 1974. The stock/use ratios for rice and coarse grain have been lower than the average stock/use ratio for all grains since 1989/90, and they have

7

been lower than 17% since 1993/94. The ratio for rice is projected to be at the extremely low level of little more than 11% in 1998/99. Grain prices rose during the first half of the nineties. Mr. J. A. Sharples, a specialist on the world grain market In the United States Department of Agriculture, mentioned in the fourth issue of “Choices” magazine in 1995 that the stockpile of major grain exporting countries such as America and Canada, which have played the role of international grain reserve stockholders since the war, has decreased in stock down to only 1.4% of the low world grain stock. The world grain market has been in a serious shortage situation. Although this reduction in grain stock/ use ratios and the increase in grain prices are partly caused by short term factors, such as reduced production of rice and coarse grain in the 1995 crop year in America, surplus investment funds flowing into the grain futures market, caused by a general slowdown in the economies of high income countries, and the increased price of feed grain, caused by an increase in the price of American beef due to mad cow disease in the UK. But, basically the ratios are affected by the long term factors, such as transformation in agricultural policies during the late eighties through to the nineties in both Europe and America, limitation in agricultural technology improvement, increasing scarcity in and degradation of natural resources such as soil and water, yield stagnation due to increase of cropping intensity in many developing countries, the world population explosion and the rapid increase in demand for feed grain, caused mainly by the high economic growth in Asia, most notably in China. In this paper, the effects of these long term factors affecting food supply and demand in the 21st century are first investigated. Then, a projection of world food demand and supply in the early 21st century is made and compared with other projections. Finally, implications of these analyses for agricultural research are presented.

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

stock/use ratios (%)

8

crop year Fig 1.1. Stock/use ratios of world major grains at the end of each crop year. Data source: USDA Database by internet and other USDA publications. est: estimates; proj: projected values.

Declining Trend in the World Grain Stock Ratio and Transformation of Agricultural Policies in Europe and America After the Last Half of the Eighties The EC and the United States have been the major holders of international reserve stock in and exporters of agricultural commodities until recently. Since the last half of the eighties, the agricultural policies of these countries have changed from protectionist, surplus producing and export dumping

policies to policies of reduced protection, curtailment of surpluses, correction of interregional income differences and environmental protection. The EC earlier achieved increased agricultural production, improving its farm size structure and increasing the farm income level through variable import levies of the Common Agricultural Policy (CAP), export subsidies and domestic price support. Until the seventies, it has been a net importer of grain, at an average of approximately 30 million tons

Food Shortage in the 21st Century and Its Implications for Agricultural Research annually. It became self-sufficient in major agricultural products by the first half of the eighties, and in 1984 it became a net exporter. It exported more than 20 million tons annually by the end of the eighties, supported by the export subsidies of CAP. The surplus agricultural products stock in the EC reached enormous levels in the middle of the eighties. 5 The financial burden for the price support and the export subsidies for the excess agricultural products reached an unbearable level. The transformation in the EC agricultural policy started with production control and decreasing the support price in 1982, and it was gradually strengthened afterward. Then it was expanded to environmental maintenance and reduction of interregional differences, whilst ensuring the objectives of improved productivity, stability of supply and farmers’ livelihoods, and sound pricing of CAP, as stated in the Prospects of the Common Agricultural Policy (Green Paper) in 1985. A comprehensive financial reform plan (Delor package) in 1987 was also heading in this direction, and was agreed upon by the European Board of Directors in February 1988. The stabilizer and set aside were introduced in 1988. Finally, a significant agricultural reform in 1992 was agreed upon that has the objectives of large reductions in the support prices complemented by decoupled income subsidy, production control, protection for the medium and small scale farms (e. g., special assistance for agricultural management by youth), the preservation of the environment by the extensification policy (e. g., special action for the disadvantaged areas and environmental preservation areas). Then, these reforms were subsequently merged with the agreements of the Uruguay Round Talks of 1993, whose major characteristics were tariffication of the variable import levies, minimum access import, reduction of export subsidies and decrease of domestic protection. Annual grain export from the United States has continued to increase, reflecting strengthened U.S. agricultural protection and increased agricultural production following the world food crisis in 1974, from 40 million tons in the sixties to a peak of 112.7 million tons in 1981. However, grain exports fell

9

sharply during the first half of eighties, reflecting excessive domestic protectionism, a strong dollar and the rapid increase of grain exports from the EC. The total U.S. grain stock increased significantly, from 50 million tons in the mid-seventies to 200 million tons by 1986. Stocks of other agricultural products also increased.6 The financial expenditure increased rapidly to an excessive level, in order to protect domestic agriculture and to subsidize export of the surplus agricultural products. To cope with these problems, the 1985 Agricultural Law introduced the 50/92 policy in order to reduce planted area of grain for the first time: reductions in the target price and in the farm support price (loan rate); the Conservation Reserve Program (CRP), which took a total of 18 million hectares of high erosion risk areas out of production through the grant of an annual average rent of $121/ hectare to the owners; and new subsidy of marketing loans, which decreased the grain export price to the international price level. These measures, except marketing loans, were production restriction policies. These policies were further strengthened in the 1990 Agriculture Law by the expansion of flexible planting and by fixing the program yield. These measures to reduce agricultural protection, surplus and financial expenditures were finally absorbed into the agreements reached in the Uruguay Round agricultural trade agreement in 1993. Thus agricultural policies of the EU and the United States have been greatly transformed from those of the mid-eighties, high protection, surplus accumulation and heavy export subsidy, to those of lower protection, less surplus, less export subsidy and more concern for environment and less advantaged areas. This transformation is clearly reflected in the long term reversal in the trend of net food (excluding fish, hereafter simply called food)7 export quantity between the developed countries and the developing countries of the world since the last half of the eighties, as shown in Figure 1.2. Before 1985, the EU and the United States dominated food exports among all developed countries. During the same period, the EU and the United States had continued to increase their food exports, while the

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

developing countries deceased their food import. The developed countries had been net importers of food during the sixties and early seventies, and they become net exporters beginning in 1977; then they increased their net export quantity rapidly during the late seventies. On the other hand, the developing countries changed from being net exporting regions of food to net importers in 1977, as shown in the figure, and their net import quantity increased rapidly. This change in the food trade balance between developed and developing regions is caused by the fall in food prices in developing countries, resulting from dumping exports of surplus agricultural products by the EU and the United States and by the policy of agricultural exploitation in the developing countries themselves. The change is unjust because rich industrialized countries dumped their surplus food to suppress food production and agricultural income in the poor agricultural developing countries, and the developing countries exploited their own poor farmers. This change is also not desirable from the viewpoint of the theory of comparative cost. A reversal of this change started in 1985, as shown in Fig. 1.2. This reversal was brought about by transformation of the agricultural policies of the EU and the US, in order to reduce the excessive financial burden from domestic agricultural protection and export subsidy for huge agricultural surpluses, to preserve the environment and to correct interregional differences in the EU and the United States, as mentioned above.8 Net food exports from developed countries decreased considerably in 1985 and 86, and declined rapidly from the late eighties. The transformation had reduced the grain stock of the EU and the US and thus the world grain stock, and raised world grain prices until recently, as previously described. This transformation will be maintained into the 21st century, since the reductions in agricultural protection in the transformation were integrated into the Uruguay Round trade agreement in 1993 and they will be executed and intensified under the World Trade Organization (WTO) system, which is the international organization for freer trade. Decoupled direct farm payment and liberalization in the kind and

acreage of grain to be planted by American farmers, with abolition of the target price in the 1996 Agricultural Law of the United States, will continue. Further decrease of agricultural protection in the CAP of the EU in the near future, as expressed in the EU’s Agenda 2000, will be executed, since without it agricultural surplus will be accumulated as more middle and east European countries will be included in the EU in the near future. Thus, the EU and the US will not hold large agricultural surplus in the 21st century as they did before. Low global grain stocks, and thus high and unstable grain prices, will be the usual condition in the early 21st century. Thus the world’s 1.1 billion poor and 800 million starving, the majority of which is concentrated in Asia,8 will face the high probability of serious food crises, as they consume grain as their main energy source. 9

Supply Factors Long term supply factors that regulate grain production are natural resources such as land, water and irrigation, and agricultural technology. The constraints on land are becoming severer. The annual growth rates of global cultivated areas for grain have been falling the last three decades, according to the FAO data. The growth rate was 0.33% for the sixties, 0.28% for the seventies and 0.18% in the eighties. The world total arable area has increased from 1.27 billion hectares in 1961 to a peak of 1.44 billion hectares in 1987, and then decreased to 1.38 billion hectares in 1996. As shown in Figure 1.3, the global per capita grain harvested area has continued to fall from 0.24 hectares in 1950 to 0.12 hectares in 1994 as population explosion has continued. The total grain harvested area in the world increased to a peak of 760 million hectares in 1977 as shown in the same figure, but it has since fallen to 690 million hectares in 1994. According to the data from the USDA, the grain harvested area in China had been falling since reaching a postwar peak of 98 million hectares in 1976, and since then has been reduced by 7% to 91 million hectares by 1992. The grain harvested area in India increased by 14 million hectares from 1961 to its peak of 106.6 million in 1983; since then it has fallen by 6.26 million hectares up to 1992.

11

100 mill $ (1966 base year)

Food Shortage in the 21st Century and Its Implications for Agricultural Research

Fig 1.2. Net food export. Data source: FAO, Trad yearbooks According to the FAO data for 1989, the total arable area in the world is approximately 1,500 million hectares, about 800 million hectares of which are in developing countries. Also, the total area of pasture and forest in the world is 7,400 million hectares, 42% of which is in developing countries. How much of this pasture and forest area in the developing countries can be turned into arable land for grain production and how much should be conserved as they are is an important issue in coping with the trade-off problem between food and local environment. An FAO report10 estimated that the potentially cultivable area in 92 developing countries, excluding China, was more than 1,800 million hectares, more than twice as much as the current arable area in the developing countries. Most of this potentially cultivable area exists in South America (48%) and sub-Saharan Africa (44%). Other research institutions and researchers came up with similar estimates during the seventies. Can this vast ,potentially cultivable area which is mostly pasture and forested land be brought into agricultural production? In the same report, the FAO estimated that actual cultivated area would increase only by 93 million hectares in these developing countries by 2010. The reasons that actual reclaimed area in Africa and

South America is estimated to be so small are such strong constraints as: 1. The need for linkage among regional food production, farm income and food demand, by which I mean that reclamation of land in a region must be done by the people in the region, in the sense that the increased income of the people in the region by the reclamation and food and agricultural production on the newly reclaimed land should be spent for increased food production, in order for the reclamation to be sustainable and equitable;11 2. Low population density in Africa and South Africa; 3. Environment and other externality (AUTHOR: externally?) related needs, to maintain forest and pasture; and 4. Economic cost for reclamation.12 In developing countries, competition for land use among economic sectors has been intensified during the last few decades. The agricultural sector has been losing ground in this competition. In my recent survey of agricultural resources on the Deccan Plateau in India in September 1998, I found that most

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Harvested area: 10 million hectares

12

Fig 1.3. World total harvested area of cereals and per capita harvested area of cereals. Note: used mainly the FAO data and additionally the USDA data. of the agricultural land along the main roads (about 500 meters on both sides) within about 50 kilometers radius from main cities had probably been purchased by some nonagricultural entities, and agricultural production there was abandoned. According to my own observations during the last twenty years, a large part of one million hectares of good paddy land in the Menam Chao Phraya Delta surrounding Bangkok, Thailand has been converted to factories, houses, roads and vast unused land. Agricultural land conversion has been rapid in China, Thailand, the Philippines, Java, India, etc. These countries do not have effective agricultural land conservation laws or institutions such as the Agricultural Land Law and supporting institutions in Japan.13 This is the main reason that these countries have lost large amounts of good agricultural land.

Soil quality has been deteriorating throughout the world. According to one study, 15% (2 billion hectares) of the globe’s total 13 billion hectares of land has been degraded.14 Of that total, it is said that 16% (300 million hectares) is severely degraded. In the intensive research interviews which I undertook in 1993 and 1994 with about thirty farmers scattered over semi-arid northeastern Thailand, every farmer without exception said that they have experienced a decrease in the yield of rice and cassava within the last twenty years, and blamed a decrease in soil fertility as the cause. Many farmers told me that they did not have enough barnyard manure to apply to their fields, as agricultural machines had replaced draft animals. They told me that they reluctantly had to start applying small amounts of expensive chemical fertilizer to non-irrigated paddy and upland crop fields in order to cope with the soil degradation. In

Food Shortage in the 21st Century and Its Implications for Agricultural Research my extensive Indian rural survey in August 1998, I found that drying cow dung cakes on farmhouse walls has decreased considerably from two decades ago. This is probably a reflection of the decrease in the number of animals replaced by machines. If so, organic materials input into Indian soil must have decreased considerably. Although the growth rate of global irrigated area was above 2% annually in the sixties and seventies, it fell to just above 1% in the eighties.15,16 An increasing trend in the irrigated area per person has reversed since 1978, and this measure decreased by 6% from 1978 to 1991. The FAO considers that these are serious limitations in food supply, for more than half of the increase in the global food production resulted from the increase in irrigated area from the mid-sixties to the mid-eighties. Causes for the declining growth rate in the irrigated areas are the post-Second World War declining trend in real world grain prices; increases in the cost of building large scale surface irrigation systems in recent decades; severe deterioration in more than half of the irrigation facilities, and shortage of government funds to build new irrigation systems, in developing countries; underutilization of irrigation systems, increasing water wastage, waterlogging, salinization and environmental destruction by dams; human diseases related to irrigation water; and external benefits derived from irrigation facilities.12,17 Soil salinization is said to occur in 10% of the global irrigated area. In 1998, I heard from an expert on Chinese irrigation systems that most of the Chinese irrigation facilities are severely deteriorated and/or were poorly constructed. But there are not enough government fund and time to build new systems against the exploding population. Thus, the main emphasis in the irrigation policy of the Chinese government is now water saving. In 1995 a high Philippine government officer in charge of irrigation told me in my interview that there is not enough government funding to construct the irrigation facilities planned, so the limited funds must be used for rehabilitating and repairing deteriorated facilities. Agriculture now uses two-thirds of the world’s fresh water supply; there are growing limitations to it. Increasing amounts of fresh water resource are being diverted to indus-

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trial and household uses as the economy grows. Water shortage is especially severe in northern China and western India. Drying up of the Yellow River is a good indication of water shortage in northern China. The drying up has been worsening every year since 1972. It reached the peak of 700 kilometers from the mouth of the river and of about 300 days in 1996. In my field survey in China in September 1998, I found that irrigation in rural Beijing was often restricted in order to supply water to Beijing City. In that month the Feng River was barely flowing at Taiyuan City in Shanxi Province, but many other rivers in that province have dried up. The underground water table in many places in Shanxi has been declining at the alarming speed of 1 to 2 meters per year during the past 30 years. Stoppage of water supply to houses in big cities was common then. 18 In my Indian survey in August 1998, I gathered that, for about 18 hours of each day in most Indian cities, water is not supplied to the city’s people. Most rivers on the Deccan Plateau were extremely polluted. In the Punjab, the grain bowl of India, the underground water table has been declining by 50 centimeters per year because of excessive pumping due to the free electricity for pumping policy. During my survey in America in the late eighties, I have personally witnessed the fear of long run water shortage in California due to difficulties in building irrigation dams because of environmental protection movements. Exhausting underground water resource by overutilization for various purposes has been occurring in the United States, Northern China and India.19 A rice farmer in Texas told me in the late eighties that he might have to abandon his rice production in 10 years because of the declining underground water table. The needed future increase in the world grain supply must rely on yield increase because of the restrictions on cultivated land, irrigated area and water. The green revolution considerably increased the yield of wheat and rice during two and half decades after 1961, as shown in Table 1.1. The yields of maize, barley and total grain have also increased considerably during the same period. However, the growth rate in the yield of grain has declined rapidly from about 3%

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

during 1961-70 to about 1% during 1985-96, as shown in Table 1.1. This long run decline in the growth rate of yield of major grain is a very serious problem in coping with the global population explosion, as constraints on agricultural land, water, and irrigation are becoming more serious, as described above. Yield of grain must grow at an annual growth rate of about 3% in order to cope with population explosion. Although the theoretical or potential yield of new varieties of rice and wheat is clearly greater compared to the ordinary varieties, it is sometimes lower in experimental fields and farmers’ fields.20 Yield of new high-yield rice varieties in the experiments at the International Rice Research Institute (IRRI), and national rice research centers and at farmers’ fields in Asian countries, have recently been static or reducing.21,22 Various factors can be considered in the postwar decline in the growth rates of, and recent stagnation in, global average grain yield. The fast grain yield increase of the green revolution was made possible mainly by the increased use of fertilizers. The world total fertilizer use started to fall from the late eighties, and it had continued to fall until the midnineties; it is expected to be stabilized during the nineties as a whole. 23 Lester Brown showed that the effectiveness of chemical fertilizers in increasing grain yield has decreased globally, and it was only one-fifth as effective for the period 1984-89 as for 195084.24 The recent yield stagnation may reflect the exhaustion of our accumulated technical knowledge of the grain varieties. The global stock of agricultural technical knowledge, which in the past had been accumulated rapidly by high research investment, and had resulted in the green revolution, has recently been exhausted because of the decline since the eighties in global research investment.21,25 Investment for agricultural/rice research in Asia has also stagnated, along with the rapid decrease in the global price of rice in real terms, since the eighties.26 Another reason for the yield stagnation is the decline of soil fertility due to the expansion of double or triple rice harvests per year in Asia and of double cropping of rice and wheat in the Indo-Gangetic region.22,27 Yields of rice and

wheat have recently seemed to be reaching a plateau and it is feared that they are near the biological limits for rice and wheat. 28,29 Global shortages in water resource for agricultural production and global deterioration of soil fertility have worsened during past decades, as mentioned previously. The significant reduction in growth rates of global grain yield and in global planted area of grain has caused stagnation or reduction in grain supply and continuous reduction in the global grain stock ratio since the mid-eighties. Let us look at the movement of crop yield and production in China, a very significant world agricultural country. According to the FAO data, grain yield has increased substantially after the Second World War, from 1.9 tons per hectare in 1961 to 4.5 tons in 1994. Although grain yield has undoubtedly increased, I thought the yield in 1994 was too high. Surprisingly, it became clear from a recent investigation by the Chinese Science Academy that statistics of cultivated area in China are 40% less than the actual amount. If we recalculate the yield in 1994 with this actual area, it is about 3.2 tons. Even the revised yield is at the same level as the average grain yield in Japan, America and Europe in the same year and is still very high. Water shortage is a serious short term and long term problem in northern China, as described above. The prices of agricultural inputs such as chemical fertilizers have increased rapidly from the early nineties, and will remain at high levels in the future. Agricultural research investment has stagnated.30,31 Consequently, rapid increase in the crop yield, such as in the recent past, will be difficult in the long term. In my recent survey in China, I gathered that superior agricultural land had been rapidly converted to nonagricultural uses because of the extremely rapid economic growth up to the early nineties. The harvested area of grain has been reduced at an annual rate of 0.462% from the postwar peak in 1976 to 1992, as described above. Grain production (including soy beans) in China has increased at an annual rate of 3.42% from 130 million tons in 1950 to a midterm peak of 407 million tons in 1984, and although it reached a historical record of 466 million tons in 1995, it has only increased at a rate of 1.27% annually from 1984 to 1994.

Food Shortage in the 21st Century and Its Implications for Agricultural Research

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Table 1.1. Long run decline in the annual growth rate of the world average yields of major cereals All Cereals

Rice

Wheat

Maize

Barley

61/70

3.02

2.72

3.61

2.17

3.46

70/85

2.41

2.13

2.50

3.12

1.26

85/96

1.20

1.22

1.44

0.90

0.63

Unit: % Data Source: FAO Production Yearbooks via FTP.

The Chinese government raised the buying prices of the grain under the quota system from the farmers by 88% in July 1994, and by 20% in 1996 as well. However, these were still a long way off the free market grain prices. It is reported that many farmers stopped rice and grain production because of the government’s low buying prices.32 What will be the global grain yield in the 21st century? I think it will not grow very fast, for the following reasons. First of all, I think stagnation in the growth of grain yield will continue into the early 21st century. Water shortage and soil degradation will worsen in the early 21st century as population explodes and economy grows. The long run decline in the growth rate of grain yield cannot be reversed in the short run. The type of technology in the near future is also relevant. The green revolution technology with high yielding varieties of grain and high inputs of chemical fertilizers and other agricultural chemicals will basically be used for the grain production of the globe. Higher input of chemical fertilizers will be needed in order to produce more grain to provide for an exploding population. But, marginal productivity of chemical fertilizer will decline. In developing countries, lesser amounts of organic matter will be input, as farmers will keep less animals and more agricultural machines will be used, and more biomass will

be used for cooking and other household uses. This will cause deterioration of soil structure and soil fertility in the long run. As more chemical fertilizers are used, more pests and diseases will attack grain. Then, increasing amounts of chemicals will be used, against which resistance will be formed in pests and diseases. 16 And, more chemicals will be needed which will destroy environment. But I think we cannot expect that an alternative technology which will increase grain yield with much less environmental destruction and soil degradation will be developed and be adopted by world farmers on the global scale by the early 21st century. Thus, the technology in the near future will be one of less yield increase and more environmental destruction. It is more difficult to increase yield of wheat, barley, sorghum and millet than rice and maize under current agricultural technological conditions.16 However, some are of the opinion that the significant differences that exist in the yield of each grain among various countries or regions in the world show the possibility of adopting existing technology and increasing yield through increased use of chemical fertilizer, and at the same time decreasing the environmental damage and soil deterioration, especially in developing countries.23 The difference in the yield, however, shows in most cases not a difference in

16

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

potentiality in the existing technology, but in the restrictions of natural conditions such as soil and climate on the yield, as is clearly seen in the big difference between the yield of wheat in Western Europe and North America. As previously described, the growth rate of grain yield has decreased significantly since the last half of the eighties, and yield of new high-yielding rice varieties has been stagnating or decreasing in Asia. The harvested area of rice, 90% of its production and consumption concentrated in Asia, has been decreasing as well. As mentioned above, a 3% annual increase in rice yield will be needed to cope with the highest level of population explosion until 2020.29 Although until now the potential yield of various crops has been raised annually by 1-2% through the efforts of genetic research,20,22 a 3% annual increase in rice yield in the long term is very difficult. How will such new technologies as biotechnology and hybrid varieties contribute to increasing grain yield? Since the appearance of hybrid corn in America in the thirties, F1 vigor has been considered the breakthrough technology that would be the best means of increasing yields. But those views are too optimistic. The only country where hybrid rice varieties have been planted on a sizable scale is China. They were planted to 55% of the country’s rice harvest area in 1992. This was possible because the high cost of hybrid seed production has been mitigated by cheap labor cost and government subsidies. It is said that yield of the hybrid rice on the average was only 20% higher than the ordinary varieties. In my field survey in China over the last few years, I heard from farmers and experts that Chinese consumers had been shifting from less delicious hybrid rice to ordinary rice and farmers had been abandoning hybrid rice planting. Although efforts have been made for the last twenty-five years, hybrid wheat seeds have not been successful, due to the prohibitively high cost of seed production.20,33 Although significant increase in crop yield has been expected from biotechnology utilizing gene transformation and gene mapping, virtually no useful result that considerably increases crop yield has been achieved so far. Many researchers now consider that it takes several decades to extend new seeds developed

by biotechnology among the majority of the farmers in the developing countries. Biotechnology is considered as an important means for genetic research and it will bring gradual increase rather than a breakthrough in yield.20 The “super rice” developed by IRRI in the Philippines using biotechnology increases rice yield by 30%. The rice has a plant type 90 cm in height with four or five short and strong stalks with big ears, eliminating stalks with no ears.33 However, I heard in my discussion with Professor T. Horie of Kyoto University, who is knowledgeable about the results of experiments with this rice in Japan, that the yield increase has not yet been achieved because of many non-filled grains. My judgement about the yield increasing potential of biotechnology in the near future, listening to discussions among the world’s leading genetic engineers at this 12th Toyota Conference, is very pessimistic.

Demand Factors According to the estimates of the United Nations, the population explosion that has occurred mainly in the developing countries after the Second World War will maintain its peak level during the period of 1990 to 2020. The world population increases annually by from 90 to 100 million during this period, and the world population will reach to 8050 million by 2020, from 5300 million in 1990. The population in the developing countries will increase at the annual rate of 1.7%, from 4080 million to 6660 million, during the same period. Population in Asia will increase at the annual rate of 1.64 % from 2900 million to 4500 million. The world population will double to 10,000 million by 2050. Grain (rice, wheat, barley, rye, corn, oats, sorghum and millet) will be the main source of the direct and indirect calorie intake for the exploding global population in the early 21st Century.26 The indirect calorie intake is from consumption of animal and fish meat produced by feeding feed grain to animals and fish. Population explosion will occur only in the developing countries, and the population there will be about 80% of the world total population in early 21st century. Both direct and indirect calorie intake, and thus grain demand or need, in the developing countries will increase very rapidly during the

Food Shortage in the 21st Century and Its Implications for Agricultural Research period from 1990 to 2020 as the population explosion occurs there, and their economies will grow relatively faster, especially in Asia. Grain demand increases because of increase in income as well as because of population explosion. When per capita income increases in the developing countries, the immediate result is an increase in direct grain demand as food. The importance of animal protein in the food consumption pattern will increase as per capita income increases in the developing countries. This will lead to a rapid increase in the demand for feed grain for animals. According to the World Bank statistics,34 the per capita GNP in Asian developing countries has grown significantly. It had grown at annual rates of 3.0% to 6.4% during 1980 and 1993, while for other developing countries as a whole it had experienced negative growth during the same period. The average per capita GNP growth throughout all developing countries was approximately 1% per year during the period from 1980 to 1993. The total GNP has grown at around 3.5% per year. This rapid per capita income increase, especially in Asia, brought about significant increases in the demand for grain, which is the staple diet of the people in the developing countries, and also rapid increases in the demand for animal protein in Asia, especially in China, resulting in explosive increases in the demand for feed grain. Let’s now investigate the actual situation for the rapid increase in grain demand within China. China had 22% of the world’s total population in 1990, and thus she is a significant influence on future food demand. According to the UN estimates, although the Chinese population growth rate has been relatively low (an annual rate of 0.9%) compared to the other developing countries, due to “the One Child Only Policy”, the population increased from 680 million to 1.2 billion during the period of 1958-92, and will continue to increase to 1.5 billion by 2020. This rapid population increase will cause a rapid increase in China’s grain need. Economic reforms started in 1978, which allow free decision by individual agricultural households, brought about a rapid increase in grain yield and production in China in the early years. According to the FAO database, the grain supply per person increased quickly from

17

230 kg in 1961 to 354 kg in 1995. However, it has remained static since 1985 due to the rapid population growth, reduction of cultivated area and slowdown in the increase rate of grain yield, as previously mentioned. Although the five year moving average of annual increase rates of grain yield has been increasing at an annual rate of over 5% during the period 19611984, it fell to between 0.3%-3% afterward until 1992. This trend is likely to continue in the future. The per capita GNP in China has seen the very high growth rate of 8.2% per year from 1980 to 1993, and an explosive growth rate of around 10% from 1992-95. This high economic growth in China will be maintained in the future, as China has planned her economic growth rate at 7-8% until 2010, as agreed at the People’s National Congress on March 5, 1996. This recent rapid income increase brought about a rapid increase in demand for meats, mainly pork, which is the most popular meat among Chinese, at an annual rate of 10%. One kilogram of pork production needs 4 kilograms of feed grain. The grain prices have rapidly risen through the explosion in the demand for feed grain. Corn prices doubled within one year in 1995. The domestic free market rice price increased 3.2 times between January, 1993 to June, 1995, and it has became more expensive than the export price of the low grade 35% Thai rice which is almost equivalent to the domestically available rice in China since May, 1994.32 The Peking government prohibited the export of corn in November, 1994, and soy beans from April 1995, to ease the domestic shortage. Grain exports from China (including soy beans) fell to almost nil in 1994 and 1995, as shown in Figure 1.4, and the net imports rose to 15 million tons annually. Actually, the import of a large net volume of grain into China is not a recent phenomenon, but it has been 10-20 million tons annually since 1977, except in 1985, 86, 92 and 93, according to the FAO data as shown in the same figure. China banned corn export again in late 1997. According to a recent publication of the USDA,32 the Peking government was forced to consider the long term demand and supply balance of food in China for foreign and domestic reasons in 1994 and 95, and conducted research within

18

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

some ministries and universities. Based on the research results, the government decided to change its food policy from a long held food self-sufficiency policy to an 88-95% self-sufficiency policy. China consumed 112 million tons of wheat, 109 million tons of corn and 129 million tons of rice in 1995. An enormous amount of grain totaling 42 million tons annually (including 15.5 million tons of white rice) needs to be imported if the self-sufficiency rate is only 88%. Since the world trade markets in wheat and corn are thick, meaning that the total trading volume is large compared to the global total production, the markets can manage the large import by China. But a catastrophic increase in the world rice trade price and resultant confusion can be expected, for the world rice trade market is thin, meaning that only 4% (15 million tons) of the world total production is traded. It can be concluded that the rapid increase in demand for grain by the developing countries which has occurred from the eighties until now, especially in China and other Asian countries, has had the effect of reducing the global grain stock ratio. This rapid increase will continue into the early 21st century, as population explosion and relatively fast economic growth in the developing countries will continue in the long run. Prospects of World Grain Supply and Demand for the Year 2020 As shown in the above analysis, the world grain stock as a trend will remain at dangerously low levels into the early 21st century, and the grain prices will remain at high levels. The reasons for this are the transformation of the European and American agricultural policies after the late eighties, the restrictions on natural resources and agricultural technology improvement, and rapid increase in the need for feed and food grain, principally in China and Asia, due to the population explosion and high economic growth. Supply and demand of grain in 2020 is projected in Table 1.2 for the world, groups of countries classified by income levels, China, India and Japan, based on assumptions according to the above analyses of long term supply and demand of grain in the world. Regarding the assumptions for demand, the production

conversion ratio from grain to meats and eggs for each region and country is estimated based on the supply and demand balance data of the FAO for 1984-86 and on expected long term increase in feed grain use in the livestock sector. Increase in the demand for feed grain is estimated by projected population growth rate, GDP growth rate and income elasticity of demand for meats and eggs. Medium estimates of the UN are used for the population and population growth rate. The year 1993 is used as the base year. The FAO statistics were used to determine demand for meats, eggs and grain in 1993. Regarding supply, the growth rates of grain production, listed in the same table, are determined so as to reflect the recent decline in the growth rates of grain production mentioned above. There are two ways to make a long term forecast of grain supply and demand. One is forecasting demand and supply independently, and analyzing their impact on grain price afterwards. I, in this paper, and Lester Brown have used this method. The other method projects supply and demand, incorporating the effect of the difference between supply and demand on the product price. This method is used by the World Bank, FAO, IFPRI and the Japanese Ministry of Agriculture, Forestry and Fishery.35-38 Lester Brown, the Ministry of Agriculture, Forestry and Fishery and I forecast a significant global grain shortfall in 2020 and/or 2030 and a considerable grain price rise, and others forecast that the world grain prices will decrease by 10 to 30 percent in the early 21st century. I think the reasons for the totally different forecasts are basically differences in the assumptions for the projections, and thus critical evaluation of the assumptions is very important. I judge the assumptions made by the World Bank, FAO and IFPRI to be too optimistic and contrary to the recent reality. The assumptions for these future surplus projections are: no limitation in arable land and water, positive price response in planted areas of grain, and considerable yield growth of grain in the future supported by technological improvements in agricultural production. According to my projection, there will be an enormous grain shortfall of 320 million tons in developing Asian countries in 2020. The breakdowns are 170 million tons for

19

net import, import, and export 10 million tons

Food Shortage in the 21st Century and Its Implications for Agricultural Research

Fig. 1.4. Chinese cereal quanitites traded: net import, import, and export. Data source: FAO and USDA estimates for recent years. China, 15 million tons for India, 33 million tons for Japan and 417 million tons throughout the world. Although the high-income countries such as the United States and the EU export 172 million tons, that is far below the projected deficit in year 2020. This shortfall is significant, especially when one considers that the total world grain trade volume was 230 million tons in 1993. The reasons for my projection of such a significant grain shortfall are: The population explosion and high economic growth in developing countries, principally in China and Asia, will bring about a fast increase in consumption of animal protein, which will lead to an explosive increase in demand for feed grain; and, increase in grain supply will be restricted by limitations in natural resources and in the improvement of agricultural technology in the developing countries, and by continuation of the agricultural policy transformation in Europe and America into the early 21st century. The projection by Brown of a significant future shortfall is for similar reasons, with an emphasis on land shortage in China. The estimated significant grain shortfall, by Brown and me, would bring about a rise

in the prices of grain in the world trade market. According to my forecast, assuming the long term price elasticity of the world grain supply and demand to be 0.15 (ratios between rates of change in supply and demand against the price change rate), the international grain trade price will increase by 50% compared to its 1993 level by 2020. This increase in the grain trade price will lead to a considerable increase in the domestic price of rice and other grain. This will cause significant difficulties to the huge hungry in the developing countries, who now number more than 0.8 billion, more than 0.5 billion of which concentrate in Asia,39and it will likely be larger in the year 2020. A significant global shortfall of grain is forecast for the year 2020. China and Japan will be important players in the long term world demand and supply of food, especially rice. Japan was forced to open her long selfsufficient rice market by accepting minimum access rice import ,under American pressure at the Uruguay Round negotiation in December, 1993. She will accept tariffication of rice import in April 1999. I think these concessions will lead to a gradual increase of Japanese rice import,

5522 4289 3058 1185 885 1228

124

Low and medium income countries

Asian developing countries

China

India

High income countires (a)

Japan

Population (millions)

World

Demand

47.80

93.10

3.96

34.50

20.70

23.90

39.50

Per capita demand for animal and chicken meat, and eggs (kg)

1993

40

767

173

366

730

1072

1830

Total demand for cereals (million tons)

Table 1.2. Projection of world demand and supply of cereals for the year 2020

52.8

100.0

6.4

91.3

54.1

58.4

58.4

Per capita demand for animal and chicken meat, and eggs (kg)

2020

42

927

298

777

1473

2342

3269

Total demand for cereals (million tons)

20 Challenge to the Crisis of the Earth's Biosphere in the 21st Century

1804 930 675 340 167 870

11

Low and medium income countries

Asian developing countries

China

India

High income countries (a)

Japan

Production (million tons)

1993

World

Supply

9

1100

283

610

1153

1752

2852

Production (million tons)

Table 1.2., cont. Projection of world demand and supply of cereals for the year 2020 2020

33

-172

15

167

320

590

417

Estimated deficits (million tons)

Food Shortage in the 21st Century and Its Implications for Agricultural Research 21

0.0164 0.0147 0.089 0.0152 0.0045

0.0014

Low and medium income countries

Asian developing countries

China

India

High income countries (a)

Japan 0.00373

0.00262

0.01794

0.03667

0.03628

0.03354

0.01458

Per capita demand for animal and chicken meat, and eggs

0.02

0.022

0.06

0.07

0.06

0.06

0.034

GDP

-0.0081

0.0087

0.0120

0.0152

0.0200

0.0237

0.0171

Cereal production (%)

1993-2020 Annual Growth Rates (%)

Note: a; US, former USSR, Eastern and western Europe, Japan, Oceania

0.0141

Population

World

Assumptions

Table 1.2., cont. Projection of world demand and supply of cereals for the year 2020

0.2

0.15

0.4

0.6

0.35

0.7

0.68

Income elasticity of demand for animal and chicken meat, and eggs

3.30

5.88

2.00

3.00

1.80

2.40

3.00

Feed grains/ animal and chicken meat, and eggs conversion rate (FAO food balance)

22 Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Food Shortage in the 21st Century and Its Implications for Agricultural Research to end up with large amounts of rice import and disappearance of the rice sector on the Japanese volcanic archipelago. Huge external benefits to Japanese people associated with self-sufficient rice production and consumption will be lost. In addition to this and more importantly, the Japanese must face a very thin, unstable and unreliable international rice trade market. Only 4 percent (16 million tons of milled rice) of world total rice production is traded. In Japan and other Asian countries, rice supplies 25 to 80 percent energy intake of the people, and thus rice is political goods in the sense that it must be supplied with certainty and at stable price. Since disappearance of the rice sector in Japan is so great a loss in external benefits and political stability, I think Japan should not have accepted minimum access and should not accept tariffication. On the other hand, large and continuous Japanese rice imports will raise and destabilize the rice price considerably throughout Asia, which will result in a crisis for the huge Asian hungry. Large Japanese rice imports will also cause environmental destruction in Asia.40 The Japanese self-sufficiency ratio for grain is forecast to decrease from 28% in 1993 to 21% in 2020, as shown in Table 1.2, if the current Japanese agricultural policy is maintained. The Japanese feel insecure with the current self-sufficiency level of 28 percent; according to a recent opinion poll, 83 percent of Japanese think Japan should maintain rice self-sufficiency even it is very expensive. The decline of self-sufficiency ratio for grain to 21 percent will increase the Japanese sense of food insecurity very much. The import of agricultural, forestry and fishery products by Japan increased much faster than the European countries’ import after the Second World War, and it caused serious euphorization problems for inland waters and nearby seas in Japan. It also caused severe environmental destruction abroad, as symbolized by Japanese shrimp import and tropical timber and wood imports. I believe Japan imports too much agricultural, forestry and fishery products, for of the following reasons: Serious environmental problems in Japan as well as in developing countries caused by Japanese import; the crisis to the huge poor in Asian

23

developing countries in the case of rice import; and the serious concerns by Japanese about loss of food security and of external values derived from the primary industries. We should reform Japan’s agricultural, forestry and fishery policy and their system, emphasizing more domestic production of the agricultural, forestry and fishery products, including rice, which will lead us to a higher self-sufficiency ratio for these products, using less environmentally destructive technology incorporating cyclical use of fertility among crops, animals and humans. This reform will lower domestic and foreign environmental destruction by Japan’s import of primary commodities, maintain external values of the primary sector and rural society and increase food security level in Asian developing countries.41 I think China will be a large importer of grain, including rice, in the near future considering her fast economic growth, large population growth, natural resource limitations and deterioration of agricultural resource base. If China continues her past growth pattern in the future, and it is likely now for China to follow it, the size of her rice import will be enormous and it will have devastating effects on Asian rice price, the Asian hungry and the Asian natural environment.

Conclusion Because of severe limitations on natural resources, environment and technological improvement against population explosion in the developing countries, and of the transformation in agricultural policies in the United States and the EU, severe food shortage is expected in developing countries for the period from now to the year 2020. The green revolution technology, which has used increasing amounts of modern chemical inputs and is still widely used in the world, had increased grain yield considerably but at the same time had destroyed environment considerably until the mid-eighties. As the effect of the new technology on grain yield has declined since then, annual growth rates of the yield have declined to about 1 percent per year from about the 3 percent before 1970 which is required to provide food for exploding populations in the developing countries. Thus, there is a strong

24

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

need for finding and applying a new technology that will realize sustainable and considerable yield increase of grain in the near future. In other words, socially optimal yield increase is needed that will increase grain yield but will also conserve the soil and water which are the basis for grain production, and will protect the environment. Genetic engineering is one approach to attaining this goal. IRRI has publicized many modern rice varieties having increasingly more resistance to insects and diseases, developed by both genetic engineering technology and conventional technology.42 These varieties have been adopted widely by Asian farmers. This type of research will decrease input of agricultural chemicals and increase yield. But, based on the information I gathered at the 12th Toyota Conference, as well as from other publications and discussions with experts, I think the genetic engineering approach alone is not sufficient for providing food to the exploding population in developing countries for the period from now to the year 2020. This type of research has not and will not be able to create a breakthrough in increasing grain yield on the farmers’ plots in the period from now to the year 2020. International and national agricultural research strategy needs a serious reconsideration. The structure of research emphases must be reevaluated. Research on research is one method for this reevaluation. First of all, the past paradigm of research strategy emphasizing modern technology must be reconsidered. I think we have to study traditional and local agricultural technologies more; many of them are natural resource maintaining and environment sustaining. Traditional rice culture in Asia, which had been followed for thousands of years without much modern input before the green revolution, is a typical example of environment-sustaining agriculture, although yield is low. In some mixed cropping fields on the Deccan Plateau, where I visited during my field survey in September 1998, about 3 to 10 crops were planted in each field at the time of my field survey. Traditional sawing and cultivating machinery is used and barnyard manure is also used. Here is another example. Dr. Ochi, a geographer, and Tanaka and Watabe, agronomists who have studied this mixed cropping

practice applied widely on the Deccan Plateau, think that this is one of the most productive and sustainable agricultural technologies on a semi-arid tropical plateau. 43,44 Shifting cultivation in northern Thailand, with long fallow periods, is another type of sustainable agriculture.45 Traditional agricultural technology has used large amounts of organic fertilizers which maintain soil structure and soil fertility in Asian countries. This is an important cyclical and sustainable relation among field crops, domestic animals and humans in traditional Asian agricultural technology. The green revolution technology has severed this cyclical and sustainable relation by using large amounts of chemical fertilizers and agricultural machinery in order to obtain short run grain yield increase. This was a necessary evil, since when the green revolution technology was introduced in the middle of sixties, world food shortage was very severe. But in the long run I think this severing has degraded soil fertility, overused natural resources and destroyed natural environment and caused stagnation in the increase of grain yield in the world after the mid-eighties. I believe that the cyclical nature of agricultural technology must be restored in both developing and developed countries, especially by more input of organic fertilizers. Combining the traditional and local technologies with modern technologies, we may be able to create a new technology that restores the cyclical relations and leads to sustainable yield increase of food. Some successful examples of this combination in various countries do exist.46,47 I think this approach is important because modern technology has been intensifying its use of modern input, and thus has been increasing environmental destruction. I was surprised to observe, in my village survey in September 1998, that many villagers in Central Java are organized by the government extension service system to perform IPM rice practices. Wide practice of IPM may be an effective approach to attaining socially optimal yield increase of rice and other grain. I believe that water shortage is the most critical factor constraining future increase in food supply in developing countries, based on my field surveys in China, India and other Asian countries. Thus, researches relating to

Food Shortage in the 21st Century and Its Implications for Agricultural Research water use institution and policies which influence water use efficiency, and to water saving technology, must be emphasized. Degradation of soil and decline in arable land are other important constraints to future food supply increase in Asia and in the world. Socioeconomic and natural science researches to these problems are also important. Socioeconomic research can increase food supply considerably. Socioeconomic factors reducing food production, increasing post-harvest losses and increasing waste in food consumption must be further studied. Firstly, socioeconomic factors to reduce food production must be identified in each country. Then, socially optimal measures balancing economic cost, environmental effects and burdens to natural resources could be found, in order to increase food production in each country. IRRI and FAO studies have estimated that about 10 to 40 percent of rice is lost at all production and marketing stages from harvest to before retail. In the current academic knowledge, there is no solid information about the magnitude of post-harvest food losses and how these losses could be reduced, but concerning staple food it seems an important problem to solve. The post-harvest loss means not only human hunger and financial loss to the farmers, but also significant environmental destruction.48 Research and policy must cope with this problem. Huge amounts of food supplied to consumers is not consumed, but rather wasted, in Japan and probably in other high income countries. Research and policy must handle reduction and recycling of this food waste. I do not have solid data with me now about how great this waste is. I personally observed in my field surveys in Vietnam and China in recent years that a lot of food wastes from households and restaurants are recycled to feed pigs and other domestic animals. In developing countries this waste seems much less. Decades ago in Japan this waste was well recycled to feed domestic animals and to crop land. Research on agricultural policy and trade rule of agricultural products is important in order to increase food supply and food security, and to reduce environmental destruction and burdens to natural resources. The people of each country, especially the huge poor and hungry in developing countries, require not

25

only a larger food supply but also stability or sure daily access to staple food. I believe liberalization of rice trade under the WTO system will destroy this stability and sure access to the huge Asian poor. This is because the international rice trade market is extremely small, unstable and unreliable compared with such important grains as wheat and maize. Also, Asian countries, which comprise about 90 percent of the world total rice production, have pursued and will maintain rice self-sufficiency policies in order to secure food security for each country. Liberalization will greatly destabilize the world rice market.49 As agricultural policy and trade rule influence environment, natural resources and income distribution, research to identify an optimal policy and trade rule is important. The agricultural trade liberalization under the WTO system will reduce food production in the north much more than food production increase in the south, and thus increase food prices, which will lead to more use of chemical fertilizer and agricultural chemicals. Liberalization will lead to more food production far away from densely populated developed and developing countries, in sparsely populated countries. Given sparse population and soft state50 in the case of developing countries, less attention will be paid to negative environmental impact and overuse of natural resources for liberalized, increased food production in these countries. These negative impacts of food production are, I believe, best controlled when more food is produced in each country. People in each country can best observe these negative impacts and can influence government policies and other measures to reduce them. More food production in the densely populated developing countries will reduce poverty and equalize income distribution by increasing income of the huge agricultural poor in these countries.51 These are the reasons why I think the free trade rule of the WTO should be modified, and socially optimal agricultural policies oriented toward self-sufficiency of staple foods in each country, and socially optimal food trade rules, should be sought by policy research. Demographic research is also critical, in order to reduce population explosions in developing countries.

26

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

References 1. Kuroda T. Considering decline in the number of children per household and aging; Japan as a leading nation, Paper presented at the Seminar: Can human survive into the 21st Century? November 13, 1998. 2. Brown L. Tough Choices. New York: Norton & Company, 1996. 3. Alexandratos N, ed. World Agriculture: Towards 2010, An FAO Study. Rome: FAO, 1995. 4. Meadows DH, Meadows DL, Randers J et al. The Limits to Growth. New York: Universe Books, 1972. 5. OECD. National Policies and Agricultural Trade. Paris: OECD, 1987:52-53. 6. OECD. National Policies and Agricultural Trade. Paris: OECD, 1987:56. 7. The net export volume of food, excluding fish, is calculated by taking the difference between the export and import values of food for the group of countries in question, deflated by the FAO agricultural export/import price indices, respectively, with 1966 as base year. FOB import value is adjusted by the coefficient of 1.07 for balance with CIF import value. 8. FAO. Food for All. Rome: FAO, 1996:14. 9. Tsujii H. The world food shortage in the year 2020 and the needed agricultural transformation in Japan. Natural Resource Economic Review 1997; 3:5-8. 10. FAO. Agriculture: Toward 2010. Rome: FAO, 1993. 11. Tsujii H. The world food shortage in the year 2020 and the needed agricultural transformation in Japan. Natural Resource Economic Review 1997; 3:10. 12. Crosson P. Future supplies of land and water for world agriculture. In Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D. C.: International Food Policy Research Institute, 1995:154-156. 13. Tsujii H. An econometric study of paddy field transactions by rice farmers in Hokkaido in Japan (in Japanese). In: Noseichosa Iinkai, ed. Research Report on Basic Agricultural Problems. Tokyo: Noseichosaiinkai, 1993:28-43.

14. Oldeman L, Hakkeling R, Sombroeck W. World Map of the Status of Human-Induced Soil Degradation: An Explanatory Note. 2nd ed., Wageningen, The Netherlands; Nairobi: International Soil Reference and Information Center and United Nations Environment Program, 1991. 15. World Bank and UNDP. Irrigation and Drainage Research: a Proposal. Washington, D. C.: World Bank, 1990. 16. Oram PA, Hojjati B. The growth potential of existing agricultural technology. In: Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D. C.: International Food Policy Research Institute, 1995:167-189. 17. Postel P. Last Oasis: Facing Water Scarcity. The World Watch Environmental Alert Series, New York: W. W. Norton, 1992. 18. This survey was supported by Research Center for Rural Economy in Beijing, Agricultural Committee of Communist Party of Shanxi and a capable research assistant, Ms. Guo Jin Ping. 19. Brown LR. Future supplies of land and water are fast approaching depletion. In: Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D. C.: International Food Policy Research Institute, 1995:161-166. 20. Duvick D. Plant breeding and biotechnology for meeting future food needs. In: Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D. C.: International Food Policy Research Institute, 1995:222. 21. Evenson R, David C. Rice research and productivity. In: OECD. Adjustment and Technology: The Case of Rice. Paris: OECD, 1990:57-84. 22. Plucknett D. Prospects of meeting future food needs through new nechnology. In: Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D. C.: International Food Policy Research Institute, 1995:208.

Food Shortage in the 21st Century and Its Implications for Agricultural Research 23. Bumb B. Growth potential of existing technology is insufficiently tapped. In: Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D. C.: International Food Policy Research Institute, 1995:191-205. 24. Brown LR. Full House, New York: Norton, 1994. 25. Author’s personal communication with Professor Takeshi Horie (a leading Japanese crop scientist), Kyoto University. 26. IRRI. IRRI Rice Almanac, 1993-95. Manila: International Rice Research Institute, 1993:8. 27. Chand R, Haque T. Rice-wheat crop system in Indo-Gangetic region—issues concerning sustainability. Economic and Political Weekly 1998; 33(26): A108-112. 28. IRRI. IRRI 1992-1993, Rice in Crucial Environments. Manila: IRRI, 1993:8. 29. IRRI. IRRI Rice Almanac, 1993-95. Manila: International Rice Research Institute, 1993:6. 30. There is an opinion that Chinese grain yield will increase much more than presently because of this underestimation, and the comparison of yield with Japan, Britain and America. 31. Gen Z. Structural analysis of Chinese food economy and supply and demand prospects. Azia Keizai 1996; 37(2):33-62. 32. USDA, ERS. Rice, Situation and Outlook Yearbook. Washington, D. C.: USDA, 1995:13. 33. IRRI. IRRI Rice Almanac, 1993-95. Manila: International Rice Research Institute, 1993:110. 34. World Bank. World Development Report 1995:162-63. 35. Brown LR. Tough Choices. New York: Norton & Company,1997. 36. Rosegrant MW, Agcaoili-Sombilla M, Perez ND. Global Food Projections to 2020: Implications for Investment. Washington, D. C.: International Food Policy Research Institute, 1995. 37. Anderson K, Dimaranan B, Hertel T, Martin W. Asia-Pacific food markets and trade in 2005: A global, economy-wide perspective. A paper commissioned for the international general meeting of the Pacific Basin Economic Council, Washington, D. C.: World Bank, 1996.

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38. Mitchell DO, Ingco MD. Global and regional food demand and supply prospects. In: Islam N, ed. Population and Food in the Early Twenty-First Century: Meeting Future Food Demand of an Increasing Population. Washington, D.C.: International Food Policy Research Institute, 1995:49-60. 39. FAO. World Food Summit—Rome Declaration on World Food Security and World Food Summit Plan of Action. Rome: Food and Agriculture Organization of the United Nations, 1996:37. 40. Tsujii H. The World Rice Warfare. (in Japanese). Tokyo: Ienohikari Kyokai, 1988. 41. Tsujii H. World Food Uncertainty and Japanese Agriculture. (in Japanese) Tokyo: Ienohikari Kyokai, 1997. 42. IRRI. IRRI Rice Almanac, 1993-95. Manila: International Rice Research Institute, 1993:113-114. 43. Ochi T. Farming practices in millet agriculture in the southern part of Deccan Plateau (in Japanese). In: Sakamoto S. ed. Agro-pastoral Culture in the Indian Sub-continent (In Japanese). Tokyo: Gakkai Center, 1991:141-172. 44. Tanaka K, Watabe T. Traditional cropping systems of small farmers in the central and southern Deccan Plateau Area. Southeast Asian Studies 1981; 19(2):205-221. 45. NRCT. Shifting Cultivation in Northern Thailand. Hawaii: University of Hawaii Press, 1985. 46. Gill GJ. Indigenous erosion control systems in the mid-hills of Nepal. In: ICRISAT. Farmers’ Practices and Soil and Water conservation Programs. Ptancheru, Andra Pradesh, India: International Crops research Institute for the Semi-Arid Tropics, 1991:17-21. 47. Gill GJ. Major natural resource management concerns in South Asia. Food, Agriculture, and Environment Discussion Paper 8. Washington, D.C.: International Food Policy Research Institute, 1995:29. 48. The World Resources Institute, UNEP, UNDP et al. World Resources 1998-99. New York: Oxford University Press, 1998:155-156. 49. Tsujii H. Characteristics of and the trade conflicts in the international rice market. The Natural Resource Economic Review 1995; 1:119-135.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

50. Myrdal G. Asian Drama—An Inquiry into the Poverty of the Nations. 3 vols. New York: Twenties Century Fund, 1968. 51. Tsujii H. International effects of emergency rice imports by Japan—the crisis to the huge poor in developing countries (in Japanese). Kokusai Mondai 1994; 416:37-52.

CHAPTER 2

Prospects for Grain Demand and Supply in the 21st Century H. Zhai

C

hina has successfully fed 22 percent of the world population by its 7 percent of the world’s arable land. Before 1978, low-priced cereals and potatoes were the staple food of the Chinese people, due to the low standard of living. Food supply has been targeted to meet the needs of this lower standard. Since China began economic reform in 1978, it has made great progress in gain production. From 1978 to 1995, the total grain output increased by 50 percent (Table 2.1). The grain production has meet the basic needs of the Chinese people. However, with rapid economic development, road and housing construction has taken over a large area of cultivated land, which resulted in a tenser situation regarding the scarce land resources. On the other hand, there is an increasing demand on feed grains due to the rising income of the Chinese people and the increased meat consumption in their diet pattern. The grain shortage resulted in grain import and the continuing growth of grain price, which inevitably caused a series of economic problems. The grain issue has a significant impact, not only on the immediate interests of the Chinese people, but also on the international grain market and price indicators. Therefore, it is not surprising to see that wide attention has been paid by both foreign and domestic experts and scholars to the grain issue in China. Herewith, I would like to give a brief analysis on the grain supply and demand prospects in China.

Food Demand Analysis Factors Influencing Food Demand The factors which influence the total demand for future grain are various, among which the following are the key ones. Population Growth China has a large population baseline. Consequently, the growth rate of the population is also noticeable. This determines not only the increase of food grain, but also of the grain demand for other purposes. Since the family planning policy was started in 1978, the population growth rate has declined steadily (Fig. 2.1). However the absolute number is still gradually increasing (Fig. 2.2). Income Level of Households The per capita gross domestic product (GDP) represents the economic development level of a country . It also has a direct influence on the structure and level of food consumption. Viewing the development of all the countries in the world, the higher the national income level, the more indirect grains will be consumed. The direct consumption of grain ration will continually decrease while the absolute demand of food will increase. According to the relevant statistics of Penn World Table 5.61 the per capita GDP in China has exploded from $567 US in 1960 to $1,493 US in 1992, with an average growth rate of 3.0

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

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Table 2.1. China’s grain yield (10,000 ton) Year

Grain gross

Rice

Wheat

1978

30,477

13,693

5,384

1979

33,212

14,375

6,273

1980

32,056

13,991

5,521

1981

32,052

14,396

5,964

1982

35,450

16,160

6,847

1983

38,728

16,887

8,139

1984

40,731

17,826

8,782

1985

37,911

16,857

8,481

1986

39,151

17,222

9,004

1987

40,298

17,426

8,590

1988

39,408

16,911

8,543

1989

40,755

18,013

9,081

1990

44,624

18,933

9,823

1991

43,529

18,381

9,595

1992

44,266

18,622

10,159

1993

45,649

17,751

10,639

1994

44,510

17,593

9,930

1995

46,662

18,523

10,221

95/78

153%

135%

190%

percent. From 1993 to 1995, the per capita GDP registered an annual growth rate of over 6 percent. With the increase of per capita gross national product, there will be a corresponding increase in the absolute demand of grains. Dietary Structure The dietary structure is mostly influenced by the per capita income level. The other factors are tradition, geographic environment and food supply situation, etc. With the rising per capita income level, the proportion of animal product consumption in the dietary structure will get higher, which means a decrease in direct grain consumption and an increased consumption of converted food

products. It is estimated that by 2010 per capita energy intake will be 2,750-2,800 calories, and 2,800-3,000 calories by 2030. The proportion of the energy intake from animal products will increase, while that of the energy intake from cereals will decline (Fig. 2.3).

Prediction of Grain Demand in the 21st Century in China The Total Demand for Grain Will Increase Firstly, we consider the increase of food grain. Grain is people’s daily necessity. It is rigid in consumption and is positively correlated with the size of population. With the increase of population and per capita GDP, the daily

Prospects for Grain Demand and Supply in the 21st Century

31

Fig. 2.1. Tendency of population growth: Growth rate. per capita energy intake gradually grows higher. The relationship between per capita energy intake and grain consumption is that every 3,500 calories of energy intake needs to consume 1kg of grain. On this basis, it is roughly estimated that the demand of per capita food grain will be 152-164 kg, and the total demand for food grain will be 213-230 million tons, by the year 2010. By 2030, the demand for per capita food grain will decline to 140-160 kg, while the total demand will be 224-234 million tons. Secondly, there is the issue offeed grains. Considering the technological progress in animal production in the future in China, we forecast that the average conversion efficiency of feed grains to animal products will be 3.5:1 by 2010, which means 3.5 kg feed grains will be converted to 1kg animal products. According to the energy intake requirements for animal products, feed conversion efficiency and the proportion of energy intake to grain consumption, we predict that the demand for feed grains will be about 260-340 million tons and 320-420 million tons, respectively, by 2010 and 2030 (Table 2.2).

In addition, the grain demand in industry, new stocks and wastage will not show big changes. Therefore, the demand in these aspects will only register as 12 percent of the total demand. The prediction of per capita grain and meat demand is shown in Table 2.3. The Structure Of Grain Demand Will Change Considerably. With the continuing increase in income of both urban and rural households, the per capita food grain will drop by a large margin in food consumption structure. Apart from this, there will be an increasing demand for feed grains, not only in the absolute volume, but also in its proportion of the total grain consumption. Consequently, feed grains will surpass grain for daily use and take the leading position in grain consumption by 2010 or so. With the continuing demand for feed grain consumption, the demand for per capita animal products and indirect grain consumption will all increase noticeably. Due to the great demand for feed grains, food grain will decrease due to large population and limited arable land. The contradiction of competion for grain between man and animals

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 2.2. (Above) Tendency of population growth: Total population.

Fig. 2.3. (Above) The proportion of caloric intake from different resources. will become increasingly sharp. To some extent, it can be said that the future grain problems in China will be the feed grain issue.

Grain Supply Potential At any point of time, total grain supply equals the sum of domestic production, imports and existing reserves. Among these three parts, the domestic grain production is

the main source of the grain supply. China is a large consumer due to its large population. The imported grain is mainly used to redistribute grain varieties and compensate the shortage of grain production. Grain reserves are generally used to smooth supply against fluctuations in production and price. Here, I would like to put the emphasis on analyzing the future supply of grain production.

Prospects for Grain Demand and Supply in the 21st Century

33

Table 2.2. Prediction of grain demand and supply (million tons) Year

Human consumption

Feed

Industry and other

Total demand

2010

213-230

260-340

12% of the total demand

554-627

2030

224-234

320-420

642-741

Table 2.3. Prediction of grain and meat capitation (kg) Year

Grain ratio

Feed

Meat

2010

152-164

186-243

53.1-69.4

2030

140-160

196-258

56.0-74.0

Cultivated Land Area and Grain Sown Area At present, China has three sources of statistics on cultivated land area (Table 2.4). Among these three sources, the data provided by the Land Survey Committee is believed to be reliable. The committee is comprised of land experts who usually spend several years in the general survey. As errors might occur during the process, it is estimated that the current cultivated land in China will be between 138 and 151 million hectares. Since 1978, the cultivated land area has decreased year after year (Table 2.5). Therefore, the Chinese government clearly states in its “Ninth Five-year Plan” and “Long-term Outlook for 2010” that land for other uses should be strictly controlled. As a result, the cultivated land area will decrease more slowly than before, with a predicted annual decrease rate of 100,000-200,000 hectares. It is estimated that the cultivated land area will be 136-149 million hectares in 2010 and 133-146 million hectares in 2030. Due to the decrease of cultivated land area and the increase of cash crop grown area through crop structure readjustment, the grain sown area continuously decreases. Its percentage in the total crop sown area is also declining. In accordance wiht the average decrease

rat of the grain sown area from 1978 to the present, the grain sown area will be about 70 percent of the total crop sown area by 2010. Considering the importance of the grain in people's livlihood, the Chinese government will take measures to encourage farmers to grow grain crops. On the other hand, farmers are not willing to sacrifice too much of their land for other uses because of the vigorous grain market and factors like changing crops, crop sequence arrangement and the selection of crop varieties, etc. It is predicted that the grain sown acreage will be about 65 percent of the crop sown acreage. Multiple crop index is another key factor influencing grain sown area. From 1980 to 1990 in China, the average multiple crop index was 115 percent. According to the sunlight condition of different areas in China, there is little potential in raising the multiple crop index in the northeast China, norhtern China and northwest China. The highest average multiple crop index can reach approximately 165 percent. Apart from the restriction of comparative advantage on growin grain crops, other factors influencing the index rise are: the extension and utilization of agricultural mechanization useful for crop sequence, the selection, breeding, extension and application

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

34

Table 2.4. Arable area statistics (10,000 ha) Office

9,510

Survey

13,800

GIS

15,100

of early-maturing varieties. As a result, China has not the capacity to tap the potential of all the multiple crop indexes even in 2030. Through our strenuous efforts, the average index can be expected to reach 150 percent and 160 percent by that time. Based on these analyses, the sown area of grain crops in 2010 and 2030 will be 143-156 million hectares and 138-152 million hectares respectively.

Analysis of the Yield Growth Possibility of Per Unit Grain Area and the Total Output From 1961 to 1995, the average growth rate of per unit area yield was 2.12 percent. Constrained by the comparative advantage of growin grain crops, the farmers slow down the growht rate of capital investment. In addition to this, the other factor which influences the decline of per unit area yield growth rate is the rising baseline grain unit yield. On the foundation of regression analysis on grain unit yield increase, it is concluded that the annual growth rate of the grain unit yield will be stable at about 1.5 percent from 1995 to 2010 and about one percent from 2010 to 2030. Based on the above analysis, it is predicted that the per unit area yield by 2010 can reach 5.25-5.85 tons/hectares and 6.47-7.05 tons/hectares by 2030 in China. Considering the changes of cultivated land area, multiple crop index and combining with the analysis of the per unit area yield growth possibility, we predict that the total grain output will be around 546-558 million tons in 2010 and 649-654 million ton sin 2030 (Table 2.6).

Forecast of the Difference Between Grain Supply and Demand Changes in population and consumption levels lead to continuing grain demand. Furthermore, various factors restrict the increase of grain supply. Therefore, the situation of grain demand and supply is not so optimistic. The above analysis tells us that the grain demand and supply is around 591 million tons and 552 million tons, respectively, in 2010. Obviously, there are approximately 41 million tons of grain demand to make up. By 2030, the grain demand will be about 692 million tons while the grain supply is about 651 million tons, which means the same amount of food demand to make up (Table 2.7).

Countermeasures Toward Making up the Balance Between Grain Supply and Demand In recent years, the Chinese government has formed a series of policies to support grain production. The total grain output has risen to a higher level. It has not only satisfied the domestic need, but also led to structural surplus. The reasons lie in, first of all, the low quality of some grain varieties, which can not meet the need of the rising living standard of consumers; secondly, the limited stock capacity; and thirdly, the low conversion efficiency of grains. Viewing the present situation, there is no problem in realizing the balance between grain supply and demand. However, from the long term point of view, there will still be a big gap to fill. due to factors like the increasing population, the decreasing land area and the rising living standard of households. Therefore, from an overall point of view, the strategies to solve the discrepancy between grain supply and demand are to rely on domestic forces, strengthen grain production, restrict demand and stick to moderate and appropriate grain import.

Support Grain Production and Increase the Overall Domestic Supply The general policy of grain production in China is to stabilize the grain production area, adjust crop structure, raise per unit yield and increase the total output. The central and the local governments should encourage and

Prospects for Grain Demand and Supply in the 21st Century

35

Table 2.5. Changes in China’s arable land (103 hectares)

Year

Land Areas Practically Used

Decreased Land Areas

Newly Increased Land Areas

Net Increased or Decreased Land Areas

1978

99,389.5

800.9

1980

99,305.2

940.8

748.0

-192.8

1984

97,853.7

1,582.9

1,077.0

-505.9

1988

95,721.8

644.7

477.8

-166.9

1989

95,656.0

517.5

451.7

-65.8

1990

95,672.9

467.4

484.3

+16.9

1991

95,653.6

488.0

468.7

-19.3

1992

95,425.8

738.7

510.9

-227.8

1993

95,101.4

732.4

408.0

-324.4

1994

94,910.0

708.6

517.2

-191.4

support the main grain production areas to industrialize their management through giving favorable treatment for finance, credit, taxes, personnel utilization and technology. On the one hand, cultivated land area should be protected by law and the grain sown area should be maintained. On the other hand, preferential policies should be made to protect the initiative of grain producers. In addition, investment should be increased from various channels to ensure meeting the needs of grain production.

Strengthen the Development of Agricultural Infrastructure and Improve the Capacity of Comprehensive Grain Production After more than 40 years’ development, the agricultural infrastructure has been improved to a certain level. However some aspects still lag behind, especially the low flood control standard of farmland embankment, as well as the underdeveloped corresponding facilities of irrigation and drainage. Every year, Chinese people suffer from a number of flooding and drought disasters. Statistics show that the average area suffering from natural calami-

ties from 1980 to 1995 was 340.16 million mu (approximately 22.67 million hectares ). Calculating for an annual decrease of 30 percent in grain yield, we can figure out that the annual output will decrease 24,945,000 tons, which accounts for 4.2-9.0 percent of the total grain output and 6.05 percent of the average output of the year. Therefore it is urgent to set up and improve a number of large scale irrigation and drainage facilities. These will play a significant role, not only in strengthening the comprehensive capacity of grain production. Apart from this, there is a great potential in the reserve resources of agriculture, though the cultivated land resource is limited in China. China has 500 million mu ( approximately 26.7 million hectares ) grass land and grass hillsides which can be developed. Consequently, cultivating undeveloped land and ameliorating fields with moderate and low output in order to protect cultivated land area and increase the per unit area yield will play a decisive role in raising grain output.

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

36

Table 2.6. Prediction of grain yield Year

Ton/ha

Grain gross (0.1 billion ton)

2010

5.25-5.85

5.46-5.58

2030

6.45-7.05

6.49-6.54

Rely on Scientific and Technological Progress to Achieve the Balance Between Grain Supply and Demand To promote agricultural development through scientific and technological progeress has become the basic feature of modern agriculture. In the past several decades, the proportional contribution made by scientific and technological progress in agricultural growth has been getting higher (Table 2.8). Research on crop germplasm in China has held the leading position in the world. Breeding research on main cereal crops has produced a higher level in the yield, quality and disease resistance of the new varieties. A large number of improved varieties bred all over the country have the capacity to replace the currently produced varieties, taking the grain output increase to 10 billion kg. Also, extending modelized cultivation and applicable technologies such as dry land farming, plastic mulch, water saving farming, scientific fertilization, integrated management of insect pests and decreasing post-harvest losses all contribute to the rapid growth of the per unit area yield and the total grain output. With the extensive application of biotechnology in agriculture, it is estimated that biotechnology and output growth measures will generate more than 80 percent of the increase in agricultural products by the end of this century. So, the application of biotechnology to breeding new crop varieties such as super-rice, the development of nitrogen-fixing technology, the improvement of crop photosynthetic efficiency, and the wide application of biological chemicals will open up new channels for grain production and provide sound technological guarantees for output growth.

Table 2.7. Prediction of the balance between grain supply and demand Year

Demand

Supply

Balance

2010

5.91

5.52

0.39

2030

6.92

6.51

0.41

Adjust Planting Structure in Accordance with the Need of Grain Ration and Feed Grains From the overall situation of planting development, particular attention should be paid to feed production, including feed grain crops and feed crops, while making overall plans on the allocation of food grain, grains for seed industry and for industrial use and various cash crops. The emphasis should be on the harmonious development of a threecomponent crop structure: cereal crops, cash crops (including crops like melons and vegetables ) and feed crops. This means that a part of the feed grain area in Southern China which is brought under grain crops should be converted to growing grain/feed crops or feed-specific varieties, such as high yield rice or corn for feed use. Meanwhile, an appropriate area of the land should be increased to grow feed/manure crops (such as some green manure crops) and feed crops (such as alfalfa, sweet clover etc.) so as to form a feed basis and stimulate the crop structure to the greater diversity of the three-component one (cereal crops-cash crops-feed crops ) compared to the dual structure (cereal crops-cash crops). According to the estimation of the Long and Medium Term Food Development Research Team of the Chinese Academy of Agricultural Sciences, the future proportion of the three crops in 2000 and 2020 is as shown in Figure 2.4.

Redistribute Grain Import and Export on the Basis of Stablizing Domestic Grain Market To avoid the grain price fluctuation generated by grain fluctuation in the domestic market, there should be a certain differentiation

Prospects for Grain Demand and Supply in the 21st Century

37

Fig. 2.4. The future tendencies of three types of crops: grain crops, feed crops and cash crops.

Table 2-8.The contribution of science and technology to yield Years

Contribution

1972-1982

27%

1983-1990

35%

1990-1996

40%

between the domestic grain market and the international market. Grain import and export should still be managed by the state-run grain industries. The national grain reserves can participate in the international grain trade to some extent. When the grain price rises rapidly in the international market, the central government can then sell a certain amount of the grain reserves to the grain business sectors for export, in order to relieve the impact of rising international prices on the domestc market. When the grain price in the international market is much lower than that in the domestic market, the central government can purchase some of the imported grains as national grain reserves. An appropriate amount of the imported grains can be used to compensate the domes-

tic grain shortages. When there is a grain shortage caused by the lower grain price in the domestic market compared to the international market, the grain importerts should be subsidized with the surplus, “the grain price in the intertnational market + tariff - the grain price in the domestic market,” apart from enjoying tariff preferences. When the grain price in the domestic market is higher than that in the international market, a target price should be set, to raise the grain purchasing price. Theoretically, the volume of grain needed to fulfill the target price should be the basis of the grain imports. Surplus grains are exported when the grain price in the domestic market is lower than that in the international market. Grain exports undoubtedly raise the grain price in the domestic market, which benefits grain producers. It will not result in raising the grain price in the market. Grain exports will also decrease the volume of grain purchased by the central government at target price and therefore lighten the financial burden of subsidies. However, one consequence should be avoided, which is that the excess exports should cause grain shortages in the domestic market.

References 1. Summers R, Heston A. An expanded set of international comparisons, 1950-1988. Quarterly J Econ, 1991; 16:327-368.

CHAPTER 3

Agricultural Science In India— Shaping for the Future M. Rai and A.K. Bawa

I

n the year 1996, from an estimated 1362 million hectares of arable land, 2050 million tons of cereals, 565 million tons of vegetables, 413 million tons of fruits and 536 million tons of milk were produced. In this global agricultural production, 88.6 kg/ha of fertilizer was used, consisting of 53 kg N, 21kg P2O5 and 14 kg K2O. An estimated 2592 million people were engaged in agriculture. In India, from 166 million hectares of arable land, 185 million tons of cereals, 65 million tons of vegetables, 39 million tons of fruits and 67 million tons of milk were produced, an average 81.8 kg/ha of fertilizer being used. Nearly 541 million people were engaged in agriculture. The projected world population of 6.17, 8.35 and 11.0 billion by 2000, 2025 and 2050 CE, respectively, would require far greater vertical agricultural growth through almost 100 per cent productivity enhancement in the next 50 years. The same would be true for India, whose population is likely to cross the 1500 million mark by 2050 CE. In order to address the multiple and interwoven problems of ensuring household food and nutritional security, enhancing productivity, sustaining production, protecting environment, conserving natural resources and improving the profitability of farming as an occupation, there is an urgent need to redefine the agricultural research agenda. The important areas of science and technology, viz., biotechnology, seed technology, post-harvest technology, hybrid technology, information technology, remote sensing, GIS-based modeling and land use planning, integrated pest management,

integrated plant nutrient management etc., are expected to provide the necessary boost to agriculture productivity in the times ahead. Indian agriculture will have to achieve ecological, economic and social sustainability, for which a change, both in agricultural research priorities and strategies, and in public policies, will be needed. It will be desirable to examine the directions of this change and to understand its multidimensional effects on farm, family, livestock, science and society. Capital formation in agriculture; critical human resource development for upstream research; institutional mechanisms; congenial policies; public/private interface; system-wide systems approach; market-oriented and demand-driven technological upgradation; technology assessment, refinement and transfer; proprietary products, processes and marketing systems processing; product development and value addition etc. will be required to bring in a much needed agricultural commerce which would withstand the globalized market based on its competitiveness in terms of cost and quality. Equity, social and economic justice and sustainable growth would be expected only with a sound development strategy that must rally around conservation of natural resources, so that these are available for rational use on sustainable basis.

Indian Scenario The “green revolution” is one of the success stories of post-independence India cited globally. It enabled India to convert its

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

“begging bowl” status into that of “selfsufficiency.” It also brought about an element of resilience in agriculture to ward off the vagaries of nature, and infused much needed confidence in the National Agricultural Research System (NARS). The green revolution ushered in an era of overall rural prosperity. This massive transformation was possible due to two basic factors, i.e., research infrastructure and trained human resource. Both of these were critical for the technological development which flourished under effective, efficient and reasonable public policies. The Indian Council of Agricultural Research (ICAR) as the apex agricultural research organization had been at the forefront in setting the national research agenda and in guiding its development. As a result, the total food grain production increased from a mere 50.8 million tons (mt) during 1950-51 to 199.3 million tons in 1996-97. The production of wheat (69.3 mt), rice (81.3 mt) and oilseeds (25.0 mt) have recorded an all time high. In the process, India emerged as the second largest producer of wheat, surpassing the USA in both production as well as productivity. Tropical, subtropical and arid fruit crops together produced 40 million tons from 3.94 m ha, and enabled India to occupy a leading position in the world. In vegetable production (64.67 mt) India is now the second largest producer, after China. In this total agricultural endeavor, 14.3 million tons of NPK fertilizers and 56 thousand tons of pesticides were used. All this was made possible due to adoption of good quality seeds, enhanced use of fertilizers and plant protection practices, and an increase in assured irrigation. The distribution of certified/quality seeds to the farmers from public sector outlets alone increased to 700,000 tons in 1996-97 from 250,000 tons during 1980-81. Interestingly, during the nineties seed production and availability through the private sector has gone up many fold. There are over 500 small and large seed companies, including several multinationals. The livestock sector, as well, continues to play an important role in India’s economy in terms of income, employment, earning foreign exchange and enhancing household nutritional security. India has a population

of 193 million cattle and 79 million buffalo, contributing about 71 million tons of milk. Besides, 28.5 billion eggs and 44.6 million kg of wool are produced in this sector. India has also achieved fish production of 5.39 million tons from marine and inland resources during 1997-98, to become one of the leading nations producing fish and fish products. The negligible imports of cereals during the last decade, and improvement in the per capita availability; per capita calorie intake closer to the accepted norms (about 2200 calories/day); the export of food grains exceeding on average the imports; considerable stability in food grain production and availability; diversified crop production increasing physical access to other commodities such as oilseeds, vegetables and fruits, sugarcane, condiments and spices in different regions; and the increase in economic access to food through increased per capita income are the positive features of an emerging strong agriculture-based Indian economy.

National Agricultural Research System The National Agricultural Research System of India is one of the largest in the world, with over 30,000 highly qualified scientists. The Central and State Governments provide most of the funds. The private sector in recent years has started to invest in agricultural research, mainly in seed improvement and production. Its share is expected to increase further with the existing and emerging congenial public policies, including appreciation for Intellectual Property Protection. Although the history of agricultural research in India goes back to the early years of the century, much of the present growth of the system has taken place in the past four decades. A significant part of it can be traced to the reorganization of the Indian Council of Agricultural Research in 1966 when, as the main executive agency, it was given responsibility and considerable autonomy to plan and coordinate research and to be the main funding body. ICAR has been described as the research arm of the Ministry of Agriculture, performing a variety of functions, including determination of national research policies and priorities, linking them with the government’s development

Agricultural Science In India—Shaping for the Future objectives, and establishing and managing a large network of research institutes and centers (Fig. 3.1).

ICAR Institutes The network of research institutions includes 4 National Institutes deemed to hold university status, 41 Central Research Institutes, 4 Bureaus, 28 National Research Centers, 10 Project Directorates and 82 All India Coordinated Research Projects (Table 3.1). These diverse institutions cover agricultural research in its wide sense, including crops, veterinary and animal science, fisheries, agroforestry, soil science, agricultural engineering, post-harvest technology, socioeconomics and other related disciplines, as shown in the table. State Agricultural Universities While the reorganization and growth of the ICAR and its institutes has been a significant development, an equally important development has been the setting up of 28 State Agricultural Universities (SAUs) on the pattern of the land grant Colleges of Agriculture in the United States, with suitable adjustments. These universities have taken over the teaching and research functions from the Departments of Agriculture in their respective states. The State Agricultural Universities, with most states having one or more of them, are the regional institutions responsible for providing technological support for the development of agriculture in the states. They receive their funding support primarily from the state governments, but also from the Indian Council of Agricultural Research in the form of coordinated projects and development grants. In recent years, the Council has set up an Agricultural University in the central sector for the northeastern states which functions like the SAUs. The State Agricultural Universities have a multifaculty and multicampus structure in order to develop location-specific technologies. They also have a network of over 120 Zonal and Regional Research Stations. The Zonal Station in an agroclimatic region coordinates and monitors the work of all the other university stations in the region.

41

Challenges Ahead Despite these favorable trends and set up, both poverty and malnutrition still remain serious problems. The average figures hide severe inequalities that prevent the poor from taking advantage of the increased supplies of food. These include lack of productive employment, particularly in rural areas, as well as lack of access to both food and non-food goods and also to services, due to insufficient infrastructure. It is estimated that one out of every five persons still does not have the means to buy two square meals a day, and around 100 million children below 5 years of age are protein energy malnourished. Further, the increase in population and agricultural intensification has brought the natural resources under considerable stress. How to protect them? How to increase their income and access to food and provide nutritional security are the key issues. Apart from the role of foodsupplier, there is an increasing awareness of the role of agricultural development as a driving force for overall economic growth, agriculture-based industrialization, employment generation, poverty alleviation, food and nutritional security and sustainability. For India, overall economic growth is inconceivable without growth in the agricultural sector. Therefore, an agriculturebased development strategy relying on increase in productivity and profitability, especially that of small holders, the availability of food at affordable cost for both poor and rich, and the provision of employment opportunities in the farm and rural non-farm sectors would be intrinsically poverty-reducing and food security- promoting. In this endeavor, some of the concerns are: 1. Recent slow pace of food grain production and plateauing of yields of major food crops like rice and wheat; 2. Practically no significant yield improvement in pulses; 3. Still limited choice of high yielding varieties and production package for highly risk prone and diverse rainfed ecologies, which account for twothirds of the cropped area; 4. Serious pest problems, which at times cause over 25% yield losses;

42

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 3.1. National Agricultural Research System in India. 5. Appearance of new virulent strains, pest resurgence and pesticide resistance; 6. Environmental safety; 7. Declining productivity factor; 8. Sharp decline in area under coarse cereals, and likely high demands on the feed front; 9. Erosion in genetic variability, soil degradation, soil erosion, waterlogging and water quality deterioration; 10. Inadequate seed production and limited quality seed availability; 11. Inadequate basic and strategic research support.; 12. Limited systems approach; 13. Limited dissemination of farmworthy agroproduction and protection technologies; 14. Limited processing, product development, value addition, marketing and trade facilities. 15. Inadequate investment in agriculture, i.e., about 0.46 percent of AGDP against its about 28 percent contribution to GDP;

Opportunities and Strategies Physical Bridging the Yield Gap A very large proportion of the area under various food crops fall under a low productivity category. The share of low productivity area varies from 57% in coarse cereals to 92% in oilseeds. Their yield levels are about 40% less than in high productivity areas. For instance, in rice and wheat the yield level in low productivity areas are respectively 2538 and 2032 kg/ha as against 2867 and 3828 kg/ha in high productivity areas. In these areas technology transfer and measures for enhancing input use holds the key. Waste Land Improvement Over 24.5 million ha remains as wasteland and 16.6 million ha as fallow lands. There are prospects of bringing under cultivation a sizable part of this large unutilized area through soil amendment and introduction of choice crop in the wastelands. Moisture conservation measures, development of facilities for life saving irrigation wherever feasible and introduction of crop species/varieties matching the available water balance in fallow lands would be the most rewarding. There is a good possibility of bringing much needed

Agricultural Science In India—Shaping for the Future

43

Table 3.1. ICAR Research Institutions in Different Disciplines

National Bureau

National Research Centers

Coordinated Projects

Disciplines

Institutes

Project Directorates

Crop Science

10

5

1

6

33

Horticulture

8

1

–

10

16

Animal Science

7

2

1

6

7

Fisheries

6

–

1

1

–

Natural Resource Management

2

1

3

Agricultural Engineering

5

–

–

–

10

Agricultural Extension

–

–

–

1

–

Agricultural Education

1

–

–

–

–

Social Science

1

–

–

1

–

Total

45

10

4

28

82

16

Agricultural

improvement to salt affected soils, which occupy over 20 million hectares. Capitalizing on Rainfed Low Lands About 8-10 million ha of saturated soils in the rainfed lowland areas of eastern India remains the least exploited. Through concerted research and development efforts, crop intensification is possible over a sizable area. In the near future about 1 million ha can be brought under winter rice in the states of Bihar, Assam and Orissa. Similarly, effective drainage could enhance production efficiency in many states, viz., Assam, West Bengal, Orissa, Andhra Pradesh, Tamil Nadu, Kerala, Madhya Pradesh, Haryana, Uttar Pradesh and Bihar.

Inter Cropping Inter-row space and time space available in crops like sugar cane, banana, cotton, sorghum etc. are least utilized. With the introduction of paired row planting and drip irrigation, the potential of using an intercrop of short duration pulses or oilseeds is enormous. When there is no room for increasing any more gross area under pulses and oilseeds, this is an opportunity to increase the area under them by 10-12 million ha over years. Water Harvesting India is fortunate to receive higher precipitation as compared to other countries of its size in the world. Watershed development

44

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

in such areas would greatly help provide protective irrigation for rainfed crops while helping recharge the wells. With in situ and ex situ water conservation, water harvesting, and water use and reuse, can provide a vast scope for enhancing cropping intensity, which has increased by only about 25% from 111% in the last four decades. Rice fallow again offer tremendous opportunity for area expansion in such crops.

Technical Conservation of Biodiversity India’s biological diversity continues to be rich, one of the 12 megacenters of plant diversity. This diversity needs to be preserved, and the immediate task will be to devise and enforce time bound plans for saving endangered plant and animal species as well as habitats of biological resources. This is considered most important, as with the advent of new tools and techniques there is no barrier to gene flow, and the living systems could be termed one gene pool with tremendous options and potentialities for new combinations. Requirement of capital resources at every level for agrobiodiversity conservation and management is critical to its success. Thus, every effort for resource mobilization, from within the country as well as from international arrangements has to be made. Intemationally, the endeavor should be to develop effective, efficient and transparent mechanisms for fair and equitable benefit sharing among nations, including technology transfer and capacity building. Consolidation of Yield Gains High yielding dwarf varieties in wheat and rice, as well as heterotic hybrids in maize, sorghum and pearl millet brought about a major advance in food grain production. Yield gap analysis reveals that a sizable part of the potential of these crops is yet to be fully tapped. Differences between experimental and farmers’ yields are quite wide. Equally, the gap is wide between potential and realizable yields. For instance, in rice about 40% of the potential available in the present day high yielding varieties is still to be exploited. In wheat, the

national average yield is 2.6 tons/ha as against the realizable 4.0 t/ha and genetic potential of 8 t/ha. In the case of hybrids, average yield of maize is 1.8 t/ha as against realizable 5 t/ha and genetic potential of 7.5 t./ha. Equally, the prospects for stepping up the yield level to 5 and 2 t/ha in sorghum and pearl millet do exist. What is required to achieve such yield targets is diagnosis and correction of factors constraining the yield increase. Insulation of all future varieties with desired levels of resistance to key pests and diseases, and tolerance to major abiotic stresses like salinity, drought, temperature extremes etc., could be the priority research area to consolidate the genetic yield potential already achieved in the plant type based varieties and hybrids. Maximization of Productivity of Rainfed Crops Over 97 million hectares (72%) of the cropped area is rainfed. It accounts for 44% of food (55% rice and 91% pulses), 90% groundnut and 68% of cotton. Even if the ultimate irrigation potential of the country is realized, about 50% of the cultivable area may continue to be rainfed. Since vagaries of weather affect production from drylands and thereby the stability of the food production in the country, strategic research on rainfed agriculture may be a priority area, to insulate the farmer from the high risks of dryland farming. The thrust areas of research could be a detailed characterization to optimize land use for rainfed crops and development of other alternate land use systems, understanding of crop/ weather/soil relationships for providing better agro-met advisory services and and rain water conservation and integrated nutrient management. Watershed development for raising productivity of rainfed crops, improvement of agricultural credit; insurance cover for risk prone areas and crops, and marketing facilities are some of the areas which need more attention. In spite of wide variation in the level of precipitation, rainfed areas are very low in productivity and predominantly monocropped. The productive potential of rainfed uplands, in particular, has deteriorated due to poor management rather than overexploitation. By developing land capacity based cropping and

Agricultural Science In India—Shaping for the Future management strategies, productivity level could be further enhanced and sustained. Four decades of experience with hybrid crops suggests that hybrids in general, in preference to varieties, have higher resilience to critical environments. Development and use of short duration hybrids/composites/varieties of millets, cotton, sunflower, castor etc. in low rainfall areas is one of the crop planning strategies based on locational advantages. Plantation of horticultural crops in some of the arid and semi-arid environments is found to pay more than some of the annual food or oilseed crops.

Varietal Improvement Following the introduction of the Norin dwarfing gene-based high yielding varieties of spring wheat Sonora 64 and Lerma Rojo in the early sixties and the Dee-Geo-Wu-Gen dwarfing gene-based high yielding rice varieties Taichung (Native) 1 and IR8 in the mid-sixties, a major breakthrough in yield was achieved. Whereas high level of response to applied fertilizer combined with non-lodging habit ensured high yields, photo-insensitivity conferred wide adaptability and early maturity helped increase the cropping intensity. India is one of the few countries to take immediate advantage of the phenomenon of hybrid vigor for improving the productivity of as many as 10 field crops. India’s more than four decades long experience in hybrid technology prompted the ICAR to explore the possibilities of exploiting hybrid vigor in non-traditional crops like cotton, rice, rapeseed-mustard, safflower, sesame, pigeonpea etc., in addition to extending the hybrid technology of traditional crops like maize, pearl millet, sorghum, sunflower etc. to new and still underexploited niches. India has become the second country, after China, to make hybrid technology in rice a field reality. Now hybrids in rainfed crops like safflower and pigeonpea are also on the ground. Success of hybrid technology in any crop plant depends on the efficiency of producing and supplying adequate quantity of quality seed. In spite of a wide choice of productive hybrids available in several crops, desired pace of growth in terms of area coverage is yet to be achieved. As hybrid culture in agriculture, cutting across crops and commodities and ir-

45

respective of the nature of the crop, is likely to prevail in the 21st century, accelerated hybrid research and development efforts are on. Intensified efforts through interdisciplinary modes of operation on tailor-made varietal development, with tolerance to multiple biotic and abiotic stresses, continue to be the priorty. In the chain of events, environmental impact assessment for realizing enhanced and sustained crop productivity are considered of importance. Biotechnology has emerged as an indispensable tool globally for crop improvement. It is essential that it become fully integrated with the conventional breeding program for achieving rapid growth in agricultural production. Recognizing this fact, resources are being mobilized to establish infrastructure facilities in different institutes/universities in the country for undertaking biotechnology research and education. Over the years, there has been rapid growth in this area and a number of organizations have graduated from tissue culture technology to recombinant DNA biotechnology. Sustaining the enhanced production potential would be possible by engineering the plants against biotic and abiotic stresses. Sterility systems, derived from alien cytoplasms by genetically engineering male sterility in crops in a non-specific manner, would enhance the exploitation of hybrid vigor. Apomixis, as an extension of hybrid technology, would offer new promise in realization of hybrid advantage on a sustainable basis. Molecular markers could be linked to apomixis, which would facilitate marker-aided selection. Reduction in cost of production by replacing costly inputs like chemical fertilizers and pesticides would be possible. Genetic modification for better processing and storage, mechanical agriculture, and appropriate size, shape, color, flavor, texture, taste etc. of the useful part would open possibilities of an icing on the cake for a holistic approach in crop improvement and production. Identification of physiological and biochemical determinants of metabolism, growth and development would be another essential component for research. Crop growth models linked with GIS and remote sensing would provide new opportunities to design eco-region specific plant type designs to extrapolate

46

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

performance across different regions. The renewed emphasis on quality of food would make it imperative to understand plant regulation of nutrient uptake as it affects tissue composition and crop nutritional quality, including the efficiency of utilization of nutrients in the soil and plants by genetic and agronomic means. Protected cultivation would be far more important, and it would be important to determine optimal environmental factors and desired crop characteristics for maximizing the production of greenhouse crops. For water-deficit environments, understanding the physiological effects of water stress and response to water availability would be necessary in order to manipulate genetically different crops for greater adoption and productivity. Understanding the mechanism of crop/ weed competition, allelopathy and host/parasite relationships to develop crop production strategies will contribute to sustainable crop management practices. The vast majority of marine microorganisms have yet to be identified. Even for known organisms, there is insufficient knowledge to permit commercial exploitation. Oceanic organisms constitute a major share of the earth’s biological resources and often possess unique structures, metabolic pathways, reproductive systems and sensory and defense mechanisms. They have adopted to extreme environments, ranging from the cold polar seas to the great pressures and temperatures of the ocean floor. Enzymes produced by marine bacteria are important in biotechnology due to their range of unusual properties. Some are salt resistant, a characteristic that is often advantageous in industrial processes. An unusual group of microorganisms from which enzymes have been isolated are the hyperthermophilic archae (archaeobacteria), which can grow at temperatures of over 1000°C and produce enzymes that are stable at high temperatures. Transferring genes of interest from marine into non-marine microorganisms would be an another area with unfathomed prospects. Knowledge of the mechanisms underlying carbon allocation and sink-source relationships could help to modify the size of desired organs in a crop. Rhizobium strains

with improved symbiotic properties for efficient nitrogen fixation, even in non-leguminous plants, is another possibility. Optimization of mineral nutrition, transport and assimilation in stressed environments to make the most of limited fertilizer input will contribute to efficient crop production. Engineering of oils for modern human diet and for use as feedstock for chemical industry is a real possibility. Production of enzymes in forage crops that will enhance the efficiency of their digestion by the livestock is another area with great promise. The value of incorporating the shortlisted physiological traits has first to be demonstrated before recommending them as selection criteria. New opportunities using biotechnology approaches are now available to create genetic variation for physiological and biochemical traits, including those for realizing enhanced photosynthetic efficiency. Water is a major constraint in many parts of India. Hence, efforts to manipulate genetically different crops for greater adaptation and productivity in water-deficit environments would be important. Water deficiency coupled with salinity/alkalinity is another paradigm for research. Abrupt weather/temperature fluctuations is yet another challenging research area.

Integrated Nutrient Management Exhaustive cropping systems like wheat/ rice, wheat/cotton, rice/rice etc. have hastened the pace of soil health degeneration. The impact is seen in the plateauing yield levels of major crop-based rotations. At present Indian agriculture is mining nearly 10 million tons of nutrients. In spite of the new technologies continuously emerging on the scenes of action, maintaining the yield growth has become increasingly difficult and costly, mainly due to inefficient input use and declining quality of resource base, manifested by increasing incidence of micronutrient deficiencies, decline in soil organic matter etc. No matter how successfully the plant potential for higher productivity is expanded, future gains would depend on meeting the nutrient requirement of plants through development of integrated nutrient management systems (IPNS) for sustainable resource management. Recent reports suggest that cereal

Agricultural Science In India—Shaping for the Future food deficient in mineral nutrients decreases the IQ of children by 10 points. The potentiating effect of protein-, energy-, mineral nutrient-deficient cereal food may adversely affect about half the population in south Asia. Therefore, promotion of nutrient management through IPNS is an important component of the Indian strategy for food and nutritional security. In the quest for greater productivity, we have a responsibility to promote the use of fertilizers, organics including farm wastes, crop residues, green manure and urban city composts and microbial inoculant to bridge the demand/supply gap of mineral nutrients. The nutrient gap has to be met by enhancing the input use efficiency through development of integrated nutrient management systems for harnessing the positive interactions of crops with growth factors in major production systems in different agro-ecological regions. In fertilizer consumption statistics, a matter of serious concern is the widening N:P:K fertilizer use ratio. A widening N:P:K ratio indicates an imbalanced fertilizer use. On a macro scale the deviation from the ideal 4:2:1 NPK consumption pattern would suggest that the greater the departure from this ratio, the more the imbalance in the N:P:K ratio. It must be fully recognized that the ideal N:P:K consumption pattern would be different for irrigated, dryland, horticultural and plantation crops. Most organics have N, P and K in proportions such that it is not possible to correct the N:P ratio through their use. Organics having low N and high K (e.g., rice straw) can at best correct the N: K ratio. It is only through the use of enriched phospho-composts that the widening N:P nutrient pattern can be corrected; otherwise, we must promote the use of phosphatic fertilizers.

Integrated Pest Management Excessive use of pesticides in some of the crops, viz., cotton has made pest management increasingly difficult because of new problems such as pest resurgence and pesticide resistance. Pollution of environment and pesticide residues at toxic levels in the food chain are other problems associated with the indiscriminate use of pesticides. It is increasingly evident that chemical pesticides alone can not provide the desired level of protection against some of

47

the key pests. For instance, management of Heliothis, a polyphagous pest having a wide host range, has become a challenge, as it resists to different degrees many known chemical pesticides. In such cases, integrated pest management involving all available control techniques, namely host plant resistance, pest-specific biocontrol agents, botanical pesticides and cultural practices has been found not only to be an effective remedy to the pest problem but also highly cost effective and environment friendly . A wide choice of resistant varieties now available against many of the pests in different crop plants, and introduction of innovative approaches, are good signs for the reduced use of toxic pesticides. Integrated pest management(IPM) utilizing the best combination of available control techniques has been found to be an effective remedy to the pest problems of major crops like cotton, rice, sugar cane, tobacco and a wide range of pulse and vegetable crops. While giving due emphasis to IPM approach as environment friendly and cost effective, it is worth mentioning that India is not among the countries that use very high levels of pesticides. Moreover, the pest is a serious constraint on tropical crops and, also, biodegradation of the pesticides is fast under tropical conditions. Integration of components of pest control and management require far greater capitalization on complementarities and synergies of crops, varieties, cropping pattern, agronomic management, chemical use, biological balance etc. This would require effective, adaptable and viable capsules, their promotion and use.

Post-Harvest Management It is estimated that food grain losses in the country are at about 10% in the case of cereals, pulses and oil seeds and up to 40% in the case of fruits and vegetables. In order to ensure Indian agriculture to be globally competitive, processing, product development, value addition, packaging, storage and marketing are considered of paramount importance. We believe that post-harvest technologies have to be an essential element of our overall strategy of enhancing productivity , production and net monetary returns per unit area, input

48

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

and time. The research intervention in this area has led to the diversification of agricultural crops/commodities/processes/ products to further boost our agricultural production, consumption and export. With the development and application of post-harvest technologies, the exports in agriculture have been steadily increasing. It is believed that wealth could be had from the waste, byproducts could become main products and full use of agricultural produce in various farms and functions could enhance substantially the competing ability of agriculture, its produce, products and processes.

Agricultural Exports In the last few years, India has been emerging as a leading exporter of agricultural produce, both fresh and processed. Our national exports of agricultural commodities during the year 1997-1998 were RS1262860 millions (million rupees). Oilmeal and oilcake, fresh fruits and vegetables and marine products are major foreign exchange earners. Fine quality basmati rice, meat and meat preparations, spices and cashew have made considerable breakthroughs in the international markets. With the globalization of markets, commerce in agriculture would be witnessed far more than ever before and hence it would be congenial for technology transfer to take place in realizing enhanced productivity and production. Efficiency would hold the key to attain and sustain advantages in terms of cost and quality locally, regionally and globally.

Agricultural Extension The increasing complexity of production environment demands efficiency, information, dissemination and training in the use of modern technologies. For this, an appropriate extension service needs to be in place to stimulate and encourage both top-down and bottom-up flow of information between farmers, extension workers and researchers. Technology transfer, in order to be effective, must be preceded and succeeded by technology assessment. How reliable an assessment has been can be judged by the effectiveness of transfer of a given technology. Therefore, technology assessment and technology transfer are complementary to each other. Technol-

ogy transfer must be based on needs and capabilities of agro-ecological settings, resource endowments, agro-production and distribution systems and farm households. Transfer of farmworthy technology is vital for harnessing the fruits of research. In this endeavor, re-orienting of agricultural extension systems to respond to the changing diverse needs of different agro-climatic situations would be important. Special emphasis will have to be given to the extension needs of hilly, tribal and rainfed areas, and especially the needs of women in agriculture. The priorities in the area of front-line extension approach would necessitate: 1. Stress on technology assessment, refinement and transfer through Institute-Village Linkage Programs; 2. Analysis of cost-risk return structure of major farming systems in different agro-eco-regions/subregions; 3. Consensus on the unified and field tested recommendations to the farmers; 4. Technology dissemination through active involvement of mass media; 5. Accelerated interface, between public/ private and research/development systems.

Programs and Policies Our development policies at times are placed on conflicting objectives.The environmental problems and policies need coordinated appraisal, as they are inextricably enmeshed in their impacts, value orientation objectives and attainments, irrespective of geo-political barriers. The specific concern at this juncture should pinpoint critical conditions for success so that development becomes truly an ally for providing social justice. Obviously, there is no simple or single solution to the complex ecological, socioeconomic and technological problems facing those engaged in promoting sustainable advances in the productivity of terrestrial and aquatic farming systems. This scientific challenge can be met through accelerated efforts in the blending wisdom of traditional technologies and modern science and technology.

Agricultural Science In India—Shaping for the Future Scientists must work in partnership with farmers, industry and entrepreneurs to bring a new culture in agriculture. Transformation of the most marginalized farmers into agents of poverty alleviation, and environmental management through the blending of traditional and frontier technologies in socially equitable, economically viable and environmentally sustainable backgrounds and through production of more food from a diminishing resource base, with new agricultural technologies and management systems providing increased productivity per unit of land, water, energy, labor and investment, would be worth perusing. Part of this will involve focusing research on neglected crops such as minor millets, grain legumes and tubers, which can perform in times of environmental stress and in neglected areas such as arid and semi-arid/ coastal and mountain areas.

Eco-Regional Planning In the present scenario, ecoregional planning will have to aim at enhancing agricultural productivity and production on a sustainable basis to meet the ever growing needs of the farm family and livestock for food, feed, fodder, fuel, fiber etc. This would imply an upscaling of research activities within the eco-regions and dovetailing research and development priorities between and within the eco-regions. This would call for an effective collaborative mechanism, i.e., responsibility for a higher level of integration in research and development efforts. Thus, a lucid distinction of collaborative mechanisms and a clear distinction between priority setting at the ecoregional level and its effective execution at the local levels would be essential. In the ecoregional approach to research and management of natural resources, a balance in development and utilization of biodiversity would be important. The research should aim at improving the productivity of scarce resources while protecting the quality of soil and water, and at the same time safeguarding biodiversity for posterity. Concerning the management issue, the following points would need attention: 1. Research on conservation and management of ecosystems that include

49

multicrop and multi-economic farming systems in a program mode; 2. Accelerated research on the management of production systems; 3. Socioeconomic and public policy research to understand farmer and community decision making processes regarding the utilization of resources and factors affecting farmers’ incentives and their adoption of improved technologies; 4. Development of capacity of NARS for far more effective understanding of the intricacies of natural resource management. This would require fresh defining of NARS as everyone who can contribute to the cause of Agricultural Research and Development as a part and partner in the national agricultural endeavor. The most important end product of an ecoregional approach may be to provide a framework for sustainability. Incorporation of social and economic components would ensure success of the ecoregional approach. It is also recognized that training in various facets of a multidisciplinary approach in program mode would be extremely important for the success of the contemplated efforts. Compiling the existing information to identify the driving forces of land use changes and resource base degradation would be important in the first instance. This could address the issues of natural resources, innovations and technological options, present use of resources, potentials for agricultural production, policy objectives and short to long term goals, research capacity building, population dynamics, farmers’ decision making processes and capabilities, and market evaluation and intervention processes.

Human Resource Development Human Resource Development is a necessary concomitant of all dynamic systems for stability and attaining equilibrium with external forces. Human resources are the most important of any research systemsm since its performance depends not only the quality of its scientific and behavioral manpower but also on their motivation and morale. The human

50

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

and organizational resources are of much greater importance than material resources. There is an incredible variation in human capability, both physical and mental. Equally incredible is the degree to which an individual or group can evolve or degenerate based on the effort put in the process. This simply underscores the need to develop the human resource irrespective of the area of activity which, in fact, is a management tool equally applicable in agricultural science as it is in other disciplines. Changes due either to the stimulus of external environment or to internal pressure, can not remain static in any dynamic system. The only alternative to change is decay. Any vibrant, forward looking organization sensitive to the forces of change impacting it has to have a set of integrated and interrelated initiatives in HRD. The problem acquires complexity because it is not only acquisition of simple skills, but also of knowledge, attitudes and, more importantly, of values. These together constitute the work culture of the organization and the nation.

Conclusion The task ahead is much more complex in nature and severe in intensity. Apart from accelerating growth in production, issues of sustainability and environment are to be addressed. At the same time, integration of our economy with the rest of the world and competitive trade environments call for much more efficient production systems. Obviously, in this emerging agricultural scenario, development and dissemination of appropriate technologies have to play an important role.

Higher agricultural growth is necessary for alleviating poverty, and for economic transformation of rural India. In spite of the progress on several fronts, from a global perspective the environment has continued to degrade during the past decade and significant environmental problems remain deeply enmeshed in the socioeconomic fabric of nations in all regions. Internationally and nationally, funds and efforts need to be enhanced substantially to halt further global environmental degradation and to address the most pressing environmental issues. To meet these challenges, agriculture has to become vibrant, productive and competitive. Science, science culminating into technology, technology realized as practices, practices translated into production, productionbased consumption and consumption impacting on health would rally around energy conversion, its cycle and recycling, which would obviously require a balanced production to consumption mode of research and development to sustain the system and society for posterity. It is hoped that increasing population pressures culminating in ever growing demands will catalyze capitalization on uncommon opportunities, through added investmentd in the area of exploitation of the vast common gene pool, in various permutations and combinations which, in conjunction with efficient utilization of added inputs, would be able to meet what the Indian population would need. A vast experience and expertise would be the most vital tool in managing the change and making it a change for the better.

CHAPTER 4

Setting Priorities for Agricultural Research: Theory and Experience D. Gollin

T

he close of the 20th century has brought new and daunting challenges to the agricultural and biological sciences. Confronted with population growth and the continual emergence of new diseases, pests, and environmental problems, researchers face pressure to develop improved technologies at an everincreasing rate. Agricultural researchers are charged with the responsibility of producing more food and fiber, lowering the costs of production, and protecting the natural environment. They are urged to design plant varieties that benefit the poor and remedy social injustice; they are encouraged to create technologies that meet the needs of women; and they are asked to target marginal environments in which producers use few inputs. For those who manage research organizations or allocate funding for agricultural research, this portfolio of responsibilities can be overwhelming. An increase in funding levels would always help, but the allocation problems remain. Should resources be spread across all the potentially useful subjects? Or should they be concentrated in a few priority areas? If so, which ones? Should they be devoted to long term projects with uncertain payoffs? Or to short term efforts with relatively modest—but predictable—returns? Should “upstream” research be a priority, or should agricultural researchers simply draw on tools and techniques developed in other fields of biological research? Should public money be used to fund research, or will the “right” technology be created in the private sector, where an emergent agro-biotechnology industry is generating new

products daily? A host of similar questions can be identified. Such questions about the efficient and equitable use of resources go to the heart of economics. Although many biological scientists are wary of economic analysis, the real strength of the discipline lies in its ability to shed light on the effective use of resources. Economics is usually defined as the study of how to achieve desired objectives with limited means, and this seems to be an appropriate way of thinking about the problems of research in the years ahead. Moreover, careful economic analysis forces research managers to set out their assumptions. This can occasionally reveal priorities that are “non-obvious” and that have previously been overlooked. Priority setting for research is complex, however. Research is an inherently uncertain process: We do not know in advance whether or not certain avenues of work will be fruitful, nor how long they may take. The people who are best able to evaluate research - the scientists involved - often differ widely in their views concerning which approaches are best. Furthermore, we typically lack good data on the payoffs associated with research success. Nevertheless, several models and techniques have been developed for attempting to prioritize agricultural research. This paper will briefly consider the relevant concepts, techniques, and models for ex ante evaluation of agricultural research. A number of examples will be considered. In particular, this paper will focus on an interesting

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine. © 2000 Eurekah.com.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

case study: the Rockefeller Foundation’s priority setting process for rice biotechnology research. It then asks whether similar priority setting methods are well suited for other decision makers, or whether different tools are appropriate in different settings. Finally, the paper speculates on what issues may emerge as central ones in the decades ahead.

Research Priority Setting: Theories and Models A substantial literature deals with the problem of allocating resources for agricultural research. Ruttan traces the literature to work by Fishel in the early 1970s.1-2 In recent years, the literature on research planning has experienced a minor boom, perhaps driven in part by decreases in funding for public sector agricultural research. Two recent books on the subject are Alston, Norton and Pardey and Evenson, Herdt, and Hossain.3-4 Numerous articles and manuscripts address specific applications of the priority setting literature. The literature distinguishes between “priority setting” and ex ante evaluation.” This paragraph, and the following one, drew heavily from work by Evenson, Herdt, and Hossain.4 Taking these categories in reverse order, the latter involves relatively detailed accounting of the expected costs and benefits of narrowly defined research projects. For example, it might be possible to conduct an ex ante evaluation of a project aimed at breeding for powdery mildew resistance in bread wheat. By contrast, “priority setting” is usually taken to be the process by which resources are allocated across broad problem areas. For example, priority setting techniques might be used to allocate research funds across different crops or regions, or between plant breeding and entomological studies. In principle, it would be possible to conduct priority setting exercises simply by aggregating information from ex ante evaluations of specific projects. In practice, however, there is seldom enough data to proceed in this fashion. Moreover, ex ante evaluations are perhaps too narrowly focused to be useful for priority setting. They typically ignore the interactions among different research projects; in some cases, there may be several projects that could achieve a particular objective.

Considered on their own, all of them may look like sensible investments, but at the aggregate level they may be redundant. Priority setting thus requires a certain breadth of perspective; it involves a kind of macro analysis that cannot be simply developed from the microanalysis of ex ante evaluations.

Basic Principles There is substantial agreement among economists about the basic principles of priority setting for research. In essence, the main economic idea is that priority setting should move a research system towards “allocative efficiency.” The notion of allocative efficiency is that resources should be allocated across research problem areas (RPAs) so that the expected net benefits of research are maximized. A necessary condition of allocative efficiency is that an additional dollar of funding will have approximately the same expected payoff regardless of the particular activity in which it is invested. In other words, the added benefit that we can expect from an increment of $100,000 in research support should be identical whether we allocate the money to plant physiology or to soil science. Moreover, the expected payoff should be the same whether we allocate the money to “upstream” research in basic science or to “downstream” research in agronomy. Note that the expected benefit is the product of: 1. The productivity gain that would be obtained if the research is successful; and 2. The probability that the research program will succeed in solving the problem. If this condition were not satisfied, there would be the potential to increase expected net benefits by reallocating resources from activities with (relatively) low expected payoffs to those activities with (relatively) higher expected payoffs. It would be efficient for such reallocation to continue until there is no further potential for increasing benefits in this fashion. How can allocative efficiency be achieved? In many contexts, competitive markets tend to result in the efficient allocation of resources. With public sector research, however, markets offer little guidance for allocating resources.

Setting Priorities for Agricultural Research: Theory and Experience Instead, research administrators typically rely on a variety of different techniques. Some of these are sensible; others are not.

Congruence Rules Research planners often cling implicitly to ideas of “congruence” or “parity” in research funding, i.e., the notion that research resources should be allocated to different RPAs proportionately to the value of production. This is an idea particularly beloved by legislators, finance ministry officials, and non-scientists. A simple “congruence” rule might allocate research funds so that research expenditures per dollar of crop production were equalized across crops. There are many other possible congruence rules. For example, other rules might allocate research funds proportionate to the number of people employed or the amount of land used. These rules share, however, the common feature that they propose allocating research funds in a way depending entirely on the “demand” for research; they do not attempt to account for differences in the “supply” of research across RPAs. It would similarly be possible to equalize research dollars per dollar of crop production across regions or ecosystems. Let Eij represent research expenditures on crop i in ecosystem j; and let Y ij represent the production of crop i in region j. Similarly, let pi give the price per unit of output of crop i (usually, though not necessarily, taken to be invariant across regions). Then, if ˆC is a constant; then the congruence rule holds that Eij / piYij = ˆC, i,j Although the congruence rule is easy to understand and has the virtue of simplicity, it is typically suboptimal in an economic sense. One reason is that there may be greater potential for successful research in some crops or regions than in others. A second reason is that there may be unusually large benefits associated with research gains in particular crops or areas; basing research expenditures on the current value of production will not necessarily capture these benefits. For example, suppose there is a simple constraint to production of maize in one geographic area, such as a particular nutrient deficiency. In the presence of this constraint, the value of production is zero. If the constraint could be removed, the area might produce a great deal

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of maize. In this situation, then, a congruence rule that is based on the current value of maize production might not allocate any research resources to this area. That might be a shortsighted decision, however, because there is a high potential for research to generate large payoffs. In general, the problem with congruence methods is that they focus exclusively on the “demand” for research and neglect differences in the “supply” of research that might lead to higher payoffs for some RPAs than for others.

Supply-Driven Resource Allocation

Ruttan (pp. 269-70)1 notes that in spite of the problems with congruence methods of priority setting, the basic notion of congruence offers a useful reference point for resource allocation. He suggests that research administrators should be prepared to justify large deviations from congruence. Ruttan notes that research administrators often prefer to focus entirely on supply-driven research agendas, in which nearly all programs are motivated by perceived scientific opportunities. He wryly observes that “there are an infinite number of interesting scientific problems, but not all of them are important.”1 To some extent, supply-driven allocation may be embodied in competitive approaches to research funding. For example, some research is funded through a competitive grant-seeking process in which grants are screened and rated by panels of experts. Although this peer review process is valuable, it may tend to reinforce a supply-driven view of research priorities. Such awards panels may tend to support “interesting” research at the expense of research that is useful but unglamorous. Similarly, in many fields there are substantial professional rewards (e.g., publication, tenure, etc.) that are based on scientific accomplishment rather than on the “importance” of research.

Economic Approaches to Priority Setting Priority setting thus involves balancing the competing pulls of “problem-driven” research (whose value is apparent in the short run) and “frontier” research that is driven by

A

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

longer term ambition to shift the scientific frontier, regardless of immediate applications. In effect, this means integrating “demand” factors and “supply” factors in considering alternative investments in research. A set of difficult questions arises: Which research areas have the biggest potential payoffs? Which are most likely to generate successful outcomes? Which are most likely to benefit the poor or to alleviate stresses on marginal environments? Which are most likely to advance the state of knowledge in ways that will subsequently generate useful applications? A number of approaches have attempted to integrate the supply and demand factors for research.5 Typically, these approaches involve the following ingredients: 1. An assessment of the benefits associated with achieving particular research goals; 2. A weighting of benefits in accordance with social objectives or other desired outcomes; 3. An assessment of the likelihood of success; 4. An estimate of the time at which benefits are likely to be realized; 5. An assessment of the costs of the research. In some cases, past experience may offer insights into the likely payoffs from different types of research. In such instances, ex post evaluations of research may serve as priority setting tools. Thus, Evenson (p. 99)6 notes that upland rice research has demonstrated few past payoffs. If nothing has changed that would alter the potential for upland rice research, a planner might want to consider the ex post evaluation data before investing funds in further upland rice research. In most cases, however, priority setting will depend on data-intensive analysis of benefits, costs, and probabilities of success. Among the methods that have been used are scoring methods, expected economic surplus models (including benefit/cost analyses), and programming models. Scoring models simply assign different weights to different criteria, allowing the planner to rank different RPAs accordingly. Expected economic surplus models attempt to quantify the gains to consumers and producers (or subcategories of

consumers and producers) from research that will alter the supply and/or demand for commodities. Finally, programming models solve an optimization problem involving the allocation of fixed quantities of scientific manpower and other resources, based on specified assumptions about the relationship between research inputs and expected outputs. One type of information critical for priority-setting exercises is data on the benefits of research. Benefits may be estimated from crop loss data; from estimates of “yield gaps” between farmers’ fields and experimental fields; or from subjective assessments of scientists working in a particular field. The benefit estimates depend on the production gains from “solving” a particular problem, the rate of diffusion of improved technologies, the duration of the gains, and the time lags until the gains are realized. The gains from a new crop variety or a new source of disease resistance will not be permanent. Typically, disease resistance depreciates over time. The duration of effective resistance is not surprsingly important for the calculation of benefits. Benefits also depend on the ways in which markets will respond to the new technology: They are sensitive to the slope of supply and demand curves for the final product. This is particularly critical for assessing the effects of new technologies on different categories or classes of consumers and producers. A second category of information critical to the priority-setting process relates to the supply of research and, in particular, the probabilities of success and the likely time to success. In many cases, such information can only be obtained from surveys of knowledgeable scientists. These scientists may have overly optimistic assessments of new technologies, but they may also fail to anticipate successes that are near at hand. In general, subjective probability estimates from scientists seem to be relatively reliable, and they are better than any alternative estimates of research time lags and success probabilities. Methodologies for eliciting scientists’ input in priority setting are now well established. Some recent studies that use scientists’ estimates to assign research priorities are Mills and Karanja for the Kenya Agricultural Research Institute’s wheat program; Mills for

Setting Priorities for Agricultural Research: Theory and Experience sorghum in Kenya; Mutangadura and Norton for the Zimbabwean agricultural sector; and Evenson, Dey, and Hossain for rice in Asia.7-10 This literature is now well established. Although researchers have encountered some difficulties in utilizing data from scientists, the methods employed have become increasingly sophisticated. Moreover, with more experience in priority-setting studies, economists are beginning to have some opportunities to check the validity of scientists’ responses. For example, Evenson examines changes over time in the subjective probability estimates of scientists participating in the Rockefeller program on rice biotechnology.11 A group of 15 scientists was surveyed in 1993 and again twelve months later. Although the sample was small, it allowed Evenson to test the hypothesis that scientists set “moving targets” for research completion dates. This hypothesis suggests that scientists will predict today that a project will come to fruition in, say, ten years; but, when they are asked about the same project two years later, they may still say that it will take ten years to achieve success. Evenson found little evidence of moving target problems, however.11 This reinforces the idea that subjective probability estimates may be of adequate quality for priority-setting studies.

From Theory to Practice: A Case Study of the Rockefeller Foundation’s Decision to Prioritize Rice Biotechnology Perhaps the most noteworthy example of research priority setting in recent years was undertaken by the Rockefeller Foundation as part of its decision to concentrate its agricultural investments in the relatively narrow field of biotechnology for rice. Since 1984, the Rockefeller Foundation has spent about $70 million to support a program for rice biotechnology research in the developing world. As Herdt (p. 19)12 notes, “rice was chosen because 90 percent or more of the world’s rice is produced and consumed in the developing world, and as a result, gains from technical change in rice will largely accrue there.” An added reason for the focus on rice was the sense that public and private research agencies in industrial countries would be unlikely to invest much in rice technologies that would be useful

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for the developing world. Although there is abundant rice research in Japan, the United States, and a number of European countries, this research may not generate very many direct benefits for developing countries because of differences in climate, photoperiod, indica vs. japonica differences, etc. Consequently, the Rockefeller program aimed at achieving two objectives: generating technology useful for developing countries, and strengthening the capacity of laboratories and scientists in the developing world to perform rice biotechnology research. To date, a network of 200 senior scientists has been developed, with 300 additional scientific trainees. Evenson (p. 328)11 notes that as of early 1994, the program had supported some 130 projects in 26 countries, including 69 projects in developing countries. More than half of these projects included “biotechnology tool development” as a goal, and more than half specified “yield-enhancement technologies” among their objectives. Disease and insect resistance also accounted for a number of projects, with grain quality technologies and stress resistance technologies accounting for most of the remainder. It is arguably too soon to see results from the Rockefeller Foundation’s investments, but some preliminary results have already been achieved. Participating scientists succeeded in transforming rice in 1988, making it the first of the cereal crops to be transformed. There are now transformed lines containing economically useful traits. Herdt reports that, in China, a rice variety produced with anther culture at the Shanghai Academy of Agricultural Sciences has incorporated genes for resistance to pathogens and to cold.13 This variety has been field tested on over 3000 hectares (ha) in Anhui and Hubei provinces, with yield improvements of 6-24 percent over the most popular current varieties. Herdt notes a number of additional attainments in rice biotechnology in Asia and predicts (p. 6)13 that “the contributions to rice yield increases from biotechnology in Asia will be on the order of 10 to 25 percent over the next ten years.” These are striking benefits, but the yield increases do not by themselves convey the full impact of biotechnology. The incorporation of disease resistance through genetic

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

manipulation can reduce the quantities of pesticides used on rice. The ability to incorporate useful traits from wild relatives raises the potential for a vast broadening of the genetic base for rice and other crops. These gains are not without dangers; many critics of biotechnology worry that hidden flaws will emerge as genetically modified plants become increasingly common. But, for now, the potential for improving human welfare through biotechnology seems to create a strong imperative. At a less controversial level, biotechnology investments have paid off by improving the speed and efficiency of conventional plant breeding. For example, biotechnology has facilitated the development of molecular markers that allow breeders to easily discern whether a plant possesses traits of interest. Such approaches can improve the productivity of conventional breeding.

2.

Priority-Setting and the Rockefeller Investments Although the decision to concentrate on rice biotechnology for developing countries was in itself an example of priority setting, the Rockefeller Foundation faced another challenge upon beginning the biotechnology program. Which areas of rice biotechnology were most worthy of support? Would the payoffs be greatest in disease and pest resistance? In breeding for abiotic stress tolerance? In the development of tools useful for breeding? In the identification of molecular markers that could be used to test for genetic diversity? In the face of such questions, the Rockefeller Foundation undertook a careful and deliberate program of priority setting. This analytic framework has continued to guide the Rockefeller Foundation’s investments in biotechnology since the mid-1980s. The priority setting experience has been documented effectively in Herdt.14 The exercise involved an eight-step process, summarized below: 1. The target environments and regions were defined. These covered six geographic regions and four cross-cutting rice agroecologies. A critical step in any priority-setting process is to decide how to break down the different potential categories of research

3.

4.

5.

into RPAs. This decision in itself requires expert opinion to delineate the boundaries of specific RPAs. The importance of different research problem areas was estimated in three different ways. First, “knowledgeable scientists” estimated the yield losses due to different problems in each environment and region. As an alternative measure, a group of scientists “scored” each problem in each region, giving the relative importance of the various problems; the scientists were also asked about the maximum benefits that could be obtained from “solving” all of the relevant problems. Third, yield losses in specific regions were compared to a “reference region”, allowing for some consistency in estimates of importance across regions. The challenge here is to arrive at useful relative measures of the importance of different RPAs while keeping sight of the aggregate plausibility of the assumptions. In other words, it is not credible to find that six insect pests each cause an average of 15 percent yield losses annually in wheat. Some adding-up constraint must be imposed if the results are to be believed. Yield losses were converted into monetary terms by multiplying by areas and prices. This is perhaps the most straightforward step in the process. Environmental effects were taken into account by creating a set of weights for each problem in each region and agroecology. These weights were designed to reflect the added benefits that could be gained from using genetic methods to alleviate a problem that currently required pesticide use, herbicide use, or other environmentally harmful practices. Equity considerations were added by making an assessment of which RPAs would have the greatest benefits to the poor. Net benefits were then adjusted to yield an “equity- weighted” measure of the payoffs from achieving success in different RPAs. The point

Setting Priorities for Agricultural Research: Theory and Experience of this step was to allow planners to place greater weight on research that might be expected to benefit the poor. 6. The net present value of research benefits was computed for each problem and each agroecology. This depended critically on the assumed time lag until the problem was solved. Time lags were based on the elicited responses of scientists, who were asked how long they would expect it to take to find a solution for a problem-region-agroecology bundle, given a rate of research investment of $0.2 million per year. Although the figures elicited in this way may not be accurate in absolute terms, they probably offer a decent index of likely time lags. In other words, all else being equal, we would be inclined to believe that scientists might do a decent job of guessing which RPAs might take longest to “solve” and which might take less time. 7. The problems, regions, and agroecologies were ranked for susceptibility to a biotechnology solution. This was based on subjective probability estimates of knowledgeable scientists. This issue arises because some RPAs may be very important but may be solvable with conventional breeding techniques more readily than through biotechnology. Since the goal here is to set priorities for biotechnology research, there seems to be little point in selecting RPAs that are equally well addressed through conventional breeding or other techniques. 8. An investment rule was developed. This was a rule converting the overall rankings into actual funding decisions. Having established “priorities”, in the sense of a ranking of the importance of alternative research problems, there is still a need to convert these priorities into some action. This list is based on Herdt.14 The Rockefeller exercise led to a detailed priority listing for rice biotechnology research. The top priority after adjusting for equity issues, environmental impacts, and the likely

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usefulness of biotechnology techniques, was the search for resistance to the rice tungro virus. Other top priorities were submergence tolerance, gall midge resistance, and a source of cytoplasmic male sterility. Some of the problems that were ranked as most serious, in terms of raw crop losses, did not emerge as high priorities after the rankings were adjusted to reflect environmental and equity concerns and their susceptibility to biotechnology solutions. For example, weed problems were the biggest single source of crop losses in the data, but they ultimately ranked 15th among RPAs in terms of priorities for research. Conversely, submergence tolerance, which ranked seventh in terms of crop losses, ended up as the second highest priority after all the requisite adjustments. This suggests that a formal prioritysetting process can lead research planners to identify priorities that would not otherwise have been obvious. Moreover, it can lead researchers to discard problem areas that seem unlikely to be worthwhile. In the case of the Rockefeller undertaking, within a relatively brief time from the beginning of the priority-setting exercise, Herdt was able to report that “rice plants transformed with various gene constructs for resistance to rice tungro virus have been produced and are being evaluated at IRRI.”15 This suggests that the priority assigned to tungro research—based in part on the expectation that research could be effective—may have been well founded. Rice tungro virus is one of the most damaging crop diseases in the world, destroying as much as 7 million tons of rice output annually.15 Such claims of success warrant careful analysis. Although it is too soon to evaluate the priority-setting exercise formally, Evenson has undertaken an interim evaluation of the rice biotechnology program. One conclusion is that the benefits appear large. A second conclusion is that interim results support the original prioritization of biotechnology research across categories. Finally, Evenson concludes that there are high payoffs to continued rice biotechnology research for developing countries.11

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Some Shortcomings of Existing Methods and Lessons for the Future The rice case study illustrates the power of the priority setting tools described above. Fairly simple economic concepts led to a dramatic result: the decision to focus resources on a relatively small range of programs with high potential payoffs. Other studies show that the same techniques can be used in a wide range of research organizations at the local, national, and international level. Still, the priority-setting techniques currently in use are limited. On some issues, they offer little guidance. The sections that follow lay out a few areas in which the priority-setting literature seems inadequate.

Managing a Portfolio of Research The priority-setting approach described by Herdt generates a ranking of alternative research programs.14 It does not, however, describe an optimal portfolio of investments. For a research manager, however, the optimal portfolio is of keen interest. Other priority-setting methodologies can generate recommendations for portfolios. For example, Evenson, Dey, and Hossain recommend research portfolios for different regions and agroecosystems.10 The portfolios described here and in similar studies, however, are based on equalizing net benefits per dollar of funding across research programs. But note that research programs involve uncertain outcomes. Some programs have low probabilities of success, but high payoffs. Others have high probabilities of success, but relatively modest payoffs. Research planners can usefully approach their task as a portfolio selection problem. Like stock market investors who choose a portfolio of investments to trade off risk against return, research planners should seek a balanced portfolio of safe and risky investments. For example, maintenance breeding for disease and pest resistance is generally low risk but has a low return. The search for drought tolerance, by contrast, is high risk but has a high return. In the economic literature, it is often argued that public actors can ignore the riskiness of investment projects because individual projects are small relative to the size of the state

sector and relative to the economy as a whole. For a research manager, however, the riskiness of a project is highly relevant. Few research programs can afford to gamble everything on a low probability, high payoff project. The Rockefeller Foundation’s investment in rice biotechnology was probably appropriate for a private foundation with limited accountability to legislators or the general public. A public research institution, by contrast, may need to invest in a less ambitious portfolio of projects, diversified across commodities and research techniques. This problem seems not to have been explored as extensively as might be warranted. For most institutions, priority setting and portfolio selection need to be based on some explicit treatment of risk.

Pursuing Comparative Advantage Just because a particular research program is worthwhile does not imply that a research organization should undertake it. There are many worthwhile programs, and no single organization should undertake them all. Instead, it makes sense for particular research organizations to focus on the activities in which they have a comparative advantage. This means simply that research organizations should specialize in the activities at which they are, relatively speaking, the best. Other research products may be borrowed or copied from other organizations or other countries. Herdt (p. 398)16 makes this point explicitly and notes that “different research organizations have different responsibilities.” It does not make sense to conduct a priority-setting exercise without thinking critically about the comparative advantages of the particular research institutions in question. To continue with the example of the rice biotechnology program, it would be foolish for every rice research establishment to imagine that it faced the same priorities as the Rockefeller Foundation. Different research organizations should specialize in different types of research. To some extent, considerations of comparative advantage can be pulled into the priority-setting process by incorporating an appropriate score into measures of the likelihood of achieving success. Even so, the

Setting Priorities for Agricultural Research: Theory and Experience priority-setting process cannot substitute for careful reflection by research administrators. These administrators need to think clearly about what their organizations can best accomplish themselves, and what can be borrowed from elsewhere. The possibilities for borrowing are discussed in the next section.

Taking Advantage of Research Spillovers In many cases, agricultural researchers can benefit from “spillovers”originating in research conducted in other crops, other countries, or other areas of science. Biotechnology offers some immediate examples. The pace of biotechnology research has been rapid over the past decade, and repeated breakthroughs have taken place in techniques, tools, and scientific understandings. Although agricultural scientists have been at the forefront of some advances, it is not clear that agriculture can or should lead in the development of upstream biotechnologies. Possibly it makes greater sense for the agricultural sciences to borrow tools and techniques that are developed for other purposes. Similarly, there may be some RPAs where progress can be made effectively by borrowing from work done in other fields. An example might be research on animal diseases such as trypanosomiasis, a disease that affects both humans and animals. If researchers are currently busy on a trypanosomiasis vaccine for humans, it might be foolish to allocate research resources to the development of a vaccine for cattle. Once a human vaccine is available, it would presumably not take much time to develop a comparable vaccine for livestock. The existence of spillovers may imply that it is optimal to delay certain research programs or to ignore others altogether, perhaps while devoting added resources to programs that are unlikely to benefit from spillovers. This issue is discussed in more detail below.

Realizing the Benefits of Delay Just because a research program is justified does not imply that it needs to be begun today. In some cases, it will be preferable to delay the start of a research program. By delaying, the researchers incur a cost, namely, the expected delay in payoffs from the research

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program. But there are benefits as well, since the research funds are available for other uses. A key question is how much it will delay the payoffs from a particular RPA to postpone the initiation of the project by one year, for example. Given the rapid improvement in biotechnologies—and especially in tools and techniques—it would seem to make sense for researchers to postpone many research projects with the expectation that projects deemed “difficult” today might become very tractable in a few years. The effort exerted today may prove in a few years to be wasted, if new techniques allow researchers to find shortcuts. A relevant question for the Rockefeller Foundation rice biotechnology program is whether there would have been any great cost associated with starting the program five years later. If the research had begun in 1998 instead of in 1988, how long would it have taken to “catch up”? Would the costs of delay have been outweighed by the benefits of having extra research funds to use for a decade?

Conclusion How useful is it, ultimately, to apply economic concepts to research planning? If priority-setting exercises are costly and time-consuming, are they worthwhile? Do they increase research output sufficiently to justify their costs? Ruttan notes that agricultural research typically displays very high rates of return in ex post evaluations. Given these returns, he asks whether the resources devoted to priority setting would be better allocated to research.1 These are difficult questions. Even “unimportant” research may generate some benefits, and it seems to be true empirically that “more research is better.” But the real cost of misallocating research funds is that the best projects will be underfunded. Suppose that priority setting allows research administrators to transfer funds from the least productive project in their portfolio to the most productive. This is a direct gain. Given the magnitudes of the benefits that emerge from agricultural research, it might not take many transfers of this type to pay for the costs of priority-setting studies.

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Whether or not a research organization undertakes formal priority-setting activities, the concepts embedded in priority setting are useful. Even if the priority-setting process is an informal one, with no data collection and no survey of expert opinion, it can provide a useful discipline for resource allocation. Research planners need to ask at every juncture where the expected payoffs from research are greatest. They should consider the demand for research—driven by the importance of different crops and commodities—and also the supply. At the dawn of a new century, with rapidly changing technologies, with new and different research needs, and with research funds ever tighter, it will be vital to manage research effectively. Priority setting can play a useful role in pointing out critical areas for agricultural research.

Ackowledgments I am grateful to the organizers of the 12th Toyota Conference for their interest in this research. I also acknowledge the helpfulness of Robert W. Herdt of the Rockefeller Foundation program on agricultural sciences, who made available his insightful and thought-provoking papers in this area. George W. Norton of Virginia Tech also provided a number of relevant and useful papers. Finally, I am indebted to Robert E. Evenson of Yale University for his profound influence on my thinking about agricultural research strategies and the economics of priority setting for research, and to the staff of the CIMMYT Economics Program for stimulating my thinking in this area.

References 1. Ruttan VW. Agricultural Research Policy. Minneapolis: University of Minnesota Press, 1982. 2. Fishel WL, ed. Resource Allocation in Agricultural Research. Minneapolis: University of Minnesota Press, 1971. 3. Alston JM, Norton GW, Pardey P. Science Under Scarcity: Principles and Practice for Research Evaluation and Priority Setting. Ithaca, NY: Cornell University Press, 1995.

4. Evenson RE, Herdt RW, Hossain M, eds. Rice Research in Asia: Progress and Priorities. Wallingford, UK: CAB International, 1996. 5. Barker R. Methods for setting agricultural research priorities: Report of a Bellagio conference. Cornell University Working Papers in Agricultural Economics No. 88-3. Ithaca, NY: Department of Agricultural Economics, 1988. 6. Evenson RE. Priority-setting methods. In: Evenson RE, Herdt RW, Hossain M eds. Rice Research in Asia: Progress and Priorities. Wallingford, UK: CAB International, 1996; 91-108. 7. Mills BF, Karanja DD. Processes and methods for research programme priority setting: The experience of the Kenya Agricultural Research Institute Wheat Programme. Food Policy 1997; 22(1):63-79. 8. Mills BF. Ex-ante agricultural research evaluation with site specific technology generation: The case of sorghum in Kenya. Agricul Econo 1997; 16:125-138. 9. Mutangadura G, Norton GW. Analysis of strategic research priorities in the department of research and specialist services. Manuscript, Department of Agricultural and Applied Economics, Virginia Polytechnic Institute and State University, Blacksburg, VA. 10. Evenson RE, Dey MM, Hossain M. Rice research priorities: An application. In: Evenson RE, Herdt RW, Hossain M, eds. Rice Research in Asia: Progress and Priorities. Wallingford: CAB International, 1996; 347-392. 11. Evenson RE. An application of prioritysetting methods to the rice biotechnology program. In: Evenson RE, Herdt RW, Hossain M, eds. Rice Research in Asia: Progress and Priorities. Wallingford: CAB International, 1996; 327-346. 12. Herdt RW. Equity considerations in setting priorities for Third World rice biotechnology research. Development: Seeds of Change. 1987; 4:19-24. 13. Herdt RW. Agricultural biotechnology in the 21st Century. Paper presented at the NABC 9 Resource Management in Challenged Environments Meeting, June 1-3 1998, Saskatoon, Canada.

Setting Priorities for Agricultural Research: Theory and Experience 14. Herdt RW. Research priorities for rice biotechnology. In: Khush GS, Toenniessen GH, eds. Rice Biotechnology. Wallingford, UK: CAB International, 1991:19-54. 15. Herdt RW. The potential role of biotechnology in solving food production and environmental problems in developing countries. In American Society of Agronomy Special Publication no. 60, Bridging Food Production and Environmental Protection in Developing Countries. Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, 1995:33-54.

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16. Herdt RW. Summary, conclusions and implications. In: Evenson RE, Herdt RW, Hossain M, eds. Rice Research in Asia: Progress and Priorities. Wallingford, UK: CAB International, 1996:393-406.

CHAPTER 5

Sustainable Agriculture and Strategies in Rice Breeding H. Ikehashi

T

wo topics are taken up on the basis of the author’s experience in rice research. The first is a renewed understanding of rice farming as a type of sustainable agriculture, a summary of many preceding views. The second is a discussion of strategies in rice breeding. In this part some of the author’s immediate experiences are cited.

Rice Cultivation as a Model for Sustainable Agriculture Ecological Stability of Irrigated Rice Farming Among prevailing agricultural systems, rice cultivation is predominant for densely populated areas with the Monsoon climate. Rice is a unique crop for flooded soils. Submergence of soil makes it possible to cultivate rice every year without any fallow land, because many pathogenic fungi do not survive the anaerobic condition. Light power is enough for cultivation. Because the weight of the soil block is decreased in water, a single cattle power, usually a water buffalo, can easily plow and paddle the soil, which is otherwise very heavy. Levees keep irrigation water in terraced farms; thus, rice cultivation is protected against soil erosion. Electrochemical changes that occur in submerged soils were earlier discussed in detail by Ponnamperuma.1 According to his paper, in normal tropical soils a set of soil conditions are achieved by submergence, where availability of nitrogen, phosphorus, potassium, calcium, magnesium, iron, manganese and silicon is high, while the supply of copper, zinc and

molybdenum is adequate. The decrease in redox potential (Eh) increases pH of acid soils and decreases pH of alkaline soils. Soil fertility can be conserved better under anaerobic conditions, because ammonium in soil from crop residues or other organic matter is not easily oxidized into nitrate compounds which are carried away by water or volatilized into air. Mineral nutrition for rice is also supplied through irrigation water. This is the reason for the traditional no-input rice farming, which consistently yields about 1.5-2.0 tons per hectare (t/ha). Nitrogen can be supplied through fixation by algae and other microorganisms. Weeds are controlled by irrigation, because many kinds are not adapted to submergence. No other crop is planted to flooded soil except rice. Besides, there are some additional merits in irrigated rice farming. Cattle can be fed with the weeds on levees. Fish culture in canals or swamps provides protein resources. The nutritional balance of rice proteins is one of the best among the staple cereals. Farms in arid regions are desalinized by a regular rotation of rice planting. For introduction of rice cultivation to new areas where rice has not been cultivated, there are a set of problems. Supply of water is a limiting factor. Accumulation of salts can be a problem in arid areas, where salts are carried from underground to the surface and water evaporates, leaving salts. The advantages of rice farming can be better understood in contrast with other farming systems like those in Europe, which

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

are characterized by the need of fallow land. No crop can be planted in consecutive years in a field plot, due to disease buildup; therefore, fallow land, pasture or rotation of crops is essential. In European types of agriculture, a major part of the nutritional proteins are supplied through animal husbandry, which requires forage crops. Uptake of proteins through animal products is estimated to require seven times more land area than relying on proteins of plant products. There is also potential soil erosion through overgrazing and rainfall. Similar problems can be seen in dryland farming, and also in nomad agriculture. In conclusion, the ecological stability of rice farming is excellent, being comparable only to some plantation agriculture of perennial plants such as tea, coconut, oil palm, rubber tree, cacao bean and so forth. Rice farming will be one of the best models for agricultural systems in the future.

Socio-Economic Aspects of Traditional Agricultural Systems in Asia Stability of farming through each production cycle has been guaranteed in rice farming. But, this system has been a subsistence farming without a marginal surplus that could have been invested for other local industries. Feudalistic land systems used to abide with the farming system. Villages provide a pool of landless farm laborers and unemployed laborers from city areas. There has been always a kind of vicious cycle: Without local industries people flow into cities to get employment; for them, food prices should be kept at a minimum; but, then there is little opportunity for villages to develop new industries. The green revolution was one of the attempts to improve such stagnant rural structures in Asia.

Green Revolution Its Start and Impacts After World War II most of the Asian countries attained independence from colonialism. At the same time, the limit for extension of farming was evident, due to population increase, particularly in Indonesia, the Philippines and Thailand. In the pre-green revolution years,

there were some experiences in varietal improvement in many countries in Asia. There was a strong motivation for intensive agriculture following the model of the countries in the Far East, where land reform was successful and recovered heavy industries were able to provide sufficient chemical fertilizers. Under such situations, international collaborative approaches were initiated for attaining higher yields of rice. The first model of a high yielding variety, Taichung Native 1 and several similar varieties were entered in cooperative trials sponsored by The International Rice Committee(IRC) of the Food and Agriculture Organization (FAO) for 1961-1963 in several countries. 2 The first crosses using such varieties were made in 1962 at the International Rice Research Institute (IRRI) in the Philippines. International collaborative approaches by scientists became easier by development of transport and communication. A new plant type of rice was identified in such a network of international testing. Then, the new plant type was further improved through breeding programs into the release of IR 8 from IRRI in 1967. The improved type was characterized by a single gene for semi-dwarfism, sd-1, which is a basis for the short stature and improved response to increased fertilizer application. This type performed best with a combination of increased fertilizers under irrigation. Similar approaches were adopted in other crops like wheat. The new technologies were adopted through the 1970s, and led to self-sufficiency of rice in chronically deficient areas. New areas for rice cultivation were explored, because sufficient return on investments for irrigation and related infrastructures was predictable. Research at national centers was also strongly supported. The intensified rice farming was further developed in Japan, Taiwan, Korea, Egypt and China.

Associated Breeding Works with the Green Revolution To stabilize the initial success, there were some immediate tasks in intensive farming, as well as new attempts to improve rice in marginal areas.

Sustainable Agriculture and Strategies in Rice Breeding First, breeding for resistance to diseases and insect pests was urgent, because the adoption of intensive rice farming, particularly of rice cultivation in the dry season, provoked an outbreak of pests and diseases which had been only minor problems in traditional systems. Breeding for resistance has been successful in wide areas, and by and large it has protected the gains from the improved plant type. Second, some resources were allocated to genetic improvement of varieties in marginal areas, which the green revolution had bypassed due to deep water, adverse soils or drought. The author was once assigned to such areas of breeding as tolerance to adverse soils, resistance to blast disease and deep water rice. But progress in these areas was not significant due to limited time, lack of scientific means and social structures, which can still be seen in the northeastern states of India. Often, the target environments are too variable to set any clear focus. Any gain expected is assumed to be marginal, even if some success is achieved. Third, improvement of grain quality is another area where steady progress has been attained. But there is a tendency for the market pressure for high quality to constrain breeders’ effort toward a higher level of yield. A decline in productivity of rice is indicated in some areas of intensive rice production. The reason for this still remains for scientific analysis. A part of this decline may be attributed to the grain quality issue. For instance, Basmati 370 and Khaw dawk mali 105, reputed varieties for high market price but with low yielding capacity, have extensively been planted to wide areas in Myanmar, Thailand and other countires.

Problems and Tasks in the Post Green Revolution Era New Problems Which Were Outside of Targets of the Green Revolution As mentioned above, the technological plateau of yield has been attained, with an emphasis on grain quality. Budget cuts have been serious for cereal production and related research. Concerns for ecological stability of agriculture and rice cultivation have been raised in light of a new concept for evaluating agricultural systems, the issue of Low Input

65

Sustainable Agriculture (LISA). The emission of methane and ‘green house effects’, as well as salt accumulation in arid regions, are indicated as adverse factors from rice cultivation, although these aspects are not adequately studied. Fear of pollution by spraying insecticides, fungicides or herbicides has led a series of new ideas, such as organic farming, to the forefront.

Socio-Economic Aspects In the post-green revolution era, a new series of socioeconomic problems have emerged with self-sufficiency of rice . The new trend of industrialization caused domestic competition for land use, labor, etc. Fragile infrastructures for grain storage, transportation and sales became clear in rural industries. There has been criticism from the point of social equity to the outcomes of the green revolution. There are also clashes between subsistence farming versus profit-seeking international agricultural business under the GATT (WTO) agreement. Fledging industries, mature societies with family farming, and workers in the middle classes are likely to be affected as well as small farmers. Those are far beyond the scope of any review by a rice breeder.

Strategies for Enhanced Yield Level Prediction of Supply and Demand While self-sufficiency in rice has been achieved in major rice-producing countries in Asia, the demand for rice is estimated to be increasing beyond the capacity of production. According to an estimate of attainable rice yield, out of eight countries surveyed, only Thailand and Myanmar will be in a comfortable position for meeting the rice needs of their populations. 3 Vietnam and India will be in a tight situation, even if they can exploit the full potential of the technologies. China, Indonesia, the Philippines and Bangladesh are likely to face severe shortages, unless there is further investment in transforming the rice area from unfavorable to favorable ecosystems and technological breakthroughs. Substantial yield loss is predicted due to various technical constraints.3 For the irrigated ecosystem, yield loss due to all technical

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

constraints is 962 kg/ha, i.e., about 20%. For rain-fed lowland and flood-prone ecosystems, such a loss is estimated to be about 33% and 40%, respectively. Yield loss due to submergence, drought and cold is estimated to be 20% and 28 %, respectively, for upland and flood-prone ecosystems. Yield loss due to insects and diseases is also predicted to be severe, particularly in the rain-fed ecosystem.

Strategies in Rice Breeding Improvement for yield level will still be the target of first priority in the coming century. According to the report of IRC of FAO in 1994, the yield of modern varieties has become stagnant. Hybrid rice is the only technology presently available to overstep these yield barriers. 4 Having been in a position to review the hybrid rice technologies, I would like to discuss the potential of this technology. Doubtlessly, hybrid rice using cytoplasmic male sterility(CMS) is one of the most significant achievements after the introduction of semi-dwarf high yielding varieties. The yield increase is estimated to be 15-20 % over the ordinary varieties. It is reported that this hybrid rice covered nearly half of the total rice areas in China. But the initial gains by hybrid rice have not been improved in China, perhaps due to the difficulty in breeding stable CMS lines and shifted emphasis on grain quality. Since the 1990s hybrid rice breeding has been one of the first priority programs in India, where hybrid rice is being increased from the initial adoption of 50,000 ha in the mid-1990s. What is interesting in hybrid rice breeding is a series of unique innovations in the technology. After the success of the CMS system, hybrid seed production along the idea of two line hybrids has been a fascinating target. With the use of environment-dependent genic male sterility (EGMS), fertile plants can be propagated by self-pollination under one set of conditions, while the same genotype can be male sterile and be hybridized with any other variety. When the new type of hybrid was first proposed in China, few scientists were confident in it. But it is now a reality showing further yield increase. Hybrid rice technology will be improved further by the further study of EGMS.

Another idea is to overcome hybrid sterility between different groups of rice varieties (Table 5.1). Partial sterility is commonly found in the panicles of F1 hybrids between Indica and Japonica groups in rice. It is known as a barrier in the use of pronounced heterosis of Indica-Japonica hybrids. I started a genetic study in the early 1980s, and found that the panicle sterility in Indica-Japonica hybrids is caused by an allelic interaction at locus S-5 on chromosome 6, where Indica and Japonica varieties have S-5i and S-5j, respectively (Fig. 5.1.) The heterozygote S-5i/S-5j produces semisterile panicles because of the partial abortion of the female gametes carrying S-5j. Some varieties such as Ketan Nangka (KN) and Dular, have a neutral allele S-5n, and the genotype S-5j/S-5n and S-5j/S-5n produce fertile panicles. S-5n is called the wide compatibility gene (WCG), and has been incorporated into Indica or Japonica varieties to overcome the sterility in Indica-Japonica hybrids. 5 In the past decade, several Indica-Japonica hybrids which have the neutral allele S-5n have been developed to determine the yield potential in China. Such hybrids showed strong heterosis, but their seed set were unstable under some environments. One way to solve the problem is to use Javanica varieties instead of Japonica. This idea has been utilized for the new generation of hybrids, inter-subspecific two line hybrids, which showed increased yield and were planted to 0.7 million ha in 1996 in China. An ultimate technology in hybrid rice breeding will be the use of apomixis, which functions in some plant species to produce genetically the same progeny via seed. In this way hybrid plants may produce hybrid seed without any artificial crossing. So far there is no basis on which further progress can be seen in rice breeding.

A New Plant Type Yield gains of 25% is envisaged by improving plant type on another front. Promising new lines with big panicles and a few thick stems are being developed at IRRI.6 So far the performance of the new types are not yet widely available. This approach reminds us of the project for ‘super high-yielding rice’ in the 1980s in Japan. A series of high-yielding lines were actually produced through this project.

Sustainable Agriculture and Strategies in Rice Breeding

67

Table 5.1. Loci for hybrid sterility Locus

Chromosome

Marker genes in order

Crosses

S-5

6

C, S-5, Amp-3; Est-2, Pgi-2, RG213, alk

indica x japonica

S-7

7

Rc, S-7, Est-9, rfs, ga-11, Acp-4

Aus x javanica

S-8

6

Cat-1, Pox-5, S-8

IR2061-481 x javanica

S-9

4

Ph, 1g, Mal-1, Est-1, S-9

Aus x javanica

S-15

12

Acp-1. Pox-2, S-15, Sdh-1

IR2061-628 x Dular (Aus)

S-16

1

Est-5

China Native Rice x javanica

S-17(t)

12

Pox-2, S-15, Sdh-1, S-17(t)

P.B. II x japonica

Revised from Wan and Ikehashi 1996.16

In any breeding program, if the yield level is emphasized by ignoring other traits, there will be potential for a higher level of yield.

Extension of Elite Breeding Lines Through International Networks A more routine approach, though no less important than those mentioned above, is systematic exchange and tests of promising lines through an international network (INGER). Further support to such a system is important. The preceding program, the international rice testing program, had enormously promoted exchanges of better lines, as well as experience among breeders in each of the national programs. Such a program, if supported adequately, would contribute significantly to food security.

Application of Molecular Biology to Rice Breeding For those plant breeders who were assigned, with conventional tools, in the 1970s to some areas of rice breeding such as tolerance to adverse soils and host resistance to blast disease, the set of new tools based on molecular

biology seems to be powerful and attractive. The following are some new challenges from our laboratory to such tasks with a renewed set of weapons.

Development of Molecular Markers in the Breeding for Tolerance Of environmental stresses in rice production, tolerance to excess of soluble iron is known to show a clear genetic difference. The severest abiotic stress in the lowlands is iron toxicity in west Africa’s lateritic soils. It has been shown that genetic tolerance to iron toxicity can contribute significantly to rice production in toxic soils. 6 We started a program to search for genetic markers for this tolerance. Initially, the testing method had to be improved. An improved screening method was developed on the basis of solution culture. Then, a search for RAPD markers which might be linked to the tolerance was started. To obtain stable PCR products as selection markers, the sequence of RAPD markers had to be determined as site-tagged sequences (STS), for which a set of new primers could be developed. This approach enabled us to identify, with a

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 5.1. Two types of allelic interaction at a hybrid sterility gene locus Si gametes are abortion in the sporophyte genetype Si/Si. high level of precision and reproducibility, a monogenic segregation in the tolerance, and can be a standard in any selection for highly variable tolerance to environmental stress.

Search for Antifungal Proteins as New Resources of Host Resistance Incorporation of exotic resistance genes had been a major means in the breeding to rice blast (Pyricularia oryzae) resistance. But such new resistances break down sooner or later.

Sustainable Agriculture and Strategies in Rice Breeding

69

Table 5.2. Pathogenesis-related proteins found in floral organs Proteins

Plants

Organs

Reference

RNAses

Rosaceae

Pistil

Sassa et al 1993.8

PR-1

Camellia

Pistil

Tomimoto et al 1999.13

Chitinase

Rice

Husk

Nakazaki et al 1997.12

Chitinase

Rice

Pistil

Takei et al 1998.14

Thaumatin

Rosaceae

Pistil

Sassa et al 1998.15

In early 1990s, we have initiated a minor study to identify self-incompatibility genes of fruit trees in Rosaceae, and identified a series of pistil-specific RNAses which corresponded to the self-incompatibility genes.8 That work suggested that plants may contain a variety of proteins against disease infection, and that such proteins may be utilized in breeding for host resistance. The proteins for self-incompatibility might be recruited from one of the antifungal proteins in floral organs, which seem to be most vulnerable to fungal infection.9 In the light of recent work to isolate host resistance genes of tomato and rice against bacterial diseases, the host resistance genes seem to function in signal transduction and do not seem to be antifungal agents for invading organisms. At the end of such responses there seem to be a set of proteins which are called pathogenesis-related proteins (PR proteins). They are initially defined as those induced by viral or fungal infection or by some chemicals like salicylic acid and benzothiadiazole.10 They can be involved as antiviral or antifungal agents in a systemic acquired resistance, because such proteins directly inhibit propagation of pathogens. 11 What, then, can be seen if such proteins are incorporated and constitutively expressed in plants ? In fact, the antifungal function of chitinases and PR-1 have been proved by gene transformation or in vitro testing. In our preliminary search for PR-like proteins, some genes which encode novel PR-1 or chitinases were isolated (Table 5.2).12,13 They are basic types and endogenously induced

in the course of development. So, their functions may be different from those expressed by other PR-l proteins, but the isolation of such types of PR proteins, and their subsequent incorporation into rice plants, are expected to reveal a new aspect of host resistance.

References 1. Ponnamperuma FN. Electrochemical changes in submerged soils and the growth of rice. In: Soil and Rice. Los Banos: International Rice Research Institute, 1978:421-441. 2. Dalrymple DG. Development and spread of high-yielding rice varieties in developing countries. Bureau for Science and Technology. Washington DC: US AID, 1986. 3. Rice Almanac 2nd ed. Rice around World. Los Banos: International Rice Research Institute and Cali, Colombia: Centro International de Agricultura Tropical, 1997. 4. FAO. Report of the international rice commision, 18 th session, FAO, Rome, Italy, 5-9 September. Rome: FAO, 1994. 5. Ikehashi H. Genetics of hybrid sterility in wide hybridization in rice (Oryza sativa L.) In: Bajaj I, ed. Biotechnology in Agriculture 14. Rice. Berlin: Springer Verlag, 1991:113-127. 6. Khush GS. Breaking the yield frontier of rice. Geo J 35. 1995; 3:329-332. 7. WARDA. Annual Report 1977. Bouake, Ivory Coast: West Africa Rice Development Association , 1998.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

8. Sassa H, Hirano H, Ikehashi, H. Identification and characterization of stylar glycoproteins associated with self-incomaptibility genes of Japanese pear, Pyrus serotina Rehd. Mol Gen Genet 1993; 241:17-25. 9. Dickinson H. Self-pollination simply a social disease. Nature 1994; 367:517-518. 10. Van Loon L C, Pierpoint WS, Boller Th et al. Recommendation for naming plant pathogenesis-related proteins. Plant Mol Biol Rep 1994; 12:245-264. 11. Friedlich L, Lawton K, Ruess W et al. A benzothiadiazole derivative induces systemic acquired resistance in tobaco. Plant J 1997; 10(1):61-70. 12. Nakazaki T, Tomimoto Y, Ikehashi H et al. A new chitinase in rice detected from husk proteins and its gene locus. Breeding Science 1997; 47:363-369.

13. Tomimoto Y, Ikehashi H, Kakeda K et al. A pistil-specific PR-1 like protein of Camellia, its expression, sequence and genealogical position. Breed Sci 1999; 49:97-104 14. Takei N, Nakazaki T, Tsuchiya T et al. Isolation of a pistil-specific chitinase gene in rice (Oryza sativa L.). Breed Sci 1998; 48 (suppl. 1):281. 15. Sassa H, Hirano H. Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR-5 like protein in Japanese pear (Pyrus serotina Rehd.). Planta 1998; 205:514-521. 16. Wan J, Ikehashi H List of hybrid sterility gene loci (HSGLi) in cultivated rice (Oryza sativa L.). Rice Genetics Newsletter 1996; 13:110-114. 17. Nakazaki T, Ikehashi H. Genomic sequence and polymorphisms of a rice chitinase gene. Breed Sci 1998; 48:371-376.

SECTION III INTRODUCTION

The Present Situation of Biological Production and the Approach to the Sustainable Production in Arid Lands Satoshi Matsumoto

M

ore than 60% of the land surface of the earth consists of arid and semi-arid soils, which are generally too dry to produce a good yield. If enough fresh water is available and the soil conditions are suitable, these soils can be irrigated and used for agricultural lands. These regions, therefore, have been expected to be the new promising land resources for food production with the progress of agricultural technologies. From this background, much attention has been paid to arid and semiarid soils, and more agricultural projects are being planned and executed. However, many projects have failed in the past and even now, because some years after irrigation the salinity or alkali hazard increases. According to the estimates of FAO and UNESCO, as much as half of all the existing irrigation agricultural farms of the world are more or less under the influence of secondary salinization, alkalinization and waterlogging. They also report that 10 million hectares of irrigated land are abandoned yearly as a consequence of the adverse effects of irrigation, mainly secondary salinization and alkalinization. This phenomenon is very common not only in old irrigation systems but also in areas where irrigation has only recently been introduced. In addition,

the recent prolonged droughts or unstable weather in arid regions is wreaking fatal damage on dry farming lands, leading to the main cause of desertification. It is debatable whether sustainable agriculture can be established in areas which are saline or alkaline, or which are potentially saline or alkaline. Aiming for sustainable agriculture in dry regions, on the other hand, many technologies for improved biological productivity in arid and semi-arid lands have been developed. They include ground water management, soil and water management, crop and resource management, and soil amelioration. However, since we postulate sustainability in dry land agriculture, we should keep an economical background in technology. In this session, we will focus on the possibilities of sustainable production in arid regions for the coming century as well as discuss, from several aspects which include the influence of climate variability on productivity, the fundamental technologies for water and soil management, the actual management for crop and resource in dry land farming, and the reasonable production of soil amendment in alkali soil.

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CHAPTER 6

Drylands and Global Change: Rainfall Variability and Sustainable Rangeland Production J.F. Reynolds, R.J. Fernández and P.R. Kemp

Introduction

O

ver a third of the land surface of the earth is composed of drylands and much of this (about 65-70 %) is seriously degraded or desertified. The two major desertification drivers—climate change and human activities— have ecological and social impacts on various temporal and spatial scales. These impacts include potential alterations of carbon, water, and trace gas budgets, loss of vegetation cover, and increased wind-borne dust, all of which may affect global biogeochemistry, radiation balance, and climate. The societal consequences of land degradation are also serious, since the fate of rural people in drylands is dependent on the effective use of natural resources, e.g., water, soils, plants, livestock and wildlife. Here we discuss how ecosystem-level predictions may be used to address issues relevant to sustainable development of these semi-arid regions. We argue that the next step in assessing sustainability is to incorporate ecological impacts into higher level models that consider direct and other human impacts on these systems. This will require further testing and evaluation of ecosystem-level models in the context of different management and land-use alternatives. We propose the incorporation of both “natural” and human factors into a spatially explicit model of landscape elements and human land-use patterns in order to develop predictive tools capable of dynamic, integrated assessments of impacts of

global climate change on human-dominated ecosystems.

Drylands and Global Change The purpose of this chapter is threefold. First, we review the extent of global change in drylands and some of its ecological and societal consequences. Second, we discuss one component of potential climate change—rainfall variability—and how it affects primary production in rangelands. We illustrate this with a case study of a rangeland in southern New Mexico using a physiologically-based ecosystem model. Lastly, we suggest that basic ecological knowledge, such as presented in our case study, should be an essential component of assessment frameworks. Doing so will not only improve assessments, but will increase its usefulness to scientists, land managers and policy makers.

Land Degradation and Desertification Arid and semi-arid drylands compose one third of the land surface of the world and are home to about 20% of the human population.1 The vast majority of these drylands consist of rangelands (~88%), whereas the rest are classified as rainfed (3%) and irrigated croplands (9%) (Table 6.1). The rapid growth of populations in many of these regions, often in conjunction with imprudent land

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

management, has led to increased social vulnerability and to rapid land degradation. Soils in drylands are especially vulnerable to wind and water erosion, loss of organic matter, decline in fertility, salinization, and compaction. This is particularly evident when the natural vegetative cover is reduced through human activities such as intensive livestock grazing, excessive cultivation, urbanization, and other land uses. While global rates of land degradation and the total areas affected are difficult to estimate,2,3 there is ample evidence that extensive areas of the world’s drylands have experienced some form of chronic degradation during the last century. It is estimated4,5 that approximately 80% of the world’s rangelands, 60% of rainfed croplands, and 30% of irrigated croplands are threatened by various types of degradation, generally referred to as “desertification.” Thus, desertified drylands make up about 65-70% of the total dryland area of the globe (arid, semiarid, and dry subhumid regions, but excluding hyperarid regions).6 All three of the developing regions of the world—Africa, Asia, and Latin America—have similar percentages of land degradation (Table 6.1). The two major desertification drivers— climate change and human activities—have ecological and social impacts at various temporal and spatial scales, ranging from local (and short term) to global (and long term) (Fig. 6.1). Carbon, water, and trace gas budgets may be significantly altered; losses of vegetation may modify regional albedo, raise air temperatures, and increase wind-borne dust; all of these changes have the potential to act in concert to affect global biogeochemistry, radiation balance, and climate. The societal consequences of land degradation are equally serious. The fate of rural people in drylands is dependent on the effective use of natural resources, e.g., water, soils, plants, livestock and wildlife. In spite of this, over large areas natural vegetation continues to be degraded, soils are eroding, and the capacity of the land to support livestock and wild herbivores is being reduced. Combined with complex political, social, and economic factors, which often tend to have equally important roles, adverse human impacts are inevitable.1

Sustainability Natural, semi-natural, and intensively managed dryland ecosystems of the world offer a wide range of different goods and services vital to human populations. The list is extensive and includes food production (humans and livestock), construction materials, climate regulation, soil maintenance, nutrient recycling, wildlife habitat, erosion control, tourism/recreation, and aesthetic enjoyment.7 When weighing the advantages and disadvantages of some particular course of action that affects these ecosystems—and hence their “sustainability”—decision-makers need quantitative assessments in order to consider these goods and services. While it is seldom possible (or desirable) to exercise complete control over a landscape, it may be possible to exercise different management regimes on parts of the landscape, and in so doing maintain a disproportionate set of ecosystem services. For example, some services (e.g., plant production) are highly dependent on “key” landscape units such as source zones for water and sedimentation, areas of reserve forage for herbivores, and fertile patch mosaics.8 Understanding the interplay between ecosystem services and ecosystem functioning and structure has urgent application to land use planning and management in dryland areas. These are examples of what constitutes the knowledge base necessary to achieve environmental sustainability. Goodland and Daly9 identify three types of sustainability—social, economic and environmental. While there are obvious linkages and overlaps, Goodland and Daly argue that their true meanings are obvious only when considered separately (Table 6.2). In the context of global change and its potential impacts on drylands, it is inconceivable that one type of sustainability could be realized in the absence of the others since the interdependencies are so strong. While the focus in this paper is on the environmental aspect of sustainability, we believe that its usefulness as a concept is limited without consideration of social and economic issues.

92 1.9 11.9 20.9 8.4

Asia

Australia

Europe (Spain)

North America

South America 43.15

1.42

5.86

1.91

0.25

31.81

1.9

Degraded

30%

17%

28%

16%

13%

35%

18%

(%)

457.7

21.4

74.2

22.1

42.2

218.2

79.8

Total

215.6

6.6

11.6

11.9

14.3

122.3

48.9

Degraded

Rainfed cropland

47%

31%

16%

54%

34%

56%

61%

(%)

4,556.4

390.9

483.1

111.6

657.2

1,571.2

1,342.4

Total

3,333.5

297.8

411.2

80.5

361.4

1,187.6

995.1

Degraded

Rangelands

73%

76%

85%

72%

55%

76%

74%

(%)

The area most affected in any individual country is a result of a combination of factors, including population density, climate and land-use history. In the United States, desertification is best exemplified in rangelands of the arid and semi-arid southwest.29 In Latin America, the majority of degraded land is found in highland pastures and grasslands of Argentina, Bolivia, Peru, Ecuador, and Colombia, the central basin of Chile, the northeastern region of Brazil, and in the central plateau of Mexico.30 In Asia, China is the dominant country that must contend with desertification, and the greatest concentration of degraded land is found in the northwestern, northern and northeastern regions.31 Three distinct regions of Africa are at most risk: Mediterranean Africa, the Sudano-Sahelian region, and the Kalahari-Namib region in southern Africa.32 Compiled from Grainger,2 Kassas,22 and Lopez-Ocaña.4

145.5

10.4

Africa

Total

Total

Continent

Irrigated cropland

Table 6.1. Amounts (millions of hectares) and percentages of irrigated cropland, rainfed cropland, and rangeland degraded in the world

Drylands and Global Change: Rainfall Variability and Sustainable Rangeland Production 75

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 6.1. Conceptual model linking changes in ecosystem properties during desertification to changes in global biogeochemistry. Reprinted with permission from Schlesinger et al,28 ©American Association for the Advancement of Science.

Climate Variability Long- vs. Short-Term Impacts Drylands are particularly vulnerable to climate variability, of which precipitation is the most important component. For example, a slight shift in seasonal precipitation and/or frequency of extreme rain events could potentially lead to overexploitation of the meager resources of drylands and contribute to further degradation of the very resource base on which human populations are so dependent. Preliminary studies with general circulation models (GCMs) projected that a doubling of atmospheric carbon dioxide (due to the rapidly expanding human population and associated activities) would result in lower precipitation, as well as shifts in the timing and frequency of rains, in the interior of large continents.10 Recent GCM studies also predict increases in rainfall intensity and longer dry periods in many dryland regions of the globe.11 Since nearly all drylands are characterized by extreme year-to-year weather fluctuations, it is often difficult to distinguish between short-term variability and long-term changes

in ecosystem appearance, as well as between temporary and permanent changes (Fig. 6.2).12 Short term variability tends to affect the range and frequency of “shocks,” whereas long term change alters the resource base.13 Shifts in vegetation may or may not be reversible, depending on the interactions of numerous climatic, edaphic, and biological factors. In the long run, however, global climate change may further exacerbate the already high natural variability of drylands, leading to permanent degradation of their productive potential, particularly since there is a lack of “buffering” by large reserves of organic matter in the soils or in woody vegetation.1

Rainfall Variability and Plant Production Ranchers and farmers in arid and semiarid regions of the world have long recognized the importance of short term rainfall variability on farm and livestock production, and rainfall variability continues to be the principal source of fluctuations in global food production, particularly in developing countries. For example, communally-owned Mexican

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Table 6.2. Comparison of social, economic, and environmental sustainability. Social sustainability (“-SS”-). Social cohesion, cultural identity, diversity, sodality, comity, sense of community, tolerance, humility, love, compassion, patience, forbearance, fellowship, fraternity, institutions, pluralism, commonly accepted standards of honesty, laws, discipline, etc., constitute the part of social capital that is least subject to rigorous measurement, but probably most important for SS. This “-moral capital”-, as some have called it, requires maintenance and replenishment by shared values and equal rights, and by community, religious, and cultural interaction. Without this care it will depreciate just as surely as will physical capital. SS will be achieved only by systematic community participation and strong civil society.

Economic sustainability (“-EcS”-) EcS is concerned with “-maintenance of capital,”- or keeping capital intact. Of the four forms of capital (human made, natural, social, and human), economists have scarcely been concerned at all with natural capital (e.g., intact forests, healthy air) because until relatively recently it had not been scarce. Economics also prefers to value things in monetary terms, so it is having major problems valuing natural capital—intangible, intergenerational, and especially common-access resources such as air, etc. In addition, environmental costs used to be “-externalized,”- but are now starting to be internalized through sound environmental policies and valuation techniques. Because people and irreversible impacts are at stake, economics has to use anticipation and the precautionary principle routinely, and should err on the side of caution in the face of uncertainty and risk.

Environmental sustainability (“-ES”-) ES means maintaining natural capital, akin to the definition of EcS. Although ES is needed by humans and originated because of social concerns, ES itself seeks to improve human welfare and SS by protecting the sources of raw materials used for human needs and ensuring that the sinks for human wastes are not exceeded, in order to prevent harm to humans. Humanity must learn to live within the limitations of the biological and physical environment (“-sources”-) and as a “-sink”- for wastes. This translates into holding waste emissions within the assimilative capacity of the environment without impairing it. It also means keeping harvest rates of renewables to within regeneration rates. Quasi-ES can be approached by holding depletion rates equal to the rate at which renewable substitutes can be created. From Goodland and Daly.9

rangelands were recently authorized to begin privatization in hopes of improving resource conditions and productivity. However, a recent study showed no differences between private and communal tenure systems in these ecosystems; instead, annual precipitation was still the most important factor related to rangeland conditions.14 Given the extreme variability of rainfall in drylands and the low primary production,

we might ask: Is there a relationship between rainfall variability and aboveground primary production? Le Houérou et al15 examined this relationship for a variety of vegetation types around the world and concluded that dryland ecosystems are highly variable in response to water inputs. They reported that variability in annual production was 50% greater than the corresponding variability in annual rainfall on sites receiving less than 600 mm (Fig. 6.3A).

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 6.2. Simplified model of various factors (natural, human-caused) that play a role in dryland desertification. Note that drought and desertification operate at different time scales (short vs. long term, respectively). Shifts in vegetation (grass-shrub) may or may not be reversible, depending on the interactions of numerous climatic, physical, and biological factors. However, the great diversity of sites evaluated (e.g., shrublands, grasslands, etc.) makes it difficult to understand what causes the high variability in plant production in relation to rainfall. For example, we might expect that arid shrub communities should show less variation in production than semi-arid grasslands because the former are deeper rooted and, therefore, less dependent on the current year’s precipitation than shallow-rooted grasslands. A number of factors could influence the relationship between rainfall and plant production, including:

1. Interactions between various aspects of water input, such as timing, frequency and intensity of precipitation events, and the particular requirements of different plant functional types (shrubs, grasses, forbs, etc.); 2. Topographic and edaphic characteristics of the landscape via their influence on the pattern of spatial redistribution and retention of water; and

3. Factors other than water availability, such as herbivory or nutrient limitations. We are conducting a series of field, laboratory, and modeling studies in rangelands to elucidate the relative importance of each of these explanations under different climate scenarios and management practices. Next, we present a brief case study that illustrates the importance of the first explanation—quantitatively, how do the different plant functional types respond to variation in seasonal and annual precipitation?

Case Study Our case study is based on work in the Jornada Basin of south-central New Mexico. The Jornada Basin is part of the Mexican Highlands Section of the Basin and Range Physiographic Province within the extreme northern portion of the Chihuahuan Desert. The Jornada Basin was once dominated by warm season perennial grasses (e.g., Bouteloua eriopoda), but much of the area is now dominated by shrubs (e.g.,

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Fig. 6.3. Relationship between the coefficient of variation in precipitation and the coefficient of variation of net productivity. (A) Based on data presented in Le Houérou et al15 (B) Results from PALS-FT based on three decades of precipitation (normal, wet, dry; see Fig. 6.4) and shown by functional types. Modified from Reynolds and Kemp.17

Larrea tridentata and Prosopis glandulosa). This transition—which was initiated in the late 1800s or early 1900s— is believed to have been driven by overgrazing and global change (Fig. 6.2). The Jornada Basin now contains

remnant grassland communities, and most areas are dominated by shrubs or codominated by shrubs, subshrubs, forbs, succulents, and

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grasses. The perennial grasses provide the most valuable forage for livestock in this region. To address the importance of rainfall variability and rangeland production, with particular emphasis on the relative effect on different plant functional types, we used the Patch Arid Land Simulator (PALS) developed for the Jornada. PALS is a physiologically-based ecosystem model that contains the principal components of ecosystem carbon, water, and nutrient cycles.16,17 The results presented below are from our phenology-based version (PALS-FT), which includes the key plant functional types (FT) of the northern Chihuahuan Desert—shrubs, cool season annuals, summer annuals, forbs, and perennial grasses. Further details of the vegetation of the Jornada Basin, PALS-FT, and this modeling study are given elsewhere.16-18 In the first series of simulations, we examined the potential impacts of historical variations in precipitation on productivity, on both a year to year basis and over periods associated with decade-length climate shifts. We selected three periods from the long term records at the Jornada Basin (1914-1997, average annual rainfall = 247 mm): a “normal” decade (1968-1977, average = 250 mm); a “dry” decade (1947-1956, 33% below normal or 166 mm); and a “wet” decade (1984-1993, 32% above average rainfall or 325 mm) (see Fig. 6.4). Grouping all of these years together, our model simulations show that rainfall has large impacts on simulated annual net primary production (ANPP), although the absolute magnitude varies with plant functional type (Fig. 6.5). The scatter for these simulated results is substantial, which illustrates that, while there is a general increase in productivity with increasing annual precipitation, there is also considerable variation associated with the timing of that rainfall within individual years and with the utilization of this moisture by different functional types. During the “dry” decade of 1947-1956, simulated ANPP was reduced by an average of 38%, but ANPP of the perennial grasses declined by 60%, whereas ANPP of the shrubs declined by only 25%. These results are consistent with the finding of Gibbens and Beck,19 who reported that above ground cover of the principal range grasses of the Jornada

Basin was reduced by 75% or more during this dry decade (they also speculated that this may have been a period favorable to increase in shrubs, and indeed our model simulations suggest that shrubs would have been less impacted than grasses—see Table 6.3). The reductions in simulated productivity of the seasonal annuals (50% for summer annuals and 20% for winter annuals) parallels the overall reductions of seasonal precipitation during the dry decade: Summer rainfall (July-September) was reduced by 60%, whereas winter rainfall (November-March) was reduced by about 25%. The “wet” decade of 1984-1993 (Fig. 6.4) was a period of slightly increased summer moisture (10%) and greatly increased winter rainfall (50%). However, the perennial grasses, in which most growth occurs in the summer, were the most impacted in our simulations, having a 500% increase in productivity over this decade (Table 6.3), which seems counterintuitive. However, during this wet decade spring rainfall increased by 85%. Spring is normally the dry period in the northern Chihuahuan Desert, and a time of severe stress and tissue loss for grasses, which break dormancy in spring. The unusually wet spring periods of the wet decade not only alleviate drought stress in the grasses, but contribute to increased grass cover, allowing them to be more competitive with the other functional types for early summer moisture. However, these increases in grass cover are contingent upon having sufficient grass biomass in the community to take advantage of this moisture, and the model does not account for grass establishment. In fact, Brown et al20 reported that shrubs were the apparent beneficiaries of increased winter/spring rainfall during this period in another Chihuahuan desert community. In our simulations, shrubs also exhibited a large increase (150%) in ANPP during this period, largely by taking advantage of several periods of deep soil moisture recharge during heavy winter rainfalls. This is also consistent with some of our experimental findings.21 To more specifically assess the impacts of year to year variability of rainfall on productivity of plant functional types, we conducted ten series of 10 year model runs using the rainfall of the normal decade (1968-1977), but

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81

Fig. 6.4. Annual precipitation at the Jornada Experimental Range (New Mexico, USA) for the last 80 years (average = 247 mm, dashed line). Maximum was 507 mm in 1984 and minimum was 79 mm in 1953. The seasonal distribution is approximately 65% in summer (July-October), 25% in winter (November-March) and 10% in Spring (April-June). The coefficient of variation (CV) of precipitation = 0.353 is identical to that reported by Le Houérou et al15 for 77 rangeland sites (see Fig. 6.3). in which each year’s rainfall was modified by a random amount varying from -30 to +30% (leaving daily distributions fixed). Thus each 10 year rainfall series had a coefficient of variation (CV) varying from a low of 0.25 to a high of about 0.45, and for the 100 year period, the CV was 0.351 (identical to that of the natural rainfall for the period 1915-1997). A plot of CV of rainfall of the decade compared with the CV of production of plant functional types (Fig. 6.3B) illustrates three somewhat distinct patterns. First, both the shrubs and forbs in PALS-FT had low CVs (~0.4) that were statistically invariant with increasing variation of rainfall (p>0.05) (Fig. 6.3B). This CV is close to the long term average CV of rainfall in the Jornada Basin (0.35). These functional types best exemplify plants capable of utilizing moisture that may occur during any season (note that the dominant shrub is a drought-tolerant

evergreen and the forbs are short-lived, herbaceous plants that can grow in any season). Second, both winter and summer annuals had production CVs that were much higher than that of rainfall (~1.45 for winter annuals and 1.25 for summer annuals) and, again, not dependent on rainfall. This high variation reflects the fact that productivity in these species depends on germination responses that are highly seasonal and rainfall specific. Small rainfall events within a season or large rainfall outside of their strict seasons result in almost no productivity because of lack of germination. Furthermore, their roots are located in the upper soil profile, which is subject to rapid drying. Thus, there are a number of seasons where substantial rain may not translate into any production; at other times, a small amount of rainfall that happens to be precisely timed for use by a seasonal annual species may result

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 6.5. Predictions from PALS-FT for the relationship between net production and annual precipitation. Based on three decades of precipitation (normal, wet, dry; see Fig. 6.4) and shown by functional types. Modified from Reynolds and Kemp.17 in substantial productivity. The third pattern of variation is shown by grasses, which are the only functional type with a significant increase (p<0.05, r2 = 0.63) in the CV of production with increasing CV of precipitation (Fig. 6.3B). Because these perennial grasses grow mainly during the summer, they appear to be utilizing the most reliable moisture resource (see legend in Fig. 6.4). However, there is competition for this moisture from other plant functional types, and high evaporative demand can quickly remove moisture from small rainfall events. Many small events are not equivalent to the same amount of rain in a single event that percolates deeper within the profile. Thus, timing and amount of individual events becomes more important in summer, when grasses are growing. The length of the spring drought period may also impact grass growth in summer, as the death of roots and shoot

tissue reduces the number of growing points capable of taking advantage of summer rainfall.

Integrating Ecological and Social Science Issues The case study presented above is intended to depict how a modeling approach can readily deal with one important aspect of desertification: plant production in an ecologically variable environment. In this section, we address the broader question of desertification in a variable socioeconomic environment.

Analysis Framework There is an immediate need for policy decisions on how to detect, prevent and/or adapt to desertification and land degradation in general.1,4,22 However, we have no convincing basis to argue why one scale of management might be preferred over another. This is due

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Table 6.3 Simulated mean and coefficient of variation of annual net primary production (ANPP) of functional types of Jornada Basin, New Mexico, in response to decadal variations in rainfall

“Normal” Decade (1968-77) (CV Rain = 0.29)

“Dry” Decade (1947-56) (CV Rain = 0.35)

“Wet” Decade (1984-93) (CV Rain = 0.26)

Le Houérou (CV Rain = 0.34)

Functional type

ANPP (g m-2)

CV ANPP

% Change in ANPP vs. “Normal”

C3 Winter Annuals

5.19

1.20

-

C4 Summer Annuals

14.57

1.16

-

Forbs

15.48

0.29

-

C4 Grass

22.39

1.12

-

Shrubs

34.92

0.45

-

Total

92.54

0.56

-

C3 Winter Annuals

4.18

1.65

- 20 %

C4 Summer Annuals

7.37

0.97

- 50 %

Forbs

10.79

0.59

- 30 %

C4 Grass

8.98

0.62

- 60 %

Shrubs

26.48

0.73

- 25 %

Total

57.81

0.60

- 38 %

C3 Winter Annuals

11.27

1.75

+ 120 %

C4 Summer Annuals

18.29

1.58

+ 25 %

Forbs

18.01

0.45

+ 15 %

C4 Grass

138.17

0.89

+ 500 %

Shrubs

89.57

0.33

+ 150 %

Total

275.30

0.52

+ 300%

77 arid rangelands

137

0.49

Based on Reynolds and Kemp.17 See Figure 6.4 for precipation patterns during each decade.

to our inability to detect with enough certainty the different levels of land degradation caused by land use practices, tenure systems, climate

variability or other factors. Furthermore, there are also few bases for predicting what institutional arrangements will best support the

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

objective of sustainable management at any given scale.23 What is certain is the need to move beyond isolated studies of various parts of the desertification problem, which has been the traditional approach.3 Analysis frameworks are needed that incorporate our best state of the art knowledge about precursors, processes, and consequences of desertification. These frameworks also need to represent the degree of uncertainty in our knowledge of the desertification puzzle and to be able to propagate these uncertainties in the analyses, thus reflecting them in the conclusions.24 The analyses presented in our case study focus attention on basic ecological feedbacks that contribute to short term ecosystem dynamics, which are precursors to longer term dynamics (Fig. 6.2). The analysis framework we endorse is one of integrated assessment (IA). There are many approaches to the development of an assessment but, in general, assessments are often designed as a collection of knowledge on related topics prepared by a group of interested parties.25 In the case of land degradation in drylands, the interested parties should comprise expertise covering key topics in social, economic, and environmental sustainability. The scientific assessment by the Intergovernmental Panel on Climate Change (IPCC) is a good example of IA, although it represents only an initial step towards gathering of knowledge from various disciplines. We need collaborative activities where interdisciplinary research is nurtured, and the interstices of knowledge are sought out as subjects of long term interactions between economists and natural and social scientists. Hence, an IA should be more than just a model building exercise—integrated assessments are a suite of methodologies for gaining insight on an array of environmental problems that span a wide range of temporal and spatial scales, from small geographical units (such as local watersheds) to larger regions. Integrated assessment models are powerful tools that can be used to explore quantitative as well as qualitative interactions between the various elements of a problem.25

Need for Cross-Disciplinary Collaboration There is a pressing need for increased collaboration among ecologists, economists, and social scientists to order to effectively address global change problems in drylands. Such collaboration is the key to integrated assessments and is imperative if we are ever to achieve social, environmental, and economic sustainability (Table 6.2). This will not be an easy task, of course, as recently noted by ecologist Bob O’Neill: “Ecologists, with some notable exceptions, perpetuate the fantasy of a ‘natural world’ where human society can be ignored … Human society and its economic activity are seen as an external driver that perturbs the natural world, not as another dynamic entity within the ecosystem itself.”26 We suspect that parallel arguments could be made for the economic and social sciences as well. It is true that the majority of ecologists trained during the 1960s and 1970s conducted research in what they would probably characterize as pristine or “natural” ecosystems. However, given the tremendous scale of current environmental concerns—and the need for fundamental knowledge in order to achieve the goal of environmental sustainability—such esoteric views of the natural world are no longer the norm in ecology. For example, in 1980 the US National Science Foundation (NSF) established the Long Term Ecological Research (LTER) network to support research on long term ecological phenomena. This network, which has now grown to 21 sites, represents diverse ecosystems and research goals, including two new sites that focus on urban ecology. These urban LTERs (in Phoenix, Arizona, and Baltimore, Maryland) represent the first attempt to invest similar resources into the study of the long term ecology of urban ecosystems. Ecologists, economists, and social scientists will be addressing questions such as: What constitutes “natural?” What is the relative importance of environmental, social, and economic factors in controlling the functioning of urban ecosystems? Do the key ecological relationships identified in relatively pristine settings operate in a similar fashion in urban landscapes?

Drylands and Global Change: Rainfall Variability and Sustainable Rangeland Production

Conclusion The case study we present in this paper illustrates how fundamental knowledge of environmental factors (rainfall variability) and ecosystem structure and function (plant functional type physiology) can be integrated within a modeling synthesis. Our results underscore the impact of short term climate shifts and year to year variability upon plant production, especially perennial grasses. Year to year variability in total plant productivity was found to be roughly 50% greater than the variability in “normal” rainfall, and variability in grass productivity was three times greater. From a management standpoint, perennial grass is one of the most important functional types because of its contribution to forage production and ground cover. Maintaining a stable grass cover is desirable in order to be able to project long term land use strategies and economic return. However, our modeling results suggest that maintaining stable grass cover may be very difficult in this semi-arid region, given the potential impacts of natural climate variability upon vegetation production. Furthermore, our results support the point of view that dryland agriculturists and pastoralists must be adaptive in terms of their response to climate variability.27 While these ecosystem-level predictions may be used independently to address issues relevant to sustainable development of these semi-arid regions, we suggest that the next step in assessing sustainability is the incorporation of the ecological impacts into higher level models that incorporate direct and other human impacts on these systems. This step will require further testing and evaluation of ecosystem-level models in the context of different management and land-use alternatives. Finally, at the highest level of integration, we propose the incorporation of both “natural” and human factors into a spatially explicit model of landscape elements and human land-use patterns. Only at this level will we have predictive tools capable of dynamic, integrated assessments of impacts of global climate change on human-dominated ecosystems. Although we are far from being able to do this, a number of programs around the world, such as the urban LTERs in the US, are striving to develop integrated approaches to

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link human and natural systems. We believe this task is essential in preparing for—and planning corrective measures to—the complex interactions between climate, human activities, and the well-being of the Earth .

Acknowledgments The authors thank Drs. M. P. McClaran and H. Dowlatabadi for helpful input. This work supported by the Carnegie Mellon University Center for Integrated Study of the Human Dimensions of Global Change (NSF SBR 95-21914). Additional support was provided by the Inter-American Institute for Global Change Research (UCAR/NCAR Award #S97-74027/IAI) and is a contribution to the Jornada Basin Long Term Ecological Research program (NSF grant DEB 94-11971).

References 1. OIES. Arid Ecosystem Interactions. Boulder: Office of Interdisciplinary Earth Studies, 1991:81. 2. Grainger A. Characterization and assessment of desertification processes. In: Chapman GP, editor. Desertified Grasslands: Their Biology and Management. Linnean Society Symposium Series, No. 13. London: Academic Press; 1992:17-33. 3. Thomas DSG. Science and the desertification debate. J Arid Environ 1997; 37:599-608. 4. Lopez-Ocaña C. Effectiveness of international regimes dealing with desertification from the perspective of the south. In: Young OR, Demko GJ, Ramakrishna K, eds. Global Environmental Change and International Governance. Hanover, NH: University Press of New England; 1996:125-135. 5. Pimentel D, Harvey C, Resosudarmo P et al. Environmental and economic costs of soil erosion and conservation benefits. Science 1995; 267:1117-1122. 6. Daily GC. Restoring value to the world’s degraded lands. Science 1995; 269:350-354. 7. Vitousek PM, Mooney HA, Lubchenco J et al. Human domination of earth’s ecosystems. Science 1997; 277:494-499. 8. Pickup G, Bastin GN, Chewings VH. Remote-sensing-based condition assessment for nonequilibrium rangelands under large-scale commercial grazing. Ecol Appl 1994; 4(3):497-517.

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9. Goodland R, Daly H. Environmental sustainability: universal and non-negotiable. Ecol Appl 1996; 6(4):1002-1017. 10. Manabe S, Wetherald RT. Large-scale changes in soil wetness induced by an increase in atmospheric carbon dioxide. J Atmos Sci 1987; 44:1211-1235. 11. Cubasch U, Waszkewitz J, Hegerl G et al. Regional climate changes as simulated in time-slice experiments. Climatic Change 1995; 31(2-4):273-304. 12. Prince SD, Brown de Colstoun E, Kravitz LL. Evidence from rain-use efficiencies does not indicate extensive Sahelian desertification. Global Change Biol 1998; 4:359-374. 13. Parry ML, Carter TR. The effect of climatic variations on agricultural risk. In: Parry ML, ed. The Sensitivity of Natural Ecosystems and Agricultural to Climate Change. Dordrecht, The Netherlands: Kluwer; 1985:95-110. 14. Coronado-Q JA. Relationship between range condition and the land tenure system in Sonora. Ph.D. Dissertation. Tucson: University of Arizona Press, 1998. 15. Le Houérou HN, Bingham RL, Skerbek W. Relationship between the variability of primary production and the variability of annual precipitation in world arid lands. J Arid Environ 1988; 15:1-18. 16. Reynolds JF, Virginia RA, Schlesinger WH. Defining functional types for models of desertification. In: Smith TM, Shugart HH, Woodward FI, eds. Plant Functional Types: Their Relevance to Ecosystem Properties and Global Change. Cambridge: Cambridge University Press, 1997:194-214. 17. Reynolds JF, Kemp PR. The relationship between rainfall variability and plant production: A modeling study at the Jornada Basin, New Mexico. Plant Ecology. (In press.) 18. Buffington LC, Herbel CH. Vegetational changes on a semidesert grassland range from 1858 to 1963. Ecol. Monogr 1965; 35(2):139-164. 19. Gibbens RP, Beck RF. Changes in grass basal area and forb densities over a 64-year period on grassland types of the Jornada Experimental Range. J Range Manage 1988;41(3):186-192. 20. Brown JH, Valone TJ, Curtin CG. Reorganization of an arid ecosystem in response to recent climate change. Proc Natl Acad Sci USA 1997;94:9729-9733.

21. Reynolds JF, Virginia RA, Kemp P et al. Impact of simulated drought on shrubs in the Chihuahuan Desert: Effects of species, season, and degree of resource island development. Ecol Monog 1999; 69:69-106. 22. Kassas M. Desertification: A general review. J Arid Environ 1995; 30:115-128. 23. Odada E, Totolo O, Stafford-Smith M et al, editors. Global Change and Subsistence Rangelands in Southern Africa: The Impacts of Climatic Variability and Resource Access on Rural Livelihoods. GCTE Working Document No. 20. Canberra, Australia: GCTE Core Project Office; 1996:99. 24. Morgan MG, Henrion M, Morris SC et al. Uncertainty in risk assessment. Environ Sci Tech 1985;19:662-667. 25. Dowlatabadi H. Integrated assessment. Encyclopedia of Global Change 1999. Oxford: Oxford University Press. 26. O’Neill RV. Perspectives on economics and ecology. Ecol Appl 96;6(1):1031-1033. 27. Ribot JC, Najam A, Watson G. Climate variability, vulnerability and sustainable development in the semi-arid tropics. In: Ribot JC, Magalhães AR, Panagides SS eds. Climate Variability,Climate Change and Social Vulnerability in the Semi-Arid Tropics. Cambridge; New York: Cam bridge University Press; 1996:13-51. 28. Schlesinger WH, Reynolds JF, Cunningham GL et al. Biological feedbacks in global desertification. Science 1990; 247:1043-1048. 29. McClaran MP, Van Devender TR eds. The Desert Grassland. Tucson: University of Arizona Press; 1995. 30. Busso CA. Towards an increased and sustainable production in semi-arid rangelands of central Argentina: Two decades of research. J Arid Environ 1997; 36:197-210. 31. Zha Y, Gao J. Characteristics of desertification and its rehabilitation in China. J Arid Environ 1997; 37(3):419-432. 32. Darkoh MBK. The nature, causes and consequences of desertification in the drylands of Africa. Land Degrad Dev 1998; 9(1):1-20.

CHAPTER 7

Sustainable Water Management and Agriculture W. Kinzelbach, D. McLaughlin and H. Kunstmann

Introduction

F

resh water is a resource that is just as important to humanity today as it was in ancient times, when civilizations such as Egypt and Mesopotamia thrived because of their access to reliable supplies. But today the stresses placed on water supplies by a large global population with rapidly growing demands raise unprecedented challenges. The challenges are particularly important for agriculture, which consumes approximately 70% of water appropriated for human use.1 Agricultural water supplies in many regions of the world are threatened both by unsustainable depletion and by degradation in quality. In this paper, we consider the role of water in agriculture and use a number of case studies to develop conclusions concerning long-term sustainable water resource management.

The Global Water Situation Water is a renewable resource which is intercepted (or “appropriated”) by man as it moves through the hydrologic cycle. This cycle, which is ultimately driven by solar energy, carries water from the atmosphere to the land and oceans and back again. The largest flux appropriated for human use is the portion of natural evapotranspiration emanating from harvested forests and dryland agriculture. The global long-term average is about 18.2 x 103 km3/yr. This is about three times the annual flow of the Amazon river. Most of the remaining appropriations are for irrigated agriculture, industry, and domestic use, totaling about 4.4

x 103 km3/yr (Global water budget data cited in this section are from Postel et al.1 and Gleick2). These are satisfied primarily by direct withdrawals from “accessible runoff,” defined as the accessible flux of water flowing from the land to the ocean as either river water or groundwater. The remainder is satisfied by nonrenewable extractions from water stored in lakes and ground water aquifers. Direct withdrawals account for about 35% of the current accessible runoff of 13 x 103 km3/yr. If “instream water uses” such as navigation, maintenance of riparian ecosystems, and dilution of contamination are treated as claims on accessible runoff, this percentage increases to about 50%. A global analysis provides useful context, but it only tells part of the story. Hydrologic fluxes vary greatly over time and space, and many regions use a much larger percentage of available water than the global average of 35-50%. An additional complication is the effect of water quality on accessible runoff. As water moves through the hydrologic cycle it picks up, transports, and deposits a wide range of substances, both natural and manmade, which influence its suitability for human uses. Domestic uses are typically the most demanding, since even trace amounts of certain contaminants can have adverse effects on human health. But, the suitability of water for industrial and agricultural uses is also dependent on the levels of solutes, especially salts, and on suspended sediment loads. In some areas, shortages can occur

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because poor quality renders a portion of accessible runoff unsuitable for particular uses. Although contaminants can usually be removed at the point of end use, this increases the cost of water, sometimes enough to make it unsuitable for price-sens- itive industrial or agricultural uses. In a sense, degraded water quality can be viewed as a depletion of accessible runoff. A variant on this theme is the concept that some portion of accessible runoff needs to be reserved to dilute natural and manmade wastes. As noted above, this has the effect of increasing the percentage of accessible runoff needed to satisfy human needs. Spatial and temporal fluctuations in precipitation, evaporation, runoff, and water quality create regions of varying water abundance and scarcity. This has the effect of making water resource management a regional rather than a global enterprise. The spatial variability of water resources can be described in many ways. As a rough indication, it is revealing to note that the runoff available per capita varies from 300 m3/yr in arid areas of Africa and Asia to 100,000 m3/ year in Canada.2 This variability has serious implications for food production in arid and semi-arid regions which depend heavily on irrigation. Irrigated agriculture requires 1000 m3/yr of diverted runoff to provide enough food to feed one adult for a year. Although only about 18% of all cropland is irrigated, irrigation accounts for 25% of all agricultural land in India, 35% in Indonesia and Iran, nearly 50% in China and Iraq, over 80% in Pakistan, and 100% in Egypt. In some of these areas there is simply not enough runoff to feed the local population. As a result, an undetermined (but probably large) fraction of irrigation water is obtained from unsustainable depletion of groundwater reserves. Once these reserves are exhausted, withdrawals will have to decrease. Water shortages will also become more common in urban areas as populations grow and traditional supplies are jeopardized by degrading water quality. However, urban water users are in a better position to invest in conservation and treatment technology than subsistence farmers. To put the issue in perspective, the minimal amount of water required for domestic use is somewhat less

than 10 m3/person/year. This is typical of consumption levels in homes which lack running water. The global average domestic consumption level is about 50 m3/person/year while per capita domestic water use in the United States is nearly 300 m3/person/year. These wide ranges suggest that improved conservation and treatment technology could absorb much of the population-driven increase in urban water demand. The potential for urban water conservation is demonstrated both by the success of voluntary and imposed reductions in domestic use during droughts and by the substantial increase in water recycling by industrial users over the last few decades. There is also room for water conservation in irrigated agriculture. Since most water withdrawals are used for irrigation, a small improvement in agricultural efficiency could provide enough extra water to satisfy growing urban demands. This argument has been convincingly applied to the California water situation, where large urban areas and productive agricultural regions compete for water from the same sources.3 Although more efficient irrigation could resolve supply problems in some areas, it may have only a modest effect in others. This is because the improvements in efficiency which are technically and economically feasible may not be sufficient to satisfy the projected increase in demand, particularly in arid and semi-arid parts of the developing world. Improvements in irrigation efficiency require investments in expensive technology (e.g., drip irrigation). Commercial farmers can afford such investments only if their operations remain profitable. Subsistence farmers are generally dependent on free or low cost local sources, and have little or no cash to invest in more efficient technology. Also, there are inherent lower limits (other than plant evapotranspiration) on the amount of water required for productive cultivation of crops. One of the most important is related to the use of water for leaching salt from the root zone in situations where the applied irrigation water is moderately saline. If the irrigation is very efficient and all the applied water is taken up by the crop, salt will accumulate in the root zone and yield will eventually start to decline.

Sustainable Water Management and Agriculture Our analysis of current and projected demands for water repeatedly reveals the critical role of irrigated agriculture in developing countries. Overall, we feel that the most serious problems of water scarcity and quality will be associated with food production in developing countries rather than with domestic and industrial use in urban areas. It will become increasingly difficult for arid and semi-arid regions in Africa and Asia to be self-sufficient in food production as their populations increase, their groundwater reserves are exhausted, and per capita runoff availability declines. This loss of self-sufficiency could be managed if: 1. Production in water rich areas of the major grain exporting countries (e.g., Canada and the United States) increases sufficiently; and 2. Local populations have the economic resources to buy food on the international market. After all, many developed countries, even those with abundant water, are not self-sufficient in food but rely on imports to satisfy their needs. Although there is considerable room for expansion of production in the exporting countries, subsistence farmers in many arid regions do not have the cash income required to participate in the international grain market. In some developing countries, a sizable portion of the gross domestic product comes from vulnerable irrigated agriculture. The economies of these areas will have to change dramatically before their food needs can be met by imports. We can anticipate that water problems will be especially serious in areas where: 1. Demands approach or exceed available fluxes; 2. Stress on limited water resources is degrading the quality of supplies; and 3. The population is highly dependent on food grown locally. As the consequences of unsustainable depletion and degradation of water resources begin to be felt, current practices will inevitably have to change. The required changes may involve transitions from dependence on local subsistence agriculture to dependence on imports, investment in improved water resource infrastructure, adoption of new technologies,

89 development of new policies which regulate depletion or insure that water is properly valued, or a combination of all of these. Careful planning and preparation will ease difficult transitions while maintaining food security and public health.

Important Water Resource Issues In this section we examine some important issues related to the sustainability of current water practices, particularly those relevant to irrigated agriculture. Although these issues are generic, they are best illustrated with specific examples. In the end, global water problems are the sum of many regional and local problems such as those discussed here.

Groundwater Overdrafts Consistently falling groundwater levels are an indicator of overexploitation. They are observed worldwide. In northern China, 4 western India5 and South Africa,6 groundwater tables are declining at rates between 1 and 3 m per year. The abstractions in the Sahara and Saudi Arabia deplete aquifers which since the last ice age have not had any recharge to speak of.7 It is estimated that by 2010 the deeper aquifers of Saudi Arabia will have only 40% of their 1985 water reserves left.2 The present net depletion by overdraft of important groundwater reservoirs worldwide is at least 200 cubic kilometers per day.8 In practically all cases, irrigation is responsible for overdrafts. The best documented case of over pumping due to irrigation is the Ogallala aquifer in the Great Plains region of the United States. It is one of the largest aquifer systems in the world, stretching across parts of eight states, South Dakota, Nebraska, Wyoming, Colorado, Kansas, Oklahoma, New Mexico and Texas, and underlying 174,000 square miles. Groundwater withdrawal on a large scale started during the 1930 drought. Besides drought conditions, reasons for the fast development of groundwater irrigation were cheap energy, improved well drilling and pumping systems, and high crop prices. Development of groundwater resources for

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

irrigation made this region one of the major agricultural regions in the United States. In the late 1970s, the abstraction rate of the Ogallala Aquifer amounted to more than a quarter of the groundwater used for irrigation in the US. Pumping rates many times in excess of the recharge rate led to a substantial decline in water levels ranging from 3 to 30 meters.9 Up to 1990, the total abstraction exceeded the total recharge by 164 km3.10 Water level declines were greatest in those parts of the aquifer where irrigation was developed first and where it was most intense. Severe depletion of the resource led to rising pumping costs, driving much of the irrigated agriculture in the region out of production by the early 1990s. In the Texas High Plains for example, irrigated area decreased by 34% between 1974 and 1989.2 A large part of the area today has reverted to rainfed agriculture with much lower yields. Increased pumping costs led to technical and institutional adaptations. Among the technical measures were increased irrigation efficiency and the practice of conservation tillage. Average water-use efficiency in the southern High Plains of Texas improved from about 50% in the mid-1970s to approximately 75% in 1990 due to improved technology. Current state of the art low pressure, full dropline center pivot systems are about 95% efficient. Buried drip lines approach 100% efficiency.11 Institutional measures include groundwater laws that made possible the institution of regionally controlled groundwater management units, which set limits on the spacing and number of wells and carried out metering of water use and promotion of water conservation.12

Salinization Salinization and related drainage problems are commonly associated with irrigated agriculture, especially in arid and semi-arid regions. In the dryland agriculture practiced in humid regions, most of the water in the crop root zone originates from rainfall, which has low salinity. The small amount of salt that enters the system is flushed through the root zone by the relatively large amount of excess water (water exceeding crop evapotranspiration and natural evaporation) available. By

contrast, the water applied to irrigated crops is obtained from surface runoff, which picks up salts or gets more concentrated by evaporation as it flows over the land and into surface waters or subsurface aquifers. If all irrigation water is taken up by the crop (i.e., there is no excess), the salt contained in this water will accumulate in the root zone until the salinity reaches levels that will inhibit crop growth. If excess water is applied, as is usually the case, it will carry the salt through the root zone to deeper soil layers or, when the soil is highly impermeable, laterally to drainage ditches. If the water table is deeper than a few meters, the salt in the deeper soil layers will be transported downwards until it reaches groundwater. In many cases, the salt carried by groundwater later enters surface waters which may serve as irrigation sources for downstream users. If the water table is within a few meters of the ground, the salt held in soil moisture below the root zone during the growing season may be drawn upwards into the root zone by evaporation (capillary rise) during the fallow season. This salt must be leached out of the root zone during the next growing season or the salinity will eventually accumulate to harmful levels. Since irrigation with slightly saline water requires, by its very nature, that water must be applied at levels exceeding crop evapotranspiration, it is important that the excess water (drainage) be removed from the crop system. Otherwise, it may raise groundwater levels and increase salt accumulation in the root zone. The problem of rising groundwater levels coupled with salinization is widespread in irrigated regions of the western United States, Asia, and our case study region in Australia. Although it is possible to install drainage systems which collect and divert much of the excess water used to leach salt, these systems can be expensive to construct and maintain. Furthermore, they just displace the problem of salt accumulation to another location (or user). Since the drainage water is more saline than the applied water, it is less desirable for irrigation. If the drainage water salinity is too high, this water must be treated as a waste product.

Sustainable Water Management and Agriculture The scope and magnitude of irrigationrelated salinization problems is well documented in Ghassemi et al.5 An informative example is the Murray-Darling river basin located in southeastern Australia. This semiarid basin covers about one-seventh of Australia and contains some of the continent’s most productive agricultural land. It is distinguished by very low runoff (only one percent of precipitation) and high evapotranspiration. Salinization problems are important throughout the basin but have received particular attention in the lower Murrumbidgee River watershed, a 40,000 ha region planted predominantly in irrigated paddy rice, wine grapes, citrus, and dryland pasture. It is estimated that approximately 25% of cropland in the lower Murrumbidgee is slightly salinized while up to 15% is severely salinized. Moreover, the amount of affected land is increasing each year. Annual rainfall in the lower watershed averages around 400 mm/yr, too little to support dryland cultivation of most food grains. Much of the irrigation water used to grow crops in this area is diverted from the Murrum- bidgee River, which rises in the more humid upland areas to the east. Irrigation related salinization problems in the Murrum- bidgee watershed are aggravated by the naturally high salinity of the shallow groundwater, which is within two meters of the surface over extensive sections of cropland, and by gradually increasing salt inflows to the river. These inflows are the result of agricultural drainage in the lowlands and dryland salinization (induced by deforestation) in the uplands. In the Murrumbidgee watershed, artificial drainage systems are generally installed in fields planted to high value crops such as grapes and citrus. The effluent from these systems goes to downstream users in the watershed and then either to the Murrumbidgee River or to evaporation ponds. The paddy rice is generally grown on low permeability soils which permit the paddies to be flooded throughout the growing season. Much of the salt entering the paddies eventually leaves when they are drained at the end of the growing season. However, the recharge to groundwater is sufficient to have created

91 extensive groundwater mounds beneath rice growing areas in the Murrumbidgee. The local water table rise has aggravated salinization due to capillary rise, especially in neighboring non-rice growing areas. Crops grown in these areas (especially grapes) generally cost more to grow than rice but consume less water and can be more profitable. As more salt accumulates in the root zone during the fallow season, more water must be used to flush the soil before planting. This puts further stress on the limited water supplies available for irrigation. Rising water tables and associated problems of salinization have induced growers, government agencies, and other groups interested in the lower Murrumbidgee watershed to develop plans to insure that agriculture in the region can be sustained over the long term. The current plan specifies that rice can be grown only in rotation on approved land (with low infiltration rates), up to a limit of 30% of total farm area. However it is not clear either: 1. That these restrictions will insure that the region’s agriculture is sustainable; or 2. That this is the most economically efficient way to achieve sustainability, particularly when external costs to downstream users and ecosystems are considered. A study currently being carried out by the authors is examining both issues in more detail.

Sea Water Intrusion In coastal areas over-exploitation of groundwater leads to seawater intrusion (see, e.g., SWIM’96).13 Due to the density difference between fresh and saline water, a saltwater wedge forms naturally in coastal aquifers. If the freshwater flow is diminished by pumping, the wedge will proceed further inland, eventually leading to the contamination of wells. Wells which are screened in the freshwater layer may still draw salt by the phenomenon called upconing.14,15 At present, six out of ten people live within 60 km of a coast, and by the year 2000 more than two-thirds of the population of developing countries will live in the vicinity of the sea. The increasing concentration of human settlements in coastal areas gives rise to excessive pressure on groundwater resources,

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

resulting in seawater intrusion and related deterioration of water quality.16 Seawater intrusion is rampant along the coasts of India, Israel, southern China, Spain and Portugal, to name only a few. In China, seawater intrusion has become more and more serious since 1970 in the eastern coastal zones. The following figures illustrate the situation on the Shandong coast, including the cities Laizhou, Weifang, Qingdao, Yantai, and Pingdu among others. The facts presented are taken from Wang et al.17 In the coastal area of Qingdao, Yantai and Weifang, the current water supply shortage in an average year is 3.1 km3. Available water resources can only meet 60% of the total demand. In a dry year, the shortage is 5.1 km3 or approximately 52% of the total demand. The well density rose from the original 10 wells/km2 to 25 wells/km2, and in some places even up to 70 wells/km2. From 1976 to 1986, overabstraction caused a large groundwater level drawdown in the northern Weifang area. Up to now, the total area with groundwater levels lower than mean sea level is 2,400 km2 in the Laizhou area. The lowest elevation is 20 m below sea level. From Yantai City to Laizhou City, the area affected by seawater intrusion is up to 400 km2 and growing fast. By the year 2000, the affected area will be 2000 km2. The intrusion, which accelerated from a frontal speed of 46 m/yr to 400 m/yr between 1976 and 1988, caused serious damage to this area, in which around 450,000 people were affected. With growing salinity the soil productivity drops or may even be lost completely. About 80% of the cultivated land of the coastal plain of Laizhou City is affected and the grain loss is up to 75,000 tons per year. To solve the problem, water resources management organizations were set up in Yantai City and water prices were increased, taking into account seasonal variation of scarcity. In order to save irrigation water, two measures were taken. First, drip irrigation and low pressure pump irrigation were introduced, which can save up to 90% and 30% of water, respectively. Second, earth canals were lined with concrete and open drains were replaced by pipes to decrease losses by evaporation and seepage.

The consequences of salinization are not seen all of a sudden, but progress gradually. Agriculture can adapt to a certain degree by planting more salt resistant, but usually less valuable, products. This happens on the Tamil Nadu coast in India, which is one of the most prominent examples for severe seawater intrusion. Rice cultivation has now given way to some more salt tolerant crops, such as trees for firewood. This allows the farmers to still procure income, but it does not reverse the trend of further degradation. Exploitation of groundwater with the help of electric and diesel pumps is continuing uncontrolled.18 The main cause is the fact that electricity is free of charge for the villages and a change in this policy is virtually taboo. This shows the political and social dimensions of harnessing a problem which on the scientific side is well understood.

Nitrate Contamination During the last few decades agricultural productivity has increased enough to keep pace with the rapid increase in global population. This dramatic increase in productivity is largely due to improvements in crop varieties and associated increases in the use of fertilizers and pesticides, technical innovations sometimes collectively referred to as the “Green revolution”. The benefits of these innovations are apparent and relatively easy to document. What is not so apparent are the costs, both to human health and to the environment in general. Concerns about these costs have generated debates and have led to a number of regulations, especially in the developed countries. Many of the issues which arise in the case of fertilizer application can be illustrated with the case of nitrate use in Germany. The increased use of nitrogen fertilizer for agriculture introduces additional nitrate into the biosphere. In order to put the issue in perspective, the nitrogen balance for Germany is summarized in Table 7.1.19 This table clearly reveals that the biological fixation of nitrogen is dwarfed by the inputs from nitrogen fertilizer and manure. On a global scale, nitrogen fixation associated with fertilizer production is already comparable to the total amount of natural (preindustrial)

Sustainable Water Management and Agriculture nitrogen fixation.20 This means that the flux through the nitrogen cycle has been doubled by human activities. The harvesting of crops removes only little more than one-half of the total input. The remainder ends up in different compartments of the environment. (Table 7.2). About one-third leaches into groundwater and eventually enriches surface waters before it gets back into the cycle in the form of nitrogen gas or is stored in the nitrogen pool of the soil. The ecological consequences of this intervention are still not fully understood. It is, however, well known that nitrogen enrichment increases the likelihood and extent of eutrophication, particularly in estuarine environments. The denitrification which occurs in the aquifers under anaerobic conditions gets rid of nitrate, but utilizes resources such as organic carbon on the aquifer matrix which when used up is no longer available to maintain the aquifer function of denitrifying filtration. On the basis of a recharge rate in Germany of about 200 mm/yr and a maximum allowable concentration of 50 mg NO3/ l, and assuming that there is no denitrification, a maximum tolerable nitrate flux to groundwater of 20 kg N/ha/yr can be computed. The actual flux of nitrate to the groundwater with about 40 kg N/ha/yr is already twice as high. The bulk of nitrate excess is due to intensive agriculture where the farmer fertilizes in the expectation of maximum yield. This yield is not achieved every year. In fact, in the majority of years the extraction of nitrogen with the crop will be less than the input of nitrogen by fertilization. Over the years, the accumulation of excess nitrate leads to a large pool of organically bound nitrogen in the soil, which makes the system more prone to produce episodically high temporary inputs of nitrate to groundwater by washout. A solution to this dilemma could come from precision agriculture, which monitors the soil’s demands much more closely and makes it feasible to apply small amounts of fertilizer frequently in an economic fashion. The time required for such technical innovations to have an effect on the scale of nitrate concentrations could be quite large. This time

93

Table 7.1. Nitrate balance for Germany (1990) Units: kg N/(ha agricultural land) Input Mineral fertilizer

135

N-fixation by legumes

14

Manure

78

Deposition from atmosphere

30

Sum

257

Output Withdrawal at harvest

141

Excess

116

includes the travel time in the unsaturated zone, the travel time in the saturated zone, and the time to deplete the pool of organically bound nitrogen in the topsoil. In a typical German watershed, this amounts to several decades The potentially adverse effects of nitrate on human health have been the primary focus of efforts to regulate nitrogen fertilizer use in Germany. The World Health Organization and the European Community have both adopted standards that require that nitrate concentrations in drinking water should not exceed a standard of 50 mg/l NO3. Concentrations in excess of this value are found frequently in groundwater. In European countries such as Germany or Denmark, about 10% of all water works are above or close to this value. The fact that the percentage has been virtually constant over the last decade is misleading. It is not due to a leveling off of the problem, but rather to the continuous shutting down of polluted wells which then do not appear in the statistics any more. If one takes the “warning value” of 25

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Table 7.2. Fate of nitrate excess (in %)

Total Nitrate Excess

kg N/ (ha agricultural land) = 100%

Direct surface runoff

9

Atmosphere as N2O

5

Atmosphere as NH3

18

N-pool of soil

17

Denitrification in soil

17

Leaching to groundwater

34

mg/l NO3 as a threshold, the percentage of water works affected is almost twice as high. In the German State of Baden-Wuerttemberg, where an extensive monitoring program has been undertaken, regions with high concentrations show a leveling off, while formerly cleaner regions are still on the rise. In Europe, non-point nitrate pollution of groundwater presents more of a problem than industrial pollution of groundwater. This is because the source is much more pervasive and the nitrate ion is very mobile and not easily removed by standard water processing techniques. Nitrate removal to meet existing standards could almost double present prices of drinking water in Germany. While such removal may be feasible in the centralized treatment plants of the industrialized world, it is much more difficult to implement in a cost effective way in the small decentralized water systems of the third world. As fertilizer use and domestic water demands increase in the developing world, it is quite possible that nitrate-induced health problems could become more pervasive. In the short term this will lead to increased costs, either for farmers who must reduce fertilizer application and accept lower yields, or for domestic water users, who must pay for nitrate removal. In the longer term, it may be possible to maintain high yields while reducing the amount of nitrogen leached into groundwater, e.g., through the use of

“recession agriculture” or genetic manipulation of crops to provide for direct nitrogen fixation from the soil air.

Pesticide Contamination Many of the issues encountered in the nitrate case study discussed above also arise with pesticides. The major differences lie in the nature of the human health and environmental costs incurred and in the types of technical solutions needed to reduce these costs. Also, the uncertainties associated with pesticide transport processes, and with the human health and ecological effects of pesticides, are probably even greater than the comparable uncertainties about nitrate. By any measure used, whether volume applied, hectares treated or market value, global pesticide use is large and still increasing. In 1995, world consumption reached 2.6 million metric tons of so-called active ingredients (the biologically active compounds) with a market value of US $38 billion. About 75% of pesticide use occurs in developed countries, mostly in North America, Western Europe and Japan, where high pesticide application rates are common. In these countries, herbicide use dominates. Herbicides are generally less toxic than insecticides, which are more widely used in developing countries. In fact, pesticide applications in developing countries are growing steadily, especially where export crops such as cotton, bananas, coffee, vegetables and flowers are predominant.21 It is estimated that annual world consumption of herbicides was around 1 million tons in 1993. Arable land and permanent crops cover around 1.4 billion hectares worldwide. Assuming that between 80% and 85% of herbicides are for agricultural use and that they are distributed uniformly on cultivated land, an average of about 0.5 kg/ha is used every year.22 However, local applications can be much higher in areas where intensive agriculture is practiced. Table 7.3 gives more locationspecific figures on the average herbicide doses applied to corn, soybeans, rice, wheat and sugarbeets in the US (Illinois), France and Italy. These are well above the global average. Cereals account for 0.7 billion ha, i.e., 50% of total global arable land, of which wheat, rice and corn are the most widespread. Among

Sustainable Water Management and Agriculture other crops, soybean is the most widely cultivated, with 56 million ha. This explains why more than 50% of herbicides are used on only a few crops. Pesticides can move through the soil with water as it percolates down to groundwater. This process is called leaching. Both soil and pesticide properties must be considered when evaluating the tendency of a pesticide to leach in a particular location. The type of degradable organic matter (OM), the soil texture, and the soil acidity (pH) are the most important parameters determining the soil leaching potential, while the mobility, the persistence (usually given in terms of half-life), the rate of application and the application method are the relevant pesticide parameters. The strong heterogeneity of natural soils makes predictions of leaching potential difficult. Homogeneous soil column studies miss the most important point of pesticide transport in the field, which is preferential flow. Therefore, laboratory studies tend to underestimate the pollution potential of a pesticide. The severity and global extent of groundwater contamination by pesticides cannot be adequately assessed. Data are only available in those isolated areas where monitoring programs have been carried out, i. e., almost exclusively in developed countries. The rapid introduction of pesticides into less developed countries has not been accompanied by monitoring, as the measurement of these compounds is still costly and requires a strong laboratory infrastructure. Although pesticides can be removed from drinking water (e.g., with activated carbon), this becomes more difficult as the pesticide (or pesticide metabolites produced by biodegradation) becomes more polar. As with nitrate, the difficulties and expenses associated with removal fall more heavily on small water suppliers who do not benefit from economies of scale. Since most pesticide contamination comes from non-point sources, the effects can be widespread. When taken together, these two factors make pesticide contamination particularly problematic in agricultural regions, such as rural Denmark, where drinking water is obtained from many small suppliers. Denmark’s decentralized water supply system consists of 3,470 waterworks. A recent

95 monitoring program examined pesticide concentration in groundwater samples from 976 monitoring wells and 2,798 abstraction wells.26 This program revealed that 3 to 4% of the samples exceeded the maximum limit for individual pesticides in drinking water (0.1 µg/l). By way of comparison, in the same survey no samples of chlorinated hydrocarbons exceeded the comparable threshold (25 µg/l). In about 10 percent of the samples, one or more of a target group of eight pesticides were detected. These pesticides were found down to a depth of 60-70 m, most frequently in younger shallow groundwaters, with the occurrence generally decreasing with depth. The pesticides dichlorprop, mechlorprop (phenoxyacids) and atrazine (triazine) were most frequently found. Phenoxy acids have been found exclusively in anaerobic aquifers, often under bedded thick till and clay layers. In unconfined aerobic sand aquifers, triazines are found.27 Some of the Danish counties analyzed groundwater samples from monitoring wells for more than the 8 pesticides in the target group. These detailed studies revealed the presence of 35 pesticides or metabolites in Danish groundwater. Moreover, 22 of these were found with concentrations above the maximum admissible concentration in drinking water.26 Understandably, these sampling results have been the source of great concern in Denmark, and wells with pesticide concentrations above the 0.1 µg/l threshold will be closed. It is worth noting that the threshold which prompted this action is based on historic detection limits rather than toxicological risk considerations. In any case, it is likely that more Danish wells will be closed in the future, as nondegradable pesticides from more remote source areas arrive at additional pumping and monitoring sites. If this process continues, it is likely that Danish water supply system will have to become more centralized. In Denmark this does not necessarily pose a great problem, since water is plentiful and it is unlikely that pesticide contamination will create serious supply shortages. This may not be the case in more arid regions in developing countries, where localized treatment and centralization of water supplies may be technically difficult

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Table 7.3. Average herbicide doses (g ha-1) applied in different crops (1988) Corn

Soybeans

Rice

Wheat

Sugarbeets

Illinois (USA)

3125

1400

-

-

-

France

2000

1600-2480

-

1140

3300-3800

Italy

3090

-

6500

1500-4100

7210

Adapted from Catizone,23 Fougeroux and Bourdet,24 Pike et al.25

and/or may impose significant economic penalties on local populations.

Conclusions Our analysis of the global water situation, as well as regional case studies such as those presented here, reveals the following general conclusions: The critical water issues of the next few decades will be regional and basin-scale rather than global. However, regional issues will have global impacts through their collective effects on world trade, political conflict, and incentives for technological innovation. The most serious problems of water scarcity will be associated with irrigated agriculture in developing countries where subsistence farming accounts for most of the local food supply. Although it may eventually be desirable for such regions to cut back on irrigated agriculture and import more food from more humid regions, this will require substantial changes in their economies. Domestic and industrial needs are a small fraction of agricultural water use and can be reduced even further with existing technology and proven conservation practices. While irrigated agriculture can certainly benefit from efficiency improvements, it is unlikely that these improvements will keep pace with increasing demands for food. Water quality problems associated with fertilizer and pesticide application will remain serious in some regions, but will probably diminish in others as agricultural practices and products become more environmentally sensitive. The technology to reduce discharges of potentially harmful agricultural chemicals is already available and will continue to improve.

The more serious problem will be allocation of the costs of using this technology. Salinization is likely to become the dominant agricultural water quality problem in much of the world. In some cases, the direct and external costs of salinization will be too high to justify continuation of irrigated agriculture. In others, existing crops will have to be replaced by substitutes which are more salt tolerant or which are more amenable to salinity control. Stresses imposed by unsustainable depletion or by degraded water quality may take effect gradually, over many years. In some areas, farmers may adapt to changing environmental conditions by changing crops, by accepting lower yields (and incomes), or by adopting new cultivation practices and technologies. Other parties, such as downstream users, may also adapt in various ways to water shortages and degraded water quality. In all of these cases, explicit and/or hidden costs can be expected to be incurred. There is a real need for more reliable and scientifically defensible assessments of regional water issues and of the prospects for the future. Uncertainties in the magnitude and quality of water fluxes, stocks and demands at the regional level frequently lead to differing opinions about the nature, severity and cause of water problems. This can, in turn, lead to the adoption of inappropriate or counterproductive policies. Scientists, engineers and economists can all play a role in making the policy process better informed and more effective. This will help to insure that we can provide secure food supplies for an increasing population while protecting our water resources.

Sustainable Water Management and Agriculture

References 1. Postel S, Daly GC, Ehrlich PR. Human appropriation of renewable fresh water. Science 1996; 271:785-788. 2. Gleick PH ed. Water in Crisis, A Guide to the World’s Fresh Water Resources. Oxford: Oxford University Press, 1993. 3. Anderson T. Water options for the blue planet. In: Bailey R, ed. The True State of the Planet. New York: The Free Press, 1995:267-294. 4. Ministry of Water Resources PRC. Department of Hydrology, Water Resources Assessment for China. Beijing: China Water and Power Press, 1992. 5. Ghassemi F, Jakeman AJ, Nix HA. Salinisation of Land and Water Resources. Wallingford: CAB International, 1995. 6. Orpen WRG, Bertram WE. Groundwater management model of the Dendron aquifer, Republic of South Africa. In: Future Groundwater Resources at Risk (Proceedings of the Helsinki Conference, June 1994), IAHS Publ. No. 222, 994. 7. Sonntag Ch, Klitzsch E, Löhnert EP, El Shazly EM et al. Paleoclimatic information from deuterium and oxygen-18 in C-14 dated north Saharian groundwaters. Groundwater formation in the past. In: Isotope Hydrology 1978, Vol. 2.Vienna: IAEA, 1979:569-581. 8. Sahagian DL, Schwartz FW, Jacobs DK. Diirect anthropogenic contributions to sea level rise in the twentieth century. Nature 1994; 367:1033. 9. Johnson, KS. Exploitation of the tertiaryquarternary Ogallala aquifer in the high plains of Texas, Oklahoma, and New Mexico, southwestern U.S.A. In: Selected Papers on Aquifer Overexploitation, 23rd International Congress of the I.A.H, Puerto de la Cruz, Tenerife (Spain), 1992. 10. Postel S. The Last Oasis: Facing Water Scarcity. New York: Norton, 1992. 11. High Plains Underground Water Conservation District No.1. The Ogallala Aquifer, http://www.hub.of the.net/hpwd/ ogallala.html , 1998. 12. Consortium for International Earth Science Information Network. Adaptation to Declining Groundwater Levels in the High Plains Aquifer. http://infoserver. ciesin.org/docs/004-071/box6-g.html, 1998.

97 13. SWIM’96. The 14th Salt Water Intrusion Meeting. Geological Survey of Sweden. Uppsala: SWIM, 1996. 14. Badon-Ghyben W. Nota in Verband met de Voorgenomen Putboring Nabij Amsterdam. The Hague: Tijdschr Kon Inst Ing, 1888:8-22. 15. Herzberg A. Die Wasserversorgung einiger Nordseebäer. Z Gasbelucht Wasserversorg 1901: 44. 16. FAO. Water Reports 11, Seawater intrusion in coastal aquifers. Guidelines for study, monitoring and control. Rome: FAO, 1997. 17. Wang, JG, Zao DS, Ji MC. Sea water intrusion and its control in the Shandong coastal zone, eastern China. In: Custodio G, Galofré F. eds. Study and Modelling of Saltwater Intrusion into Aquifers. Barcelona: CIMNE, 1993. 18. Unnikrishnan K. Sea water intrusion along Tamil Nadu coast, India: An underestimated risk. In Proceedings of the 8th Stockholm Water Symposium, Stockholm, 1998:142-144. 19. Wendland F, Albert H, Bach M, Schmidt R.eds. Atlas zum Nitratstrom in der Bundesrepublik Deutschland. Berlin: Springer Verlag, 1993. 20. Kinzig AP, Sokolow RH. Human impacts on the nitrogen cycle. Physics Today 1994; 11:24-31. 21. WRI. Intensification of Agriculture. Washington DC: World Resources Institute, 1998. 22. Vighi M, Funari E. eds. Pesticide Risk in Groundwater. Boca Raton: CRC Press, 1995. 23. Catizone P. Diserbo. In: Agricoltura e Ambiente. Bologna: Edagricole, 1991: 481-540. 24. Fougeroux A, Bourdet M. Les produits phytosanitaires. Evaluation des surfaces et des tonnages par type de traitement en 1988. La Defence des Vegetaux 1989; 259:3-8. 25. Pike DR, McGlamery MD, Knake EL. A case study of herbicide use. Weed Technol 1991; 5. 26. GEUS. Grunddvandsovervagning 1997. Geological Survey of Denmark and Greenland. Kopenhagen: GEUS, 1997. 27. Brüch W. Groundwater quality. In: Water Supply No 6, Kopenhagen: Danish Water Supply Association, 1997:284-290.

CHAPTER 8

Crop and Resource Management for Improved Productivity in Dryland Farming Systems O. Ito and M. Kondo

Introduction

A

ccording to the agroecological zone system proposed by FAO,1 the terms arid and semi-arid, representing dry-land, cover areas with less than 75 days length of growing period (LGP) and 75 to 180 days of LGP, respectively. Dryland occupies 30% of global land area. Two-thirds of dry land exists in Asia and Africa. About one-third of dry-land in Asia and Africa belongs to the semi-arid region, where various management options are still available to the farmers to increase crop productivity and resource use efficiency, depending on environmental and socioeconomic situations. The typical soil types found in the dry land are Entisols, Aridisols, Mollisols, Alfisols and Vertisols with unfavorable chemical and physical characteristics. The semi-arid tropics (SAT) hold onesixth of the world population, half of which subsists on less than a dollar a day. The agricultural productivity in the region should be improved to meet an ever-growing population which will reach 8.5 billion by 2025 and exceed 10 billion by 2500, according to the prediction made by the United Nations. The major constraints to agricultural production in SAT are unpredictable weather and poor accessibility to agricultural resources, which forces the farmers to take an option of low input farming systems. Among several options available at the farm level, N fertilizer management would be most suitable to

achieve an immediate increase in crop production in the area where most of the farmers have no access to irrigation facilities. The SAT soils are usually low in organic matter as compared with temperate soils. Since organic matter is a source of available N in the soil, many soils in the SAT can satisfy only a part of the crop N requirement, even at a low yield level, and therefore N fertilization is necessary to improve crop yields and land productivity. The fertilizer application is a risky investment for the farmers in SAT because of the heavy rainfall at the onset of each growing season, which leads to a considerable leaching loss of the nutrient element applied, and unreliable rainfall, which periodically results in a fatal crop failure. Fertilizer application does not always bring more income by increasing crop yield; instead, it sometimes incurs a huge monetary debt and consequently puts the farmers in a more difficult financial situation. In contrast, the option to select a suitable cropping system does not require investment and needs only appropriate judgement based on agronomic knowledge obtained by long practical experience. Among various cropping systems, intercropping seems to be a very attractive system which may improve the stabilization of crop production in harsh conditions.

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The crop combination in intercropping is usually determined so as to capture more solar energy, in other words, to achieve more light interception than monocropping by considering the canopy structure and growth duration of the crops. To improve the utilization of soil resources, the root system structure should be also considered. Combining crops with shallow and deep root systems may increase resource capture from extended soil layers where limited amounts of water and nutrients exist. In the case of a legume and cereal combination, it is reported that biological nitrogen fixation of the legume is enhanced by the presence of the cereal.2 The proper crop combination, and careful resource management in intercropping, will certainly improve the resource use efficiency and, ultimately, land productivity. This paper focuses on crop and resource management in relation to cropping systems widely practiced in the SAT region. The management of crops and resources should be considered within the framework of farming operations as a whole, considering the flexible decision making adopted by farmers in harsh environments for their survival. This flexibility has created numerous cropping patterns at the farm level.

Agriculture in the Semi-Arid Tropics The semi-arid tropics stretch over 48 counties on four continents, covering around 2,000 million hectares. It is inhabited by 700 million people. The soil types in the region are usually low in organic matter, N and P. The mean annual temperature is above 18oC and precipitation exceeds potential evapotranspiration for only 2 to 7 months (Fig. 8.1), with moisture balance being kept at a deficit for the rest of the year.4 Annual precipitation ranges from 400 to 1000 mm with erratic distribution, which often results in either no rainfall for extended periods or heavy local downpours. Because of repeated wet/dry periods and periodical heavy rainfall, nutrient depletion from the soil is the main biophysical problem in most of the SAT.

Despite the harsh environmental conditions, the rate of population increase has been high in the past and will be kept high(er) in the near future. To feed the growing population, more foods should be produced, by improving productivity of the lands and crops and by developing more sophisticated and sustainable ways of resource management. Due to the unpredictable weather and to poor accessibility of agricultural resources such as fertilizers, the cropping options for farmers are restricted to low input farming systems. High rainfall, which often comes at the onset of each growing season, greatly reduces the efficiency of fertilizer for crop uptake. The fear of a fatal crop failure due to drought makes the farmers reluctant to apply sufficient fertilizer for crop growth. Most crops in the region are grown with no fertilizer or insufficient amounts of fertilizer, due to the consideration of safe investment. Sorghum and pearl millet are the two major cereals grown in the region, followed by rice, wheat and maize. Rice is grown either irrigated or upland. Pulses and oilseeds are important cash crops, on which the household largely depends for its cash income. There is a variety of cropping systems practiced by the farmers for these crops. Monocropping is not the farmers’ only choice. They move flexibly to other cropping systems. The cropping options available to the SAT farmers include shifting cultivation, pioneer cultivation, alley cropping, monocropping, multiple cropping, intercropping, mixed cropping, mixed row cropping and relay cropping.

Intercropping as One Cropping Option To reduce the risk of a crop failure in a drought year, intercropping is a common practice of resource-poor farmers in the SAT. Intercropping can be defined as simultaneously growing two or more crops, with different canopy structure and growth period, in alternating rows or sets of rows.5 Although a wide range of crop combinations are practiced in the SAT, a combination of cereals and legumes is recommended, mainly due to the

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Fig. 8.1.Monthly changes in temperature, potential evaporation and precipitation at Patancheru, Hyderabad, India, 17˚38’N, 78˚21E.3 economic value of legumes, high potential yield from the cereals and favorable effects of BNF on soil fertility. Among legumes as component crops in intercropping, pigeonpea is considered to be a promising choice 6 because of its: 1. Having deep rooting systems to pump water and nutrients from deep soil layers; 2. Fixing substantial amounts of N in low fertility and dry environments; 3. Contributing high quality residue that recycles N and P benefits to subsequent crops; 4. Enhancing P availability by solubilizing Fe-P, which allows it to grow under low-P status soil. The productivity of intercropping is normally expressed with a land equivalent ratio (LER), which can be obtained by summation of yield ratio of each component crop in intercropping over monocropping. Under the

appropriate crop combination and row arrangement, the LER can exceed unity, which means increased land productivity over monocropping. In the case of cereal/legume intercropping, in order to increase the LER the aim should be to minimize reduction in yield of the cereal, so that any yield from the legume will bring additional benefit over monocropping. Intercropping is a system to increase resource utilization above and below ground by growing two or more crops with different growth patterns of shoots and roots. In monocropping, there may be times during the crop growing period when aerial resources (mainly light and CO2) and soil resources (mainly water and nutrients) are not properly utilized by crops. Because all the crops in a field grow at the same rate in monocropping, the crops may not be ready to utilize the resources at the initial stage, although they are available,

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and too much mutual competition may take place for too little resource at the later stage. Intercropping has been extensively examined in relation to solar radiation utilization. Appropriate planting densities and row arrangements have been proposed for every possible cropping combination to minimize competition for solar radiation. When it comes to resource sharing below ground, however, little work has been done, mainly because of technical difficulties in measuring resource dynamics and root system development in soils. Intercropping should be more closely researched from this aspect to increase utilization of soil resources. The interactions between component crops in intercropping takes place not only above ground but also below ground. In pigeonpea-sorghum intercropping, there is evidence that the presence of sorghum in neighboring rows enhances N input from biological nitrogen fixation (BNF) into pigeonpea (Fig. 8.2). This would be explained by root interaction.2 Since sorghum is very active in N uptake from soil, it depletes most of the N from soils close to pigeonpea roots when sorghum roots are developed to the pigeonpea row. Because of lowered N content in soil, inhibition of BNF by inorganic N would be relieved. This finding suggests that the N balance in the system can be improved by proper management of the cropping systems through increased input from BNF, without requiring any investment.7

Interaction Among Water, Nutrients and Roots There is no doubt that water is a scarce resource in the SAT, but this does not mean that water always limits growth and production of crops. It should be more accurate to say that the natural supply of water does not match the crop demand in time and space. Water cannot be properly supplied to crops when and where they require it, because of erratic rainfall pattern, loss from soil surface through pan evaporation and loss into deep soil layers through leaching. Water is a resource beyond management for most of the SAT

Fig. 8.2. Proportion and amount of N derived from atmosphere (Ndfa) by pigeonpea when intercropped with sorghum, pearl millet, groundnut and cowpea. farmers who have no access to reservoir or irrigation system. On the other hand, nutrients, particularly nitrogen, are manageable by the farmers to some extent, though the natural supply is almost identical with the case of water. A considerable amount of N, mainly NO3 (roughly 100 to 200 kg ha-1) is usually found in the soil solution at the time of planting (Fig. 8.3). The entire amount of N disappears from the soil solution within 50 to 100 days, suggesting active dynamics of N in the soil solution during an initial cropping period. Since N accumulation in the crops is much less, and slow, as shown in Fig. 8.3, most of the N disappears from the system without being utilized by crops.8 This finding raises a question for the traditional farming practice of basal application of N fertilizer. It leads to the expectation that delayed N application will enhance the dependency of sorghum on native soil N, thereby increasing N use efficiency of the system. The soil solution is the aqueous liquid phase of the soil, which provides the immediate source of nutrients for plants and microorganisms and acts as a temporary sink for some of their products. Nutrient levels in soil solution have been related to plant growth

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Fig.8.3. Changes in soil solution N (50 cm depth) and plant accumulated N in pigeonpea/sorghum intercropping. in many studies. Nitrogen from soil and fertilizer is finally released into the soil solution and becomes available to the crops. In soil solution, mineral N is subjected to a dynamic state, which means that there is always turnover of N in and out of the soil solution, even though its concentration may remain constant. The major inflow processes into this pool include fertilization, mineralization, rainfall and flow from neighboring layer of soils. The outflow of NO3 from it is mainly due to plant uptake, immobilization, volatilization, denitrification and leaching. Although we can only observe the balance of those complex processes, plant uptake seems to affect its pool size most intensively,9 especially near the rhizosphere. Nitrate is a major form of N under upland conditions, and its fluctuation in soil solution could more clearly reflect the

root development and nutrient uptake activity of roots than NO3 extracted with KCl, which is commonly used to assess available N to plants. Under resource-limiting conditions ubiquitous in the semi-arid tropical environments, crops with deep root systems may have an advantage in exploiting soil resources. To understand root distribution within the soil profile, roots are collected with various methods such as auger, monolith, trench wall and minirhizotron, and expressed in terms of length or weight of a unit soil volume against soil depth. This normally gives an exponential pattern skewing heavily toward deep soil layers.9 The cereals develop more roots near the soil surface and less in deep layers than the legumes. Even within the cereals, this is more evident in rice than in corn. To achieve

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more efficient exploitation of limited soil resources, it would be ideal to combine two crops that display different developmental patterns of root systems. In addition to root system architecture expressed by root distribution along the soil profile, root function is also important.10,11 Even though the crop has a well developed root system, it would be rather a waste of carbon allocation if it does not function properly for uptake of nutrients and water. Germ plasm improvement and crop management should pay attention to both architecture and function of root systems. There is a significant interaction between root development and nutrient status in soils. The application of N and/or P as fertilizer enhances root development in terms of length in almost all layers of soil profile. Through this interaction, it can be expected that fertilizer application may increase water uptake, resulting in less volumetric soil water content in the soil profile compared with unfertilized soils. This observation suggests that the proper management of nutrients will increase crop production not only through direct effects on crop growth but also through enhancement of water utilization due to improved root development.

Crop and Resource Management in Low Input Farming Systems Nitrogen fertilizer application is a management practice that can be easily modified by farmers in terms of time and method of application. The method of application would considerably affect N fertilizer use efficiency (NFUE), which indicates how much proportion of N applied as fertilizer is utilized by the crop. To minimize the amount of N fertilizer which is not utilized by crops, in other words to increase NFUE, timing of application should be well synchronized with patterns of N supply from the soil and with crop requirement. In regions where intercropping is commonly practiced, most farmers do not apply, or apply very low doses (less than 25 kg N ha-1), of N fertilizer, because of economic, logistic and social reasons. When N is applied, the farmers prefer basal to delayed applica-

tion because they believe the crops require N for early growth. As shown in Fig. 8.3, however, an appreciable amount of N is available to the crops at the time of planting. Obviously a small dose of N at planting will be diluted by the soil N pool, leading to low efficiency for crop utilization. Thus, timing of nitrogen application becomes very important in low input farming systems.12 Delayed urea-N application results in a higher NFUE in sorghum than a basal application (Fig 8.4). The NFUE of sole crop pigeonpea is higher (14.6) than that of intercrop pigeonpea (1.8-3.9), because fertilizer is usually applied only to the sorghum rows in the case of intercrop treatment. Delayed N fertilization also enhances the dependency of pigeonpea on atmospheric N2. Grain yields and total N of sorghum in sole crop and intercrop are increased by delayed N application.8 Water and soil nutrients, especially nitrogen, are the two major natural resources whose internal and external supply are limited in SAT. For stabilization and increase in crop productivity, it is important to improve the utilization efficiency of these limited resources. In the case of nitrogen, approaches to increase nitrogen use efficiency may be: 1. To improve nitrogen uptake efficiency of crops through conventional and molecular breeding; 2. To increase the gain from biological nitrogen fixation; and 3. To alter the balance between uptake and other components of nitrogen loss by denitrification, volatilization, runoff and leaching. The crop and resource management options for improvement of nitrogen use efficiency in SAT will include: 1. Changing fertilizer application methods such as timing and placement; 2. Selecting varieties with deeper roots; and 3. Developing a cropping system best suited to the local environments. It is an extremely difficult task to improve productivity of the dry-land agriculture, but reconsideration of crop and resource

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Fig. 8.4. Effect of basal (BAS) and delayed (DEL) applications of urea on nitrogen use efficiency of pigeonpea and sorghum in monocropping and intercropping.

management in relation to the cropping system may provide resource-poor farmers with a new option, which may at least stabilize crop production and hopefully increase return on the investment.

References 1. Pingali PL, Hossain M, Gerpacio, RV. Asian rice bowls: The returning crisis? Wallingford, CAB International, 1997:341. 2. Katayama K, Ito O, Matsunaga R, AduGyamfi JJ, Rao TP, Anders MM. Nitrogen balance and root behavior in four pigeonpea-based intercropping systems. Fert Res 1995; 42:315-319. 3. ICRISAT (International Crops Research Institute for the Semi-Arid Tropics). Soil, crop, and water management systems for rainfed agriculture in the Sudano-Sahelian Zone. ICRISAT Sahelian Center, Niamy,

Niger. Patancheru, India: ICRISAT, 1989:385. 4. Troll C. Seasonal climates of the earth. In: Rodenwalt E, Justkatz H, eds. World Mapsof Climatology. Berlin: Springer Verlag, 1965:28. 5. Willey RW. Evaluation and presentation of intercropping advantages. Exp Agric 1985; 21:119-133. 6. Ali M. Pigeonpea: Cropping systems. In: Nene YL, Hall SD, Sheila VK eds. The Pigeonpea. Wallingford: CAB International, 1990:279-302. 7. Tobita S, Ito O, Matsunaga R et al. Field evaluation of nitrogen fixation and Nfertilizer utilization by sorghum/pigeonpea intercropping on an Alfisol in the Indian

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semi-arid tropics. Bio Fert Soils 1993; 17:241-248. 8. Adu-Gyamfi JJ, Ito O, Yoneyama T, Gayatri Devi, Katayama K. Timing of N fertilization on N2 fixation, N recovery and soil profile nitrate dynamics on sorghum/pigeonpea intercrops on Alfisols on the semi-arid tropics. Nutrient Cycling in Agroecosystems 1997; 48:197-208. 9. Nielsen Ne, Hensen HE. Nitrate leaching from loamy soils as affected by crop rotation and nitrogen fertilizer application. Fert Res 1990; 26:197-207. 10. Gayatri Devi, Ito O, Matsunaga R, Tobita S, Rao TP, Vidya Lakshmi N, Lee KK. Simulating root system development of short-duration pigeonpea. Exp Agric 1996; 32:67-78. 11. Ito O, Matsunaga, R, Tobita, S, Rao, TP, Gayatri Devi. Spatial distribution of root activity and nitrogen fixation in sorghum/pigeonpea intercropping on an

Indian Alfisol. Plant Soil; 1993 155/ 156:341-344. 12. Ito O, Katayama K, Adu-Gyamfi JJ, Gayatri Devi, Rao TP. Root activities and function in component crops for intercropping. In: Ito O, Johansen C, Adu-Gyamfi JJ et al, eds. Roots and nitrogen in cropping systems of the semi- arid tropics. Japan International Research Center for Agricultural Sciences (JIRCAS), Ibaraki, Japan. JIRCAS International Agriculture Series No. 3. Tsukuba: JIRCAS, 1996:453-468. 13. Ito O, Matsunaga R, Tobita S et al. Nitrogen fertilizer management in pigeonpea/sorghum intercropping on an Alfisol in the semi-arid tropics. In: Ando T, ed. Plant Nutrition For Sustainable Food Production and Environment. Dordrecht: Academic Press, 1997:689-890.

CHAPTER 9

Sustainable Irrigated Agriculture in Arid Lands: Kazakstan Case Study T. Yano and S. Wang

Introduction

D

esertification in arid regions is caused mainly by misuse of soil and water resources. In the 1960s, the intensive land development program in the Aral Sea region promoted by the former Soviet Union, triggered a series of environmental and socioeconomical problems. In Kazakstan, economical gains in cultivating agricultural crops on the original pastoral land by using water from the Syr Darya and Amu Darya Rivers have been achieved at a great expense, degradation of the environment in these river basins. In the Kzyl Orda region, rice cropping is popular and the total rice area in the region is about 93,000 ha, which is equivalent to 36% of the total irrigated area.1 Due to inappropriate water management, however, salt accumulation has occurred in farmlands, resulting in the increase of abandoned lands. It is estimated that, in the Kzyl Orda region of Kazakstan, about 60% to 70% of the total irrigated area has been salt-affected. Since salt accumulation in abandoned lands worsens the soil environment of surrounding cultivated lands due to salt transport, they should be reclaimed as much as possible. Although reclamation research and practice has a long history throughout many areas of the world, there is a great necessity to establish siteoriented techniques because of the diversities in the climate, composition of soil parent material, soil texture, salt types, and drainage conditions in the targeted area. Even after salt-affected lands are improved through reclamation practices, they are subject

to secondary salinization, provided suitable management measures are not taken. In this paper, we discuss the situation of salt accumulation in the Kzyl Orda region, a reclamation study aimed at achieving the optimum reclamation techniques for this region, and a soil permeability study to clarify the effect of water quality on hydraulic conductivity, information necessary to improve current water and soil management to avoid secondary salinization of reclaimed lands.

Present Situation of Salt Accumulation in the Study Area Outline of the Study Area A collective farm (kolkhoz) located in the Kzyl Orda region was selected as a study area. The region is in a typical arid continental temperate zone, characterized by a high solar radiation of 0.16 to 0.19 kW/m2, a mean annual temperature of 9°C and annual precipitation of less than 150 mm. Though the farm has a gross area of 19,000 ha, only 1,900 ha or 10% has been reclaimed for agricultural purposes. Out of the 1,900 ha of reclaimed land which have been cultivated for farming, an area of 600 ha has been abandoned without cropping due to severe salt accumulation. This situation is commonly seen in all the collective farms in the region. An eight year crop rotation system has been widely practiced in the region. The system is composed of rice cropping for the first and

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second year, wheat cover-cropped with alfalfa for the third year, alfalfa for the fourth and fifth year, rice for the sixth and seventh year and corn (forage) or fallow for the eighth year. As the soil fertility which was lost during rice planting can be replenished through alfalfa planting, as well as the alfalfa fields being irrigated through percolated water from adjacent rice fields under the system, this system has been recommended by researchers and adopted by farmers in this region. In most rice producing areas in Kazakstan, this cropping pattern has been the basic for design and construction of new rice land development. Growing rice minimizes salt accumulation in the soil profile through leaching effects in the ponding period. However, there is a possibility that this system has induced waterlogging and salt accumulation in upland areas adjacent to rice fields in and/or out of the irrigation block. Two sites in an alfalfa field and a nearby deserted field were intensively sampled. Soil samples were measured for electrical conductivity (EC), particle fractions and chemical compositions. Part of the result is shown in Table 9.1.2 Due to high evaporation and less rainfall, the abandoned field was covered with a layer of white salt crust, whereas there was no visual salt accumulation on the soil surface in the alfalfa field. However, EC of the soil saturation extract (ECe) of the alfalfa field was in a range of 15-30 dS/m (decisiemens per meter =millimhos/cm, mhos = ohms-1), around 4 times higher than the critical value demarcating a saline soil. In the deserted field, ECe was as high as 80-100 dS/m, and at least 5 times higher than that in the alfalfa field. The sodium adsorption ratio (SAR = Na/(Ca + Mg)1/2, unit in nmoles per liter) of the saturation extract was higher than 50 for the deserted field and 30 for the alfalfa field. From the data presented above, the soils both in cultivated and abandoned lands were highly saline. Among other factors that attributed to soil salinization, such as high evaporation, less rainfall and low drainage capacity, the current cropping pattern has its own share of blame. The irrigation water from the Syr Darya River has 1.6 dS/m in EC and 4.5 in SAR. However, in the rotation, paddy rice is usually supplied with excess water with the intention to leach salt from the soil profile, whereas

upland crops in the adjacent fields receive no irrigation water from the surface. Thus, ground water is replenished and elevated during ponding when cultivating paddy rice, but concentrated during non-irrigation periods. This allocation of irrigation water has made the ground water more concentrated after each rotation. Affected by the high evaporation rate on the soil surface, salts accumulate in the profile through capillary rise, resulting in soil salinization in the non-rice growing fields.

Water Balance and Salt Balance There are four major irrigation blocks and some minor blocks in the farm, and the irrigation season starts at the end of April and ends at the end of August. One irrigation block was selected for the study on water and salt balance. In this block, an area of 350 ha was under cultivation for rice, whereas an area of 330 ha was under cultivation for alfalfa. Since this block was recently developed, the area of abandoned fields was only 10 ha. Water and salt balance was analyzed to clarify the influence of water management on salt accumulation, for developing a proper water management technology. Water balance in the block was analyzed by measuring flow discharge both at the inlet of the irrigation canal supplying water to the block and at the outlet of the main drain from which drainage water from the farmlands is collected. The water balance situation in the block for 89 days during May 19 to August 16, 1997 shows that, during this period, 18 million m3 of water were introduced to the block. Of this amount, a little less than 9 million m3 of water (a little less than 50%) was consumed and the rest, a little over 9 million m3 of water (a little over 50%), is drained off to the river. When the water volume is converted to water depth by dividing by 680 ha of the total area of the irrigation block, the amount of water intake to the block is equialent to 30 mm/d, water consumed in the whole block is approximately 15 mm/d, and the balance of 15 mm/d of water is drained out. Thus, a large quantity of water is introduced, consumed and drained in the block, notwithstanding the water-scarce arid land. The water consumption in the block consists of crop consumption for rice and alfalfa,

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Table 9.1. Some physical and chemical properties of the two soils Deserted field Depth (cm)

Clay fraction (%)

pH

ECe (dS/m)

Major ion content (cmolc/kg) Ca2+ Mg2+ Na+ ClSO42-

SAR

0-5

13.5

7.1

99.3

0.3 22.7

20-40

17.3

7.1

100.0

60-80

37.2

7.6

91.2

pH

ECe (dS/m)

Major ion content (cmolc/kg) Ca2+ Mg2+ Na+ ClSO42-

97.8

103.0

18.1

200.6

0.9 6.3

17.6

18.1

6.6

63.2

1.5 12.6

31.2

30.5

15.5

57.6

Alfalfa field Depth (cm)

Clay fraction (%)

SAR

0-5

41.8

7.6

24.2

1.1

4.6

11.5

6.7

10.6

40.3

20-40

33.2

7.7

16.5

1.1

2.9

8.1

7.1

7.6

35.2

60-80

27.2

6.9

16.1

1.2

3.0

7.6

5.6

7.2

39.4

seepage and percolation in farmlands, ground water outflow to the adjacent areas and canal seepage. Since evapotranspiration (ET) from paddy rice is 8-10 mm/d at maximum, and ET from alfalfa is less than that from rice, excessive water results in ground water rise, due to inadequate functioning of the existing drainage systems. Water quality analysis was carried out at the farm, and its spatial as well as seasonal changes were identified. The EC shows higher values on the downstream side as compared with that of the upstream side in the irrigation and drainage system. For instance, at the beginning of May, EC was 1.3 dS/m in the Syr Darya River, 1.4 dS/m at the head of the main canal, 1.5 dS/m in the rice field of the block, and 2.6 dS/m at the end of the drain, respectively. Since water quality becomes worse in the Syr Darya River in the latter part of irrigation season, the values of the EC increase throughout the water system in the latter stages of crop growing. The amounts of salt inflow and outflow were calculated by multiplying the total dissolved solids (TDS) by the observed flow discharge. TDS was estimated using the linear relationship between EC and TDS. Salt balance in total volume for 89 days from May 19 to

August 16, 1997 showed that total salt inflow to the block amounted to 26,000 tons (38 ton/ ha or 427 kg/ha/d), whereas total salt outflow was 22,000 tons (32.6 ton/ha or 366 kg/ha/d). Thus, the balance amounting to 3,700 tons (5.4 ton/ha or 61 kg/ha/d) remained in the block. This can be regarded as the amount of the annual accumulation of salt. The places where salt accumulation occurs are not rice fields, but adjacent areas outside the block and alfalfa fields where percolated water is supplied from rice fields. Especially, salt accumulates in soil layers where water is supplied by capillary rise from the ground water. This tendency could be explained by variations in ground water level and EC value of the alfalfa field located at 300 m from the edge of the paddy fields. Ground water rose from the initial level of about 2 m depth below the surface before the irrigation season to a maximum level of 1-1.5 m. The initial EC values of 6-7 dS/m before irrigation decreased quickly to a value of 1.5 dS/m, equal to the EC of irrigation water, and increased gradually up to 6.5 dS/m at the end of irrigation season. Ground water levels as shallow as 1-1.5 m promote evaporation due to capillary rise and become a continual source of salts to the crop root zone. Although a high ground water level has the advantage of

110

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

providing moisture supply for those upland crops without any surface irrigation facility, the salinity problem cannot be controlled without lowering the ground water table to the permissible depth of at least 2 m in most soils.

Reclamation of Salt-Affected Soils Reclamation of salt-affected soils by leaching is commonly used, and considered to be one of the most feasible and economical choices. Leaching efficiency is a function of: 1. Soil texture; 2. Water application modes (intermittent ponding or sprinkling or continuous ponding); 3. Types and amount of salt present, etc. Leaching efficiency has been comparatively studied in both field and laboratory conditions. In the field study, the traditional leaching method was considered; ponding water continuously on the soil surface was tested; whereas, in the laboratory, both continuous ponding and intermittent ponding were examined in terms of leaching efficiency. The abandoned field of 2.5 ha that was previously investigated was selected as the site for soil reclamation study for the farm. Four subplots, each 10 m wide and 150 m long, were prepared in the experimental site for a leaching study in 1997. Irrigation water was conveyed into each plot and ponded on the soil surface continuously, at water depths of 372, 677, 937, and 1,248 mm, respectively. Soil was sampled in each plot at three locations before and after leaching, at the depths of 5, l0, 20, 40, 60, 80, 100, 120, 140, and 160 cm, respectively. The air-dried soil samples were analyzed for EC of extracts at soil to water ratio of 1:5 (EC1:5), and for chemical composition. In the laboratory experiment, soil samples from the A horizon of the experimental site (sandy loam) were used for soil columns (50 mm in diameter and 100 mm long). Irrigation water simulating the composition of the irrigation water on the site was applied to the columns from the top by two methods— intermittent ponding and continuous ponding. In intermittent ponding, water was ponded at 0.1 pore volume (PV) per day. One pore volume was 90.7 ml for the soil (46.2 mm in depth). Under continuous ponding, a constant

water head was kept at 10 mm and a fraction collector was used to collect the effluents. All the effluents were measured for volume and EC. The experiment was terminated after 1.8 PV of the leaching water passed through the soil columns. Then, the soil columns were sliced at 20 mm intervals and the sections were oven dried at 105°C. EC and soluble salts were measured in the saturated soil solution. Fig. 9.1 shows the relationship between the soil EC change after leaching and the amount of leaching water.1 EC1:5 and ECe were used as the soil EC in the field and laboratory experiments, respectively. The ordinate axis was expressed by the fraction (ECf - ECw)/(ECi -ECw), where ECw is the EC of irrigation water, ECi and ECf are the soil EC (dS/m) before and after leaching, respectively. The abscissa axis was expressed by the amount of water leaching through the profile per unit depth of soil, Dw/Ds. The fitting equation, showing a power relationship, is given in Fig. 9.1(a). In the field experiment, when Dw/Ds= 1, the reduction in soil EC1:5 was 50%, which means that only half of the initial salt in the soil was removed. Over the whole profile, a high reduction in soil EC was obtained within 50 cm, and around 60% of the initial salinity still remained in the deeper layers irrespective of the amount of leaching applied. The leaching efficiency apparently was lower than the value of 70% or more shown by Hoffman.3 In the laboratory experiment, there was no observed difference in leaching efficiency with continuous ponding (CP) and intermittent ponding (IP). This was in agreement with Hoffman’s result that leaching efficiency was less affected by leaching methods on the sandy loam soils. 90% of the initial salt was removed when Dw/Ds = 1, which is significantly higher compared with the field result. The different leaching efficiencies of the field and column experiments possibly resulted from the different pore-size distribution; soil was homogeneous in the column, but heterogeneous in the field. In both cases, around 10% of the initial soil salinity was remained even under high water application. Since the soil contained considerable amounts of gypsum, gypsum dissolution may be the main reason for the decline in leaching efficiency.

Sustainable Irrigated Agriculture in Arid Lands: Kazakstan Case Study

A

111

more uniform. Therefore, unsaturated water flow occurred under both intermittent ponding and continuous ponding. As a result, leaching efficiency was similar under both intermittent ponding and continuous ponding.

Permeability of Salt-Affected Soils

B

Fig. 9.1. Leaching curves from both field and laboratory experiments. (a) y = 0.544x-0.524 represents the fitting equation for the graph, with the square correlation efficient, R. (b) IP, intermittent ponding; CP, continuous ponding. Desalinization of soil and leaching efficiency depend on salt transport processes which are affected by water application modes (saturated or unsaturated flow), soil texture, initial water content etc. Intermittently ponding water on the soil surface usually causes unsaturated water flow in the soil. On the other hand, continuous ponding creates saturated water flow. Salt leaching is efficient where unsaturated water flow prevails, and vice versa. In soils where more aggregates exist, soil pore size distribution is more diverse, and consequently large differences exist in water flow caused by the various water application methods. In the column study with sandy loam, because of the low clay content, 14%, there are few aggregates and pore size distribution is

The rate of infiltration, which refers to the rate of water entry into the soil, generally increases with increasing salinity and decreases with either decreasing salinity or increasing sodium content relative to calcium and magnesium (SAR). A column study on the effect of water quality on saturation hydraulic conductivity was conducted in the laboratory. Silty clay that contained 40% clay from the B horizon of the experimental plot was tested. Soil samples were packed into plastic cylinders with a length of 50 mm and an internal diameter of 50 mm at the bulk density of 1.4 g/cm3. Columns were first saturated with a solution that contained 0.5 molc/l (moles of charge per liter) Cl- (CaC12 and NaCl) at SAR = 10. After saturation, the column was leached consecutively with solutions of the same SAR but diluted concentrations (0.05, 0.01 molc/l) and distilled water. EC of the solution was 50 dS/m for 0.5 molc/l, 5 dS/m for 0.05 molc/l and 1 dS/m for 0.01 molc/l solution, respectively. The effluent was collected with a fraction collector and saturation hydraulic conductivity (HC) was measured. Figure 9.2 shows the relative HC as influenced by water quality and the cumulative effluent volume. The relative HC is the ratio of treatment HC and HC at 0.5 molc/l concentration. HC of the soil at 0.05 molc/l concentration was not changed with the increase of effluent volume, and kept constant at 10 mm/ hr. HC was only 60% and 40% a with leaching solution of 0.01 molc/l and distilled water compared with the 0.5 molc/l solution, respectively. This decrease in the soil HC resulted from clay dispersion and/or swelling. When the concentration of soil solution becomes lower than a certain value, clay swelling and/or dispersion occur, causing soil pores to be narrowed or clogged, and HC of the soil will decrease. Clay swelling is the major cause for the decrease of soil HC when soil ESP (Exchangeable Sodium Percentage) >15,

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 9.2. Effects of concentration and SAR of water on the relative hydraulic conductivity. The lines represent SAR of 0.05 molc/l, 0.01 molc/l and distilled water (D). whereas clay dispersion is responsible for the decrease of soil HC when ESP <15.4 As leaching continues, the soil ESP decreases, owing to desodification of adsorbed salts. Thus, the decrease in soil HC was considered to be due to clay dispersion. Since the decrease in soil permeability affects the irrigation efficiency as well as the leaching efficiency, use of flocculant as a soil conditioner to improve the soil permeability may be promising for water and soil management.

Conclusion Based on a series of on site water and salt balance studies, it was suggested that the ordinary eight year rotation system and overirrigation to rice fields have accelerated salt accumulation and concurrently increased abandoned farmlands in the farm. In order to improve the present undesirable situation, it is necessary to lower the ground water table and increase the surface water supply for upland crops. To reclaim the highly saline soils, leaching experiments were conducted both in the field and laboratory. In both conditions, soluble salts cannot be removed completely from the soil, possibly due to gypsum dissolution. 50% of the initial salinity remained in the soil when Dw/Ds = 1 in the field, whereas in the column experiment with sandy loam soil, only 10% of the initial salinity remained in the soil. Leaching efficiency was similar between intermittent ponding and continuous

ponding, and significantly higher than the field result. However, it is expected that higher leaching efficiency will be obtained by ponding water intermittently on the soil surface in the field where the soil texture varies from sandy loam to heavy clay. The soil saturated hydraulic conductivity was measured using leaching solutions with different concentrations: 0.5, 0.05, 0.01 molc/l (SAR = 10) and distilled water. Similar results in the soil HC were observed when leached with solutions of 0.5 and 0.05 molc/l. However, a decrease of the soil HC occurred with continuing leaching with 0.01 mol c/l or distilled water. Clay dispersion possibly caused this decrease. To prevent clay dispersion, the application of flocculant is considered to be promising for keeping the leaching efficiency and irrigation efficiency high.

Acknowledgments We thank Dr. A. Rau of the Kazakstan Academy of Sciences for conducting the field experiments in Kazakstan. This research was partly supported by the Global Environmental Research Fund from the Environment Agency of Japan.

References 1. Ogino Y, Hatcho N, Tsutsui H. World irrigation (VIII): Salinization and water management in arid regions with emphasis on Kazakhstan. Rural and Environmental Engineering 1998; 34:5-24. 2. Wang S, Kitamura Y, Yano T. Salt accumulation and reclamation of soils in Kzyl-Orda, Kazakstan, Proc. International Symposium on Arid Region Soil. IzmirTurkey, Sept. 21-25, 1998:120-125. 3. Hoffman GJ. Guidelines for reclamation of salt-affected soils. Appl Agr Res 1986; 1:65-72. 4. Keren R, Shainberg I. Colloidal properties of clay minerals in saline and sodic solution. In: Sahinberg I, Shalhevet J, ed. Soil Salinity under Irrigation. Berlin: Springer-Verlag, 1984:32-45.

CHAPTER 10

Distribution and Amelioration of Alkali Soils in Northeast China S. Matsumoto

Introduction

A

lkalinization of soils has expanded rapidly in many irrigated areas of the world due to man’s misuse of the land. Alkalinization changes soils to a much poorer support for vegetation than is the case with salinization. Their adverse effects on plant growth can be attributed to impaired aeration, restricted rooting depth, interference in nutrient uptake and plant metabolism, corrosion of root surface, and sodium toxicity.1 In the same way as soil salinization, long periods of dry and warm or hot weather are important factors in the formation of alkali soils. Another important factor is hydrology: high groundwater level, intensive irrigation, and inundation by the sea or brackish water. Relief is also often important, because alkalization is most severe in depressions or at the edge of depressions. Alkalinization of soils is characterized by the formation of soils with a high percentage of exchangeable sodium; often sodium carbonate and sodium bicarbonate also dominate, increasing the pH to beyond 8.5, often to 9 or 10. Clay particles and humus are easily dispersed and eluviated from the A to B horizon under alkali reaction, and accumulate as a natric subsurface horizon. A natric horizon is characterized by a very hard pan with prismatic structure formed by clay and humus coatings on the ped surfaces.2 The total area of the alkali soils in the arid and semi-arid regions of China reaches to 320,000 km2. The very important fact that more than 50,000 km2 of Mollisol distributed

in semi-arid regions of China suffer from alkalinization has been revealed by the recent soil surveys carried out by Japanese and Chinese soil scientists.3 Therefore, it would contribute to the more desirable condition of self sufficiency for food in China to rehabilitate these Mollisols suffering from alkalinization, because normal Mollisol generally shows high productivity. To reclaim alkali soil, a two step process has been used. The first step involves the replacement of exchangeable sodium with calcium; the second step is to leach the resulting sodium from the soil. Leaching alone of a calcareous soil on which a crop is growing can reclaim alkali soils, but the process is slow. Of the amendments used to bring about exchangeable sodium replacement with calcium, gypsum (hydrated calcium sulfate) is far and away the most used material, because it has the advantages of being nontoxic to plants, easy to handle, and moderately soluble. However, using gypsum as an amendment material for alkali soils has been barely put into practice in China. The main reason is that demands for gypsum in China are pressing, and its agricultural utilization as a soil amendment is not common due to the market price. Incidentally, the acid rain and air pollution originating from sulfur dioxide released from coal combustion furnaces is one of the serious environmental problems in China. Hence, it would be considered truly a case of killing two birds with one stone if we can

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

produce gypsum from the desulfurization process of each coal combustion facility at power stations and factories, and use it as an improvement reagent for sodic soil. In this report, the possibility of using the byproduct from the desulfurization process for amelioration of alkali soil will be examined for its possible incentive to the construction of desulfurization processing in China.

Alkali Soil Formation, Characterization and Its Distribution in China Alkali soil is formed when soil colloidal materials are inundated for a long period with groundwater whose chemical composition is predominantly sodium carbonate or sodium bicarbonate. Fig. 10.1 shows the concept of alkali soil formation. These sodium salts in the groundwater are produced when carbon dioxide in soil or atmosphere is absorbed by sodium hydroxide, which is hydrolyzed from sodium ions. With ionic absorption on the surface of soil colloids, predominantly by sodium ions, soil colloidal materials become dispersed, forming very compact, hard structures of high pH, beyond 9, as they dry. The layer cohered from sodium colloids is called a natric horizon, and will become a soil so undesirable as a medium for plants that almost all of crops even weeds cannot grow at the presence of the horizon. Alkalinization of soils once formed change to much poorer soils for vegetation than the case of salinization because soil condition changes to impaired aeration, restricted rooting depth, interference in nutrient uptake and metabolism, corrosion of root surface and sodium toxicity. The existing hard horizon with prismatic structure formed by clay and humus coating on the ped surfaces makes also very difficult to leach excess salts from soil. Alkali soils distribution in China are found in the arid and semi-arid regions of Inner-Mongolia, Jillian, Heilonjiang, Liaoning, Xinjiang-Uygur Autonomous, Heibei, Gansu, Shaanxi and Qinghai provinces and especially the distribution in the northeast and north China of latitude 40 degree north is intensified. And its acreage is estimated more than

100,000 km2.4 According to the criteria of saline soils,1 the saline soils are classified to three types due to TSS (Total Soluble Salts), pH and ESP (Exchangeable Sodium Percentage) as shown Table 10.1. And Tables 10.2 and 10.3 give the chemical properties of non-alkali soils (Haplic Solonchak) and alkali soils (Alkali Solonchak) distributed in northeast China. The most characteristic difference between non-alkali soil and alkali soil is found in the content of calcium, magnesium, and carbonate and bicarbonate ions in the solution extracted from soil by distilled water. In northeast China, the distribution of alkali or alkali-saline soil is also related to the topographical characteristics of the land; they usually are found in the areas with high groundwater tables, or in low lying ground. These areas are favorable for forming sodium carbonate or bicarbonate, because the sodium ion is more mobile than other cations, and because carbonate and bicarbonate originate from microbial decomposition of the organic matter which predominates in wet locations. Corn cultivation was originally very popular among Chinese farmers because of it produces a large biomass as well as seeds. However, its recent crop cultivation in upland soils of China has been more popular than before. The reason lies in the fact that the consumption of meat in China has been increased largely in the past decade owing to Chinese economic growth, and that corn cultivation has been stimulated by subsidy from the Chinese government. As water consumption of corn cultivation is estimated at 7 to 8 times that of wheat,5 a large amount of irrigation water has applied to cornfields during the vegetative stage in the summer season. The observed data from groundwater levels of wells in the Kangpin district, Liaoning province, which lies in the middle of the corn cultivation area, show that the groundwater level changed from 4 to 12 m in the last eight years.6 The large, rapid declines of groundwater levels are also found in many upland areas not only in northeast China but also in China as a whole; this fact suggests that there are still high possibilities of expanding soil alkalinization.

Distribution and Amelioration of Alkali Soils in Northeast China

Fig. 10.1. Concept of alkali soil formation.

Table 10.1. The classification of saline soils EC (dS/M)

pH

ESP (%)

Saline soil

>4

<8.5

<15

Alkali soil

<4

>8.5

>15

Saline-

>4

>8.5

>15

alkali soil EC: electric conductivity; ESP: exchangeable sodium percentage.

Reclamation of Alkali Soils

115

maintenance of hydraulic conductivity by providing a sufficiently high electrolyte concentration in the soil solution to counter the influence of exchangeable sodium. The reason it is necessary to apply a sufficiently high electrolyte concentration is that low salinity water such as rainwater makes clay swell, and swelling clay leads to low impermeability which impedes the leaching salts. Figure 10.2 illustrates the concept of alkali soil amelioration by calcium sulfate. Generally, the higher the electrolyte concentration, the higher the exchangeable sodium fraction at which a relatively high permeability can be maintained.7 The electrolyte concentration affects the hydraulic conductivity less when the content of soil water is low.8 According to our laboratory experiments,9 among the amendments used to bring about exchangeable sodium replacement with calcium, gypsum (CaSO4nH2O, hydrated calcium sulfate) is far and away the most effective amendment material.

Concept and Principles

The Chemical Composition of Byproducts from Desulfurization Processes

Reclamation of alkali soils usually involves a two step process. The first step is replacing exchangeable sodium ions with calcium ions. This calcium may originate from the dissolution of Ca-containing minerals in the soil, such amendments as gypsum and calcium chloride, or irrigation water with calcium ions. A second step involves leaching the resulting sodium salt from the soil. A significant factor in reclaiming alkali soils is the

The most widespread desulfurization process in the developed countries is the wet lime slurry method, whose byproduct is mostly pure gypsum. In the developing countries, on the other hand, a semi-dry process tends to be adopted, owing to its low construction and running costs. However, the byproducts from a semi-dry process contain Ca(OH)2 (calcium hydroxide), CaCO3 (calcium carbonate) and fly ash, as well as gypsum. Among several

8.1

8.3

7.9

7.7

7.9

0-32

32-55

55-90

90-100

100-150 70.7

66.0

69.0

89.0

171.1

TSS g/kg)

0.01

0.05

0.09

0.09

0.04

CO32-

0.45

0.30

0.27

0.20

0.22

HCO3-

TSS; Total soluble salts; ionic determination was done by water extraction in soil: water=1:5.

pH

Depth (cm)

56.4

40.0

30.0

28.2

224.0

15.6

17.7

19.9

17.4

53.0

ClSO42cmol/kg soil

Table 10.2. Salt composition of soil profile of Haplic Solonchak (non-alkali soil)

24.6

20.9

14.6

13.9

29.1

Ca2+

30.2

29.8

28.0

30.0

34.9

Mg2+

30.2

30.6

29.7

30.5

190.0

NA+ + K+

10.9

9.5

10.06

10.5

12.8

ESP (%)

116 Challenge to the Crisis of the Earth's Biosphere in the 21st Century

10.6

10.3

10.4

10.6

10.4

9.0

0-2

2-17

17-31

31-57

57-80

80-100 1.9

3.9

4.5

6.9

9.9

20.9

TSS (g/kg)

1.5

2.0

2.6

1.9

2.1

21.9

CO32-

2.0

3.3

4.0

3.8

3.8

3.5

HCO3-

TSS; Total soluble salts; ionic determination was done by water extraction in soil: water=1:5.

pH

Depth (cm)

1.1

2.1

3.3

4.9

5.3

8.4

0.2

0.4

0.4

0.6

0.9

3.6

ClSO42cmol/kg soil

Table 10.3. Salt composition of soil profile of Alkali Solonchak

0.07

0.20

0.10

0.02

0.03

0.02

Ca2+

0.09

0.19

0.15

0.07

0.04

0.07

Mg2+

9.9

17.5

10.5

19.4

18.9

38.7

NA+ + K+

20.9

42.9

50.1

70.4

60.3

78.9

ESP (%)

Distribution and Amelioration of Alkali Soils in Northeast China 117

118

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 10.2. Concept of reclamation of Alkali soils by calcium sulfate. semi-dry processes, the byproducts from the ash recovery activation method (ARAM) were examined, because the Japanese government has already installed a working plant at Qingtao City, Shandong province in China. ARAM is the process by which a slurry of the polycrystal substance called Ettringite, which is formed through hot water treatment of a mixture of CaO (calcium oxide), fly ash and gypsum, is sprayed into the flue gas to absorb SO2 (sulfur dioxide) as gypsum.10 The chemical properties of byproducts from desulfurization processes are shown in Table 10.4. The byproduct from the wet process is almost pure gypsum, while the byproduct from the semi-dry process includes only 20 wt % gypsum and 20 wt % calcium hydroxide, which would increase the pH of the soil. Until now, there have been very few applications of the byproduct from the semi-dry process to the alkali soils in China, because it is considered to be only waste.

The Effect of Byproduct Application from the Semi-Dry Process to Alkali Soil Using soil samples classified as alkali soil (Table 10.5), pot cultivation experiments were conducted. Air dried alkali soils were mixed thoroughly with the byproduct from wet process at levels of 0, 0.1, 0.3, 0.4, 0.5, 0.7 and 0.9 wt %, and with the byproduct from semi-dry process at the levels of 0, 0.5, 1.5 and 2.5 wt %, respectively. These eleven mixtures were placed in separate pots (1/5000a; "a" is

Table 10.4. Chemical properties of the by-products Wet process

Semi-dry process

7.6

12.6

wt%

wt%

CaSO4

88

20

CaSO3(%)

0

35

ph

equivalent to 100m2, so 1/5000a is 0.02 m2.) and wheat grains were sown in the upper layer of each pot. The pot cultivation experiments were undertaken by triple repeat tests in the green house using to the normal cultivation method. After harvesting, wheat grains obtained from each pot were measured by weight; the results are shown in Figures 10.3 and 10.4. The productivity of alkali soil was increased at each level by the addition of the byproduct from wet process; an effect of increasing additive was not recognized. On the other hand, an amelioration effect to alkali soil by byproducts from the semi-dry process was recognized only at the level of 0.5 wt % addition, and addition of byproduct over 0.5 wt % showed much lower productivity of wheat than that of alkali soil. This result seems to be attributable to the fact that the pH level was

Distribution and Amelioration of Alkali Soils in Northeast China

119

Table 10.5. Chemical Properties of the soil used ph

9.8

EC(dS/M)

0.84

CEC (cmol(+)/kg soil)

6.5

Exchangeable cation (cmol(+)/kg soil) Ca

10.7

Mg

1.7

K

1.1

Na

7.6

ESP (%)

36

Fig. 10.3. Effect of calcium sulfate (wet process by product).

EC: electric conductivity; CEC: cationexchangeable capacity; ESP: exchangeable sodium percentage

increased with the addition of calcium hydroxide included in the byproducts from the semi-dry process. Therefore, I evaluate that in the case of amelioration of alkali soil by byproducts from the semi-dry process, the maximum addition should be 0.5 wt %. Results of soil analyses after harvesting the wheat are shown in Table 10.6. In the table, data of two 0.5 wt % plots in the pot experiment are given to show the amelioration effect by the byproduct from the semi-dry process, and the lasting effect of the amendment as well. The table gives us the facts that the byproduct from wet process have decreasing effects over time on pH and exchangeable sodium percentage (ESP) of alkali soil, and that the addition of an amount over 0.5 wt % of the byproduct from semidry process has little decrease over time in effects on pH and ESP of alkali soil. The persistence of the amendments increased remarkably over the cultivation period.

Fig. 10.4. Effect of byproducts from the semidry process.

Conclusion The possibility of using the byproduct from desulfurization processes for amelioration of alkali soil is examined, to provide incentives for the construction of desulfurization processing facilities in China. It was shown that applying mixtures of 0.5 wt % of the byproduct from semi-dry process, which tends to be adopted owing to its low construction and

wet

semi-dry

control

control

wet

semi-dry

wet

semi-dry

1

2

3

4

5

6

7

8

No.

Process source of Gypsum

1.5

0.9

0.5

0.5

0

0

0.5

0.5

wt(%)

8.65

8.00

8.03 88.05

8.32

7.70

9.10

9.20

5.23

7.09

9/24

10.35 10.05 9.90

9.55

10.10 8.20

9.80

10.37 9.60

10.52 9.47

8.42

10.12 8.20

5/20 8/2

ph

5.20

5.45

6.29

6.24

5.18

4.83

8.34

5.15

8.23 8.75

8.49 8.22

8.65 9.212

7.39 8.26

5.99 4.82

5.16 5.72

8.66 4.57

8.38 6.28

5/20 8/2 9/24

Ca2+

Table 10.6. Chemical analyses of soil after harvesting of wheat

3.61

5.34

3.35

2.13

2.40

2.81

3.08

4.60

5/20

2.32

2.99

1.95

1.41

1.69

2.30

3.00

2.59

8/2

Mg2+

2.28

2.47

1.44

0.27

1.74

2.50

0.128

1.99

9/24

1997 8/2 9/24

0.014 0.226 0.232

0.112 0.271 0.63

0.056 0.346 0.219

0.126 0.265 0.230

0.000 0.205 0.170

0.030 0.727 0.336

0.274 0.246 1.99

0.064 0.207 0.266

5/20

K+

1.49

1.05

2.50

2.52

2.91

4.36

0.84

2.20

5/20

0.06

0.00

0.86

0.62

3.27

3.48

0.00

0.42

8/2

Na+

0.17

0.00

0.40

0.00

2.35

2.13

16.7

0.00

9/24

14.5

8.0

20.5

22.6

27.7

36.3

6.7

17.8

12.2

0.0

7.4

6.4

29.3

29.9

0.0

3.6

5/20 8/2

12.9

0.0

3.6

0.0

25.9

20.0

9.72

0.0

9/24

ESP(%)

120 Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Distribution and Amelioration of Alkali Soils in Northeast China running costs, to alkali soil have the same amelioration effect on such soils as the byproduct from the wet lime slurry used in developed countries. However, application to alkali soils of more byproduct than 0.5 wt % from the semi-dry process has a deteriorating effect on amelioration, due to the persistent increased pH of the soil.

References 1. Buringh P. Introduction to the Study of Soils in Tropical and Subtropical Regions. Wageningen: Center for Agricultural Publishing and Documentation, 1970:29-44. 2. Tanji KK ed. Agricultural Salinity Assessment and Management. New York: American Society of Civil Engineers, 1990:410-431. 3. Matsumoto S, Zhao QG, Yang J et al. Soil salinization and its environmental hazard on sustainable agriculture in east Asia and neighboring regions. Global Environment 1998; 1:75-81 4. Wong CL. Saline Soils in China. Xian: Shaanxi Academic Press, 1992:25-50.

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5. Arnon I. Agriculture in Dry Lands, Principles and Practice. Amsterdam: Elsevier, 1992:585-630. 6. Chinese Water and Power Authority. Changes of Groundwater Level in Northeast China. Beijing: Academia Sinica of Agriculture (in Chinese), 1997:15-30. 7. Quirk JP, Schofield RK. The effect of electrolyte concentration on soil permeability. J Soil Sci 1955; 6:163-178 8. Russo D, Bresler E. Effects of mixed Na/ Ca solutions on the hydraulic properties of unsaturated soils. Soil Sci Amer J 1997; 41:713-717 9. Iino F, Aoki M, Nitta Y. Effects of alkali soil amelioration by several kinds of calcium materials. J of the Japan Institute of Energy 1997; 76:119-124 10. Hattori H, Kumagai H, Ishizuka T et al. Ettringite formation through the hot water treatment of mixture of CaO, fly ash and gypsum. Beijing: The 3rd ChinaJapan Sympodium on Cool C-P Chemistry 1990:385-390.

SECTION IV INTRODUCTION

Conservation and Contribution of Plant Genetic Resources Kazuo N. Watanabe

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lant genetic resources (PGR) for food and agriculture are broadly defined to include resources which contribute to people’s livelihoods by providing food, medicine, feed for domestic animals, fiber, clothing, shelter and energy, etc. The use of plant genetic resources to enhance crop productivity and sustainability is a high priority worldwide because of its: 1. Historical contribution; 2. Significance to environmental protection; 3. Balancing of socioeconomic and cultural aspects with development; and 4. Future potential. PGR have contributed to the daily life of people in the world through the process of their domestication and through their involvement in the evolution of civilization. PGR should be regarded as essential subjects, the same as water, air, and soil, which are important components of the global environment. In the same way, PGR can easily deteriorate or be lost unless we use extra caution in how we live with them. In olden times, more numbers of plant species were used for food, feed, fiber, remedy, energy, construction, manufacturing and/or environmental protection, compared with the limited number of species used at present. PGR have supported the development of traditional knowledge and culture; in contrast these are now diminishing very rapidly along with PGR. With modern agriculture supported by plant breeding, many crop species have been modified for the needs of human beings,

increasing their diversity. On the other hand, an extensive use of particular species and cultivars has caused a decrease in the diversity of plant species for use and for conservation in nature. As science and technology are being further developed, there is a constant increase in possibilities for enhancing the potential capabilities of crop species to synchronize sustainability and productivity. On the other hand, without the contribution of PGR as a platform for application of such science and technology, there would not be much future, nor an outcome to be expected, for agricultural applications of modern science and technology. Learning from the past, the rejuvenation of traditional knowledge and cultural information, and the exploitation of under-utilized PGR from elsewhere, should alleviate the diverse spectra of pitfalls confronting the needs of human beings. In this session, four topics will be covered by the distinguished speakers. Dr. Masaru Iwanaga of IPGRI overviews the management and uses of PGR associated with sustainable agriculture; Dr. Masahiro Nakagahara of NARC-Japan covers the issues associated with ex situ conservation, mainly those of genebank management; Dr. Ayfer Tan of AARI-Izmir, Turkey, presents a specific case in PGR conservation and utilization; and, finally, Professor K. V. Raman of Cornell University highlights the links between PGR and biotechnology applications, providing a specific example.

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine. ©2000 Eurkeah.com.

CHAPTER 11

Integrated Plant Genetic Resources Management Systems for Sustainable Agriculture M. Iwanaga, P. Eyzaguirre and J. Thompson

Introduction

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gricultural systems are faced with complex challenges in ensuring increased productivity to meet the rising demands of an ever increasing population. The safe conservation and sustainable use of plant genetic resources are the keys to ensuring food security, eliminating poverty and protecting the environment. Two agricultural production systems, for favorable and marginal areas, are facing different but equally complex challenges and therefore should be integrated in terms of genetic diversity management, instead of having two separate agriculture paradigms. This paper proposes an integrated genetic resources management system to address the global task of safe conservation, sustainable use and equitable sharing of benefits of plant genetic resources. Key issues for the development of such an integrated system are analyzed in the three main areas, namely: system overview and institutional linkages; the technical and scientific base; and policy framework.

Biodiversity: A Foundation for Food Security, Poverty Elimination and Environmental Protection The dynamic processes which sustain the biosphere are based on biological diversity. Biodiversity is essential for food security, for the elimination of poverty and for the sustainable use of environmental resources. It is the

foundation of social and political stability and one of the key elements of economic development. Whether biodiversity’s ability to continually regenerate itself is compromised in any way is in part determined by the degree and type of pressures placed on it by human activities.

Population Pressure One of the major human activities is agriculture, providing sustenance for the majority of humankind for 10,000 years. The growing efficiency and productivity of agriculture is also responsible for the precipitous increase in human population in the last 500 years. While the rate of population growth is now expected to begin leveling off, most projections nonetheless estimate that there will be an additional three billion people to feed in the next 25 years. By far the major part of this growth will be in developing countries already affected by poverty and undernourishment. At present, despite all this century’s improvements in production technology, eight hundred million people are estimated to be undernourished. In the 21st century the expanded global population will compete ever more intensely for food and other resources that agriculture provides. The social, environmental, economic and political repercussions of a failure to meet these demands on the world’s agricultural resource base are staggering.

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At the same time, an overall decline is being registered in the per capita surface area available for food production. This decline, combined with population increase, means food security is precarious. This is a challenge facing all of humanity, and requires concerted global efforts to find solutions through agricultural research. The major imperative is to find sustainable, cost-effective means of augmenting food supplies, primarily through reliable improvement of crop yields, to meet the inevitable increased demand for resources. The World Food Summit, which took place in Rome in 1996, mandated the conservation of agricultural biodiversity as an essential element in ensuring future food security and underlined the urgency of providing this security to a burgeoning world population. There is now a consensus that growth in agricultural productivity has to be achieved in ways that conserve natural resources and protect the biodiversity of the Earth’s precious life support systems, which are already under stress.

Challenge of Sustainable Agriculture To increase food production, three avenues are available: 1. Extensification of agriculture; 2. Intensification of agriculture; and 3. Reduction of crop losses. Advances in the direction of extension of arable land, however, appear to have reached the limits of our present scientific and technological capabilities. In addition, there is growing competition for land for nonagricultural uses such as housing, industry and recreation. In poor countries with large agrarian sectors, population pressure on the land is degrading the basic biological, soil and water resources upon which agriculture depends. Further expansion of agricultural areas can only be made at unacceptable environmental costs and loss of sustainability. A second means of increasing food production would be to intensify cultivation and increase yields from lands which are presently agriculturally viable. This intensification is achieved through greater energy inputs, irrigation, chemical fertilizers and pesticides, combined with genetic materials that are responsive to high inputs. These were the

objectives of agricultural policies and research programs in the last few decades, when the development and spread of high yielding varieties of major crops resulted in large increases in production (the green revolution). These gains may have had costs which were not foreseeable at the time. One major cost was environmental, including pollution, agricultural wastes and reduced sustainability of agricultural ecosystems. Monoculture of high yielding varieties has made crops more vulnerable, in the long run, to the resilience of endemic pests and diseases, which adapted to the expanded and homogenized host environment. This leads to an increase in pest infestations and increased use of pesticides in an escalating treadmill of action and reaction, and higher production costs and damage to the environment.1 The biggest danger of monoculture lies in the reduction of genetic diversity. Crop failures due to genetic uniformity are well documented. To cite a few of the major examples of overdependence on a narrow genetic base, leading to devastating crop diseases: The potato blight in Ireland in 1846 wiped out the entire crop upon which Ireland’s poor depended, contributing to the famine which followed; crop disease led to the destruction of three million tons of rice in Indonesia in 1974; and one billion dollars’ worth of the US maize crop was lost in 1970 due to the corn leaf blight.2 More recently, taro leaf blight has eliminated entire populations of this traditional staple food of the South Pacific nations. The third means of increasing food production tackles this problem of reducing crop losses. It proposes to secure and increase agricultural productivity through means which break or circumvent the “pesticide treadmill” pattern and which restore a sustainable balance of organisms in the ecosystem. It has been suggested that by adopting integrated pest management approaches, including post-harvest management, global food availability could be doubled. Techniques include the judicious and reduced use of pesticides, multicropping to avoid monoculture and the introduction of pest-resistant cultivars.1

Integrated Plant Genetic Resources Management Systems for Sustainable Agriculture

Biodiversity: A Key Weapon The number of higher plant species is estimated to be between 300,000 and 500,000, only a half of which have been identified.3,4 Of these, about 30,000 are edible and an estimated 700 food species have been cultivated or managed by humans for food.5 Only three – rice, wheat and maize – account for almost 60% of the plant-derived calories consumed by humans.6 A diversified genetic base for these crops is essential for ensuring protection against disaster in the form of a common threat to which the few major varieties are susceptible. Diversity in terms of a wide range of species within sustainable ecosystems is even better insurance. A diversity of crop and animal species adds to social and economic stability through reducing reliance on a single or few species. Such diversity can also contribute to a more efficient use of natural resources and provide a buffering effect against losses to diseases, pests and weather fluctuations, because species have differing traits which make them more or less susceptible to these variables which affect survival, growth and yield. This differential ability to withstand micro- or macro- environmental stresses is based on the genetic diversity both within and among species. It is genetic diversity which allows species to adapt to changes in ecosystems through natural or human selection.

Challenges of Agricultural Systems Throughout the world, since the birth of agriculture, farmers, plant breeders, foresters and gardeners have used the genetic variation in plants to develop new types and varieties of crops and other useful plant species. They have developed an immense range of different plant genotypes adapted to widely varied environments. Since 1945, world crop yields have increased between 200% to 400%, depending on the crop. Today, more than ever before, the safe conservation and sustainable use of plant genetic resources are the keys to ensuring food security and, by extension, poverty alleviation and the promotion of peaceful development. The prevention of genetic erosion and thus loss of diversity of these resources is essential to

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future food security. This is the challenge facing agricultural systems today. To meet the challenge, both national governments and international bodies must collaborate in increasing productivity on all available agricultural land while employing environmental safeguards to protect natural resources for the future. Of these natural resources (i.e., soil, water, plant genetic resources), genetic resources offer us the greatest benefits in terms of return on scientific, technological and economic inputs and therefore require the most focused attention of researchers.

Favorable and Marginal Agricultural Areas The introduction of monocultures with large external inputs and standardized high yielding varieties of a few major crops has intensified the division between land favorable to agriculture and lands which are only marginally adapted for agriculture. This division has made the battle against genetic erosion a two-front one. Favorable agricultural areas have accounted for most of the food production increases. They are well suited to large scale agricultural enterprises, fertile soils and level terrain amenable to agricultural activities. The farming system is one requiring high inputs of energy and depends largely on mechanization, irrigation and major crop species. Favorable agricultural areas tend to have good access to markets and export facilities with resulting options for diversification. Marginal agricultural areas, on the other hand, are characterized by a range of factors which serve to limit their capacity for agricultural production. These may include infertile soil, adverse climatic conditions, hilly terrain, wetlands, difficulty of transportation, distance from markets, poor infrastructure and unfavorable output/input ratios which make large scale investment in agriculture unattractive. Many of these marginal areas occur in developing countries where resources for developing agricultural potential are few or lacking, often in tropical, arid or mountainous zones. Farming systems are often rainfed at subsistence level, with low inputs, difficulty of mechanization and centered on

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minor, locally-important crops. These zones provide the livelihood for 1.2 billion people. In the struggle of dealing with difficult and diverse constraints, traditional farmers have domesticated many species and created a wide range of intra-specific diversity (i.e., traditional landraces).

Challenges in Favorable Agricultural Production Areas In modern agroecosystems in favorable agricultural areas there are two main challenges. First, the current practice of high external input agriculture is not sustainable. The resulting high outputs (i.e., high yield) often come at the expense of the natural resources base (water, soils, biodiversity), both on-farm and off-farm. Yield plateaus of major crop species in high production areas have been reported, causing concern regarding the sustainability of on-farm production. The heavy dependence on agrochemicals (including pesticides and chemical fertilizers) in uniform production systems can result in the destruction of a wide array of susceptible species in the ecosystem7 and pollution of water sources. Intensive cultivation with irrigation can lead to rapid water depletion and salinization of the soil. Future as well as present generations are affected, since valuable stores of genetic resources in natural habitats may be irretrievably lost.8 Second, genetically homogenized production fields are vulnerable to significant risks and losses over time, especially from increased vulnerability to pests and diseases. A plant pest or disease can be devastating if it infests a uniform crop, especially in large, homogeneous plantations. Producers have suffered serious economic losses from relying on monocultural varieties,2 as when a root disease destroyed vineyards in France and California in recent decades and a virulent disease devastated banana plantations in Central America in this century.

Challenges in Marginal Agricultural Areas In marginal agricultural areas, genetic erosion has been accelerated by abandonment of traditional crops in favor of new “improved” varieties, often imported from outside the

marginal area, the push to substitute cash crops for traditional ones, deforestation and land clearance, overgrazing, war and civil strife and ecological reverses such as drought and flooding. By concentrating on high input, high producing, genetically uniform varieties and the agricultural systems which support them, agricultural researchers have failed to strengthen smallholder agriculture in marginal areas. The complex, heterogeneous agricultural environments, crucial sources of genetic variation, are being weakened. As the farming systems in marginal environments are replaced, these traditional “cauldrons” of genetic diversity will no longer generate new combinations, leaving only what small portion of this diversity the formal breeding and genetic resources conservation institutions have been able to collect and maintain. This change is often compounded by the exclusion of minor crops or local staples, which are replaced by high yielding varieties of major crops developed outside the marginal agricultural zone. Formal breeding often requires large economic returns through wide scale adoption under uniform conditions of the improved cultivars produced by breeders. The less favored agricultural environments have thus been bypassed by more formal breeding efforts. As the use of high yielding varieties of crops spreads into marginal areas, often through local governmental policy, farmers are often encouraged to adopt these varieties to the detriment of locally adapted traditional landraces. As these landraces fade, so does the traditional knowledge associated with them. Both may be irreplaceable. When communities are displaced or when smallholdings give way to large scale monocultures, there is often a migration of labor to growing urban areas. Those left in marginal areas to carry on agricultural activities are often older farmers, who may represent the last repositories of traditional knowledge about local plants. Agricultural researchers and formal breeding programs often do not consult with or address the needs and resources represented by these farmers, who in many marginal areas are predominantly women. The feminization and aging of the smallholder workforce present

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additional challenges for the formal sector’s efforts in marginal areas.

Favorable Agricultural Production Area Systems

Plant Genetic Resources Systems View: Avenues for Meeting Agricultural Challenges

Ex Situ Conservation To support favorable agricultural areas, germplasm is stored in ex situ genebanks where diversity can be conserved under controlled conditions and protected from the stresses and pressures encountered in their native habitats. The germplasm stored in genebanks also provides the raw material for breeders to use in developing crop varieties with traits selected for their adaptive value to specific biotic and abiotic conditions and market needs. There are more than 1300 genebanks worldwide, with total germplasm accessions numbering more than 5.5 million.9 The Consultative Group of International Agricultural Research Centers (CGIAR) maintains approximately 500,000 accessions in trust for the world community.

Sources of Diversity in Genepools The total genetic diversity in a crop genepool is the result of: 1. Natural processes unaided by humans; 2. (Wild relatives of crops) crop evolution; 3. Environments; and 4. Formal breeding to create new genetic combinations according to predetermined criteria. An important feature of genetic diversity within traditional farming systems is that it allows for geneflow between crops and their wild relatives. One way of conceptualizing the geneflow within a crop genepool is as a plant genetic resources (PGR) system with interactions and flows between the three sources of genetic variation. Each of the three sources of diversity, wild relatives, landraces and formal breeding, are characterized by an increasing degree of human control over the process of exchange of genes, and by the reduction of complex environmental factors in the selection process.

Existing Systems for Conserving, Improving and Using PGR In both favorable and marginal agricultural areas, we have seen that sustainable agriculture is under threat from genetic erosion. The causes of these losses are traced in large measure to human intervention and can only be corrected by reverse intervention. Where genepools are narrowed, some food crops forgotten and crop varieties lost, actions can be taken to monitor and measure agrobiodiversity, conserve it and promote its sustainable use. Both favorable and marginal agricultural areas have systems in place for ensuring these actions.

Agricultural Research Systems Methods and technologies for the conservation and use of genetic resources are being developed and improved by a range of agricultural research systems on both national and international levels. In favorable agricultural areas, the national agricultural research programs are often in the forefront of efforts to strengthen sustainable agriculture through the safe conservation and judicious use of genetic resources. On an international level, the 16 centers grouped together under the umbrella of the CGIAR are the most prominent agricultural research centers currently at work to develop practices and policies for ensuring sustainable agriculture. Essential for the continued safe conservation and sustainable use of plant genetic resources, agricultural research efforts are nevertheless often among the casualties when economic conditions dictate the reduction of public funding. Private and Formal Sector Crop Improvement Programs Crop improvements are carried out by public sector germplasm improvement programs throughout the world in national agricultural research units, in universities, in regional agricultural research institutes such as AVRDC in Taiwan and CATIE in Costa Rica, as well as in international agricultural centers

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such as those of the CGIAR. Crop improvement programs are also run by the private sector in efforts to develop new crops with traits which increase their yield and marketability. Crop improvements have, since 1945, increased world crop yields between two- and four-fold depending on the crop. An estimated 20-40% of this increase has been achieved by genetic modifications and breeding.1,7 The contribution of genetic resources through the introduction of new genes and genetic modifications through crossing with wild relatives is approximately $115 billion per year worldwide in crop yield increases.10 Seed Supply Systems Seed supply systems also serve to enhance the use of plant genetic resources and are concerned with the health of conserved germplasm and with the safe movement and exchange of germplasm. These systems are increasingly involved with deployment of new varieties developed by application of modern molecular genetics and transgenics and are increasingly controlled by large global companies. In consequence, there is a growing concern over future seed supply systems with respect to principles of equitable access and sharing of benefits.

Marginal Agricultural Production Systems Farmer Breeding There are now substantial data and evidence that smallholder farmers in marginal areas maintain and select among their landraces, and that this can be considered traditional breeding or management of diversity 11 as well as conservation of that diversity. In the crops they plant, they select for those criteria that allow for greatest resistance across several competing characteristics: straw versus grain, hardiness/rusticity versus yield, cultural preferences in flavor and appearance over total calories. Examples are adaptation to microenvironments, environmental stresses or biological hazards such as pests. In genetic terms, this adaptation is often not based on single characters but is multilocus, with complex inheritance or co-

adapted gene complexes. Breeding this type of diversity is something that farmers do well. Community-Based Conservation Systems Agricultural land, grazing lands, agroforestry areas and village gardens are often held in common by a community, especially in marginal areas of developing countries. The rural poor may derive as much as 30% of their food for consumption as well as market income from community land. In the process, the community farmers are involved in the in situ conservation of landraces, agroforestry species and wild species. Often small community-based in situ conservation sites are the sole repositories of particular crop varieties adapted to their specific environments. The use of unique, diverse and adapted genetic resources is something that marginal areas are noted for. By taking agriculture into the extremes or limits of their growing areas, farmers have provided the world with unique adaptive characters. The main vehicle for ensuring the conservation of many landraces and forestry species is through the maintenance of these species in their native habitats or in farmers’ fields, protected areas and home gardens. The value of these methods of in situ conservation of plant genetic resources lies in the fact that they involve conservation not only of species and within species diversity, but also of ecosystems. It allows for continued evolution and adaptation of plant populations in their native habitats. It can also be an effective component of sustainable development strategies and can increase control of traditional farmers and communities, especially in marginal areas, over their own resources. Seed Exchange And Supply Systems In marginal agricultural areas, seed supply systems are often a “home-grown” affair. Formal, large scale commercial seed suppliers, while their reach is now extending into the less favored agricultural production areas, are not the primary means of seed supply to smallholders and poor farmers in developing countries. These farmers have traditionally created their own seed supplies by saving seeds from one harvest to sow for

Integrated Plant Genetic Resources Management Systems for Sustainable Agriculture the next season. They have also traditionally formed informal seed exchange networks, which are particularly prevalent for the minor and underutilized crop species which are neither cost effective nor profitable for formal commercial seed development and distribution systems to handle. It is estimated that “80 to 100% of the production of planting material takes place in the informal sector in developing countries.”12

Global Challenges for Realizing Equitable, Sustainable Harvests The realization of an agricultural harvest which is both: 1. Sustainable in terms of protection of the environment and renewability of resources; and 2. Equitable in terms of costs and benefits to producers and consumers, is a global challenge. The urgency of this challenge is evident in the increasing burden of a burgeoning population to feed, a deteriorating resource base to protect and standards of living to be improved. Solutions to these problems lie with the extant agricultural systems which dominate the human food supply. The two systems, at once overlapping, disparate, conflicting and complementary, have in recent decades been characterized by an almost parasitic, one way relationship in genetic resources management, with gene flow going from landraces and wild relatives of crops in marginal zones to the formal, centralized breeding programs (Fig. 11.1). To harness what both systems can offer, modern agricultural researchers must be urged to emphasize the complementarity of the two systems over their disparateness. This requires the integration of the two systems in a single, forward looking one which aims above all at equity and sustainability (Fig. 11.2). The three ovals in Figure 11.1 represent the three sources of genetic variation in a crop genepool. The three boxes on the right denote the institutionalized processes that use, maintain or transform genetic resources in the formal germplasm management system. Enclosed by the thin line in the upper left of the figure are the components of the traditional plant genetic resources manage-

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ment system: wild relatives of crop plant and traditional cultivars (landraces) which are maintained in marginal areas. The arrows in Figure 11.1 illustrate the unidirectional geneflow from traditional agricultural systems to the formal, institutional-based system, culminating in the introduction into favorable agricultural areas of improved modern cultivars. In Figure 11.2, this geneflow has been altered by a proposed direct feedback in the middle of the cycle to link agricultural research and germplasm improvement directly to the traditional farming systems in marginal areas to improve the productivity of farmers’ production systems, thus ensuring the continuity of this important source of new variation in genetic diversity.

System View and Institutional Framework The system view considers the links in agriculture from breeding through production, distribution and use and looks at the underlying institutional framework, technical and scientific base and policies.

Proposed Changes of Gene Flow and Institutional Relationship Wild relatives and landraces still account for the bulk of genetic diversity within a crop genepool. Formal breeding reaggregates existing genetic variation from these two sources. While new techniques employed in mutation breeding programs and some engineered genes (e.g., those with enhanced herbicide resistance) may actually introduce new variations, isozyme and molecular data on the amounts of diversity in wild relatives, landraces and modern cultivars indicate that wild relatives and landraces remain the main sources of genetic diversity in crop genepools.13 Conserving these sources is therefore crucial to the future of crops. Our existing system of genetic resource conservation and use, however, may need to be redirected in order to maintain this diversity. The current unidirectional flow of genetic resources into formal centralized breeding of elite varieties with high yields and specific resistance offers little that is useful to small scale farmers who are engaged in maximizing genetic diversity and who maintain landraces. For poorer farmers on marginal lands, a growing

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Fig. 11.1. Maintenance and use of genetic diversity: existing system. reliance on high yielding varieties of improved crops reduces their options for coping with variable environmental conditions and exploiting niches and micro-environments in their farming systems. New varieties of crops able to meet the challenge of marginal areas must come from the use of the genetic resources conserved from these areas. We need to conserve not only the genes themselves but also the farming systems and agroecosystems that produce and maintain genetic diversity. This requires strong positive feedback in the germplasm improvement and conservation system directed to traditional farming systems which use and maintain landraces. Those inputs need to be in a form that farmers can use as part of their own system with its particular practices of selection, breeding and management of crops. In this way they can continue to use and develop genetic diversity in crops as an integral part of their own social and economic development.14 Many genebanks are finding that their holdings are increasingly dominated by advanced cultivars, with landraces poorly rep-

resented and inadequately documented.15 In the final analysis, we may be losing significant portions of the crop genepool by not feeding back into the sources of genetic variation in crops, namely, farmers and their interactions with complex environmental pressures. In Africa and India, for example, “cassava (Manihot esculenta) yields increased up to 18 times after genes from wild Brazilian cassava, conferring disease resistance, were incorporated into local varieties.”16 Other minor and locally important crops benefit from similar inputs.

Participatory Breeding and Research Perhaps the most important key to promoting an integrated and dynamic agricultural system is in participatory interactions between marginal and favorable agricultural production areas. Empowerment of small scale farmers in marginal areas is crucial. Where formal and informal systems must converge, experience has shown us that in an interactive approach, with farmers taking the lead in the decision making and organization of agroecological initiatives, improves the

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Fig. 11.2. Integrated PGR management: proposed system. chances that agrobiodiversity enhancement efforts will succeed. 2 It has likewise been demonstrated that scientists can improve the relevance of their research by drawing on farmers’ own informal methods of experimenting with unfamiliar cultivars and practices.17

Formal and Informal Sector Linkage; Public-Private Sector Linkage Participatory interactions involve those between the formal and informal sectors (e.g., international research organizations and poor women farmers in marginal zones) and those between public and private sectors (e.g., national Departments of Agriculture and commercial seed suppliers). The widely applicable lesson is that close, effective and long term collaboration is required among different players, including germplasm curators, plant breeders, farmers, molecular biologists, entomologists, ecologists, community groups and social scientists. Linkage of efforts at national, regional, and international levels is also critical to deal with globally important agricultural

problems, thus to increase the world’s food security by maximizing existing resources and production potential.

Technical and Scientific Base To make a lasting impact in eradicating poverty and hunger, current and future systems must operate from technological and scientific bases which seek to preserve genetic diversity and not simply exploit it. Technological means must be sought for improving the safe conservation of genetic diversity in crop plants in both ex situ genebanks and in in situ contexts. Importance must also be given to the technological aspects of the deployment and use of genetic diversity in both marginal and favorable agricultural areas. The scientific basis on which technological means are built will increasingly be targeted by researchers, policy makers and others whose responsibility it is to ensure food security.

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Understanding Traditional Knowledge and Management Systems The traditional farming systems which sustain the conservation of this genetic raw material are the result of generations of experience with landraces and their native ecosystems. The cultures of which these systems are part are as important to conserve as the genetic resources themselves. Formal institutions dealing with agricultural research and plant breeding must ensure that the agrobiodiversity of the traditional farming system remains within the cultural context in which it developed and which continues to sustain it. Agrobiodiversity developed in tandem with man, the farmer, and must be regarded as a whole with all its elements: environmental, genetic and cultural. Farmers have an intimate knowledge of useful plant characteristics such as which seeds require less fertilizer, which varieties are able to outgrow weeds, which are less susceptible to pests, and, not least, which taste better. As we pay greater attention to the role that farmers play in the conservation and use of genetic resources in situ, we will need to consider social and cultural factors such as decision making patterns, local institutions, indigenous knowledge and value systems. Gender analysis can provide an understanding of the critical role that women play in the management and use of genetic resources at the farm level. Equitable and ethical use of local knowledge of genetic resources requires a system for recognizing and supporting traditional resource rights and local systems for the maintenance and exchange of knowledge.

Genepool and Genomics Recent progress in genomics study has been impressive, offering us much better knowledge of gene structure and function in a genome. This provides a strong scientific base for crop improvement efforts. Crop genepools have adapted to and sustained the demands of agricultural systems for thousands of years.18 A genepool represents a dynamic system of genetic diversity in the form of the allelic diversity of species or groups of species structured over space and time. It is often used as a unit of thinking for global

genetic conservation and use efforts. Yet our understanding of its dynamic structure over time and space is very limited. Molecular markers are powerful tools to enhance our knowledge of genepools, especially when combined with other data in geo-referenced form. Adaptive traits are of key interest for crop improvement efforts, and molecular genetics applied to genomics and genepool study will help us to understand how such traits develop (functional genomics) and are maintained in genepools.

Diversity Management in Production Fields Researchers are seeking to address areas of natural resources management in which the genetic diversity aspect is highlighted, working on the premise that the ultimate use of genetic resources occurs when they are deployed or managed in ecosystems over space and time. Sustainable agroecosystem management strategies include: 1. Intraspecific diversity (genetic vulnerability, farmers’ fields favoring coadaptation dynamics, populations mixtures, multilines, genetic base of cultivars or pedigree complexity, etc.); 2. Interspecific diversity (monoculture, intercropping, rotations, agroforestry, neglected and underutilized species, home gardens, species interaction dynamics, etc.); and 3. Ecosystem management (sustainable natural resources management, unfavorable agricultural production systems, geneflow, etc.)

Biodiversity as a Key Element for Natural Resources Management Research Attempts to increase productivity of agricultural land has contributed to the degradation of the natural resources base through: 1. Extension of agricultural lands into marginal zones, resulting in deforestation and fragmentation of habitats; and 2. Intensification of agriculture, which results in contamination of soils and water supply through concentration of chemical fertilizers and pesticides.

Integrated Plant Genetic Resources Management Systems for Sustainable Agriculture Both types of pressure on the natural resources base tend to decrease genetic diversity. Reversing the process would mean using an increase in genetic diversity to bolster the natural resources base and contribute to its sustainable management. Careful and selective intercropping and mixed cropping in marginal zones, for example, can reduce the stress on soils, slow the erosion of watersheds and eliminate the need for heavy application of pesticides. Sustainable biodiversity-based intensification of agriculture, including agroforestry systems, requires a considerable degree of skill in natural resources management but results in greater sustainability of productivity increases. Such agricultural systems lend themselves to biodiversity conservation and protection of natural resources by “mimicking more diverse natural ecosystems.”2 Understanding how ecosystems are altered by intensive agriculture, and developing new strategies that take advantage of ecological interactions within agricultural systems, are crucial to the continuance of high productivity agriculture in the future.19

Biotech-Based Germplasm Development Biotechnology is making vast progress in many areas, and perhaps genetic engineering is the most obvious area of advance and in which the largest investment has been made by both the public and private sectors. Biotechnology-based germplasm improvement is producing genetic materials which are different from those of traditional breeding methods in terms of genetic nature and deployment strategy. Agricultural research will now need to also look in greater depth into the following areas. Implications of Biotechnology for Deployment of Genetic Diversity Use of a standard variety for genetic engineering work aimed toward the addition of a few genes means that there has been no real broadening of the genetic base. There has also been a strong push by breeders of such a variety to promote it over a wide area of cultivation in order to recover the economic investment. Only major crops tend to get

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attention from biotechnology research investment, and concentration on these major crops might result in the neglect of other minor species which are less competitive, due to the increasing focus on research investment into crops with large market potential. Living Modified Organisms (Lmos) and Ecology New crop cultivars have already been produced using genetic engineering, and large areas of genetically modified plants are now being grown in many countries. Agricultural research in the future will address concerns that these techniques and resultant products may have a negative impact on biodiversity (e.g., gene flow to related wild or weedy species) and on the amount and nature of diversity in crops under production.

Policy Framework Developments in international, regional, and national law and policy over the past five years have significantly changed the policy environment relating to the management and control of genetic resources. And the situation continues to evolve rapidly. National policy makers with an interest in plant genetic resources and agrobiodiversity are currently faced with a combination of multiple and often conflicting national interests and a bewildering array of international interests and obligations with direct and indirect effects on the conservation and management of PGR. Some of the most significant recent developments include: The Convention on Biological Diversity The Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPs) The WTO Agreement on Agriculture The International Undertaking on Plant Genetic Resources (IU) The 1994 FAO/CGIAR Agreements and 1998 External Review of CGIAR The Union for the Protection of New Varieties of Plants (UPOV) The World Intellectual Property Rights Organization (WIPO) Indigenous and local communities including farmers and farm communities National legislatures and court systems.

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Policy makers are faced with a legal and policy environment under active debate in multiple intergovernmental forums and with legal regimes for ownership, control and intellectual property rights over genetic resources in a state of flux. The challenge of developing good, coherent, consistent policy on the conservation, development, use and exchange of genetic resources in this context is great.

Conclusions The redirected system envisioned for conserving and using plant genetic resources is one that is open to farmers. It makes germplasm directly available to them in a form that is enhanced and documented through the work of plant genetic resource programs, but not necessarily as products of a national breeding program. Plant genetic resources programs, which have heretofore regarded breeders as the primary direct users of the genetic resources they maintain, will increasingly look to farming communities which maintain landraces as direct users as well as contributors of plant genetic resources held in genebanks.14 The integrated system of plant genetic resources management would no longer be based on a unidirectional flow of genetic material from marginal areas, where finely adapted landraces and wild relatives of crop plants are concentrated, to formal crop improvement programs designed to develop high yielding standardized varieties of crops for high input, large scale agriculture in favorable agricultural areas. Instead, the proposed system would recognize the value of conserving the genetic diversity located in marginal areas and ensuring that it is used in such a way that benefits are returned to the smallholder farmers in these areas. This will ensure a fundamental role of favorable agricultural areas for food security. The proposal envisages that the flow of genes and the resulting economic, social and technological feedback are bidirectional between the formal research and breeding programs and the caretakers and users of genetic diversity in marginal areas. The technical and scientific bases of the proposed system, as well as the policy framework supporting it, give importance to the traditional knowledge

systems in marginal areas and to participatory approaches which link across favorable and marginal agricultural areas. In the long run, the integrated system would strengthen bonds between conservation and use and thus offer a foundation for maintaining agrobiodiversity.

References 1. Iwanaga M. Foreword. In: Clement S, Quisenberry S eds. Global Plant Genetic Resources for Insect-Resistant Crops. Boca Raton: CRC Press, 1999:xii-xv. 2. Thrupp LA. Cultivating Diversity: Agrobiodiversity and Food Security. Washington DC: World Resources Institute, 1998. 3. Wilson EO. The current state of biological diversity. In: Wilson E, ed. Biodiversity. Washington DC: National Academy Press, 1988:15. 4. Heywood VH. ed. Global Biodiversity Assessment. Cambridge: Cambridge University Press, 1995. 5. Wilson EO. The Diversity of Life. London: Penguin, 1992. 6. Prescott-Allen R, Prescott-Allen C. How many plants feed the world. Conservation Biol 1990; 4 (4):365-374. 7. Pimentel D, Stachow U, Takacs D et al. Conserving biological diversity in agricultural/forestry systems. BioScience 1992; 42:360. 8. World Resources Institute. World Resources Report 1996-1997. New York: Oxford University Press, 1995. 9. Food and Agriculture Organization of the United Nations. The State of the World’s Plant Genetic Resources for Food and Agriculture. Rome: Food and Agriculture Organization of the United Nations, 1998. 10. Pimentel D, Wilson C, McCullum C et al. Economic and environmental benefits of biodiversity. BioScience 1997; 47 (11): 750. 11. Riley KW. Decentralized breeding and selection: A tool to link diversity and development. Presented at a working seminar on using diversity: Enhancing and maintaining genetic resources on-farm. Ottawa: IDRC, 1998.

Integrated Plant Genetic Resources Management Systems for Sustainable Agriculture 12. Delouche JC. Seed Quality Guidelines for the Small Farmer. Cali: CIAT, 1982. 13. Miller J, Tanksley S. RFLP analysis of phylogenetic relationships and genetic variation in the genus Lypersicon. Theoret Appl Genet 1990; 80:437-448. 14. Eyzaguirre P, Iwanaga M. Farmers’ contribution to maintaining genetic diversity in crops, and its role within the total genetic resources system. In: Eyzaguirre P, Iwanaga M, eds. Participatory Plant Breeding. Rome: International Plant Genetic Resources Institute, 1996:9-18. 15. Evenson R, Gollin D. Genetic resources, international organizations, and rice varietal improvement. In: EconomIc Development and Cultural Change. In press.

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16. Prescott-Allen R, Prescott-Allen C. Genes From the Wild: Using Genetic Resources for Food and Raw Materials. London: International Institute for Environment and Development, 1982. 17. Haugerud A, Collinson M. Plants, Genes and People: Improving the Relevance of Plant Breeding. Gatekeeper Series 30. London: International Institute for Environment and Development, 1991. 18. Lee M. Genome projects and gene pools: New germplasm for plant breeding? Proc Natl Acad Sci 1998; 95:2001-2004. 19. Matson P, Parton W, Power A, Swift M. Agricultural intensification and ecosystem properties. Science 1997; 277:504.

CHAPTER 12

Genebank Management of Crop Genetic Resources M.Nakagahra, S. Miyazaki and D.A. Vaughan

Introduction Crop Genetic Resources

C

rop genetic resources can be described as germplasm which represents past building blocks of present day crops, and germplasm which can furnish the genetic material for future new crop varieties. In this biotechnological age the second category, genetic material for future new crop varieties, can encompass all germplasm, plant, animal and microorganism, since gene transfer across kingdoms, and even the introduction of manmade genes, is now a reality. Crop genetic resources can be categorized into four groups (Fig. 12.1): 1. Germplasm from natural habitats; 2. Germplasm from cultivation and cultivators; 3. Germplasm from breeding programs; and 4. Gene sources from research laboratories. For the purposes of this paper we will focus only on the first three categories, while realizing that the fourth category may become increasingly important for crop improvement in the future.

Genebank Management The Genebank As our understanding of crop genetic resources has expanded, so has our understanding of genebank management. While the

genebank is generally envisaged as a building holding conserved germplasm, in reality the genebank is the sum total of the reserves of conserved genetic resources. Thus, if we consider the situation in Japan as a case of the system in a resources-rich developed country, it would include: 1. Active collections (which may include parts of breeders’ working collections); 2. Base collection (of conserved materials); 3. Seeds stored in cold storage rooms; 4. In vitro collections of cell cultures; 5. Cryogenically preserved germplasm; 6. Living collections of growing plants (which may or may not be part of an active or base collection); 7. Preservation areas and nature reserves; 8. Botanical gardens; and 9. Research institutes, e.g., fruit tree collections. Management Management is an all encompassing word covering everything from staff motivation to output efficiency and cost effectiveness. However, for the context of this paper in relation to genebanks and crop genetic resources, we will focus on the genebank system established by genebank managers and we will use the Japanese Ministry of Agriculture, Forestry and

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine. ©2000 Eurekah.com.

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Fig. 12.1. The scope of crop genetic resources. Fisheries (MAFF) genebank system as an example.

Features of Genebank Management in Japan The working party of the Science Council of Japan is the coordinating committee in Japan concerned with technological infrastructure for biological genetic resources (Fig. 12.2). The nodal ministry conserving agricultural genetic resources is the Japanese MAFF, and for crop genetic resources the National Institute of Agrobiological Resources (NIAR) is the central bank. Since 1983 NIAR, in coordination with fourteen other agricultural organizations in MAFF has been developing and improving the crop genetic resources genebank system of Japan. 1,2 Key to the system is strong central leadership and devolved responsibility in a matrix type system. The current structure of the MAFF genebank system for crops is shown in Figure 12.3, and some of its major activities are highlighted below.

Introduction and Collection The MAFF has been very active in collecting crop genetic resources within Japan and abroad. For example, eight domestic and seven international collecting missions were undertaken in 1996. 3 National efforts to collect native crop genetic resources have been on going for many decades. An early comprehensive collecting effort for native rice germplasm was conducted in the 1960s.4 Currently, traditional Japanese upland rice germplasm that was first collected in the 1960s has been the focus of intensive genetic analysis to determine the quantitative trait loci in this germplasm for blast resistance.5 Recently, greater emphasis has been placed on collecting wild relatives of crops in Japan. Internationally, in recent years multiple collecting trips have been made with collaborators in Pakistan, Russian, the Central Asian republics and Vietnam.6,7 These collecting trips are often followed up by analysis of collected diversity. Thus, for example, the inter- and intra-species diversity of Aegilops

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Fig. 12.2. The Japanese Genetic Resources System. species in Central Asia and northern Caucasus has been determined based on germplasm collected in 1993 and 1994.8

Preservation Preserving the original genetic constitution of germplasm is very difficult because regeneration of germplasm can shift the population genetic structure, and different genotypes and species require particular preservation conditions. Detailed research is required to find the optimum storage conditions for species with non-orthodox storage behavior. Considerable advances have been made in recent years in applying new technologies to store such species. Among these technologies, cryoperservation has the advantage of not requiring frequent subculturing of in vitro cultures. Recent reviews of this topic can be found in Ashmore9 and JIRCAS/ IPGRI.10 In Japan the potential of storing tuber and tree crops with non-orthodox seeds by in vitro and/or cryopreservation is being pursued so that labor and land devoted to collections of these crops can be reduced.

Characterization and Evaluation The first phase of this activity occurs in the field and represents the passport data. The location at which germplasm is collected is, perhaps, the single most important information on germplasm since it can guide further evaluation as well as re-collection. For example, the highlands of Indo-China are known as one place where genes for resistance to blast have been found; thus, new genes for resistance to this pathogen may be expected in germplasm from this region. The use of global positioning systems and increasingly sophisticated geographic information system software has greatly enhanced passport data. The MAFF genebank system has different levels of characterization and evaluation, level one being the simplest and easiest to record and level three the most complex. Within each level, there are optional and required traits for recording. On average more than fifty traits per accession are recorded; the aim is to have a broad base of useful information on each accession.

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Fig. 12.3. The Ministry of Agriculture, Forestry and Fisheries crop genetic resources system and national and international linkages.

Database Development and Information Management The MAFF genebank database is now available on the internet and can be read in either Japanese or English (http://www. gene.affrc.go.jp). This database is comprehensive and includes both information on germplasm as well as germplasm management data such as seed viability.

To add a new dimension to the genetic resources database, an illustrated database has recently been developed to enable scientists and students to learn more about germplasm. The first phase of this database has been devoted to legumes and can be read at http:/ /www.gene.affrc.go.jp/ image/legume.html (for the English version).11 Database development and management requires significant

Genebank Management of Crop Genetic Resources resources, both financial and human, as germplasm collections increase in size.

In Situ Conservation—Research and Collaboration Recent global attention on the topic of in situ conservation has resulted in various efforts to protect germplasm in its natural habitat. Red data books devoted to various species and habitats in Japan have been prepared (e.g., Japan Society of Plant Taxonomists,12 NACS-Japan and WWF-Japan13). The topic of in situ conservation is at present in the inventory phase, and measures to actually conserve germplasm in situ are beyond the ability of the genebank manager since these involve social and political decisions. The MAFF genebanks’ role in in situ conservation has been devoted to research, both within Japan and internationally. In recent years, Japan has conducted in situ conservation research with scientists in Chile on tomato wild relatives, the Philippines on sweet potato and Vietnam on rice. Currently, collaboration with Nepal on in situ conservation of buckwheat genetic resources is in progress.

Distribution and Exchange of Plant Genetic Resources Annually the MAFF genebank distributes about 8,000 accessions within Japan and abroad. Genebank managers need to be active in promoting the use of germplasm and to facilitate germplasm use by improving databases and access to them and organizing germplasm into useful sets of materials for evaluation and research, such as core collections.14

Prebreeding and Breeding During systematic evaluation, many potentially useful traits are found. Only a few of these are actually used in breeding, and an even smaller number find their way into released varieties. An array of useful traits that have been used in prebreeding and breeding in Japan is presented (Table 12.1).

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Human Resources Development A key role of genebank managers is to foster human development. MAFF has various training programs to help upgrade the ability of its genetic resources staff in various fields, including statistics and data management. In addition, MAFF scientists have been involved in training foreign scientists both in Japan and overseas in various aspects of crop germplasm conservation and research. One major training course held at NIAR in conjunction with JICA is the “Plant Genetic Resources Training Course”. This training course has trained more than 140 students from over 30 countries over the past 16 years. This training course has been one way to build up lasting international linkages. One key aspect of the course is the hands on nature of the course, with each trainee conducting research in an area, and on germplasm, of their interest.

International Activities Genebank managers are part of an international network of scientists who as a whole are trying to comprehensively conserve global genetic resources. The MAFF genebank system participates in the major forums for discussion of international crop genetic resources issues, such as the FAO and IPGRI regional network for East Asia. MAFF also has promoted international dialogue through an annual International Workshop on Genetic Resources, two of which have been devoted to crop genetic resources.15,16 In addition, active international collaboration on collecting, training, in situ conservation and database development among other areas is ongoing. The experience of developing and improving the MAFF genebank project has led to collaboration in helping to develop, through the Japan International Cooperation Agency, genebank systems in many countries, such as Chile, Bangladesh, Myanmar, Pakistan and Sri Lanka.

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Table 12.1. Examples of conserved germplasm used in prebreeding and breeding in Japan Crop

Useful trait

Rice

Jukkoku, a native variety from Kyushu island has been the principal semi-dwarf gene source for rice in Japan. Exotic varieties of rice have mainly been used in Japan to breed for pest and disease resistance, for instance Modan, a Pakistan variety which furnished resistance to rice stripe virus.

Food legumes

The Japanese soybean variety Keburi has an altered protein content, and a wild soybean from Kumamoto prefecture was found to be a group A acetyl saponin-deficient mutant. These varieties have been used to study soybean chemical composition so that soybeans with improved quality can be produced.40,41

Cereals

A search among about 2000 wheat accessions found that one variety, ‘Bai-huo’ from China, lacked the Wx protein on the D genome. Using the haploid breeding method, the first waxy bread wheat has been developed from the cross between ‘Kanto 107’ and ‘Bai-huo’.42 The wheat variety Nobeokabozu has polygenic resistance to scab (Gibberella zeae (Schw.) Petch). This variety has been used as a parent in breeding.43 The barley variety Mokusekko 3, from China, has two resistance genes to barley yellow mosaic disease (Ym, Ym(t)). This variety is being used to breed for resistance to the disease.43,44

Tuber crops

The Japanese sweet potato variety Hichi-fuku, which is resistant to black and stem rot and has excellent storage ability, has been used in breeding many varieties, including the variety ‘Hi-Starch’ popular in Japan.45 An accession of the wild relative of sweet potato (Ipomoea trifida), from Mexico, has been used to increase starch production. This wild species also has resistance to root knot nematode and root lesion nematodes. These traits have been transferred to improved varieties.46

Forage/lawn grasses

Japanese accessions of the lawn grass Zoysia japonica have been found which stay green longer in autumn. The commercial use of this germplasm is being exploited. Apomictic Guinea grass (Panicum maximum) from Africa has been used to develop a new variety of Guinea grass forage called “Natsukaze.”

Small grains/ industrial crops

Japanese native buckwheat has been found with a high rutin content The sesame cultivar H65 from China has higher anti-oxidation activity than Japanese cultivars and is being used as a parent in breeding.

Fruit

Japanese pear (Pyrus pyrifolia) is not resistant to scab disease (Venturia nashicola). However, resistant clones have been found in Pyrus aromatica. This resistance is being analyzed to determine its value in breeding.

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Table 12.1. (cont'd) Examples of conserved germplasm used in prebreeding and breeding in Japan Crop

Useful trait

Tea

One Japanese accession (MAK ZAI 17-1) has been used to produce lower caffeine type tea and an Indian accession (MAK IND 113) has been a source of high caffeine.

Vegetables

Late bolting Chinese cabbage, Brassica campestris, has been an objective of breeders in Japan. The Japanese local variety, Oosakashirona, has been a source of this trait. 47

Success Stories in Management of Crop Genetic Resources As discussed above, crop genetic resources management consists of many steps. In the following examples the role of Japanese scientists in some of these different steps is highlighted.

Table 12.2. Analysis of genetic diversity in different populations of the Vigna angularis complex19 Groupa

In Situ Conservation Few major crops were domesticated in Japan. Most of Japan’s cultigens were introduced initially from China and Korea, and subsequently from other regions. One major crop which could have been domesticated in Japan is the azuki bean, Vigna angularis var. angularis. The fact that today Vigna angularis consists of a wild/weed/crop complex17 is one reason to think that this crop could have been domesticated in Japan. Recent research at the NIAR has focused on the population structure to be found in this crop complex from different parts of Japan.18 This has highlighted four types of population: wild, weedy, cultivated and complex. Complex populations consist of a mixture of morphological types. It was found that these complex populations have the most genetic variation and are thus the most suitable for in situ conservation and long term monitoring (Table 12.2).19 This research has also found that the present day center of diversity of Vigna angularis in Japan are the prefectures surrounding Osaka, where weedy

Genetic diversityb Nc

Average

Range

Cultivated

66

0.274

0.0950.510

Weedy

861

0.341

0.00.574

Wild

1431 0.388

0.00.660

Complex

2346 0.399

0.00.665

a: Type of population. Complex populations consist of a variety of different plant types; b: Genetic diversity was measured using Jaccard’s dissimilarity coefficient; c: Number of individuals in each group.

and complex populations are most frequently found (Fig. 12.4).20 Detailed population level analysis is useful to help elucidate evolutionary pathways and, based on DNA polymorphism, weedy azuki

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appears to usually have evolved from wild azuki rather than being a hybrid between wild and cultivated azuki or an escape from cultivation.19 Genebank managers’ role in in situ conservation is to facilitate research and monitoring which can help policy makers determine what measures are necessary to efficiently protect genetic resources.

Collecting In 1958 the late Dr. H. I. Oka, then of the National Institute of Genetics, Mishima, Japan, was collecting rice in different parts of Thailand. Along a forest trail near Sukothai, Dr. Oka and Thai counterpart scientists found the wild rice O. officinalis. Seeds collected from this population were taken to Japan and several years later a sample was sent to the International Rice Research Institute, the Philippines, for research and conservation. For almost 20 years this accession was conserved before entomologists evaluating wild rices for insect resistance found this population from Thailand to be resistant to brown planthopper, green leafhopper and zigzag leafhopper.21 O. officinalis has a different genome from rice, and producing hybrids with rice is difficult. However, in 1984, because it had useful traits, hybrids with elite rice lines were produced using embryo rescue techniques. Subsequent backcrossing to rice resulted in lines with improved resistance to insects and high yield. These improved lines were tested in international trials in the late 1980s. In Vietnam, where brown planthopper is a serious pest, three of these lines with O. officinalis insect-resistant genes were selected for release to farmers. These three lines were named MTL 98, MTL 103 and MTL 105 in Vietnam.22 From originally being collected to finally providing useful genes to the farmer, more than 30 years had passed. During that time the O. officinalis accession had been multiplied, preserved, distributed and evaluated before finally being used.23

Evaluation The landrace variety Silewah was collected from the highlands of northern

Sumatra in 1974 by Dr. A. T. Perez and collaborators. This upland variety grew to 1300 meter high and was “highly diseased” and had “high sterility” according to the collecting notes of Dr. Perez. Thus it is not surprising that when Indonesian and Japanese scientists visited the area in 1988 they did not find Silewah.24 Two years later, when germplasm collectors visited the village where Silewah was originally collected, Silewah was remembered by one villager but he said it was no longer grown (D. A. Vaughan, 1990, collecting notes) Cold tolerance/ resistance is a very important trait in areas of high altitude. Cold tolerance is a complex trait since, depending on the location, cold weather may adversely affect rice at any stage. At the International Rice Research Institute (IRRI) 24,158 accessions were screened for cold tolerance. From these, only eleven accessions were selected as cold tolerant varieties.25 These germplasm accessions were subsequently re-evaluated in Japan and Korea. In Hokkaido it was found that Silewah was one of the most tolerant varieties at the booting stage.26 Silewah, a tropical japonica variety, was crossed with a japonica breeding line, Hokkai 241, and indica variety IR38 at IRRI. After four years of selection, cold tolerant breeding lines were tested in international trials. In Japan, Norin PL8, with genes from Silewah, has been registered under the Seed and Seedling Law of Japan as an important cold tolerant breeding line for temperate and northern areas such as Hokkaido. This example highlights both how evaluation of seemingly unpromising landraces can reveal useful traits, and also the continual replacement (erosion) of varieties in centers of diversity.

Fundamental Genetic Resources Research as the Foundation of Comprehensive Rice Genome Analysis Three areas of fundamental rice research, diversity analysis, mutation and isogenic line development and linkage analysis, have provided a foundation for the rice genome

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Population Status Wild
Weed  Complex 

Fig. 12.4. The distribution of components of the Vigna angularis complex in Japan collected between 1996-1998.20 Wild and weedy relatives of the Vigna angularis complex have not been reported from Hokkaido. Wild population. Weedy population: Complex population composed of different plant types. Triangle represents the present center of diversity of Vigna angularis complex in Japan. project (Fig. 12.5). These studies have depended on the genetic diversity of conserved rice germplasm.27 In Japan, initial research into rice varietal diversity worldwide was initiated by Kato and coworkers.28 Since then, Japanese researchers have applied a variety of techniques to understand the varietal diversity of rice in greater depth. The use of isozyme analysis confirmed data from morphological and historic data that southern China and northern Southeast Asia is the center of rice diversity. Clinal variation for different esterase isozyme genotypes was found to radiate from this center.29 Application of RFLP analysis to

varietal diversity analysis has helped to provide much greater detail of the varietal differentiation in rice. Kawase et al30 found three distinct clusters of japonica varieties based on RFLP analysis, and these corresponded to varieties from Japan, Southeast Asia and Nepal. Development of genetic stocks such as mutants and isolines has provided an early foundation for subsequent linkage analysis. In depth linkage analysis began in the early 1960s when Nagao and Takahashi 31 first proposed twelve linkage groups corresponding to the haploid chromosome number in rice. Now all of the twelve linkage groups are

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known and have been associated with respective chromosomes using reciprocal translocation and primary trisomics.32,33 The development of aneuploids in rice and advances in linkage analysis (see reviews by Iwata,34 Kinoshita,35 Khush and Kinoshita36) provided the basis to which molecular marker maps—an early product of the rice genome projects—could be integrated.37,38 Now the rice genome project has entered a new phase, with a wide variety of important genes now being rapidly and accurately located on the rice genome (for reviews of this area see Sasaki and Moore39).

Evolving Issues Related to the Management of Crop Genetic Resources Many difficult and some contentious plant genetic issues are being discussed both at national and international levels, and these need to be resolved. Some of these issues are: 1. What is the scope of crop genetic resources? 2. How can access to crop genetic resources be guaranteed? 3. What is the appropriate response to the issues of farmers’ rights and benefit sharing? 4. To what extent do intellectual property rights exist for crop genetic resources and information on this germplasm? 5. What are sovereign rights and how broadly should they be interpreted? 6. How can new technologies best be transferred to developing countries? Crop genetic resources managers have to keep abreast of the evolving issues related to germplasm. They must ensure that all aspects of crop genetic resources system are up to date and in accordance with common internationally accepted policies and practices.

Fig. 12.5. Basic rice research which has provided the foundation for the rice genome project in Japan.

References 1. Nakagahra M. The national program on plant genetic resources system and the perspectives. In: Education and Research for Sustainable Development of Agriculture and Conserving Nature and Agroecosystem in Asian and Pacific Ccountries. Series 4. Tsukuba: University of Tsukuba, 1995:131-141. 2. Miyazaki S. Plant genetic resources: Their conservation and evaluation. Farming Japan 1997; 36(6):12-17. 3. NIAR. Annual Report on Exploration and Introduction of Plant Genetic Resources. Volume 13. Tsukuba: National Institute of Agrobiological Resources, 1997. 4. Omura T. Collection of indigenous rice cultivars. Ikushugaku Saikin no Shinpo (Advances in Breeding) 1970; 11:23-26 (in Japanese). 5. Fukuoka S, Okuno K. QTL analysis for field resistance to rice blast using RFLP markers. Rice Genet Newsl 1998; 14:98-99 6. Okuno K, Katsuta M, Takeya M et al. Collaboration of Pakistan and Japan in collecting genetic resources in Pakistan. Plant Genet Res Newsl 1995; 101:16-19 7. Okuno K, Seki-Katsuta M, Nakayama H et al. International collaboration on plant diversity analysis. In: Japanese MAFF International Workshop on Genetic Resources: Plant Genetic Resources: Characterisation and Evaluation. Tsukuba: Ministry of Agriculture, Forestry and Fisheries, 1998:157-169.

Genebank Management of Crop Genetic Resources 8. Okuno K, Ebana K, Noov B et al. Genetic diversity of central Asian and north Caucasian Aegilops species as revealed by RAPD markers. Genet Res Crop Evol 1998; 45:389-394. 9. Ashmore SE. Status report on the development and application of in-vitro techniques for the conservation and use of plant genetic resources. Rome: International Plant Genetic Resources Institute, 1997:67. 10. JIRCAS/IPGRI. Proceedings of the International workshop on Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Applications. Tsukuba: Japan International Research Center for Agricutural Sciences, 1999; in press. 11. Takeya M, Tomooka N. The illustrated legume genetic resources database on the World Wide Web. Misc Publ Inst Agrobiol Resour 1997; 11:1-93. 12. Japan Society of Plant Taxonomists. Red data book for Japan—Plants. Tokyo: Noson—bunkasha. (In Japanese); 1993:143. 13. NACS-Japan and WWF-Japan. Red Data Book of Plant Communities in Japan. Kamakurashi : Aboku Printing Company, 1996. 14. Miyazaki S, Carter TE, Hattori S et al. Identification of representative accessions of Japanese soybean varieties registered by the Ministry of Agriculture, Forestry and Fisheries, based on passport data analysis. Misc Publ Natl Inst Agrobiol Resour 1995; 8:1-17. 15. MAFF. Root and tuber crops. Proceedings of the International Workshop on Genetic Resources. Tsukuba: Ministry of Agriculture, Forestry and Fisheries, 1995. 16. MAFF. Plant genetic resources: Characterization and evaluation. Proceedings of the International Workshop on Genetic Resources. Tsukuba: Ministry of Agriculture, Forestry and Fisheries, 1998. 17. Yamaguchi H. Wild and weed azuki beans in Japan. Econ Bot 1992; 46(4):384-394. 18. Tomooka N, Vaughan DA, Xu RQ et al. Wild relatives of crops conservation in Japan with a focus on Vigna s. Annual Report on Exploration and Introduction of Plant Genetic Resources. Tsukuba:

149 National Institute of Agrobiological Resources, 1999; In press. 19. Xu RQ, Tomooka N, Vaughan DA et al. The Vigna angularis complex: Genetic variation and relationships revealed by RAPD analysis, and their implications for in-situ conservation and domestication. Genet Res and Crop Evol 1999; in press. 20. Vaughan DA, Tomooka N, Xu RQ et al. The Vigna angularis complex in Japan. Japan Agric Res Quartly 1999; in press. 21. Heinrichs EA, Medrano FG, Rapusas HR. Genetic evaluation for insect resistance in rice. Los Banos: International Rice Research Institute, 1985. 22. Brar DS, Khush GS. Alien introgression in rice. Plant Molec Biol 1997; 35:35-47. 23. Vaughan DA, Sitch LA. Gene flow from the jungle to farmers. BioScience 1991; 41:22-28. 24. Oka M, Akana Y, Kikuchi H et al. Joint exploration for collecting rice varieties in Sumatra, Indonesia, 1988. In: Annual Report on Exploration and Introduction of Plant Genetic Resources, Vol. 5. Tsukuba: NIAR, 1988:153-191. 25. Vergara BS, Visperas RM. Adaptability and use of indica varieties in high-altitude areas. Potential productivity and yield constraints of rice in east Asia. In: Proc Intl Crop Sci Symp. Tokyo: Crop Science Society of Japan, 1994:39-52. 26. Satake T, Toriyama K. Two extremely cool-tolerant varieties. Intl Rice Res Newsl 1979; 4:9-10. 27. Nakagahra M, Okuno K, Vaughan DA. Rice genetic resources: History, conservation, investigative characterization and use in Japan. Plant Molec Biol 1997; 35:69-77. 28. Kato S. On the affinity of cultivated varieties of rice plants, Oryza sativa L. J Dept. Agric Kyushu Imp Univ 1930; 2:241-276. 29. Nakagahra M. Geographic distribution of esterase genotypes of rice in Asia. Rice Genet Newsl 1984; 1:118-120. 30. Kawase M et al. Intraspecific variation and genetic differentiation based on restriction fragment length polymorphism in Asian cultivated rice, Oryza sativa L. In: Rice Genetics II. Los Banos: International Rice Research Institute, 1991:467-473.

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31. Nagao S,Takahashi M. Genetical studies on rice plant. XXVII. Trial construction of twelve linkage groups of Japanese rice. J Fac Agric Hokkaido Univ 1963; 53:72-130. 32. Iwata N, Satoh H, Omura T. Relationship between the twelve chromosomes and the linkage groups. (Studies on the trisomics in rice plants, Oryza sativa L). Jpn J Breed 1984; 34:314-321. 33. Khush GS, Singh RJ, Sur SC et al. Primary trisomics of rice: Origin, morphology, cytology and use in linkage mapping. Genetics 1984; 107:141-163. 34. Iwata N. The relationship between cytologically identified chromosomes and linkage groups. In: Rice Genetics. Proceedings of the International Rice Genetics Symposium. Los Banos: International Rice Research Institute, 1986: 229-238. 35. Kinoshita T. Report of committee on gene symbolization: Nomenclature and linkage analysis. Rice Genet Newsl 1995; 12:9-153. 36. Khush GS, Kinoshita T. Rice karyotype, marker genes, and linkage analysis. In: Khush GS, Toenniessen GH, eds. Rice Biotechnology. Wallingford: CAB International, 991:83-108. 37. Kurata N et al. A 300 kilobase interval genetic map of rice including 883 expressed sequences. Nature Genet 1994; 8:365-372. 38. McCouch SR, Kochert G, Yu ZH et al. Molecular mapping of rice chromosomes. Theor Appl Genet 1988; 76:815-829. 39. Sasaki T, Moore G, eds. Oryza: Molecule to Plant. Dordrecht: Kulwer Academic Publishers, 1997:254.

40. Kitamura K. Spontaneous and induced mutations of seed proteins in soybean (Glycine max L. Merrill). Gamma Field Symposium 1991; 30:61-69. 41. Tsukamoto C et al. Group A acetyl saponin deficient mutant from the wild soybean. Phytochemistry 1992; 31:4139-4142. 42. Hoshino T, Ito S, Hatta K et al. Development of waxy common wheat by haploid breeding. Plant Breed 1996; 46:185-188. 43. Yamada T. Genetic resources and breeding of wheat and barley in Japan. In: Textbook for the Group Training Course in Plant Genetic Resources. Tokyo: Japan International Cooperation Agency and Tsukuba: National Institute of Agrobiological Resources, 1993. 44. Seko H. Mechanisms for the evaluation of plant genetic resources in Japan. In Plant Genetic Resources: Characterization and Evaluation MAFF International Workshop on Genetic Resources. Tsukuba: MAFF, 1998:189-198. 45. Shiotani I. Use of root and tuber crop genetic resources in Japan. In: MAFF International Workshop on Genetic Resources: Root and Tuber Crops. Tsukuba: MAFF, 1995:23-36. 46. Komaki K. Sweetpotato genetic resources and breeding in Japan. In: MAFF International Workshop on Genetic Resources: Root and Tuber Crops. Tsukuba: MAFF, 1995:115-120. 47. Ishiuchi D. Genetic resources and breeding of vegetable crops. In: Textbook for the Group Training Course in Plant Genetic Resources. Tokyo: Japan International Cooperation Agency and Tsukuba: National Institute of Agrobiological Resources, 1993.

CHAPTER 13

Biodiversity Conservation Ex Situ and In Situ Conservation: A Case in Turkey A. Tan

Introduction

T

urkey is a country significant for its rich plant genetic resources/plant diversity. Two of Vavilov’s centers of origin (i.e., the Near Eastern and Mediterranean Centers) extend into Turkey. This, of course, indicates that Turkey is a center of origin and/or center of diversity of several crop plants and many plant species. Turkey is endowed with a rich diversity of families, genera and species of plants (163 families, 1225 genera, 9000 species). 3000 plant taxa, out of 9000 species, are endemic to the area. This rich biodiversity (primitive landraces, wild crop relatives and other wild plant species) of Turkey continues to provide new sources for important traits to improve agricultural production and introduce new sources of efficiency worldwide. The potential and the reasons for this richness can be described by these characteristics:1 1. Meeting place of three phytogeographical regions; 2. Center of origin and center of diversity of many crop/plant species; 3. Domestication center for many crops; 4. High species endemism; 5. Bridge between Europe and Asia, apparently having served as a migration route for the penetration of other elements. Factors such as environmental destruction, overexploitation, replacement of traditional cultivars, and modernization of

agriculture result in the erosion of genetic diversity. Some regions of Turkey are now undergoing some degree of change in terms of trade, exports, urbanization and market driven farming. Despite the positive aspect of such changes, these have greatly contributed to the decrease, even loss, of agrobiodiversity.2 There is no disagreement among plant scientists about including Anatolia in the two centers of diversity and center of origin, the Near Eastern Center and the Mediterranean Center, which overlap in Turkey. Paleoethnobotanical findings have shed light on the origins and development of plant domestication and confirmed the center of origin, based on plant exploration studies. Early sites for finds of domesticated plants are Catalhoyuk, Can Hasan, Aceramic Hacilar and Late Neolithic Hacilar, Mersin and Cayonu dating to about 7000-5000 BC.3-6 The early Neolithic findings in Anatolia are shown in Table 13.1. Turkey is one of the centers of origin of some cultivated plants like Linum, Allium spp., Hordeum, Secale, Triticum, Avena, Cicer, Lens, Pisum, Vitis, Amygdalus, Prunus, Beta etc. The potential of plant diversity in Turkey has been determined and recognized by well known plant scientists.7-10 Turkey is also described as a microcenter for Amygdalus spp., Cucumis melo, C. sativus, Cucurbita moshata, C. pepo, Lens culinaris, Lupinus spp., Malus spp., Medicago sativa, other annual

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Table I3.1. Early Neolithic findings in Turkey2,6 Location and Period

Plant finds

Cayonu 7200-6500 BCE

Wild einkorn, einkorn, naked (free threshing) wheat, wild barley, emmer, wild emmer, pea, lentil, vetch, flax

Hacilar 6750 BCE

Wild einkorn, emmer, naked barley, lentil

Can Hasan 6500 BCE 6500 BCE

Wild einkorn, einkorn, emmer, naked wheat, two rowed barley, lentil, vetch

Catalhoyuk 6000-5000 BCE

Einkorn, emmer, naked wheat, naked barley, pea, vetch

Erbaba 6000-5000 BCE

Einkorn, emmer, naked wheat, two rowed barley, naked barley, pea, lentil, vetch

Medicago spp., Onobrychis viciifolia, Phaseolus vulgaris, Pistachio spp., Prunus spp., Pyrus spp., Trifolium spp., Vicia faba, Vitis vinifera and Zea mays.11 Turkey’s wealth in plants is apparent in the fact that 3000 out of the 9000 plant species are endemic to the area.12,13 Endemics are scattered throughout the country, but few are found in Trace. The largest number of endemics occurs in the Irano-Turanian Region and the Mediterranean region.13 The endemics show definite areas of concentration throughout the country, predominating in the mountainous parts of south and southeast Anatolia. Wild relatives and wild ancestors of cereals include those of wheat (wild einkorn, Triticum boeoticum; wild emmer, T. dicoccoides; goat grass, Aegilops spp.), barley (Hordeum spontaneum, H. bulbosum, H. marinum and H. murinum), oats (Avena spp.) and rye (Secale spp.).14 Five wild species of lentil (Lens orientalis, L. nigricans, L. ervoides, L. montbretii, L. odemensis), the wild and weedy forms of Pisum (primary progenitor of the pea, P. humile; P. elatius ) and wild progenitors of Cicer (C. pinnatifidum, C. echinospermum, C. bijugum, C. reticulatum) occur in Turkey.15 An extremely rich variety of medicinal, aromatic and ornamental plant species are

found in the flora of Turkey.16 Within the ornamental plants great numbers of bulbous or tuberous plants, woody and herbaceous perennials, biennials and annuals are found. Most of the ornamental species grow in wild habitats among deciduous shrubs and under deciduous trees or scattered among bushes and/or rocks. The diversity of ornamental plant species is related to the diverse topography and climate of Turkey. Medicinal and aromatic plants in Turkey present almost the same situation. The rate of endemics is also high within those plant groups. A number of vegetables have their origin in Anatolia. The wild relative of Brassica, B. cretica, is found in south Anatolia (in the south Aegean and Mediterranean belt). Wild Raphanus raphanistrum also has a distribution in the west and south coastal parts. Wild celery, Apium graveolens; the wild beet B. maritima and other Beta spp.; wild carrots, Daucus spp.; wild rockets, Eruca spp.; wild lettuce, Lactuca spp.; and wild mustard, Sinapis spp., are some of the wild vegetables commonly used as vegetable or salad plants. Many other wild plant species are used as salad and vegetable plants, but most of those species are not utilized for development and/ or are neglected.2

Biodiversity Conservation Ex Situ and In Situ Conservation: A Case in Turkey Indigenous fruit trees are also found in Turkey. These woody plants are valuable genetic resources as food crops. Because of their resistance to insects and disease, and their natural adaptability to an array of sites, such species as chestnut (Castanea sativa), olive (Olea europea) and walnut (Juglans regia) are valuable fruit genetic resources. Wild relatives of apple (Malus spp.) pear (Pyrus spp.) and plum (Prunus spp.) are also found in Turkey.17 The wild pistachios P. terebinthus and P. lentiscus; wild hazel nuts, Corylus spp.; wild plums Prunus spinosa and P.divericata; wild cornel cherry, Cornus sanguinea; wild pears Pyrus elaegrifolia and other Pyrus species; and wild almonds, Amygdalus spp., are some of the wild relatives of fruit trees found in Turkey. Sweet and sour cherries are also indigenous; various wild types are found, especially in North Turkey. Most of these wild relatives of fruit trees are utilized as rootstock. There are also wild relatives of other fruits like wild strawberry, Fragaria spp., and wild blackberries, Rubus spp.2 Wild relatives of forage grasses and legumes commonly occur in Turkey. The natural pastures and meadows show high genetic diversity. This has led to ecological populations of forage which are superior to those currently used and can be released as commercial cultivars with a minimum of further selection and breeding. But, most of them are threatened with genetic erosion, mainly due to overgrazing. Landraces are found in the areas where crop species first arose through domestication. Turkey also lies within a broad region of of crop domestication. Therefore, there are highly variable domesticated crops, as well as landraces with unique characteristics, in Turkey. The traditional agricultural systems used in the backyard gardens to grow vegetables, especially in remote areas of Turkey, have been important in bringing together some species that have subsequently hybridized. Some industrial crops like flax have a history of ancient cultivation in Turkey. Turkey is the junction between primary and secondary centers of diversity of some crops, like sesame; different forms of those crops are found.18 Although Turkey is not a center of

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origin of tobacco, sunflower and corn, these crops also have diverse landraces which are adapted to different ecological conditions. Various local types of fruits are found in Turkey.17 Prunus species are represented by different fruit types such as almond, plum, cherries, apricot etc. Almond types may differ widely in vigor, yield, nut and kernel quality, and flowering time. Various plum types are found having very ancient cultivation and wide distribution. Different types of sweet cherry have also grown for centuries throughout Turkey. Spontaneous seedlings are occasionally allowed to develop into bearing trees, especially those of apricot, almond and cherry plum (P. ceracifera), which increases the rate of existing diversity.

Biodiversity Conservation Activities in Turkey The plant genetic resources activities were started by the establishment of the Crop Research and Introduction Center (CRIC) in 1964 (a more recent name of the institute is the Aegean Agricultural Research Institute, AARI). The plant genetic resources activities reorganized in 1976 within the framework of the National Plant Genetic Resources Research Program (NPGRRP). The objective of NPGRRP is the exploration, collection, conservation (both ex situ and in situ) and evaluation of existing plant genetic resources and plant diversity of Turkey for today and the future. AARI has been designated as a coordination center for the national program.1,2,19

In Situ Conservation The recent application of in situ conservation projects within the framework of GEF aims to maintain the wild crop genetic resources in their natural habitats in existing state-owned lands. This project is the first of its kind in the in situ world to address both woody and non-woody crop relatives from an integrated multispecies and multisite approach. 20 This has been done through conducting ecogeographical surveys and inventories to provide bases for establishment of in situ Gene Management Zones (GMZs) in selected pilot areas that are rich in target wild crop relatives. The highest priorities have

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been given to globally significant non-woody crop species which are in the first gene pool of cereals (wheat and barley) and legumes (Vicia and Lens), as well as important woody species such as chestnut, plums, and selected forest species. The project has initiated and developed a mechanism to foster the ongoing National Plant Genetic Resources Research Program for identifying, designating and managing areas specifically for in situ conservation of nationally and globally significant wild crop relatives which have originated in Turkey.2 The project has also aimed to integrate in situ conservation with existing ex situ conservation programs in Turkey. The project uses the complementary strengths of the Ministry of Agriculture and Rural Affairs (MARA), with experience in genetic resources activities, especially in ex situ conservation, the Ministry of Forestry (MOF), which has experience in land management, and the Ministry of Environment (MOE), which has a strategic outlook on resource management. MARA and MOF are the implementing ministries of the In situ Conservation Project. The lead institute of MARA, the Aegean Agricultural Research Institute (AARI) coordinates activities for in situ conservation projects and collaborates with other related research institutes. The pilot areas have been selected, and designated as: The Kaz Dag Area of the northwestern Aegean Region; Ceylanpinar in southeastern Turkey; the mountains of southern Anatolia on the southern part of the Anatolian diagonal. The project has been designated around the following five components: 1. Site survey and inventories; 2. Designation of GMZs; 3. Data management; 4. Development of a National Plan for in situ conservation; 5. Institutional strengthening within and between MARA, MOF and MOE. The project started in 1993 with training of the project staff of MARA, MOF and MOE. The survey activities have been completed at three designated areas, and the GMZs have already been identified according to the results of survey and inventory and genetic variation analysis.21-27

In 1995, IPGRI, together with national programs in nine countries, formulated a global project to strengthen the scientific basis of in situ conservation of agricultural biodiversity. Nine countries involved in the project are: Burkino Faso, Ethiopia, Nepal, Vietnam, Peru, Mexico, Morocco, Turkey and Hungary. The main objectives of the project are: 1. To support the development of a framework of knowledge on farmer decision—making processes that influences—in situ conservation of agricultural biodiversity; 2. To strengthen national institutions for planning a new implementation of conservation programs for agricultural biodiversity; and 3. To broaden the use of agricultural biodiversity and participation in its conservation by farming communities and other groups.

Ex Situ Conservation Ex situ conservation activities have been undertaken since 1964. They are still in progress within the framework of NPGRRP. Collection by sampling maximum variations and determination of the interspecific, agroecological and phytogeographical distribution of plant species are the first steps of the project. Data of former surveys and expeditions are compiled, and priorities of locations and plant species are considered to eliminate duplicate efforts, during planning of the collection missions. The missions each year are programmed to collect the existing plant genetic resources for eight plant groups (cereals, forage, food legumes, vegetables, industrial crops, ornamental, medicinal and aromatic plants, fruit and grapes) and endemic plant species. The collections in each plant group consist of landraces, wild relatives and other wild plant species considered to be in the plant group. The endemics are collected specifically, separately from the plant groups. Ex situ conservation is implemented both for generative and vegetative collections, which are preserved in seed gene bank and field gene banks, respectively. The vegetatively propagated material, mainly fruit genetic resources, are kept in field gene banks at 13 in-

Biodiversity Conservation Ex Situ and In Situ Conservation: A Case in Turkey stitutes (including AARI). Garlic, some medicinal and aromatic plants and ornamental collections are also kept as field collections at AARI. The national collection contains the landraces, wild and weedy relatives (both for seed and vegetative collections) and other wild species which are especially economically important plants and endemic species. The main users of the material are the plant breeders and researchers both from Turkey and abroad. There are some research activities on the in vitro storage techniques of some vegetative plant species. The storage facilities of the Izmir Gene Bank (at AARI) for seed collection have been designed for the needs of long term and medium term storage for both base and active collections, respectively.28 Cold rooms work at minus 18°C for long term and 0°C for medium term storage for base and active collections. For temporary storage, aluminum laminated foils are used. All conditions in the gene bank comply with internationally recommended standards. For safe duplication of the base collection, other storage facilities are available in Ankara (at CRIFC).

Documentation Documentation is one of the main functions of the NPGRRP for both ex situ and in situ activities. A Database Management System exists for documentation of both ex situ and in situ conservation information.23 Since the in situ conservation program is complementary to ex situ conservation, the two databases are linked and complementary to each other. The Geographic Information System (GIS) is available to evaluate the quantitative and spatial data gathered, especially from survey and inventory activities.30

Evaluation and Characterization NPGRRP makes a clear distinction between the processes of characterization and evaluation of the genetic resources material holding at the gene banks (seed and field gene banks). The characterization activities are coordinated by NPGRRP and carried out within the framework of NPGRRP. The evaluation programs are conducted in cooperation with the National Plant Breeding Programs. The data resulting from evaluation carried out

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by users of the samples are returned if the evaluation and/or characterization work is cooperatively planned by NPGRRP. The annual report of the characterization/evaluation project also contains the results.

National Legislation, Policies and International Agreements The national plant genetic resources collections sre protected by legislation. The regulations on collection, conservation and utilization of Plant Genetic Resources (PGR Code of Conduct) were published in the Official Gazette on 15 August 1992. The responsibilities of related institutions, including the institutes of MARA, universities or the institutes of other ministries and other related institutions working on related aspects of PGR are described, as well as cooperation with foreign institutions, IARCs and CGIAR centers. The plant genetic resources exchange mechanism has also been regulated according to specific principles. Turkey is also a member of FAO commission on Plant Genetic Resources for Food and Agriculture and adheres to the International Undertaking on Plant Genetic Resources. Turkey has actively contributed to the preparation of the Global Plan of Action (GPA) and the Report on the State of the World’s Plant Genetic Resources, presented in a national report, and participated in the intergovernmental meetings and Fourth International Technical Conference that culminated in the formal adoption of GPA. Turkey has had close cooperation with IBPGR/IPGRI since its establishment, and is a member of the European Cooperative Program for Conservation and Utilization of Plant Genetic Resources (ECP/GR) and of the West Asia and North Africa Plant Genetic Resources Network (WANANET) of IPGRI. Turkey is a signatory of the Bern Convention and RAMSAR Convention. The Convention on Biological Diversity (CBD) was signed in 1992 and ratified by Turkish Government in 1996. Turkey has also signed CITES.

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References 1. Tan A. Plant diversity and plant genetic resources in Turkey. Anadolu 1992; 2: 50-64. 2. Tan A. Current status of plant genetic resources conservation in Turkey. In: Zencirci N, Kaya Z, Anikster, Y Adams WT, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996, Antalya, Turkey. Rome: IPGRI and Ankara: Central Research Institute for Field Crops, 1998:5-16. 3. Flannery KV. Origin and ecological effects of early Near Eastern domestication. In: Ucko PJ, Dimbleby GW, eds. The Domestication and Exploitation of Plants and Animals. London: Duckworth, 1969:75-100 4. Renfrew JM. The archaeological evidence for domestication of plants: Methods and Problems. In: Ucko PJ, Dimbleby GW, eds. The Domestication and Exploitation of Plants and Animals. London: Duckworth, 1969:145-172. 5. Zohary D. The progenitors of wheat and barley in relation to domestication and agricultural dispersal in the old world. In: Ucko PJ, Dimbleby GW. eds. The Domestication and Exploitation of Plants and Animals. London : Duckworth, 1969:47-66. 6. Harlan JR. The Living Fields: Our Agricultural Heritage. Cambridge: Cambridge University Press, 1995. 7. Zhukovsky PM. Agricultural Turkey. Moscow: Acad. Sci. USSR, 1933. 8. Davis PH. Flora of Turkey and Aegean Islands. Vol 1. Edinburgh: University of Ediburgh, 1965. 9. Bennet E. Adaptation in wild and cultivated plant population. In: Frankel OH, Bennet E, eds. Genetic Resources in Plants-Their Exploration and Conservation. Oxford: Blackwell Science Publishers, 1970:115-130. 10. Zagaja SW. Temperate zone tree fruits. In: Frankel OH, Bennet E, eds. Genetic Resources in Plants-Their Exploration and Conservation. Oxford: Blackwell Science Publishers, 1970:327-334. 11. Harlan JR. Anatomy of gene centers. Am Nat 1951; 85:97-103. 12. Ekim T, Koyuncu M, Erik S et al. R. List of Rare Threatened and Endemic Plants in Turkey, TTKD-18. Ankara: TTKD, 1989.

13. Tan A.. Türkiye’de bitkisel çesitlilik, endemik tür dagilimi ve muhafazasi Biodiversity and endemism in Turkey). Tarim ve Köy 1992; 74:22-24. 14. Firat AE, Tan A. Ecogeography and distribution of wild cereals in Turkey. In: Zencirci N, Kaya Z, Anikster et al, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Ankara: Central Research Institute for Field Crops, 1998: 81-86. 15. Acikgöz N, Sabancy CO, Cinsoy AS. Ecogeography and distribution of wild legumes in Turkey. In: Zencirci N, Kaya Z, Anikster, Y Adams WT, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Ankara: Central Research Institute for Field Crops, 1998: 113-122. 16. Ulubelde M, Ekim M, Tan A. The aromatic and medicinal plants in Turkey. In: Raychaudhuri SP ed. Recent Advances in Medicinal Aromatic and Spice Crops. New Delhi: ISMAP, 1991; 1:28-31. 17. Gönülsen N. Bitki Genetik Kaynaklari Meyve ve Bag Envanteri (Fruit Genetic Resources Inventory of Turkey). Ege Böl Zir Ara Ens Yay. Menemen: AARI, 1986; No.79. 18. Tan AS, Tan A. Morphometric variation analysis on Turkish sesame (Sesamum indicum L.) J Anadulu 1996; 6:1-23. 19. Firat AE, Tan A. Turkey maintains pivotal role in global genetic resources. Diversity 1995; 11:61-63. 20. Firat AE, Tan A. In situ conservation of genetic diversity in Turkey. In: Maxted BV, Ford-lloyd BV, Hawkes JG, eds. Plant Genetic Conservation. The In situ Approach. London: Chapman and Hall, 1997:254-262. 21. Eser V, Göcmen B, Erisen S et al. Determination of biochemical variation in an Aegilops tauschii population collected from Ceylanpinar. In: Zencirci N, Kaya Z, Anikster, Y Adams WT, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Central Research Institute for Field Crops, 1998:93-98.

Biodiversity Conservation Ex Situ and In Situ Conservation: A Case in Turkey 22. Kitiki A, Tan A. Vegetation survey in the southern part of the Anatolian diagonal and possible gene management zones for wild crop relatives. In: Zencirci N, Kaya Z, Anikster Y, Adams WT, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Central Research Institute for Field Crops, 1998:129-134. 23. Karagöz A. In situ conservation of plant genetic resources in the Ceylanpinar State Farm. In: Zencirci N, Kaya Z, Anikster Y et al, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November,1996. Antalya, Turkey. Rome: IPGRI and Ankara; Central Research Institute for Field Crops, 1998:87-92. 24. Kucuk SA, Tan AS, Sabancy CO et al. Ecogeographical and floristic differentiation of chestnut gene management zones at Kazdag. In: Zencirci N, Kaya Z, Anikster Y et al, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Ankara: Central Research Institute for Field Crops, 1998: 135-148. 25. Onal MK, Sabanci CO, Kucuk SA, Cinsoy AS . The pomological variation patterns of wild plum (Prunus divaricata Ledeb.) and chestnut (Castanea sativa Miller) in Kazdag. In: Zencirci N, Kaya Z, Anikster, Y et al, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November,1996. Antalya, Turkey. Ankara; Central Research Institute for Field Crops, 1998:149-154.

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26. Sabancy CO, Onal MK, Tan AS et al. Ecogeographical and floristic differentiation of plum gene management zones at Kazdag. In: Zencirci N, Kaya Z, Anikster, Y Adams WT, eds. The Proceedings of International Symposium on In Situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Rome; IPGRI and Ankara: Central Research Institute for Field Crops, 1998:155-162. 27. Tan AS, Ulubelde M. Selection criteria and planning of gene management zones (GMZs) for in situ conservation. In: Zencirci N, Kaya Z, Anikster, Y Adams WT, eds. The Proceedings of International Symposium on In situ Conservation of Plant Genetic Diversity. 4-8 November, 1996. Antalya, Turkey. Rome: IPGRI and Ankara; Central Research Institute for Field Crops, 1998:363-372. 28. Tan A. Gen bankalari ve tohum muhafaza (Gene banks and seed conservation). Tarim ve Köy 1992; 80:35. 29. Tan A, Tan AS. Data collecting and analysis: for in situ on-farm conservation. In: Jarvis JI and Hodghin T, eds. Strenthening the Scientific Basis on In Situ Conservation of Agricultural Biodoveristy On-Farm. Options for Data Collecting and Analysis. Proceedings of a workshop to develop tools and procedures for in situ conservation on-farm, August 25-29, 1998:31. 30. Tan A, Tan AS. Database management systems for conservation of genetic diversity in Turkey. In Zoncirci N, Kayo Z, Anikster Y, et al, eds. The Proceedings of International Symposium on In Sity Conservation of Genetic Diversity. November 4-8, 1996, Antalya Turkey. Ankara Central Research Institute for Field Crops, 1998:203-321.

CHAPTER 14

Plant Genetic Resources for Food and Agriculture: Status and Future Prospects K.V. Raman and K.N. Watanabe

P

lant genetic resources for food and agriculture (PGRFA) is broadly defined to include resources which contribute to people’s livelihoods by providing food, medicine, feed for domestic animals, fiber, clothing, shelter and energy, etc.1 The use of plant genetic resources to enhance crop productivity and sustainability is considered a high priority subject today. In this article we discuss: 1. The contribution and value of PGRFA in modern varieties, including its role in integrated pest management (IPM); 2. The state of utilization of plant genetic resources for food and agriculture, including genetic vulnerability and erosion; and 3. Genetic improvement of crops, including the current and future use of biotechnology applications. Partnerships between the public and private sector are recommended in order to further promote the use of PGRFA. The strengths of US scientists could be harnessed for PGRFA application through active networking and partnerships. An example of such efforts is demonstrated in the newly developed project, The Cornell-Eastern Europe-Mexico (CEEM) International Collaborative Project in Potato Late Blight Control. This project aims to exploit the full potential of potato genetic resources to develop cultivars with durable

resistance to the disease-causing fungus responsible for the Irish potato famine. Through such partnerships, comparative advantages are fully exploited. The lessons learned from these programs as they mature will set the stage for further development of successful global programs to address critical PGRFA associated problems.

Contribution and Value of PGRFA The current world population of 5.6 billion is expected to double to 11 billion by 2050. Ninety-seven percent of that increase will occur in developing countries, where 90% of the population will be living 50 years from now. Asia will be, by far, the most populous continent. Food demands will more than double by the year 2025 and could triple by 2050.2 By the year 2025, the grain requirements of the developing countries will be more than three times that of the entire USA harvest. The enormity of the food security issue is best illustrated by the fact that in the next fifty years the global population will consume twice as much food as has been consumed since agriculture began 10,000 years ago.3,4 The challenge for the agricultural sector is to double food production by 2025, and triple it by 2050, on less land, with less water, and under increasingly challenging conditions.5 Food security and

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biodiversity will continue to be our most pressing challenges. For the developed nations, population increases can be accommodated in part by eating lower on the food chain and consuming grains directly, but the developing world is already doing that. Because new arable land in the developing world is becoming steadily more scarce, higher yields can come with better agronomy dependent on a combination of more fertilizer, plowing, water lifting energy and improved plant material. All but the last are agricultural inputs that compete for meager resources available in developing countries. Therefore, breeding for better crop plants will be the central focal point around which all strategies to increase crop yields will develop. Watanabe and Pehu6 in a recent book titled, “Plant Biotechnology and Plant Genetic Resources for Sustainability and Productivity”, outline several case studies on different crops/ commodities that look closely at the introduction of a particular crop or commodity. They also cover topics on the contribution of plant introduction to the global economy. Nevertheless, it is relevant to take a look at the contribution and other values of PGRFA to modern varieties, including its role in integrated pest management (IPM). The improvements in agricultural production brought about through the use of modern varieties have been possible because of the rich and varied genetic diversity in farmers’ landraces, and of wild and weedy species. There are now several examples of the introgression of valuable agronomic traits from landraces, and wild relatives of crops.1 The green revolution of the 1960s is a good example that permitted spectacular increases in yields of rice and wheat, without which it is unlikely that the food needs of rapidly expanding populations would have been met. In wheat, rice and maize, about half of the increase in production has been ascribed to breeding new varieties through the use of plant genetic resources. The remaining increase was derived from the use of fertilizers, pesticides and improved crop management. Over the past 25 years, irrigated rice

production has increased at 3% per year. Nearly 60% of that growth is the result of increases from breeding. Other successful products of this era through plant breeding are hybrid corn and changes in the photoperiod response of soybeans, making it the most important legume and oil crop in the world.7,8 There is no doubt that plant genetic resources are very valuable. Estimates of the global value associated with the use of these resources vary from hundreds of millions to tens of billions of dollars per year. For example, the contribution of rice landraces from South Asia, assembled in the region’s genebanks, is estimated to be about $150-200 million per year.9 Similarly, estimates for the core wheat collection, maintained at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico, to agriculture in the OECD countries range from $300 million to $11 billion per year.10 These ranges indicate the difficulty in assessing their value. Most estimates do not usually give the value of the genetic material per se, but rather aggregate value of both the genetic material and the work of plant breeders and other research inputs. Host plant resistance to pests and diseases is the first line of defense, and is an essential component of integrated pest management (IPM) programs worldwide. Several successful IPM programs depend on the development of plant varieties resistant to both biotic and abiotic stresses. Plant genetic resources (wild, primitive and cultivated gene pools) provide the essential genes needed to develop resistant crop plants. Successful programs in rice, potatoes, beans, wheat, maize, vegetables, fruits and other crops have been developed using conventional breeding strategies. However, as a result of the last twelve years’ using tools of genetic engineering, it is now possible to add specific genes (transgenes) to many crop plants, most microorganisms, and some insects. During the period from 1986 to 1997, approximately 25,000 field trials of transgenic crops were conducted globally on more than 60 crops with 10 traits in 45 countries. The most frequent crops featured in transgenic crop field trials were maize, tomato, soybean,

Plant Genetic Resources for Food and Agriculture: Status and Future Prospects canola, potato and cotton, and the most frequent traits tested were herbicide tolerance, insect resistance, product quality and virus resistance.11,12 Plant genetic resources continue to play a major role in genetic improvement of crops. Strong international collaboration is already underway in: 1. The characterization of important genes and gene products; 2. The relationship between gene structure and function; 3. Regulatory mechanisms of gene expression; 4. Alteration and use of germplasm resources; and 5. The cellular and molecular mechanisms underlying human nutrient requirements. 2 Research in these areas is essential to meet the needs of sustainable food and agriculture development.

Utilization of PGRFA Including Genetic Vulnerability and Genetic Erosion Not all genetic resources seem to have the same immediate utility. Public and private breeders use germplasm mostly from adapted and productive commercial varieties. A survey conducted by the International Plant Genetic Resources Institute (IPGRI) indicated that only 6.5% of germplasm used by breeders came directly from an exotic source. Of this small portion, two-thirds were sourced from landraces conserved ex situ in genebanks. One-third was sourced from germplasm found in situ, with a predominant usage of landraces over wild species. Another survey in Germany gave similar percentages (6.9% of commercial varieties containing materials from genebanks). Surveys of maize germplasm in the United States have given even lower figures for use of exotic germplasm resources. (http://www.worldseed.org/-assinsel/osl-ass.htm). At the global level, world food security depends, to a large extent, on the 30 or so crop species that provide most of the dietary energy or protein, and in particular on the three crops—rice, wheat and maize—which

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together provide more than half of it. Efficient utilization of plant genetic resources is a key to improving agricultural productivity and sustainability and can contribute to socioeconomic development, food security and the alleviation of poverty.14 These vital resources are seriously threatened by genetic vulnerability and erosion. Genetic vulnerability is defined as the condition that results when a widely planted crop becomes uniformly susceptible to a pest, pathogen or environmental hazard as a result of its genetic constitution, thereby creating a potential for widespread losses.15 One of the main causes of genetic vulnerability is the widespread replacement of diverse varieties by homogeneous varieties. Examples include the growing of the rice variety “IR 36.” In 1982 this variety was grown on 11 million hectares in Asia.16 Similarly, over 67% of the wheat fields in Bangladesh were planted to a single wheat cultivar, “Sonalika”, in 1983, and 30% of Indian wheat fields to the same cultivar in 1984.17 Reports from the USA in 1972 and 1991 indicate that, for each of the eight major crops, fewer than nine varieties made up between 50% and 75% of the total acreage grown.18 Ireland’s Country Report cites 90% of its total wheat area sown to just six varieties. The dangers of planting large areas to a few genetically uniform crop varieties must be recognized, as these varieties could suddenly become uniformly susceptible to new pathogen races and be wiped out. The most famous example of this is the potato famine of 1845-1848, when a epidemic of late blight (Phytophthora infestans) wiped out the potato crop in Europe and North America (for additional details see section on the CEEM project). Some other examples include: 1. Severe epidemics of Shoot fly and Karnal bunt in the 1970s in modern wheat varieties in India;17 2. The new race of corn leaf blight in the US which destroyed more than 15% of the crop in 1970;18 and 3. In 1975, white clover varieties in the UK had to be totally abandoned for several years when a new pathogen, Scerotinia trifoliorum, killed off white

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clover populations throughout much of Britain—all recommended varieties were susceptible.1 Similar examples are cited for the loss of wheat in Russia due to the severe winter of 1972 and the rust attack on sugar cane during 1979/80 in Cuba. This attack on the sugar cane, which covered 40% of the country, resulted in the loss of more than a million tons of sugar, worth about US $500 million. Genetic erosion also leads to the loss of genetic diversity. This includes the loss of individual genes as well as the loss of particular combinations of genes present in locallyadapted landraces. The main causes of genetic erosion are: replacement of local varieties; changing agricultural systems; overexploitation of species; reduced fallow; overgrazing; land clearing; environmental degradation; pests/weeds/diseases; population pressure; and legislation policies .1 It is now clear that these forces need to be better understood so that the problem can be addressed more effectively. Further, the relationship between genetic uniformity and genetic vulnerability needs to be better understood. Both genetic vulnerability and genetic erosion are poorly documented, and more research needs to be done for the development of better indicators and measurements. At the global level, there is now a consensus to develop an integrated approach to the conservation and utilization of PGRFA in major crops. This includes the need to: 1. Reduce genetic erosion in the field and the need to promote in situ conservation; 2. Ensure that the genetic diversity of major crops is adequately represented in ex situ collections, and that these collections are secure and available for use; 3. Use genetic diversity effectively, inter alia through improvement programs; and 4. Promote sufficient levels of genetic diversity in crops and breeding lines. These issues are examined on a crop by crop basis in a recent publication.1

Genetic Improvement and Use of Biotechnology Applications in PGRFA As the 21st century begins, scientists are close to being able to identify and manipulate individual complexes of genes in plant genetic resources that act together to produce specific plant traits. The wild germplasm and agronomically unadapted relatives contain great genetic wealth. Tools such as DNA markers and mapping, developed in molecular genetics, allow scientists to identify useful genes from such germplasm and transfer them efficiently to improved cultivars. DNA based technologies can be used to measure genetic variation quickly and accurately. They can identify novel types and extract genetic variation quickly and accurately that would otherwise remain hidden among the thousands of accessions in germplasm collections. Recent studies using this approach in wild relatives of rice, tomatoes, wheat and soybeans have led to the discovery of novel genes that can boost the output of these crops. Without the application of new DNA mapping technologies, these valuable genes would have remained undetected. Genetic linkage maps have made it possible to study the chromosomal locations for improving yield and other complex traits important to agriculture. Additional details on the use of these techniques can be found in Tanksley et al19 and Tanksley and McCouch.20 Molecular marker technology can only produce knowledge about important economic traits in combination with intelligently designed field experiments. Significant accomplishments in molecular biology are now enabling scientists to better understand the genetic control of complex and quantitative traits. DNA marker technology also provides an accurate estimation of genetic diversity and helps in the identification of duplicates in germplasm banks. It also enables the accurate identification of commercial varieties or other germplasm, subject to concerns about associated intellectual property rights. Evaluations with this technology provide knowledge on general genetic variation existing among and between groups of accessions using multivariate analysis. Then, a well documented group

Plant Genetic Resources for Food and Agriculture: Status and Future Prospects of accessions can be characterized as a core collection which represents a known degree of general genetic variation in given populations. This work is essential for genebanks that must keep many accessions at the same time in long term storage, including cryopreservation, while a genetically representative working collection should be continuously ready for use by clients.21 In a few years the cost of DNA marker technology is likely to reduce further, much as what happened with computer hardware. Much more critical will be handling data efficiently. In any central genotyping service, laboratory automation of sample handling will be desirable for high throughput. Today, the ability to identify and use genetic information is doubling every 12 to 24 months. This exponential growth in biological knowledge is transforming agriculture, nutrition and health care in the emerging life sciences industry.22 The field of genomics, which is the combination of an organism’s genome and the informatics tools needed for data acquisition, storage and analysis, has two major components: the molecular genetics laboratory (data producers) and the computational laboratory (data handlers). The latter is know as bioinformatics.23 Major developments in molecular genetic research and breeding are moving in the direction of acquiring data ever more quickly and cheaply. Often overlooked is the fact that data are not information, and that extracting information from complex data sets and applying it to breeding goals are skills often outside the domain of either molecular biologists or crop breeders. A main point of this discussion is that future advances in crop breeding lie as much in information science, or bioinformatics, as in biotechnology. Yet, throughout the biotechnology industry, professional level expertise in the area of bioinformatics, or computational management of complex biological data, is rare and highly sought. At present, the most avid consumer of such expertise appears to be the pharmaceutical industry and the most highly desired skills are that of database mining and computational analysis of DNA sequences.

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Demand for such expertise in plant sciences is now on the increase as genomic approaches to plant breeding and engineering of plant products are being applied toward selective breeding. A variety of data bases, built and maintained at universities, largely in North America, Europe, Australia, and Japan, offer free electronic access to genome mapping data and genetic studies on the world’s food and fiber crops. An Internet entry point to these is maintained. 24 The increased focus on bioinformatics eventually will create: 1. An efficient database system uniting diverse sources of genetic information for breeding; 2. Computer software for visualization, analysis, and application of molecular data to breeding; and 3. Superior crop varieties, often incorporating genes from wild relatives, with a genetic complexity contributing to stable performance and embodying molecular selection expertise. Many of the benefits of this work will be realized indirectly through the development and release of superior varieties. Rao and Iwanaga8 provide an excellent review of the role of biotechnology in plant genetic resources conservation. These techniques have been effectively applied in germplasm collecting, characterization, evaluation and conservation. Despite all the advantages of having these modern biotechnological tools, there is a great amount of debate on their use, especially in developing countries, in terms of access, property protection etc. There is a growing awareness that the profits generated from the exploitation of plant genetic resources, especially through biotechnology, are not shared equitably. These issues continue to be debated at the global level and are not discussed here, but it is important to remember that the bulk of the work in biotechnology, unlike the green revolution, is financed largely by the private sector. Hence, legal and property protection issues and sharing of profits come to the forefront. There is therefore an urgent need to

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develop procedures to link commercial benefits from the exploitation of plant genetic resources through biotechnology (and other methods of exploitation) to conservation of plant genetic resources.25,26

Potato Late Blight as a Case Study The Irish potato famine began in 1845 when the potato late blight disease decimated crops there. One hundred and fifty years later, in 1995, the famine was commemorated by many events in Ireland and elsewhere. It seems ironic that after a general subsidence of the disease, due to increasingly effective integrated pest management programs, the disease is once again causing concern in potato growing regions around the world. The first appearance had terrible consequences for Ireland and birthed the science of plant pathology. Fortunately the current resurgence is not as devastating as the first occurrence; nonetheless it increases human misery. This disease remains the world’s most devastating agricultural disease. The United States and Canada were initially spared the effects of the most recent outbreaks. However, the situation changed dramatically in the early 1990s. Migrations of the oomycete pathogen Phytophthora infestans initiated the calamity in the 1840s and migrations are once again causing current problems—thus illustrating the point that pathogens of plants as well as of humans are an increasing concern in a world of rapid mass transport.27 During the 1980s and 1990s, problems due to late blight began to worsen worldwide. Late blight is still the potato crop’s most devastating disease. More chemicals are applied annually to potatoes worldwide than to any other food crop. It now costs the world $1.8 billion US a year to control potato late blight. By regions, the former Soviet Union tops the expense list at $620 million, followed by Europe at $479 million, Asia at $461 million, Africa at $78 million, North and Central America at $74 million, South America at $91 million, and Oceania at $5 million. Potato harvests worldwide are now severely

affected, and the fungus is responsible for losses of 15%, costing about $3.25 billion in lost yields.28,29 The center of origin of the late blight pathogen is in central Mexico, where a highly diverse sexual population with both the A1 and A2 mating types exist. The A1 genotype was the only one that initially spread worldwide. It was not until 1984 that A2 mating types were reported in western Europe. Since then, they have appeared in an increasing number of countries in Asia, North and South America and in Africa. Current scientific information on population dynamics of the fungus and its genetic structure indicates that the frequently reported A2 mating types belong to a new sample of the sexual population that carries both A1 And A2 and which escaped from central Mexico in the late 1970s. This new migration is rapidly displacing the old population. It seems to be more diverse, more aggressive, and is more fit than the old one that is represented mainly by a few clonal lineages of A1. Reports from several countries indicate that the disease has become more severe. It appears earlier in the season, and despite heavy sprays, control is difficult.27,30 The potential threat of this renewed disease may lie in: 1. The spread of new strains with increased fitness, aggressiveness, capacity to produce inoculum (sexual and asexual), and potential to initiate disease earlier; 2. The shortage of chemicals and effective integrated control measures; and 3. Lack of widely accepted, resistant commercial potato varieties. Given this scenario, a rapid commitment to support research in this area, particularly by industrialized countries, is the most positive option to prevent a potential catastrophe.

Background and Need for Networks Current potato production depends on a very narrow genetic base comprised of few varieties. For example, in North America there are only four or five varieties which dominate

Plant Genetic Resources for Food and Agriculture: Status and Future Prospects production. All the major potato varieties grown worldwide are susceptible to late blight. Developing late blight resistant potatoes is, therefore, crucial because potatoes are an important food and income source for poor people. If current trends continue, 40% of the world’s potatoes will be harvested in the developing world by the year 2000. In these and other regions of the world, late blight is indeed a serious and increasing threat to the future of world food security. Worldwide migrations of this fungus and concurrent worsening of late blight problems during the 1980s and 1990s have mandated a much more serious attempt to solve the late blight problem worldwide. Global collaboration through the development of networks to promote research and technology transfer on late blight control are important. There are many activities all over the world; one of them is the Global Initiative on Late Blight (GILB) which was conceived in 1995 by the International Potato Center (CIP) along with its national collaborators.31 However, eastern Europe is not yet fully involved in this initiative. A seriously underfunded initiative is PICTIPAPA (International Cooperative Program for Potato Late Blight) based in Mexico. Eastern Europe is perhaps in greater need than any other region worldwide of establishing a stable, environmentally benign late blight management program. Potatoes are an important food crop, and late blight has been especially troublesome in the last decade. Although late blight can be controlled with massive amounts of protectant fungicides, the economies of eastern Europe may not allow such expenditures and the effects of fungicides on the environment are not completely known. Thus, knowledge of late blight epidemiology and the use of resistant potato germplasm are crucial. The Toluca Valley in the highlands of central Mexico is the ancestral home of Phytophthora infestans, and contains a fungal population that is remarkably diverse and has been sexual for millennia. In contrast, the fungus was not reported from any other location until the mid-19th century, and until

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very recently, populations outside of Mexico were exclusively asexual. Recent worldwide migrations have suggested that the sexual reproduction which has occurred in the Toluca Valley will soon become a component of production systems worldwide. There is an urgent need to understand the basic biology of P. infestans in this sexual population. The results of these studies will contribute to the development of disease management programs worldwide. The tremendously diverse population of P. infestans in Toluca is an underutilized resource due to lack of infrastructure in Mexico. This location should be utilized as a field lab for evaluating the stability of late blight suppression programs (including host resistance, cultural controls, biocontrols and fungicides). Funding is needed to strengthen the infrastructure in the Toluca Valley that can facilitate the activities of scientists worldwide and to initiate research done by resident scientists. Once an improved infrastructure is available, public and private agencies will want to utilize this infrastructure for addressing the stability of various breeding lines, fungicides and management procedures for late blight control.

Cornell-Eastern Europe-Mexico (CEEM) International Collaborative Project in Potato Late Blight Control Cornell University in Ithaca, NY, USA, houses one of the largest concentrations of potato scientists in the world. Their expertise includes potato breeding with emphasis on disease and insect resistance; epidemiology, biology and management of P. infestans, the cause of late blight; production of pathogenfree seed potatoes; potato physiology; potato insects; nematodes; molecular genetics; production; biotechnology and integrated pest management. CEEM was established in 1996 through support from donors representing private foundations, private sector companies and bilateral/multilateral agencies. The project began efforts to build support and define objectives through individual contacts with other major researchers around the world, both in developed and developing country

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national programs. Representatives of the private sector were consulted regarding their interest in participating in this project. During 1996, CEEM organized a international planning conference involving representatives from several countries to: 1. Review and identify priority research topics to be addressed in the CEEM project; and 2. Determine the potential of technical cooperation and financial support. Workshop participants agreed that the CEEM project should link with relevant activities of GILB and PICTIPAPA. An Executive Committee, made up of four internationally known individuals, with wide representation of partners from Mexico, Poland, Russia, Peru and the USA, was agreed to as a guiding component for the project, and was appointed. In addition, the project has an executive director and a project associate as its management team. The scientific objectives and associated activities of the project are to: 1. Develop and make broadly available long day adapted potato germplasm with resistance to late blight; 2. Initiate basic investigations on the epidemiology, biology and life history of P. infestans; 3. Help evaluate breeding lines, genotypes and other disease management components in the Toluca Valley; 4. Provide short term training at Cornell for eastern European, Mexican and other scientists; and 5. Help support graduate students from Mexico and eastern Europe at Cornell. Successful pursuit of these objectives is expected to: 1. Yield potato cultivars adapted to long days with high levels of late blight resistance; 2. Increase the understanding of the basic biology, epidemiology, and life history of P. infestans in a sexual population; 3. Provide an infrastructure that will enable plant breeders and plant pathologists from all over the world

to conduct experiments in the Toluca Valley; 4. Increase the understanding of factors influencing late blight in New York, Mexican and eastern European production systems; and 5. Enhance our knowledge of the potential stabilities of new and traditional methods of managing late blight.

Partnerships Within CEEM The project recognizes the enormity of the late blight problem and realizes that successful achievement of the goal requires effort from institutions/scientists worldwide. CEEM works with other institutions to achieve a common goal. Each institution plays an important and non-duplicative role. An important goal of CEEM is to facilitate the sharing of information and biological materials/technology that might contribute to a solution of the late blight problem. The formation of CEEM is now fostering partnerships with programs in Peru (GILB), Mexico (PICTIPAPA), Russia (Moscow State University and the N. I. Vavilov All-Russian Research Institute of Plant Industry), Poland (Mtochów). Research Center, Plant Breeding and Acclimatization Institute), and India (Central Potato Research Institute). Other members from other countries are also likely to join soon. Several private sector firms such as Nature Mark Potatoes, DuPont, Zeneca, Frito-Lay, Rohm and Haas, and federal agencies in USA (such as the United States Department of Agriculture (USDA), are also cooperating with CEEM.

Program Activities and Specific Outcomes Several important areas of investigation have been determined which are basic to the success of developing resistance to late blight and can be achieved only through international cooperation. In Mexico, the focus of PICTIPAPA/CEEM collaboration works in five research modules. Module 1 deals with evaluation of international potato materials for durable late blight resistance in the Toluca Valley. Module 2 identifies new sources of durable genetic resistance to late blight and

Plant Genetic Resources for Food and Agriculture: Status and Future Prospects conservation of the Mexican potato germplasm by the national potato program. Module 3 deals with support of basic research on the etiology of P. infestans, with special emphasis on the A2 mating type as well as studies on genetic structure of populations, epidemiology and biological control of P. infestans. Module 4 distributes new potato cultivars with durable resistance and studies their impact on sustainable agriculture via small subsistence farming. And, Module 5 focuses on establishment of standardized international field trials to facilitate evaluation and accelerate worldwide introduction of new potato cultivars with durable resistance. In eastern Europe the projects focus on development of evaluation methods for late blight resistance; assessment, comparison and analysis of breeding achievements; comparison of naturally occurring P. infestans populations and resistance evaluation results; and exchange of cultivars most resistant to late blight. Such materials have already been exchanged among Russia, Poland, Mexico and the USA. Resistant materials identified through these efforts will aid in the development of durable resistance to late blight. In Russia, studies on the pathogen are revealing important differences in mating types of the pathogen and its resistance to the fungicide Metalxyl. Such findings are important to the development of integrated control programs. In the USA, CEEM facilitates basic research in all areas. This includes pathogen characterization, the soil ecology of P. infestans, the epidemiological role of oospores, population structure, breeding, evaluating the durability of “resistant’ cultivars/breeding lines, pathogen diversity for biologically important traits, and developing integrated management strategies. Other activities such as training, workshops or conferences are also important. These collaborative projects are now fostering high priority research and is enhancing communications between all stakeholders. CEEM continues to assist, promote and catalyze technology transfer, focusing on areas where there is great need and potential impact. While it is too early to document the impact of CEEM, the specific outcomes expected are many, and include:

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1. Facilitation of cooperation and research on late blight at an international level; 2. Establishment of a clearing house mechanism that provides current data and information to participating members on late blight management; 3. Increasing the absorptive capacity of national programs to acquire, transfer and adopt new control components; 4. Training of core national scientists and policy makers to pursue late blight-specific research at Cornell and with private sector and public sector institutions in industrial countries; 5. Strengthening of both basic and applied research at Cornell, eastern Europe and Mexico; 6. Transferring Cornell’s expertise to eastern Europe, the Newly Independent States (NIS), and Mexico; 7. Implementation of a few carefully thought out projects which will serve as a demonstration model for effective late blight control; 8. Establishment of a international advisory committee which will promote late blight research worldwide; 9. Creation of crosssector institutional linkages through the establishment of new mechanisms for late blight management and technology transfer between the private and public sectors; 10. Adoption of improved management strategies to combat late blight and thereby increase potato productivity, particularly for poor farmers.

Conclusions The increasing demand for food production has decreased our biodiversity and altered the landscape and environment. As food production is population driven, the demand for increased productivity can come by augmenting existing yields through conventional or non-conventional methods of plant breeding. The global community depends on PGRFA as a least expensive input

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to increasing crop plant productivity. More than ever before, international efforts are required to help slow genetic erosion, establish and encourage activities of international genebanks, and help prevent epidemics of plant diseases, pests and other abiotic factors in the developing countries where the greatest threats of genetic vulnerability and germplasm erosion now exist. Networking and cooperation at the global level to promote effective use of plant germplasm from diverse breeding programs, and from centers of origin of plant species, to develop new crops and crop varieties to support low-input agricultural and agroforestry programs should receive high priority. The Global Plan of Action (GPA) for PGRFA aims to promote the conservation, sustainable utilization, and fair and equitable sharing of benefits of plant genetic resources. It is designed to contribute to the implementation of the Convention on Biological Diversity (CBD) in the field of food and agriculture. Major issues such as intellectual property rights, farmers’ rights, biosafety, food safety and socioeconomic issues are further discussed in this GPA and in several forums related to the CBD. It is hoped that these debates will finally develop a clear consensus in these areas. How to conserve invaluable PGRFA from around the world, while promoting their widespread and effective use to feed a hungry world today and provide food security for the future, continues to be a challenge of many national and international programs. International collaborations and networking on PGRFA are now being promoted by International Centers such as the International Plant Genetic Resources Institute (IPGRI) in Rome, Italy. Most of the activities funded in this area are restricted to specific crops and do not include activities in the human resource development area.26 In the case of the potato, many national programs worldwide depend on genetic resources maintained in gene banks. One important gene bank is the one currently maintained at the N. I. Vavilov All -Russian Research Institute of Plant Industry (VIR), in Russia. VIR is the world’s oldest plant genetic

resources institute and holds one of the world’s largest national genebank collections, containing over 345,000 accessions of cultivated plants and their wild relatives. Unfortunately, economic difficulties in recent years have led to a severe reduction in staff and lack of funds with which to renew old equipment and facilities. The more recent changes that have occurred in Eastern Europe have also threatened the security of the collection. One significant problem caused by the breakup of the Soviet Union means that six of the 17 former VIR experimental stations are now located in the Newly Independent States (NIS). Work on maintenance of VIR collections is now affected. To alleviate this situation, some emergency assistance has been allocated by IPGRI to VIR, and further support is still needed for germplasm conservation and utilization at VIR.32 The CEEM project is an example that encourages germplasm sharing, testing and evaluation among programs in Russia, Mexico, Poland and the USA to produce potato varieties with durable resistance to late blight. It also tries to promote training and research activities in potato germplasm conservation and use for developing late blight resistance. At VIR, some moderate support is being provided to rescue important wild potato germplasm of use to developing late blight resistant cultivars. Global and national programs, such as those being promoted by CEEM, GILB, PICTIPAPA and others, who have broad participation from International Agricultural Research Centers (IARCs), allow research and technology transfer to take place in a cost effective manner. Participants in such programs benefit in many ways, including: 1. Global prioritization of research needs; 2. Improved possibilities for funding for program participants through the recognition of the program by donor agencies; 3. Close interaction with, and knowledge of, other research teams within their area of specialization;

Plant Genetic Resources for Food and Agriculture: Status and Future Prospects 4. Opportunities for interdependent research projects (i.e., projects requiring interdisciplinary and complementary partnerships); 5. Improved access to information and resources; and 6. Participation in program meetings and conferences.31 The development of cultivars durably resistant to pathogens and pests should be a high priority. With late blight, despite phytosanitary hygiene and good agronomic practice, sources of inoculum have never been entirely eliminated, even in the sophisticated farming systems of Europe and the USA, where late blight remains an annual problem and fungicides are the only effective control. A combination of conventional and molecular techniques should enhance the development of durable resistance. The use of novel approaches such as DNA fingerprinting and the development of resistant plants through genetic engineering needs collaboration with the private sector. Private and public partnerships need to be further fostered. The link between world food security and the conservation and sustainable use of plant genetic resources is important. The development of an endowment fund exclusively for research and technology transfer to promote late blight control is a high priority of CEEM. The development of such a fund will contribute to increased research in the maintenance of useful potato germplasm, the development of late blight-resistant cultivars, and the use of integrated pest management strategies to control late blight on a global scale. The participation of companies such as Toyota and others in these efforts should strengthen germplasm conservation and utilization efforts on a global scale. Global programs serve as a valuable mechanism for universities and other public and private sector organizations to increase their involvement. Programs such as the one on late blight promote innovative and worthwhile approaches to resolving global problems. The advantages are considerable.

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Acknowledgments Our thanks to Dr. Ripusudan Paliwal, Visiting Scientist, Cornell University and Dr. Richard Tenney for editorial corrections and useful comments.

References 1. FAO. State of the World’s Plant Genetic Resources. Rome: FAO, 1996. 2. GREAN. Global Research on the Environmental and Agricultural Nexus: The report of the Taskforce on Research Innovations for the Productivity and Sustainability, Gainsville and Ithaca: University of Florida and Cornell University, 1997:160. 3. Raman KV. Public and private research: Promoting synergies. In: Chopra L, Singh RB,Vera A, eds. Crop Productivity and Sustainability, Shaping the Future. Proceeding of the 2nd International Crop Science Congress, 1996. New Delhi: Oxford and IB. Publishing Co., 1998:937-947. 4. Watanabe KN, Raman KV. Plant biotechnology and plant genetic resources: A global perspective. In: Watanabe KN, Pehu E, eds. Plant Biotechnology and Plant Genetic Resources for Sustainability and Productivity. Austin: R.G.Landes Co., 1997:1-13. 5. Vasil IK. Biotechnology and food security for the 21st century: A real-world perspective. Nature Biotechnology 1998; 16:399-400. 6. Watanabe KN, Pehu E, eds. Plant Biotechnology and Plant Genetic Resources for Sustainability and Productivity. Austin: R.G. Landes Co., 1997. 7. Wilkes G. Strategies for Sustaining Crop Germplasm Preservation, Enhancement, and Use. Issues in Agriculture, No. 5. Washington DC: Consultative Group on International Agricultural Research. 1992:62. 8. Rao VR, Iwanaga M. Utilization of plant genetic resources. In: Watanabe KN, Pehu E. eds. Plant Biotechnology and Plant Genetic Resources for Sustainability and Productivity. Austin: R.G. Landes Co., 1997:29-69.

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9. Peeters JP, Williams JT. Toward better use of genebanks with special reference to information. Plant Genet Resource Newsl 1984; 60:22-32. 10. Hodgkin T. The core collection concept. In: Crop Networks-New Concepts for Genetic Resources Management. International Crop Network series 4, Rome: International Board for Plant Genetic Resources, 1991. 11. APHIS. http://www.aphis.usda.gov/bbep/ bp/. Washington DC: USDA, 1998. 12. James C. Global Status of Transgenic Crops. ISAAA Briefs No. 5. Ithaca: ISAAA, 1997:31. 13. Worldseeds. http://www.worldseed.org/ -assinsel/posl-ass.htm. Washington DC: Worldseeds-CGIAR, 1998. 14. Swanson TM, Pearce DW, Cervigni R. The appropriation of the benefits of plant genetic resources for agriculture: An economic analysis of the alternative mechanisms for biodiversity conservation. FAO commission on Plant Genetic Resources, Background Study Paper No. 1. Rome: FAO, 1994. 15. Anonymous. Genetic Vulnerability of Major Crops. Washington DC: National Academy of Sciences, 1972. 16. Plucknett DL, Smith NJH, Williams JT et al. Genebanks and the World’s Food. Princeton: Princeton University Press, 1987. 17. Dalryample DG. Development and Spread of High-Yielding Wheat Varieties in Developing Countries. 7th ed. Washington DC: US Agency for International Development, 1986. 18. National Research Council. Genetic Vulnerability of Major Crops. Washington DC: National Academy of Sciences, 1972. 19. Tanksley SD, Grandillo S, Fulton TM. Advanced backcross QTL analysis in a cross between elite processing line of tomato and its wild relative L. pimpinellifolium. Theor Appl Gent 1996; 92:213-224. 20. Tanksley SD, McCouch SR. Seed banks and molecular maps: Unlocking genetic potential from the wild. Science 1997; 277:1063-1066.

21. Watanabe K. Potato molecular genetics. In: Bradshaw J, Mackay G, eds. Potato Genetics. Wallingford: CAB International, 1994:213-235. 22. Monsanto. Annual Report. St. Louis: Mosanto Co. 1997:62. 23. NCGR. http://www.ncgr.org/ag/essay/ html. Washington DC: NCGR, 1998. 24. Yandell B. WWW page maintained by Dr. Brian Yandell at the University of Wisconsin: 1997. 25. Rao R, Riley KW. The use of biotechnology for conservation and use of plant genetic resources. In: Loh CS, Lee SK, Lim TM, eds. Proceedings of the International Conference on Agrotechnology in the Commonwealth: Focus on the 21st Century. Singapore: Singapore Institute of Biology, 1994:89-94. 26. Watanabe KN, Rao VR, Iwanaga M. International trends on the conservation and use of plant genetic resources. Plant Biotechnology 1998; 15:115-122. 27. Fry WE, Goodwin SP. Resurgence of the Irish potato famine fungus. BioScience 1997; 47:363-371. 28. CIP. CIP in 1995. The International Potato Center. Annual Report. Lima: International Potato Center, 1995:56. 29. Kleiner K. Save our spuds. New Scientist 1998; 2136:24. 30. Fry WE, Goodwin SP. Re-emergence of potato and tomato late blight in the United States. Plant Disease 1997; 87:1349-1357. 31. Frison EA, Collins WW, Sharrock SL. Global Programs: A New Vision in Agricultural Research. Issues in Agriculture, No. 12. Washington DC: Consultative Group on International Agricultural Research, 1997:29. 32. IPGRI. World bank to Rescue VIR. IPGRI News Letter for Europe. No. 3. Rome: International Plant Genetic Resources Institute, 1994.

SECTION V INTRODUCTION

Improvements of Plant Function with Conventional Methods and Biotechnology T. Fujimura

T

his section coordinates with plant genetic engineering for: 1. Elucidating the fundamental questions of plant metabolism by knocking out key physiological processes of plants; 2. Coping with external environmental stresses by improving physiological functions; and/or 3. Improving qualitative and/or quantitative traits for specific industrial uses.

All these categories of endeavor are very challenging and extremely intriguing in their potential toward developing new plants for enhanced production of specific metabolic products and/or developing ideotypic plants against the possibility of serious global environmental changes. These studies and their outcome will make up an important component of an integrated approach in alleviating environmental concerns, food security and health issues.

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CHAPTER 15

Engineering Carbohydrate Metabolism in Transgenic Plants A.G. Heyer

Introduction

L

ooking at plants as factories for the production of food and industrial raw materials, one sees three aspects of prime importance. The first is the overall biomass production; second is the proportion of biomass that is accessible to human consumption, which is given by the so-called harvest index; the third is the quality of the harvestable material. Plant biotechnology offers a wide array of possibilities for modifying the physiology and metabolism of crop plants, and it will be a key question to the future prosperity of man whether we will be able to exploit these possibilities to optimize plant production with respect to human needs. As biomass production by plants is initially the fixation of energy as carbohydrates, modifying carbohydrate metabolism is one of the main aspects of plant biotechnology.

Source Capacity Focusing on limiting factors for biomass production, we encounter the question of possible limitations of primary energy fixation in the source organs of the plant. Certainly energy uptake by the absorption of light is not a limiting factor, as photosynthetic fixation already reaches a maximum at moderate photon flux densities. If abiotic factors like water availability or CO 2 supply are not restrictive, regulatory features of carbohydrate metabolism could become limiting. During photosynthesis, photosynthates are exported from the chloroplast as triose phosphates that are condensed to hexoses and

ultimately converted to sucrose. The sucrose is usually exported to sink organs of the plant such as growing leaves, the root system and storage organs. In terms of regulation, two metabolites are of central importance. The first is inorganic phosphate, which is released from fructose bisphopsphate and in the last step of sucrose synthesis, and is needed as a transport equivalent for every triose phosphate that is exported from the plastid. The second is fructose 2,6-bisphosphate (F-2,6-PP), which is produced from fructose 6-phosphate (F6P) and is a regulator of the activity of the enzyme frucosebisphosphatase (FBPase), which catalyzes the main regulatory reaction of sucrose synthesis. Under conditions when sucrose accumulates in the cytosol, fructose 6-phosphate (F6P) concentrations increase and as a consequence, F-2,6-PP is produced and causes inhibition of F6P production. This restrains triose phosphate withdrawal from the plastid and initiates transitory starch production in most plants. Alternatively, the sucrose accumulation can be circumvented by import into the vacuole (Fig. 15.1). The fact that metabolites control the reactions complicates genetic modification of the pathway. Attempts to increase flux by hydrolyzing pyrophosphate to force the reaction of fructose-6-phosphate 1-phosphotransferase (PFP) towards F6P production failed, because pyrophosphate is needed in later steps of sucrose transport to sink tissues.1,2

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Fig.15.1. Regulation of carbohydrate metabolism in mesophyll cells (source tissue). Metabolites: F-6-P: fructose-6-phosphate; F-1,6-PP: fructose-1,6-bisphosphate; F-2,6-PP: fructose-2,6bisphosphate; G-1-P: glucose-1-phosphate; G-6-P: glucose-6-phosphate; P: phosphate; Pi: inorganic phosphate; PPi: pyrophosphate; S: sucrose; S-6-P: sucrose-6-phosphate; Triose-P: triose phosphates; UDP: uridine diphosphate; UTP: uridine triphosphate. UDP-G: UDP-glucose. Enzymes: FBPase: fructose-bisphosphatase; PFK: phosphofructo-kinase; PFP: fructose-6-phosphate 1-phosphotransferase; SPS: sucrosephosphate-synthase. Another means of controlling sucrose production is the activity of the last enzyme in the pathway, sucrosephosphate-synthase (SPS), which usually has a low activation status and is regulated allosterically. Posttranslational phosphorylation of SPS reduces affinity for the substrate UDP-G. Again, genetic modification is hampered, because overexpression of the enzyme does not necessarily increase its activity.3

Sink Capacity The fact that sucrose accumulation in the mesophyll cell occurs when photosynthesis is saturated implies that the removal of photosynthates from the sources, but not the source capacity, is limiting. So, consequently, the question is: “Can we increase sink demand?” We attempted to increase sink strength by expressing an enzyme that metabolizes sucrose in sink organs to enhance the sucrose gradient between source and sink. This was achieved by expressing an invertase from baker’s yeast in the cytosol of potato tubers (Fig. 15.2). To our surprise, the consequence was a strong reduction of sink strength and a strong increase in the concentration of glucose (Table 15.1).4

We concluded that the phosphorylation of glucose, which is necessary to allow partitioning into starch, is limiting in the tubers. Consequently we added a glucokinase from Zymomonas mobilis to overcome this limitation. The result was an even stronger reduction in starch synthesis in the tubers. Table 15.2 shows the density of the tubers, which is a direct measure of starch content.5 The reason for the further reduction in starch accumulation is that additional G6P was not diverted into starch formation but into glycolysis and respiration. Measuring CO2 release from the tubers of invertase/glucokinase plants revealed a 4-fold increase in respiration as compared to the untransformed wild type (Table 15.3). Currently we do not know the reason for the partitioning into glycolysis, but it is to be supposed that glucose itself or a metabolite acts as a signal to increase respiration. A completely different result with respect to sink strength was achieved when the invertase was targeted to a different compartment: Expression of a chimeric gene construct that led to apolastic localization of the invertase caused a significant increase in the fresh

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Fig 15.2. Regulation of carbohydrate metabolism in potato tubers (sink tissue). Metabolites: ADP: adenosine diphosphate; ATP: adenosine triphosphate; F-6-P: fructose-6-phosphate; G-1-P: glucose-1-phosphate; G-6-P: glucose-6-phosphate; P: phosphate; Pi: inorganic phosphate; PPi: pyrophosphate; UDP: uridine diphosphate; UTP: uridine triphosphate; UDP-G: UDP-glucose. Enzyme: SuSy: sucrose-synthase. weight of individual tubers, whereas total yield per plant was unchanged (Table 15.4).4 This demonstrates that manipulating the capacity of a single sink organ like a potato tuber does not result in increased biomass production of the whole plant, but it is a means toward influencing the harvest index. Furthermore, it shows that the harvest index is influenced by the sink strength of an individual sink organ, which itself can be modulated by changing osmotic potential of cells or the surrounding apolastic space.

Transport Processes Attempts to modify sink strength revealed that the harvest index seems to be strongly dependent on transport processes. This hypothesis is confirmed by the following examples. Expressing an apoplastic invertase that increases the tuber size when expressed in potato tubers, in source but not in sink tissues, leads to a very strong reduction of biomass production, because the carbohydrates that are usually transported as sucrose cannot leave the source organs.6 The same is achieved by inhibiting the expression of the sucrose transporter that is responsible for loading sucrose into the vascular system.7 It is there-

fore very likely that transport of photosynthates from the source to the sink organs is the limiting step in biomass production. Consequently the question would be whether we could increase production by raising the transport abilities. Unfortunately, the question cannot be answered yet, because to date increased transport capacity has not been achieved. But, nevertheless, there is good evidence for the hypothesis of transport limitation. Potato plants react very sensitively to the reduction of transporter activity, thereby demonstrating that export of sucrose from the sinks is likely to be a rate limiting step in the control of photosynthetic production. In transgenic plants showing antisense inhibition of transporter expression, photosynthesis is reduced and soluble sugars accumulate in the mesophyll (Table 15.5). From the experiments described, we can conclude that biomass production can be influenced by means of genetic modification that alters sink strength, as demonstrated by expression of cytosolic invertase and glucokinase, or by modifying transport processes as shown for antisense inhibition of sucrose transporter expression. In the given examples, genetic modification negatively affects production, thereby demonstrating which steps are

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Table 15.1. Soluble sugars in potato tubers in µmol/g fresh weight ± standard deviation Sucrose

Glucose

Fructose

Control

15.6 ± 0.7

2.1 ± 0.4

1.1 ± 0.1

U-In2-34

1.4 ± 0.3

24.2 ± 0.9

0.5 ± 0.2

U-In2-17

0.6 ± 0.1

40.6 ± 3.4

0.8 ± 0.1

Control: untransformed wild type (Var. Désirée). U-In2-34: plant No. 34 expressing cytosolic yeast invertase. U-In2-17: plant No. 17 expressing cytosolic invertase.

candidates for rate limiting steps. This gives an idea of what would be needed to increase biomass production, but so far there are no convincing strategies for increasing photosynthetic carbon fixation in transgenic plants.

Altering Carbohydrate Composition: Starch Completely different is the picture for approaches to modifying carbohydrate composition in an attempt to improve plant quality. There are many reports on successful modification of starch composition in potato, pea or other species, and also on the production of fructans in transgenic plants that are normally not capable of fructan production. Starch is a glucan polymer that is synthesized from activated glucose units that are synthesized from G6P and ATP by ADPglucosepyrophosphorylase. The activation of glucose is the rate limiting step in the pathway.8 Four different starch synthases are involved, one of them being tightly bound to the growing starch granule, the others being soluble enzymes. The α-(1,4)-glucan chain produced by the synthases is subject to modification by branching enzymes that introduce α-(1,6) branches that characterize the amylopectin portion of starch. Besides branching enzymes, debranching enzyme and disproportioning enzyme, as well as starch phosphorylases and the degradative amylases, are involved in the metabolism of starch. All these enzymes have been characterized and the genes are cloned from different species. Work in our institute focused on potato, which, behind corn and

wheat one of the most important starch producing crop plants and yields a qualitatively interesting starch because of its high phosphate content. By means of antisense reduction of enzyme activity of single or multiple enzymes, a set of starches with different properties has been generated.9 The antisense repression of branching enzyme in combination with repression of the so-called R1 enzyme, which is involved in starch modification in a way not yet completely understood,10 yields a starch that has a dramatically enhanced gelation capacity and is therefore expected to be superior in the production of films. A very important feature of starch with respect to the production of films is the color of the starch gel. A very turbid gel can be obtained in plants with reduced activity of starch phosphorylases, but a completely colorless gel results from repression of granule bound starch synthase (GBSS), which leads to loss of the amylose portion of starch. The pure amylopectin starch obtained in these plants shows other interesting features like reduced retrogradation and is already a very interesting raw material for use as a food additive in production of soups and sauces. Not only the turbidity, but also the gel strength, can be manipulated. For example, repression of starch phosphorylases as well as repression of branching enzyme and R1 give turbid and very stiff gels, the latter of which can only be obtained by gelatinization of starch at high temperatures.

Engineering Carbohydrate Metabolism in Transgenic Plants

Table 15.2. Density in g/cm3 of potato tubers as direct measure of starch content

177

Table 15.3. Release of CO2 from potato tubers in nmol C/g fresh weight

Density

CO2 Production

Control

1,082

Control

18

Inv

1,074

Inv

58

GK-41

1,066

GK-41

71

GK-29

1,061

GK-29

79

GK-38

1,057

GK-38

81

Control: untransformed wild type (Var. Désirée). Inv: plant expressing cytosolic yeast invertase. GK-41, -29, -38: cytosolic invertase plants expressing glucokinase from Zymomonas mobilis.

Control: untransformed wild type (Var. Désirée). Inv: plant expressing cytosolic yeast invertase. GK-41, -29, -38: cytosolic invertase plants expressing glucokinase from Zymomonas mobilis.

The production of pure amylopectin starch by repression of GBSS that is essential for amylose synthesis has already been mentioned. It is also possible to reduce amylopectin synthesis and raise the amylose content of starch. This is achieved by repression of branching enzyme and R1 gene expression, whereas the repression of either one of the enzymes has no significant effect. Amylose is an interesting polymer because it is mostly linear and therefore interesting for film production. One of the most intersting features of potato starch is the high content of covalently bound phosphate that adds chemical functionality to the polymer matrix. The way in which phosphate gets integrated into starch is not fully understood, but it is mainly associated with the amylopectin fraction of starch. Consequently, phosphate content can be lowered by repression of branching enzyme and R1, which leads to a higher amylose content. Astonishingly, the lowest phosphate content is observed for R1 repression that does not significantly reduce amylopectin production. The desired increase in phosphate content is achieved via inhibition of GBSS, linked to increased amylopectin content. But most

interesting is the additional repression of branching enzyme, which leads to a more than 3-fold increase in the phosphate content of starch.

Altering Carbohydrate Composition: Fructan A goal oriented to improving food quality is the production of fructans in transgenic plants. Fructans are regarded as “prebiotic” because of antitumoral effects that seem to be associated with their stimulative effects on Bifidobacteria in the human intestine. This stimulation causes an increased propionate and butyrate fermentation and lowers the concentration of tumor promoting substances like ammonia and β-glucuronidase. In a mutant mouse strain which spontaneously develops colon tumors because of a genetic defect in the APC gene, a fructo-oligosaccharide-containing diet significantly reduces the number of colon tumors, whereas starch and wheat bran have no effect on total tumor number, as reported by Pierre and his coworkers.13 Besides, fructans are interesting low calorie fibers because the β-linkage of the fructose moieties cannot be cleaved by human enzymes. Bacterial fermentation and resorption of

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178

Table 15.4. Mean fresh weight per tuber in g and total tuber yield per plant in kg ± standard deviation of potato expressing apoplastic yeast invertase

Table 15.5. Maximal photosynthetic rate of potato plants in mmol O2/m2*h ± standard deviation Max Photosynthetic Rate

Mean fresh weight/ tuber (g)

Total yield per plant (kg)

Control

74 ± 7.2

Control

143

1.21 ± 0.08

aSp13

71 ± 7

U-In1-3

192

1.30 ± 0.10

aSp43

54 ± 7.5

U-In1-41

179

0.91 ± 0.07

aSp5

46 ± 7.1

U-In1-33

209

1.23 ± 0.09

aSp34

38 ± 12.7

Control: untransformed wild type (Var. Désirée). U-In1-3, -41, -33: different transgenic plant lines expressing yeast invertase targeted to the apoplast.

Control: untransformed wild type (Var. Désirée). aSp13, 43, 5, 34: different transgenic plant lines expressing an antisense RNA to the sucrose transporter transcript.

fermentation products yields an energy value of 1 kcal/g, which is about 30% of that for the free monomers. The texture of the fiber gives a fat-like feeling in the mouth, and therefore fructans are excellent bulking agents for low calorie foods. Fructans are fructose polymers that are synthesized from sucrose as substrate by transfer of the fructose moiety from sucrose to a growing chain. The glycosidic C-2 hydroxyl group can be transferred to the C-6 position; in this case, a levan type fructan is synthesized. Alternatively, it can be transferred to the C-1 position, leading to an inulin type fructan. In both cases the fructan chain contains a terminal glucose and is therefore a non-reducing sugar. Fructan synthesis is widespread in evolutionary terms: It occurs among bacteria and plants and there are also some reports of fungal fructan production. Nevertheless, bacterial and plant metabolic pathways of fructan production have not much in common. The responsible enzymes are different and so are the synthesized carbohydrates. Bacteria need only one enzyme for the synthesis of fructans, and this enzyme is

capable of synthesizing a fairly huge polymer having a molecular mass of several million. In most cases bacterial fructans are of the levan type; only one high molecular weight inulin has been described. Plant fructans are of low molecular weight and their linkage type depends on their origin. Monocotyledonous plants usually synthesize levan type fructans, whereas the typical dicot fructan is inulin. All studies on beneficial effects of fructans on human health rely on low molecular weight fructans that are either isolated from plants like chicory or Jerusalem artichoke, which are both inulin producers, or can be produced with the help of fungal invertases. This method is very important in Japan. Fructan synthesis in plants is dependent on at least two enzymes, one of them producing the trisaccharide kestose, the other being a transfructosylase that uses fructans as donor and acceptor of fructosyl residues. We chose artichoke as the source for the fructosyl transferase genes. Artichoke produces the largest inulin known among the plant kingdom. We believe this can possibly influ-

Engineering Carbohydrate Metabolism in Transgenic Plants

179

Fig.15.3. Inulin isolated from artichoke and transgenic potato plants expressing artichoke SST and FFT. The inulin preparation was analyzed by high pressure anion exchange chromatography (HPAEC) with pulsed amperometric detection. ence yield, because longer chains would cause lower osmotic load on storage organs than short ones, considering that fructans are—in contrast to strach—water soluble carbohydrates. We have cloned both genes needed for inulin synthesis in artichoke and expressed them in potato plants. 11, 12 Transformation of potato with the SST and FFT genes was performed in two steps. At first, we transformed potato with an SST construct under control of the CaMV (cauliflower mosaic virus) 35S promoter. The plants produce the trisaccharide 1-kestose and also nystose, which is the next higher homolog, in substansial amounts. Oligofructans are located in the vacuole and would be subject to degradation by invertases. Fortunately, invertase activity is low during loading of tubers with

photosynthates. Under conditions of cold storage, only longer chains would be resistant to invertase activity. Transformation of the SST-expressing potato with an FFT-construct led to the accumulation of inulin in tubers. The inulin resembles the artichoke inulin in size (Fig. 15.3; see previous page), and the yield reaches up to 1% of the fresh weight, which is high considering the low concentration of sucrose in potato. A closer look at the carbohydrate composition of the potato tubers reveals that fructan synthesis might take place at the expense of starch, but as starch content is about 20-fold higher, the reduction is not significant, at least in greenhouse experiments (Table 15.6). We are now performing field tests to better assess biomass production.

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Coming to a conclusion, we can summarize that it is possible to modify carbohydrate composition by manipulating activities of endogenous enzymes. This allows the production, for example, of starches with new properties that are normally not found in nature and gives access to a wide array of renewable resources for industrial production and also of food substances with improved quality. Introducing new synthetic pathways by sequential transformation with genes encoding heterologous enzymes allows the production of carbohydrates that are uncommon to a given plant species and substantially alters its nutritional value.

References 1. Sonnewald U. Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning. Plant J 1992; 2:571-581 2. Lerchl J, Geigenberger P, Stitt M. et al. Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants. Plant Cell 1995; 7:259-270 3. Herbers K, Sonnewald U. Manipulating metabolic partitioning in transgenic plants. TIBTECH 1996; 14:198-205 4. Sonnewald U, Hajirezaei MR, Kossmann J et al. Increased potato tuber size resulting from apoplastic expression of a yeast invertase. Nature Biotech 1997; 15:794-797 5. Trethewey RN, Geigenberger P, Riedel K et al. Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis. Plant Journal 1998; 15:109-118 6. Buessis D, Heineke D, Sonnewald U et al. Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol. Planta 1997; 202:126-136 7. Riesmeier JW, Willmitzer L, Frommer WB. Evidence for an essential role of the sucrose transporter in phloem loading and assimilate partitioning. EMBO J 1994; 13:1-7

Table 15.6. Soluble sugars in untransformed potato (Control) and a transgenic plant expressing artichoke SST and FFT (SST/FFT) in µmol/g fresh weight ± standard deviation µmol/g FW

Control

SST/FFT

Glc

3.73 ± 4.4

7.99 ± 5.65

Frc

0.36 ± 0.16

0.4 ± 0.2

Suc

34.2 ± 6.5

28.1 ± 6.2

starch

836 ± 167

651 ± 110

fructan

n.d.

41.9 ± 7.09

Starch and fructan content is expressed in umol hexose equivalent/g fresh weight. Glc: glucose; Frc: fructose; Suc: sucrose; n.d.: not detectable.

8. Mueller Roeber B, Sonnewald U, Wilmitzer L. Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J 1992; 11:1229-1238 9. Mueller-Roeber B, Kossmann J. Approaches to influence starch quantity and starch quality in transgenic plants. Plant Cell Env 1994; 17:601-613 10. Lorberth R, Ritte G, Willmitzer L et al. Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nature Biotechnology 1998; 16(5):473-477 11. Hellwege EM, Gritscher D, Willmitzer L et al. Transgenic potato tubers accumulate high levels of 1-kestose and nystose: Functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossom discs. Plant J 1997; 12:1057-1065

Engineering Carbohydrate Metabolism in Transgenic Plants 12. Hellwege EM, Raap M, Gritscher D et al. Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs. FEBS Letters 1998; 427: 25-28.

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CHAPTER 16

Super-RuBisCO for Improving Photosynthesis A. Yokota, S. Okada, C. Miyake, H. Sugawara, T. Inoue and Y. Kai

Introduction

A

present and urgent worldwide issue is the global warming caused by an increase in the atmospheric concentration of CO2. The concentration is expecteded to increase 2-fold in the next century and the global atmospheric temperature would increase to 2 to 3 degrees higher than its present value. Under the estimated circumstances, the next generation of people will lose land for cultivation and the ecosystem that supports their lives will be changed. It should be essential for present scientists to devise technologies to relieve our ecosystem from these crises. Photosynthetic organisms have contributed to adsorption of atmospheric CO2 for over the last 4 billion years. The enormous economic and social activities of human beings, however, are releasing CO2 at a much higher rate than that of CO 2 fixation by photosynthesis of plants. This causes our ecosystem to be polluted and damaged. The deforestation in the tropical regions promotes the destruction of the ecosystem. One of the most plausible approaches to halting ecosystem destruction would be increasing the land area for plantation and greening arid, unused areas and regions for sequestration of the atmospheric CO2 into long lived or undegradable plant organic materials. This may be done by changing our woody and crop plants to live under severe habitat conditions. Plants that can grow on poor, arid lands may be created by improving their physiology in growth performances. Photosynthesis converts high energy photons to chemical

energies in chloroplasts. The photosynthetic carbon reduction (PCR) cycle utilizes the energies for reduction of CO2 incorporated by ribulose 1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) from the atmosphere (Fig. 16.1). This review refers to the importance of the maintenance of the energy balance between capturing photon energy and its utilization, and discusses how we are able to fortify the capacity of plants to achieve balance in arid lands. The target is RuBisCO.

Why is RuBisCO the Target? Balancing the rate of the conversion of photon energy into chemical energies such as NADPH and ATP, and those of the inflow of CO2 from the atmosphere and of the reduction of the fixed CO2, is the critical point for land plants. If the rate of the energy utilization is lowered by the decreased inflow of CO 2 through the stomata, chloroplants are obliged to direct energies to photorespiration and reduction of oxygen molecules to superoxide radicals (O2-).1 O2- is dismutated to hydrogen peroxide by superoxide dismutase. Hydrogen peroxide is a potent oxidant of some PCR enzymes, which lose their activity by oxidation of the functional vicinal sulfhydryl groups. Plants have a machinery to decompose hydrogen peroxide in chloroplasts.1 It has been found recently, however, that the machinery itself is very labile under droughtstress conditions.2 To over come this liability of plants, we introduced a bacterial catalase into tobacco chloroplasts, where hydrogen

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Fig. 16.1. Overall reactions of photosynthesis. PQ, plastoquinon; Cyt, cytochrome; Fd, ferredoxin; FNR, ferredoxin:NADP+ reductase; MDAR, monodehydroascorbate reductase; SOD, superoxide dismutase; APX, ascorbate peroxidase; RuBP, ribulose 1,5-bisphosphate; PGA, 3-phosphoglycerate; TP, triose phosphate; CF, coupling factor. peroxide is formed in abundance. The introduced catalase decomposes the active oxygen to greatly protect chloroplasts from oxidative damage. The above study clearly shows that it is possible to improve the endogenous active oxygen-scavenging system by introducing bacterial catalase into plant chloroplasts. However, one should not ignore the fact that the transformants can be alive for longer periods without any growth. This kind of approach to creating aridity-philic plants would not meet the desired goal by changing present plants into ones that can sequestrate atmospheric CO 2 by growing on unused, deforested and arid lands. The plants we should seek will be ones that are still productive in photosynthesis under these growth conditions. A plausible target for this purpose is RuBisCO.3 The CO 2 fixation step catalyzed by RuBisCO in photosynthesis is the important rate limiting step. The control coefficient of the enzyme in photosynthesis is over 0.5 in the presence of full sunlight. This fact tells us that improving the enzymatic efficiency is a meaningful direction to take for improvements

in plant water use efficiency and crop productivity. RuBisCO, even that of higher land plants, has several disadvantages as an enzyme.3 The reaction turnover rate is up to 3/sec/reaction site; 1/100 to 1/1000 that of most enzymes found in nature. The affinity of the enzyme for CO2 is 10 to 15 µM; just a quarter of the enzyme in chloroplasts can participate in photosynthesis. Much worse is the occurrence of the unavoidable oxygenase reaction. Plant RuBisCO, well adapted to the present oxygenic atmosphere, still fixes O2 once for every 2 to 3 CO2 fixations in chloroplasts. A part of the reaction product is oxidized to CO2 in the subsequent glycolate pathway. In total, the oxygenase reaction reduces the productivity of plants up to 60%.

Is Plant RuBisCO the Most Evolved Enzyme? We have been trying to improve the enzyme based on the molecular and biochemical mechanisms of the evolution and adaptation of the enzyme to the present atmosphere after the appearance of the enzyme in nature. Information on the

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structure-function relationship of RuBisCO evolution will be highly likely to give us various approaches useful for the improvement of the enzyme. Particularly, removing the oxygenase reaction will render plants resistant to drought, and the plant with such a RuBisCO will show significant net CO 2 fixation in photosynthesis under drought conditions.3 The biphasic reaction course, fallover, of carboxylation catalyzed by RuBisCO has been known as a characteristic of the enzyme from higher land plants. 5 Fallover consists of hysteresis in the reaction, seen during the initial several minutes, and a subsequent, very slow suicide inhibition by inhibitors formed from the substrate ribulose-1,5-bisphosphate (RuBP).6 We have examined the relationship between occurrence of fallover, the putative hysteresis-inducible sites (Lys-21 and Lys-305 of the large subunit in spinach RuBisCO), and the relative specificity in the carboxylase and oxygenase reactions among RuBisCOs from a wide variety of photosynthetic organisms. Figure 16.2 shows the evolutionary relationships among them.6 The phylogenetic tree for the evolution of the gene for the large subunits of RuBisCO, rbcL, has been well accepted.6 Occurrence of fallover and the hysteresis-inducible sites followed well the sequence of adaptation of photosynthetic organisms to a terrestrial habitat, or the increase in the relative specificity of RuBisCO. From this line of study, we expected that introduction of the hysteresis-inducible sites into the photosynthetic bacterial (γ) enzyme would give rise to an increase in the relative specificity of the bacterial enzyme. However, this was not the case. The mutant Chromatium vinosum RuBisCO, having lysine residues at 21 and 305, showed fallover, but its relative specificity was very similar to that of the wild enzyme. Another interesting point in Figure 16.2 is the occurrence of hysteresis-inducible sites in the RuBisCOs of β-purple bacteria and non-green algae. The relative specificity between carboxylase and oxygenase reactions (Sr) of the non-green algal enzyme was much higher than that of higher C3 plants. Interestingly, red algae are divided into two groups

in the phylogenetic tree of rbcL. The group including Porphiridium and Porphyra live at moderate temperatures in the presence of salts. The other group contains Cyanidium and Galdieria, which grow at higher temperatures. The relative specificity of RuBisCOs from the latter group were the extremes of RuBisCOs examined so far.7 The higher specificity for CO2 fixation in these RuBisCOs was due partly to their higher affinities for CO2 (6.6 µM) and partly to a higher activation energy in the oxygenation reaction (28.6 kcal mol-1).7

Structural Analysis RuBisCO is composed of 8 large subunits and 8 small subunits. The peptide of the large subunit has two domains; the N-terminal domain from residues 1 to 150 and the C-terminal domain from residues 151 to 475 in spinach RuBisCO. In the C-terminal domain, the C-terminal end of a β-sheet is connect to an α-helix with a loop. In total, 8 β-sheet–loop–α-helix structures are linked by 7 loops. Eight β-sheets construct a barrel structure which is covered by 8 α-helices. The amino acid residues involved in the binding of the substrate RuBP are located on the C-terminus sides of some β-sheets. Two large subunits form an L2 dimer, in which two dimers associate similarly to the number 69. The circular parts of the numbers 6 and 9 are the barrel structures of the C-terminal domain. The catalytic residues are from both the C-terminal domain of one large subunit and the N-terminal domain of the other subunit. Four L2 dimers construct an L8 core around the 4-fold axis. Two L2 dimers interact at several amino acid residues from both dimers. One of the interactions is formed by Arg-258 from one large subunit and Glu-259 from the other. The large space formed between two L2 dimers is occupied by a small subunit. Accordingly, four small subunits locate on one end of the L8 core and the remaining four on the other end. All amino acid residues from the small subunits are very far from the catalytic sites and have been deduced to participate, not in catalysis directly, but in supporting catalysis on the large subunits. Figure 16.3 compares the primary structures of the small subunits of spinach,

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Fig. 16.2. Evolution of structure and function of RuBisCO among photosynthetic organisms. Amino acid residues of the hysteresis sites are given by one-character designations. Synechococcus and Galdieria.8 As has been noted for the phylogenetic tree of rbcL, the gene for the small subunits, rbcS, is divided into three groups. The most apparent difference among them is the lack of a loop composed of spinach residues 52 to 63 in the cyanobacterial and non-green algal small subunits. This loop is conserved in the subunits of green algae and higher plants and located in the central solute channel formed in the L8 core in spinach RuBisCO.9 There are two types of small subunit in isolated spinach RuBisCO. One has histidine at residue 56. The imidazol residue of SHis-56 (the superscript S indicates that this residue is located on the small subunit) interacts with the oxygen atom of the caroxyl group of LGlu-259 (L indicates that this residue is from the large subunit) to influence interdimer communication

(Fig. 16.4). The other small subunit has leucine, which cannot have a similar interaction with the L8 core. Since the two types of small subunit occupy their positions one by one at one end of the core, and with 90˚C rotation around the 4-fold axis at the other end, spinach RuBisCO may have a structure of (L2SI2)2(L2SII2)2, where SI is the small subunit with histidine at residue 56 and SII is the other type of subunit. The L2 dimers carrying SI might have different catalytic properties than those of the dimer with SII. The small subunits of Galdieria RuBisCO do not have the loop from residues 52 to 63 (Fig. 16.3). Instead, the C-terminal end of the small subunit has a long extension, with two β-sheets. The ε-amino group of SLys-139 interacts with the main chain oxygen of L Glu-259, the carboxyl oxygen of which does

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Fig. 16.3. Alignment of the primary structures of the small subunits of RuBisCOs of spinach, Synechococcus (cyanobacteria) and Galdieria (non-green alga). The loop conserved in plant and green algal small subunits is marked by a solid line, and the C-terminal extension specific to nongreen algae by double lines. not interact with LLys-258 from the large subunit of the neighboring L2 dimer, unlike spinach RuBisCO (Fig. 16.4). An ionic bond between the carbonyl oxygen of LGlu-259 and S Lys-139 may block such a direct interaction between L2 dimers. Instead, the peptidyl nitrogen of SSer-135 interacts with the main chain carbonyl oxygen of LLys-258. Thus, all C-terminal extensions of the eight small subunits in a Galdieria RuBisCO holoenzyme occupy the central solvent channel, as shown in Figure 16.5, in contrast to the case of the spinach enzyme. These extensions also make extensive interactions between the small subunits in the channel. In some bacteria and dinoflagellates, RuBisCO functions without the small subunits. The Sr values of these RuBisCOs range from 10 to 20. It has been postulated that attainment of the small subunits may be the cause of the observed great increase in the Sr value. In fact, the Sr value of RuBisCO with small subunits of some bacteria is 2 to 3 times higher than the enzyme without the small subunits in the same organisms. Among RuBisCOs with the small subunits, there is a strong variation in the Sr value: 40 for the

cyanobacterial enzyme to 240 for the redalgal enzyme. This great difference may be also related to the structure around the small subunits. Figure 16.5 depicts the structures of RuBisCOs of Synechococcus, spinach and Galdieria viewed from the end of the 4-fold axis passing through the center of four small subunits at one end.8 The small subunit of the cyanobacterium lacks both part of the loop conserved in the subunits of green algal and plant enzymes and the C-terminal end of the Galdieria enzyme. As a consequence of this lack, the central solvent channel of the Synechococcus enzyme is very wide. The other extreme is represnted by Galdieria RuBisCO, where the channel is almost completely buried by the C-terminal extension of the small subunits. The order of the occupation level of the space of the central solvent channel among the three RuBisCOs is very similar to that of the Sr value. It might be possible to postulate that not only the existence of the small subunits, but also the close interaction between the small subunits in the channel, is related to the increase in the Sr value of RuBisCO.

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Fig. 16.4. Superimposition of the structures around LGlu-259 (E259) of spinach and Galdieria RuBisCOs. Amino acid residues of spinach RuBisCO are colored in blue and cyano and those of the Galdieria enzyme are depicted in pink and red. L and S are the subunit origin of the residues. Yellow dotted lines represent ionic and hydrogen bonds having distances of not more than 3.3 Å. Details of structural analysis are given in reference 8.

Fig. 16.5. Comparison of the structure of the central water channel of RuBisCOs of spinach, Synechococcus and Galdieria. The large and small subunits are colored in brown and green, respectively. Details of structural analysis are given in reference 8.

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Physiological Implications Figure 16.6 shows the A/Ci curve for ordinary C3 plants, calculated by the equations of Farquhar and von Caemmerer10 from the reported kinetic values for plant RuBisCO. The CO2 compensation point is 50 ppm intercellular CO2, and the CO2 fixation shifts from the RuBisCO-limiting phase to the RuBP-regeneration-limiting phase at 170 ppm CO2. If Galdieria RuBisCO is substituted for plant RuBisCO, the realizable transgenic plants will have the CO2 compensation point at 16 ppm CO2. The phase transition will occur around 70 ppm CO2. This predicts that the introduced Galdieria enzyme will utilize the photosynthetic chemical energies efficiently even in the presence of low concentrations of CO2. These considerations teach us that changing the enzymatic properties of RuBisCO of C3 plants is a meaningful direction for improve- ment of plant productivity.3 Particularly, increasing the relative specificity and the affinity for CO2 of RuBisCO is a meaningful direction in plant biotechnology.

Acknowledgments This work was partly supported by PEC/ MITI of Japan.

References 1. Asada K. Radical production and scavenging in the chloroplasts. In: Barker NR, ed. Photosynthesis and the Environment. Amsterdam: Kluwer Academic Press, 1996:123-150. 2. Shikanai T, Takeda T, Yamauchi H et al. Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts. FEBS Lett 1998; 428:47-51. 3. Yokota A. Super-RuBisCO: Improvement of photosynthetic performances of plants. In: Inui T, Anpo M, Yanagida S et al, eds. Advances in Chemical Conversions for Mitigating Carbon Dioxide. Amsterdam: Elsevier, 1998:117-126. 4. Yokota A. Ribulose bisphosphateinduced, slow conformational changes of spinach ribulose bisphosphate carboxylase cause the two types of inflections in the course of its carboxylase reaction. J Biochem 1991; 110:246-252

Fig. 16.6. Calculation of the photosynthetic CO2 gas exchange rates of ordinary C3 plants and of realizable C3 plants in which Galdieria RuBisCO is functioning in place of the original enzyme. Calculations were done using the kinetic parameters for spinach and Galdieria RuBisCOs,7 and equations for photosynthetic gas exchange.10

5. Yokota A, Wadano A, Murayama H. Modeling of continuously and directly analyzed biphasic reaction courses of ribulose 1,5bisphosphate carboxylase/oxygenase. J Biochem 1996; 119:487-499. 6. Uemura K, Tokai H, Higuchi T et al. Distribution of fall-over in the carboxylase reaction and fall-over-inducible sites among ribulose 1,5-bisphosphate carboxylase/oxygenases of photosynthetic organisms. Plant Cell Physiol 1998; 39:212-219. 7. Uemura K, Anwaruzzaman, Miyachi S, Yokota A. Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic red algae with a strong specificity for CO2 fixation. Biochem Biophys Res Commun 1997; 233:568-571. 8. Sugawara H, Yamamoto H, Shibata N, et al. Crystal structure of carboxylase-oriented ribulose-1,5 bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita. J Biol Chem 1999; 274:15655-15661. 9. Shibata N, Inoue T, Fukuhara K et al. Orderly disposition of heterogeneous small subunits in D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach. J Biol Chem 1996; 271:26449-26452.

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10. Farquhar GD, von Caemmerer S, Berry JA. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 1980; 149:78-90.

CHAPTER 17

Molecular Physiology of Nitrogen Recycling in Rice Plants T. Yamaya, S. Kojima, M. Obara, T. Hayakawa and T. Sato

Introduction

I

n japonica type rice (Oryza sativa L. cv. Sasanishiki) plants, approximately 80% of total nitrogen in the ear is the nitrogen which is remobilized through the phloem from older, senescing organs. Senescing leaf blades contribute about 50% of the total nitrogen in the ear. Thus, the process of nitrogen recycling is very important in determining both the productivity and quality of rice. However, little attention has been paid to the mechanisms of nitrogen remobilization from the senescing organs or to the re-utilization of the remobilized nitrogen for biosynthetic reactions in developing organs. Major forms of nitrogen in the phloem sap of rice plants are glutamine and asparagine. The asparagine is probably synthesized from glutamine. Thus, the synthesis of glutamine in senescing organs, as well as the utilization of glutamine in developing organs, are the key steps for nitrogen recycling in rice plants. Our immunocytological studies with a japonica type rice at the reproductive stage show that cytosolic glutamine synthetase (GS1; EC 6.3.1.2) is important for the export of leaf nitrogen from senescing leaves, because the GS1 protein was detected in companion cells, which are important for phloem loading of solutes, and vascular parenchyma cells. NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in developing organs, such as expanding leaves and developing grains, is involved in the utilization of

glutamine that is transported through the vascular system, because the protein was located in the cells which are important for solute transport from the phloem and xylem elements. Transgenic rice plants expressing antisense RNA for NADH-GOGAT at T0 generation markedly reduced the weight of 1,000 grains, indicating that NADH-GOGAT is indeed a key step for nitrogen recycling and a possible target for the improvement of plant productivity. The content of NADH-GOGAT protein in the developing organs alters dramatically and its expression is regulated in a cell type-specific manner. We recently isolated both genomic and cDNA clones for rice NADH-GOGAT. Regulation of NADHGOGAT gene expression is currently being studied more precisely using these molecular tools. When various cultivars of japonica, indica, and javanica type rice plants were tested to determine the contents of NADHGOGAT in expanding leaves and those of GS1 in senears were found to contain less NADHGOGAT protein and more GS1 protein than a typical japonica, Sasanishiki. If the functions of GS1 and NADH-GOGAT also hold true in indica type cultivars, the content differences may be related to the morphological characteristics different from japonica. Molecular physiology, together with the use of genetic resources, could provide targets in nitrogen recycling to improve productivity in rice plants.

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine ©2000 Eurekah.com.

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Physiology and Biochemistry of Nitrogen Recycling in Rice Plants Under sufficient light conditions, the assimilation of inorganic nitrogen and efficient recycling of the assimilates within plants are the most important processes for determining the productivity and quality of many crops. The major source of nitrogen for developing leaves and spikelets in rice plants is the nitrogen remobilized, via the phloem, from older, senescing organs.1 In particular, senescing leaf blades are the major source of nitrogen; and they contribute about half of the nitrogen in the developing spikelets (Fig. 17.1). This intricate process of nitrogen recycling, from the senescing organs to the developing organs, is very important in determining the productivity and the quality of rice plants. Nitrogen recycling consists of at least the following four major steps: 1. Degradation of nitrogen-containing macromolecules, such as RuBisCO and chlorophyll, during senescence; 2. Conversion of the hydrolyzed nitrogen to compounds for transport in the senescing organs; 3. Long distance transport of the nitrogen via phloem; and 4. Re-utilization of the transported nitrogen in developing organs for many biosynthetic reactions. The first step, mechanisms for the degradation of RuBisCO for example, is largely unknown at this moment. During natural senescence of rice leaves2 or wheat leaves,3 degradation of RuBisCO occurs prior to the breakdown of chlorophyll, indicating that RuBisCO is possibly hydrolyzed in chloroplasts. Although the presence of proteolytic enzymes in chloroplasts is not clearly reported, recent findings in which active oxygen splits the RuBisCO large subunit into two polypeptides4 could be a clue to understanding the mechanisms for RuBisCO degradation in chloroplasts. In the phloem sap of rice plants, glutamine and asparagine, which is synthesized from glutamine,5,6 are the major forms of nitrogen.7 Therefore, synthesis of glutamine is important in senescing organs, whereas the re-utilization of the transported glutamine is necessary in developing organs. Glutamine synthetase (GS;

EC 6.3.1.2) and glutamate synthase (GOGAT) are candidates for performing the synthesis and utilization of glutamine in plants. There are two isoenzymes of GS in many plants:5,6 a minor isoenzyme located in the cytosol (GS1) and the main isoenzyme in the chloroplast (plastid) stroma (GS2). As for GS, two molecular species of GOGAT are found in both green and non- green tissues,5,6 one requiring NADH as reductant (NADH-GOGAT; EC 1.4.1.14) and the other requiring reduced ferredoxin (Fd- GOGAT; EC 1.4.7.1). In leaves, these two GS isoenzymes and two GOGAT species have distinct functions. Elegant studies with mutants lacking either GS28 or Fd-GOGAT9,10 show that a major role of GS2 and of FdGOGAT, both located in the chloroplast stroma, is the reassimilation of ammonium ions released from photorespiration. Because the mutants are able to grow normally under nonphotorespiratory conditions,8-10 GS1 and NADH-GOGAT in leaves could be important in the synthesis of glutamine and glutamate for normal growth and development.

Export of Glutamine from Senescing Leaves Our immunological and immunocytological studies have suggested that GS1 is important for the export of nitrogen from senescing rice leaves. 11-13 During natural senescence of a leaf blade of rice plants, the contents of GS2 declined in parallel with other chroloplastic enzymes such as RuBisCO and Fd-GOGAT. In contrast, the GS1 protein remained relatively constant throughout the senescence period.12 When various positions of leaves are tested, the relative content of GS1 protein was found to be highest in the oldest leaf blade on the main stem of rice plants and gradually declined toward the youngest unexpanded blade.14 Tissue print immunoblots clearly show that GS1 protein is detected in large and small vascular bundles, whereas GS2 protein is in mesophyll cells.11 When cross sections from the lowest position of the attached leaf blade were immunostained with an affinity purified anti-GS1 IgG, strong signals for GS1 protein were detected in companion cells and parenchyma cells of vascular bundles.13 The signals for GS1 were barely detected in mesophyll cells, where the

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Fig. 17.1. Origin of nitrogen in ear of rice plants. Hatched area indicates the nitrogen remobilized from senescing organs into the ear, whereas the open area represents newly absorbed nitrogen originating from soil/medium. The values are adopted from Mae et al.1 GS2 protein was mainly located. Companion cell-specific localization of GS1 was also shown using immunogold labeling by a Portuguese group working with tobacco15 and potato16 plants. The spatial localization of these GS isozymes indicates distinct and non-overlapping roles in nitrogen metabolism in rice plants. The companion cells and vascular parenchyma cells are active in the transport of solutes, since they contain abundant mitochondria and endoplasmic reticulum.17 The companion cells are important in the regulation of phloem loading.17,18 The localization of GS1 in these cells of rice leaves further supports our hypothesis11-13 that GS1 is important in the synthesis of glutamine for nitrogen export. Conclusive evidence to support our hypothesis could be obtained either by creating mutants lacking GS1

or transformants having reduced amounts of the GS1 gene product. We are currently working on the reduction of GS1 protein in transgenic rice using techniques to express the antisense RNA driven with a companion cell-specific promoter. GS1 protein was detected not only in the senescing leaf blades but also in the younger blades of rice plants, although the content in the developing leaf blade was less than that in the senescing blade.14 When the cellular localization of GS1 was tested in the different leaf positions of the blades, the signals in companion cells were less striking in the younger green leaf blades and were hardly detected in the non-green portion of the unexpanded blade.13 In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and

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xylem parenchyma cells of vascular bundles.13 Thus, the cellular localization of GS1 in vascular bundles of rice plants changed during leaf development. When the metabolic route for generation of ammonium ions is considered in these cell types, there is no immediate answer as to a function for GS1. Biosynthesis of lignin polymer in these compartments would be one explanation for the generation of ammonium ions by a reaction of phenylalanine ammonia-lyase (EC 4.3.1.5) and GS1 might be important for the assimilation of generated ammonium ions.

Re-Utilization of the Transported Glutamine in Developing Organs NADH-GOGAT is a minor form of GOGAT species and little has been known concerning its cellular and subcellular localization, gene structure and expression, or function in higher plants. In 1992, a specific antibody for NADH-GOGAT from rice cell cultures was prepared as the first instance to crossreact NADH-GOGAT protein in rice plants.19 Antibody for NADH-GOGAT in alfalfa root nodules had been obtained prior to the rice, but the antibody hardly recognized NADH-GOGAT in plant organs other than nodules.20 Using the antibody for rice NADHGOGAT, immunochemical and immunocytological experiments have been performed. A high abundance of NADH-GOGAT protein was detected in the non-green, developing leaf blade 14 and in the developing grains. 21 Immunocytological results showed that the NADH-GOGAT protein was present in vascular parenchyma cells and mestome sheath cells of vascular bundles of the developing leaf blade, as well as in vascular parenchyma cells, nucellar projection, and nucellar epidermis of dorsal vascular bundles of the developing young grains.22 On the other hand, Fd- GOGAT protein is mainly present in mesophyll cells of leaf blades and in the chloroplast-containing cross cells of the pericarp of the grains.22 The spatial localization of these GOGAT proteins indicates distinct and non-overlapping roles, as in the case of GS isoenzymes. The cells containing NADH- GOGAT protein in leaf vascular bundles and the dorsal vascular bundles of grains are considered by the anatomical studies to be active in the solute transport

from phloem and xylem.17 In addition, the content of NADH- GOGAT protein, as well as its activity, in the apical spikelets increased several-fold in the first 2 weeks after flowering.21 Thus, it is likely that NADH-GOGAT is involved in the re-utilization of glutamine transported through the vascular system, and the synthesized glutamate is further utilized for biosynthetic reactions in these young organs. We have recently determined the gene structure for NADH-GOGAT in rice plants.23 To obtain direct evidence to support the possible role of NADH-GOGAT in nitrogen recycling, a cauliflower mosaic virus 35S promoter was fused with a fragment of NADH-GOGAT cDNA in the antisense orientation, and the chimeric construct was introduced into rice calli by the methods of Agrobacterium-mediated transformation.24 The experiments to look at the effects of expression of the antisense RNA in these transformants are now in progress. We have just obtained results which show that the weight of 1,000 grains from some of the transformants at the T0 generation is significantly reduced when compared to the grain weight of the control transformants introduced to vector without the NADH-GOGAT cDNA fragment (Fig. 17.2).

Primary Assimilation of Ammonium Ions in Rice Roots In rice roots, the mRNA and protein for NADH-GOGAT increase dramatically within 12 h of supplying a low concentration (<50 mM) of ammonium ions.25,26 Immunolocalization studies clearly showed that two cell layers of the root surface, i.e., epidermis and exodermis, are responsible for the rapid increase in NADH-GOGAT protein.27 Recent anatomical studies indicate that there is a Casparian strip between the epidermis and the sclerenchyma cells, which locate one cell layer outside the first layer of cortex in rice roots,28 indicating that solute transport between these cell types should be a symplastic process. Because cytosolic root GS is also localized in these two cell layers, NADH-GOGAT could have a responsibility to supply glutamate to the GS reaction for the rapid assimilation of ammonium ions in these two cells. Our recent

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Fig. 17.2. Grain yields of transgenic rice at T0 generation, introduced either with a cDNA fragment for NADH-GOGAT in the antisense orientation (A1 to A5) or with the pIG121Hm-vector alone (G1 and G2). intracellular localization studies indicated that NADH-GOGAT protein is located in the plastids of rice roots.29 The localization in plastids might be expected in developing leaf blades and young grains, since NADHGOGAT occurs as a single gene in rice.23 The presumed transcribed region (11.7 kbp) of the NADH-GOGAT gene consists of twenty-three exons separated by twenty-two introns with a range in exon size of from 65 bp to 1,530 bp.23 This gene structure is slightly different from that found in alfalfa nodules,30 which comprises twenty-two exons and twenty-one introns, and apparently lacks the first exon seen in the rice gene. Most of the corresponding exons show more than 60% similarity in the nucleotide sequence between the two species. Sequences for a putative amide transfer, FMN binding, [3Fe-4S] cluster, and NADH binding regions were detected,23 as in the NADH-GOGAT gene from alfalfa nodules.30 The genomic clone for rice NADH-GOGAT covered a 3.7 kbp 5'-upstream region from the first methionine. The transcriptional start sites were identified with primer extension analysis and S1 nuclease protection experiments. Rice NADH-GOGAT is synthesized as a 2,166 amino acid protein that includes a 99 amino acid presequence.23

By using the 3.7 kbp 5'-upstream region as a promoter for NADH-GOGAT, a chimeric gene construct having this promoter and the β-glucuronidase (GUS) structural gene as a reporter was introduced into rice calli that had been generated from germinating seeds, and transgenic rice plants were produced. Expression of the NADH-GOGAT gene is now monitored by GUS activity staining in various organs of the regenerated rice at the T0 generation. Preliminary results show that GUS activity is detected in vascular bundles of various organs of the T0 rice plants (Kojima et al, unpublished results). The 3.7 kbp upstream region from the translation start possesses the promoter activity for the NADH-GOGAT gene, with element(s) which determine the cell type specificity for gene expression in rice plants. Further transformation with a series of deletion clones of this region is now in progress to identify cis-elements related to the agespecific, cell-specific, and nitrogen-responsive expression of NADH-GOGAT in rice plants. In the roots of rice seedlings, the mRNA for NADH-GOGAT was markedly accumulated within 3 h after the supply of a low concentration of ammonium ions.26 Cycloheximide in the presence of ammonium ions had no

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effect on the increase in mRNA, suggesting that protein synthesis is not required for the accumulation of mRNA. This could be related to the fast response of the accumulation after the supply of ammonium ions. Methionine sulfoximine, an inhibitor of GS, completely inhibited the accumulation of NADHGOGAT mRNA and the supply of glutamine replaced the effect of ammonium ions. 26 These results show that ammonium ions are not a direct inducer of this process. Glutamine or its metabolite might be the signaling factor for the inducible accumulation of NADHGOGAT mRNA in rice roots. A similar response would be expected in the young developing organs to regulate the expression of a NADH-GOGAT gene caused by glutamine or its metabolite in solute transported through vascular tissues.

Variation in the Amounts of GS1 and NADH-GOGAT Protein in Rice Plants The use of the genetic resources of rice plants is another approach for the improvement of the productivity and quality of rice. If the role of GS1 in senescing organs and that of NADH-GOGAT in developing organs were also true for indica and javanica, some differences in the contents of these enzymes would be expected, because of their great biomass productions. When several indica and javanica cultivars of rice plants were grown in a greenhouse at Sendai, located in northern Japan, typical characteristics of morphology were observed.31 Indica and javanica cultivars generally show the characteristics of high productivity of biomass, i.e., large plant height, great leaf length and weight, or large numbers of tillers, when compared to those of Sasanishiki or Nipponbare, typical cultivars of japonica. Relative contents of the GS1 protein in the lowest senescing leaf blades of most indica cultivars were much higher than those of Sasanishiki, when the contents were calculated on the basis of leaf fresh weight.31 When the relative amounts of GS1 protein were plotted against the total leaf nitrogen on a unit of leaf dry weight, to roughly eliminate the effect of leaf age,5 cultivars of japonica and two cultivars of javanica were plotted as one regression line

(Obara et al, unpublished). Four cultivars out of a total of eight indica cultivars contained approximately twice as much GS1 protein as the japonica and javanica on the basis of total leaf nitrogen, although a regression line was not drawn for these indica cultivars. On the other hand, relative contents of NADHGOGAT protein in unexpanded developing leaf blades of most indica cultivars were significantly lower than those of japonica and javanica. Cellular localization of GS1 and NADH-GOGAT proteins were identical among all cultivars tested. These results indicate that several indica cultivars possess an enhanced capacity to export leaf nitrogen, whereas japonica and javanica cultivars have an efficient system to utilize glutamine transported via vascular tissues.

Conclusion To increase crop yield per unit of land area, an efficient nitrogen use within the plants is very important under conditions of sufficient irradiation. Because most of the nitrogen in rice grains is transported from senescing organs, the process of nitrogen recycling is very important in determining both the productivity and the quality of rice. Our immunolocal- ization studies clearly show that these GS isoenzymes or GOGAT species are individually accumulated in different cell types of tissues, and hence each enzyme has a distinct function in nitrogen metabolism. The localization of GS2 and Fd-GOGAT in mesophyll cells of rice leaves is in good agreement with the excellent studies using mutants which show that the major function of these enzymes is in the reassimilation of ammonium ions released during photorespiration.8-10 On the other hand, GS1 could be important in the synthesis of glutamine, which is a major form of nitrogen exported from the senescing leaves. In association with this, NADH-GOGAT has a role in re-utilizing the glutamine transported from senescing organs. The fact that some lines of the antisense transformants reduce the weight of seeds further supports the important role of NADH-GOGAT in nitrogen recycling in rice plants. Analyses of the promoter for the NADH-GOGAT gene will provide information on the regulatory mechanisms for the cell type-specific, age-specific, and

Molecular Physiology of Nitrogen Recycling in Rice Plants nitrogen-responsive expression of the NADHGOGAT gene in rice plants. As far as genetic resources are concerned, more than half of the indica cultivars tested possess approximately two-fold greater content of the GS1 protein in senescing leaves than most japonica and javanica cultivars, while most of the indica cultivars contain lower amounts of NADH-GOGAT protein in unexpanded young leaf blades. Most cultivars of indica and javanica show a greater biomass productivity than japonica, even when cultivated in northern Japan. The great production of biomass of indica might be related to the enhanced system for exporting leaf nitrogen to the developing organs. If our hypothesis were true, enhancement of GS1 protein in senescing leaves of japonica and that of NADH-GOGAT in developing leaves of indica cultivars would possibly provide more nitrogen in the grains. At the same time, to increase the source capacity, genetic approaches to enhance the amounts of total nitrogen in leaves and also to delay the senescing period would be targets for genetic manipulation to improve the productivity of rice plants. Because the expression of NADH-GOGAT protein, as well as GS1 protein, is regulated in an age-specific, cell type-specific and nitrogenresponsive manner, mechanisms for the fine control of NADH-GOGAT gene expression should be studied in detail in the near future. The mapping of genes related to nitrogen recycling in chromosomes and analysis of quantitative trait loci (QTL) related to these characteristics will be tested by using recombinant inbred lines between a japonica and an indica. Information from these experiments will provide direction for genetic manipulation to improve the productivity and quality of rice plants.

Acknowledgments This work was supported in part by a program of Research for the Future from the Japan Society for the Promotion of Science (JSPS-RFTF96L00604) and in part by Grantin-Aid for Scientific Research on Priority Area (Nos. 09274101 and 09274102) from the Ministry of Education, Science, Sports and Culture of Japan. Critical reading by Dr. A. K. Tobin, University of St. Andrews, UK, is greatly acknowledged.

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References 1. Mae T, Makino A, Ohira K. The remobilization of nitrogen related to leaf growth and senescence in rice plants (Oryza sativa L.). Plant Cell Physiol 1981; 22:1067-1074. 2. Makino A, Mae T, Ohira K. Photosynthesis and ribulose 1,5-bisphosphate carboxylase in rice leaves. Changes in photosynthesis and enzymes involved in carbon assimilation from leaf development through senescence. Plant Physiol 1983; 73:1002-1007. 3. Mae T, Kamei C, Funaki K et al. Degradation of ribulose 1,5-bisphosphate carboxylase in the lysates of the chloroplasts isolated mechanically from wheat (Triticum aestivum L.) leaves. Plant Cell Physiol 1989; 30:193-200. 4. Ishida H, Shimizu S, Makino A et al. Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves. Planta 1998; 204:305-309. 5. Sechley KA, Yamaya T, Oaks A. Compartmentation of nitrogen assimilation in higher plants. Int Rev Cytol 1992; 134:85-163. 6. Lea PJ, Robinson SA, Stewart GR The enzymology and metabolism of gluta- mine, glutamate and asparagine. In Miflin BJ, Lea PJ, eds. The Biochemistry of Plants, Vol 16. Intermediary nitrogen metabolism. San Diego: Academic Press, 1990:121-157. 7. Hayashi H, Chino M. Chemical composition of phloem sap from the upper most internode of the rice plant. Plant Cell Physiol 1990; 31:247-251. 8. Wallsgrove RM, Turner JC, Hall NP et al. Barley mutants lacking chloroplast glutamine synthetase. Biochemical and genetic analysis. Plant Physiol 1987; 70:827-832. 9. Kendall AC, Wallsgrove RM, Hall NP et al. Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase. Planta 1986; 168:316-323. 10. Somerville CR, Ogren WL. Inhibition of photosynthesis in Arabidopsis mutants lacking in leaf glutamate synthase activity. Nature 1980; 286:257-259. 11. Kamachi K, Yamaya T, Hayakawa T et al. Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves. Plant Physiol 1992; 99:1481-1486.

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12. Kamachi K, Yamaya T, Mae T et al. A role for glutamine synthetase in the remobilization of leaf nitrogen during natural senescence in rice leaves. Plant Physiol 1991; 96:411-417. 13. Sakurai N, Hayakawa T, Nakamura T et al. Changes in the cellular localization of cytosolic glutamine synthetase protein in vascular bundles of rice leaves at various stages of development. Planta 1996; 200:306-311. 14. Yamaya T, Hayakawa T, Tanasawa K et al. Tissue distribution of glutamate synthase and glutamine synthetase in rice leaves. Occurrence of NADH-dependent glutamate synthase protein and activity in the unexpanded non-green leaf blades. Plant Physiol 1992; 100:1427-1432. 15. Carvalho H, Pereira S, Sunkel C et al. Detection of a cytosolic glutamine synthetase in leaves of Nicotiana tabacum L. by immunocytochemical methods. Plant Physiol 1992; 100:1591-1594. 16. Pereira S, Cavalho H, Sunkel C et al. Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves of Solanum tuberosum L. Protoplasma 1992; 167:66-73. 17. Chonan N, Kaneko M, Kawahara H et al. Ultrastructure of the large vascular bundles in the leaves of rice plants. Jpn J Crop Sci 1981; 50:323-331. 18. Van Bel AJE. Strategies of phloem loading. Annu Rev Plant Physiol Mol Biol 1993; 44:253-281. 19. Hayakawa T, Yamaya T, Kamachi K et al. Purification, characterization, and immunological properties of NADHdependent glutamate synthase from rice cell cultures. Plant Physiol 1992; 98:1317-1322. 20. Anderson MP, Vance CP, Heichel GH et al. Purification and characterization of NADH-glutamate synthase from alfalfa root nodules. Plant Physiol 1989; 90:351-358. 21. Hayakawa T, Yamaya T, Mae T et al. Changes in the content of two glutamate synthase proteins in spikelets of rice (Oryza sativa) plants during ripening. Plant Physiol 1993; 101:1257-1262. 22. Hayakawa T, Nakamura T, Hattori F et al. Cellular localization of NADH-dependent glutamate-synthase protein in vascular bundles of unexpanded leaf blades and young grains of rice plants. Planta 1994; 193:455-460.

23. Goto S, Akagawa T, Kojima S et al. Organization and structure of NADH-dependent glutamate synthase gene from rice plants. Biochim Biophys Acta 1998; 1387:298-308. 24. Goto S, Ishii Y, Hayakawa T et al. Agrobacterium-mediated transformation of Sasanishiki, a leading cultivar of rice (Oryza sativa L.) in northern Japan. In: Ando T et al., eds. Plant Nutrition for Sustainable Food Production and Environment, Dordrecht: Kluwer Academic Publishers, 1997:839-840. 25. Yamaya T, Tanno H, Hirose N et al. A supply of nitrogen causes increase in the level of NADH-dependent glutamate synthase protein and in the activity of the enzyme in roots of rice seedlings. Plant Cell Physiol 1995; 36:1197-1204. 26. Hirose N, Hayakawa T, Yamaya T. Inducible accumulation of mRNA for NADHdependent glutamate synthase in rice roots in response to ammonium ions. Plant Cell Physiol 1997; 38:1295-1297. 27. Ishiyama K, Hayakawa T, Yamaya T. Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions. Planta 1998; 204:288-294. 28. Morita S, Lux A, Ebstone DE et al. Reexamination of rice seminal root ontogeny using fluorescence microscopy. Jpn J Crop Sci 1996; 65:37-38. 29. Hayakawa T, Hopkins L, Peat LJ et al. Quantitative intercellular localization of NADH-dependent glutamate synthase protein in different types of root cells in rice plants. Plant Physiol 1999; 119:409416. 30. Vance CP, Miller SS, Gregerson RG et al. Alfalfa NADH-dependent glutamate synthase: Structure of the gene and importance in symbiotic N 2 fixation. Plant J 1995; 8:345-358. 31. Yamaya T, Obara M, Hayakawa T et al. Comparison of contents for cytosolicglutammine synthetase and NADHdependent glutamate synthase proteins in leaves of japonica, indica, and javanica rice plants. Soil Sci Plant Nutr 1997; 43:1107-1112.

CHAPTER 18

Agrobacterium-Mediated Cereal Transformation: Low Glutelin Rice Development T. Kubo, Y. Hiei, Y. Ishida, Y. Maruta, J. Ueki, N. Nitta and T. Komari

Introduction

A

n efficient transformation method is needed for genetic studies and plant improvement. A large flow of putatively useful genes is now being supplied from genomics research on various living organisms. Final evaluation of such genes has to be done in transgenic plants, into which each gene is incorporated. Each gene should be properly integrated into a plant. There is a wide variation in quality of transformants in terms of level, time and site of expression; somaclonal variation; and physical state of transgenes. A number of transformants have to be created for selection of appropriate transformed plants. Transformation is therefore one of the key processes in plant biotechnology. Genetic transformation delivers DNA into plant cells and regenerates plants from such cells. Physical delivery includes gene gun or electroporation methods; biological delivery uses Agrobacterium-mediated transformation. Monocotyledons, including most major cereal crops such as rice, wheat and maize, are generally transformed by physical methods; dicotyledons such as tobacco, tomato, oilseed rape and potato, are transformed by Agrobacterium. Agrobacterium-mediated transformation offers a number of advantages, including a defined transgene, low copy number and easy handling, but it was long believed that this method is applicable, with a few exceptions,

only to dicotyledons, which are the natural hosts of this soil-borne pathogen. However, stable and efficient transformation of economically important cereal crops by Agrobacterium has been reported. This report discusses the factors to be noted in Agrobacterium-mediated transformation of various cereal crops. The development of low glutelin rice for better quality in sake brewing and cooking is then presented as an example of rice improvement in biotechnology.

Agrobacterium-Mediated Genetic Transformation of Cereals Genetic transformation mediated by A. tumefaciens in cereal crops was confirmed first in rice. Chan et al.1 produced transgenic rice plants by inoculating immature embryos with A. tumefaciens. They proved the inheritance of the transferred DNA to the progeny by Southern blot analysis, although they analyzed the progeny of only one plant. Hiei et al2 subsequently reported a method for efficient production of a number of transgenic rice plants from calluses of japonica cultivars that had been cocultivated with A. tumefaciens. They also clearly showed the large scale inheritance of transgenes in the progeny. In addition to these successful results in rice, the efficient transformation of maize was shown by Ishida et al.3 This achievement was

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then followed by successful reports in barley4 and wheat.5 Now Agrobacterium-mediated cereal transformation is recognized as an unquestionable fact. Moreover, it is becoming the preferred method, at least in rice and maize.

Key Factors Involved in Agrobacterium-Mediated Transformation of Cereals The key factors generally involved in Agrobacterium-mediated transformation of cereal crops are the type and stage of recipient plant tissue, the strain and vectors of Agrobacterium, and the cocultivation conditions. The tissue culture protocol for smooth regeneration of plants from transformed cells may also play a key role in production of normal, fertile transgenic plants. Every factor mentioned above has to be properly adjusted for successful transformation. This multiplicity may explain why it was initially so difficult to apply this technology to cereals. Tissues showing active growth are suitable for transformation. In monocotyledonous species, such tissues are limited to shoot apex, root apex, scutellum of mature and immature embryos, and their calluses or suspension-cultured cells. Hiei et al2 reported that more than 90% of calluses induced from rice scutellum showed transient expression after cocultivation with A. tumefaciens for three days. They also observed that immature embryos are a suitable tissue for transformation of indica rice.6 Immature embryos are also used for Agrobacterium-mediated transformation of maize, barley, and wheat. Transformation efficiency is highly dependent on the genotype of a germplasm to be transformed, particularly in recalcitrant cereal species. In rice, indica varieties are generally harder to transform than japonica varieties. The problem was solved by a slight modification of the cocultivation medium, and a high efficiency of transformation has been shown in a number of economically important indica varieties.6 Ishida et al3 succeeded in transforming a maize inbred, A188, at high efficiency. With the same protocol, however, they obtained no transgenic plants from

another five inbreds, and only a small number of transformants of five hybrids parented by A188. Thus, choice of a suitable germplasm, for which a protocol for plant regeneration has been established, is a key factor for success. A new vector system involving a superbinary vector has been developed.7 It is based on a Ti plasmid, pTiBo542, harbored by a supervirulent strain of A. tumefaciens, A281, which exhibits a wider host range and high transformation efficiency. In this system, a DNA fragment including a part of the virulence region of pTiBo542 is introduced into a small T-DNA-carrying plasmid in a binary vector system, in which the disarmed Ti plasmid has its own full set of virulence genes. Super-binary vectors definitely showed very high efficiency in the transformation of rice and maize.2,3 Choice of vectors and strains of A. tumefaciens are important for transformation. Hiei et al 2 tested the efficiency of four combinations of bacterial strains and vectors. The strains were an ordinary strain, LBA4404, and a supervirulent strain, EHA101. The vectors were an ordinary binary vector, pIG121Hm, and a super-binary vector, pTOK233. In rice transformation experiments, LBA4404(pTOK233) was definitely more effective than LBA4404 (pIG121Hm) or EHA101(pIG121 Hm). EHA101(pTOK233), which is the combination of a supervirulent strain with a super-binary vector, was not very effective. In maize, efficient transformation was possible only with super-binary vectors. 3 Therefore, superbinary vectors proved to be more useful in transforming these cereal crops. The conditions during cocultivation of plant tissues with Agrobacterium significantly affect the efficiency of transformation. Media containing acetosyringone and 2,4-dichlorophenoxyacetic acid and solidified with a gelling agent are suitable in rice and maize. In addition to acetosyringone, glucose and some surfactants were effective in enhancing transformation of wheat.5 In barley, however, Tingay et al.4 used immature embryos from which the embryonic axis was removed. The embryos were then shot with gold microprojectiles and cocultivated with Agrobacterium

Agrobacterium-Mediated Cereal Transformation: Low Glutelin Rice Development on medium without acetosyringone. Tingay et al observed an appreciable level of transformation. Because wounding the cultured barley embryos enhanced the recovery of transformed cells, they reported, such a pretreatment may release some phenolic compound, instead of added acetosyringone, which induces the vir genes of Agrobacterium. The bacterial concentration was adjusted to 1 to 5 x 109 cfu/ml in rice, maize, and barley, and cocultivation was done at 24–25°C for three days. Selectable marker genes and their promoters often greatly affect the efficiency of transformation. A hygromycin resistance gene, hpt, driven by the CaMV 35S promoter was highly effective in rice.2 In maize3 and barley,4 a phosphinothricin resistance gene, bar, driven by promoters 35S (maize) and Ubi1 (barley) gave good results. In wheat5, the nptII gene driven by an enhanced 35S promoter was used, and selection was done on media containing G418 as the selectable agent.

Transformation Efficiency and Quality of Transformants Transformation efficiency is sometimes crucial for the success of research, because a great number of transformed plants, even for one gene construct, need to be created to obtain transformants that incorporate a single, highly expressing transgene and show little somaclonal variation. There is wide variation between transformants in expression level of a gene, due probably to the state of gene integration in the plant genome. Different promoters combined with a single gene may change the level, position, and timing of gene expression. The copy and locus numbers of a transgene may vary from one plant to another. Transformants may show some somaclonal variation, including low fertility and abnormal morphology. The transformation efficiency is calculated as a percentage based on the ratio of the number of independent transformed plants to the total number of explants, calluses, or immature embryos infected by A. tumefaciens. Hiei et al.2 and Ishida et al.3 reported high percentages (18.5% and 15.1%) in rice and maize

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in experiments involving about a thousand explants. These high numbers explain why this technology is reliable. Efficiencies of 4.8% and 1.8% have been reported in barley4 and wheat,5 respectively. The number of copies of an incorporated gene in a transformed plant is estimated by Southern blot analysis, and the number of loci is determined by the segregation ratio in its progeny. Hiei et al2 estimated that 35% of transformed rice plants had a single copy, 40% had two copies, and only 25% had more. They also observed single locus segregation in the progeny from 60% of original transformed plants and two locus transformation in 25%. In maize, Ishida et al.3 reported that about 70% of positive transformants, which is amazingly high, showed a single copy incorporation, and more than 90% possessed the expected length of transgenes. In wheat, Cheng et al5 observed that 35% of transformants possessed single copies of transgenes, which was significantly higher than with the gene gun method. These results are consistent with the introduction of a few copies of genes, showing a simple Mendelian inheritance, into the genome of dicotyledonous species. This is one of the advantages of this method over direct DNA delivery. Agrobacterium-mediated transformation of rice is now being extensively used as an efficient method for biotechnological research in both japonica and indica varieties. In barley, relatively good efficiency of transformation has also been reported, but more effort is needed in practical application. Transformation should improve in wheat as experiments are repeated.

Development of Low Glutelin Rice Rice improvement by Agrobacteriummediated transformation has a wide range of possibilities for developing traits. As an example, low glutelin rice is being developed for the improvement of rice quality for sake brewing and cooking.8

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Glutelin and Rice Quality Rice seeds contain about 8% storage proteins by weight; the content has been reported to be negatively correlated with the quality of rice for sake brewing and cooking.9,10 During brewing, proteins are decomposed to peptides and amino acids, some of which are assimilated by yeast and give sake its full, heavy taste. But the excess often gives sake a rough taste and accelerates the deterioration of its quality.11 Glutelin, which accounts for 80% of the rice storage proteins, is localized in Protein Body II (PBII). PBII is readily digested during brewing,12 and a negative correlation has been reported between the amount of PBII and the quality of rice for sake.9 An increase in the amount of total proteins also negatively affects the eating quality of cooked rice.10 Deterioration in the eating quality of protein-rich rice is attributed to a change in the physical properties of cooked rice due to increased glutelin.13 Thus, suppression of glutelin accumulation in rice grains may improve the quality of rice for both sake brewing and cooking.

Gene Construct and Transformation of Rice Plants A full length glutelin cDNA and its promoter region were isolated from a Japanese cultivar, Sasanishiki. A partial fragment at the 5' end of the cDNA was cut out and assembled to make eight tandem repeats of the fragment. This was done because a preliminary study with in vitro translation implied that antisense RNA transcribed from the repeats can inhibit the translation of the glutelin gene more effectively than single, full length, antisense RNA. The antisense construct was assembled from the glutelin promoter region, the first intron of the castor bean catalase gene, the eight tandem repeats in antisense orientation, and the NOS terminator, in that order. The assembled construct was transferred to a super-binary vector of A. tumefaciens carrying two T-DNA regions, one of which incorporates a hygromycin resistance gene, hpt.14 Calluses derived from mature embryos of japonica rice (cv. Tsukinohikari) were trans-

formed, and hygromycin resistant plants were obtained as described by Hiei et al.2 Out of 106 hygromycin resistant regenerates, 14 plants were selected for low copy number of the antisense gene, normal plant shape, and high fertility. The selfed seeds of these plants were harvested in a greenhouse experiment, and their glutelin content was determined by density measurements of glutelin bands made visible with CBB in SDS-PAGE analyses of total seed proteins. Two plants that showed considerable reduction in the glutelin content were selected for further examination, and their progeny formed lines H39 and H75. H39 showed two locus segregation for the antisense gene, one of which was not linked with the hpt gene. Plants possessing the antisense gene in a homozygous state and lacking the hpt gene were selected. H75 showed one locus segregation of the two linked transgenes, and homozygous plants were selected. The progeny of H39 and H75 were grown in an isolated field experiment. Northern blot analysis revealed that these two lines showed considerable decrease in glutelin mRNA in immature grains. Mature seed proteins of these lines were also analyzed. H39 and H75 showed thinner glutelin bands than those of wild type plants, but thicker bands of prolamin, another storage protein group. The total nitrogen of the glutelin fraction of H39 was lower than that of the wild type plants, although the total nitrogen in its whole grains was almost the same as that of the wild type plants. These results imply that glutelin synthesis and accumulation were inhibited by the antisense gene but that prolamin possibly increased, resulting in little change overall in total protein content in H39. It is possible that inhibition of synthesis of one protein results in a compensatory increase in other proteins.

Safety Assessment Under Greenhouse Conditions H39 and H75 were grown for safety evaluation experiments in a contained greenhouse (Stage I) and in a semi-contained greenhouse (Stage II). These experiments were conducted

Agrobacterium-Mediated Cereal Transformation: Low Glutelin Rice Development according to the guidelines established by the Science and Technology Agency of Japan. No significant differences were observed between transformants and wild type plants for reproductive traits, although there were slight differences in plant shape, possibly due to somaclonal variation. No specific peak was observed in gas chromatographic or HPLC analyses of chemical compounds in leaves of the transformants and in areas surrounding the roots. No appreciable effects on soil microflora were exerted by growing the transgenic plants. No difference was observed in their ability to become weeds, either.

Safety Assessment in Isolated Fields After approval from the Ministry of Agriculture, Forestry and Fisheries, H39 and H75 were grown in an isolated field (Stage III). The experiments were conducted according to guidelines established by the Ministry. Although there were slight differences in the shape of a part of H39, there were no differences between the transgenic and wild type plants in outcrossing frequency, ability to become weeds, or influence on the environment. Results from these three stages of safety assessment indicate that this transgenic material is substantially equivalent to an ordinary rice cultivar developed through conventional breeding in all traits measured except the target trait, which is the reduction of glutelin in the grains. Open field experiments are being conducted at three locations for agronomic performance. Yield and grain quality will be further evaluated in detail. Food safety will be evaluated following guidelines established by the Ministry of Health and Welfare.

References 1. Chan MT, Chang HH, Ho SL et al. Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene. Plant Mol Biol 1993; 22:491-506. 2. Hiei Y, Ohta S, Komari T et al. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 1994; 6(2):271-282.

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3. Ishida Y, Saito H, Ohta S et al. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnol 1996; 14:745-750. 4. Tingay S, McElroy D, Kalla R et al. Agrobacterium tumefaciens-mediated barley transformation. Plant J 1997; 11:1369-1376. 5. Cheng M, Fry JE, Pang S et al. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 1997; 115:971-980. 6. Hiei Y, Komari T. High efficiency transformation of indica rice varieties mediated by Agrobacterium tumefaciens. Breed Sci 1997; 47(Suppl 2):99. 7. Komari T. Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulent region of pTiBo542. Plant Cell Rep 1990; 9:303-306. 8. Maruta Y, Ueki J, Saito H et al. Experience in the development of low-protein japonica rice. Proceedings of the 5th International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, 6-10 Sept. 1998, Braunschweig, Germany. In press. 9. Wakai Y. Relationship between brewing suitability and various traits of rice grains. 17th Seminar on Sake-Rice 1993:2-13. 10. Ishima T, Taira H, Taira H et al. Effect of nitrogenous fertilizer application and protein content in milled rice on organoleptic quality of cooked rice. Rep Natl Food Res Inst 1974; 29:9-15. 11. Yoshizawa K, Kishi S. Rice in brewing. In: Juliano BO, ed. Rice: Chemistry and Technology. St. Paul, Minnesota, USA, American Association of Cereal Chemists, Inc., 1985:619-645. 12. Kisaki K, Takeda N, Okazaki N et al. Purification of protein in sake-rice and digestability by Koji enzyme. Nippon Nogeikagaku Kaishi 1990(3); 64:123. 13. Yamashita K, Fujimoto T. Studies on fertilizers and quality of rice. IV. Relationship between protein content of milled rice as affected by nitrogen fertilization and eating quality. Bull Tohoku Natl Agric Exp Stn 1974; 48:91-96.

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14. Komari T, Hiei Y, Saito Y et al. Vectors carrying two separate T-DNAs for cotransformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 1996; 10(1):165-174.

SECTION VI INTRODUCTION

Environmental Adaptation and Generation of Resistant Plants H. Uchimiya

A

number of abnormal environment parameters such as drought, salinity, cold, freezing, high temperature, anoxia, high light intensity and nutrient imbalances etc. are collectively termed as abiotic stresses. Abiotic stresses lead to dehydration or osmotic stress through reduced availability of water for vital cellular functions and maintenance of turgor pressure. Plants have evolved mechanisms to respond to various abiotic stresses at morphological, anatomical, cellular and molecular levels. Some responses to tolerance or adjustment are highly species-specific, whereas others are fairly common even among plants belonging to different families and orders, microorganisms and animals. In response to dehydration or osmotic stress, a series of compatible osmolytes are accumulated for osmotic adjustment, water retention and free radical scavenging. Attempts have been made

to understand the molecular basis of tolerance to certain abiotic stresses. Enzymes responsible for the production of compatible osmolytes have been cloned and characterized and used for genetic transformation of stress susceptible genotypes or mutants to confirm their unequivocal role in stress protection and relief. In the absence of well documented and reliable sources of genetic resistance to abiotic stresses in the germplasm of crop plants and their related wild species for sound breeding programs using conventional approaches, improvement of tolerance to the abiotic stresses through genetic engineering may be the second best available alternative. These developments have also resulted in precise understanding of the genome organization and regulation of gene expression in higher plants. Notable innovation along this line of research is the subject of this session.

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine ©2000 Eurekah.com. .

CHAPTER 19

Stress Tolerance in Crops—How Many and Which Genes? H.J. Bohnert, H.-X. Li and B. Shen

Introduction

F

resh water is unequally distributed over the planet and must supply the needs not only of agriculture but also of an increasing human population. This poses problems in areas where water is a precious commodity, most significantly in Australia, countries of the Mid-East, north Africa, the west and mid-west of the United States, or parts of the Indian subcontinent and central Asia. Even in areas with typically ample precipitation, transient drought can lead to economic hardship for farmers or inconvenience urban populations. At the same time, predictions of global climatic changes seem to indicate that, for the foreseeable future, precipitation and the distribution of rain could become more erratic than in the past. Lack of water prolongs the agricultural growing cycle, reduces yield and results in diminished value. Also, agricultural practice jeopardizes productivity in many irrigated, highly productive areas, because long term irrigation leads to the buildup of sodium and other salts in the soil. In fact, up to half of the irrigated land may be in jeopardy.1 The threat of excessive salinization is real and avoided only by careful management of the soil. What has happened in many growing areas with elaborate irrigation schemes is reminiscent of events in the past that led to the decline of ancient civilizations. How will we provide a stable supply of food, feed and fiber for human population which will reach maybe 9 billion people within the next two generations? There are several reasons for concern. First, breeding programs for many established

crop species approach, or may have already reached, saturation level with respect to increased productivity under normal growth conditions. Also, breeding programs in general have not generated significantly drought tolerant, high yield crops, and practically none that are salinity stress resistant. Breeding specifically for environmental stress tolerance has been severely hindered by the fact that the tolerance or resistance traits are multigenic and quantitative. In addition, these traits conflict with another similarly multigenic trait, productivity, which must be the ultimate objective of all breeding programs. 1,2 Remedies can surely result from better land management and improved harvesting, storage, transportation and distribution systems, as well as from incorporation of new concepts in plant stress tolerance breeding. These rely on the application of existing technologies, resource management and capital expenditure. We advocate another direction. We suggest using genetic knowledge and technology to embark on large scale plant metabolic and developmental engineering for stress tolerance. We maintain that improvements through engineering of the sensing and signaling of stress and engineering of metabolism in crop plants is not only conceivable, but that such engineering is now possible. The immediate goal, however, is not to generate crops that can be grown in the sea or in true deserts, which lack rain completely, but rather to improve plant performance under moderate

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stress situations. Protection of crops in traditional growing areas, many of which experience stress episodes, should be the goal. Much would be achieved if salinity sensitive (~1-3 ppt of sodium) crops could be productive at 8 ppt (equivalent to ~100 mM Na+ or approximately 20% seawater), or if crops that cease to grow after one day of drought would continue to grow for a week without irrigation. Engineering such changes requires, first, more knowledge about basic plant metabolism and development. We need to know which and how many genes are important for water stress tolerance caused by drought, salinity and temperature, where and when they need to be expressed in the plant, and how genetic engineering schemes might integrate these genes into the endogenous metabolism of the chosen crop. What constitutes tolerance or resistance to environmental stresses has many facets (Table 19.1). For continued vegetative growth and development of reproductive organs under stress, plants must obtain water for photosynthesis. Each one of the many diverse mechanisms, which evolved in an order-, family- or species-specific fashion, must be subordinate to this essential goal. We will review molecular mechanisms for which the evidence seems clear: 1. Scavenging of radical oxygen species;3-9 2. Controlled ion and water uptake;10-12 3. Management of accumulating reducing power (see below) and adjustments in carbon/nitrogen allocation.13-14 The progress brought by molecular genetic analysis is ultimately based on past physiological observations and biophysical and biochemical principles that have been outlined by generations of researchers preceding the advent of molecular biology. The conclusions we can make now go beyond correlation, because a few principles have emerged over the last ten years through genetic and molecular genetic analyses.15-16 Now is the time to test these principles and integrate the results from physiological studies for the genetic engineering of crop species.

Osmotic Adjustment Most organisms increase the cellular concentration of osmotically active compounds, termed compatible solutes (Table 19.1), when in danger of becoming desiccated by either drought conditions or external lowering of the osmotic potential.17-20 The accumulating compounds are “compatible” with normal cellular metabolism at high concentrations.21 Often these are hydrophilic, which gave rise to the view that they could replace water at the surface of proteins, protein complexes, or membranes. The biochemical mechanisms through which compatible solutes protect are still unknown, but this does not necessarily preclude working on application strategies. Our focus has been on physiological mechanisms of compatible solute action. Also, we wish to understand how compatible solutes are integrated into a whole plant stress response that includes maintenance of ion homeostasis and water relations, C/N partitioning, reserve allocation and management of reducing power.22-25 There may be more than one function for a particular solute (see Shen6-7) and, based on results from in vitro experiments,26-28 different compatible solutes may have different functions. The importance of solute accumulation, interpreted as “osmotic adjustment”, had been recognized long ago (e.g., refs. 21, 29-30). A correlation between solute amount and tolerance has been documented. Plant transformation had to be developed before experiments could be designed to replace correlative relationships by proofs. The logical next step has been the engineering of plants to express enzymes that lead to the synthesis of such solutes and subsequent physiological analysis of these plants. Different compounds can function as compatible solutes. Potassium, if available, serves this function. Also, amino acids and some amino acid derivatives, sugars, acyclic and cyclic polyols, fructans, and quaternary amino and sulfonium compounds frequently have been identified.19-20,30-32 Recently, genes have been characterized leading to ectoine (1,4,5,6-tetrahydro-2-methyl-4pyrimidinecarboxylic acid), a zwitterionic metabolite found in a number of halobacteria which

Stress Tolerance in Crops—How Many and Which Genes?

209

Table 19.1. Breeding objectives and mechanisms for enhanced stress tolerance Plant breeding term

Physiological term

Mechanistic term

Suggested gene complex

Vigor

Growth

Meristem activity

Cell cycle, cyclins

Photorespiration

ROS scavenging Stomatal

conductance One-carbon metabolism Signal transduction

Protein kinases/ Phosphatases

Osmotic adjustment

Osmolyte accumulation

Internal osmotic pressure decrease Water transport

Polyol,proline Gly-bet synthesis Water channels Metabolite facilitators

Osmoprotection

Osmolyte accumulation

Radical oxygen scavenging

SOD, ASX, Cat, Asc/GS cycle

Protective proteins

Protein solvation Membrane integrity Enzyme complex stabilization

LEA/ dehydrins Lipid saturation

Sodium exclusion Sodium partitioning

Proton pumping

HKT, Cation activitychannels P-ATPase; V-ATPase PPiase;

Ion homeostasis

shows exceptional protection of protein function in in vitro assays.33-34 Typically, pathways leading to osmolyte synthesis are connected to pathways in general metabolism with high flux rates.32 Examples are the proline biosynthetic pathway,20 glycinebetaine synthesis,19 and the pathway leading to the methylated inositol, D-pinitol. 2,32,35-38 We present a description of pinitol biosynthesis, which highlights essential features that seem to characterize what is required of a compatible solute. Biosynthesis of D-pinitol in the halophyte Mesembryanthemum crystallinum L. (common ice plant) requires an increased flux of carbon from glucose 6-phosphate to myo-inositol

Vacuolar sink size Na/H-antiport

1-phosphate and then myo-inositol.36 The first gene in the pathway, encoding inositol-1P synthase, is transcriptionally upregulated, and increased protein amounts can be detected.36-37 The second enzyme, inositol monopos phatase, is not regulated under stress conditions in the ice plant (D. E. Nelson, personal communication). Utilizing increased amounts of inositol following stress, the enzyme myo-inositol O-methyltransferase (IMT) generates D-ononitol.35 In the ice plant, IMT is only expressed following salt stress, i.e., the protein is virtually absent in unstressed plants and increases dramatically within one to two days of stress.35,37 Finally, D-ononitol is converted into D-pinitol by an epimerization reaction which

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

may include more than one enzyme. This activity, which we term OEP (ononitol epimerase), has not yet been characterized biochemically or genetically. There are two signature features of this pathway. First, the pathway is connected to inositol synthesis and phospholipid biosynthesis, pathways which are tightly controlled in organisms in which they have been studied,39 and beyond that the synthesis of the methylated inositols is connected to the major flux of carbon in photosynthetic cells. The second feature is that the pathway includes additional enzymes which remove the product from general metabolism. D-pinitol is an extremely stable end product.40 The activities of the IMT and OEP enzymes have not been detected in tobacco and Arabidopsis.36,41-42 In fact, genes for these enzymes may be missing in these species.

Functions of Compatible Solutes Compatible solutes are non-toxic to protein structure and function, and alleviate inhibitory effects of high ion concentrations on enzyme activity. Some, such as trehalose, do not respond to osmotic stress by accumulating but are protective even at low concentrations.43-44 The majority of the compatible solutes, however, also seem to have osmoregulatory functions and accumulate in response to osmotic stress. They may function as osmolytes as well as osmoprotectants. The net increase of solutes lowers the osmotic potential of the cell, which supports the maintenance of water balance under osmotic stress. The main function of compatible solutes may be stabilization of proteins, protein complexes or membranes under environmental stress. In in vitro experiments, solutes at high concentrations have been found to reduce the inhibitory effects of ions on enzyme activity.17,45-47 Solute addition increased thermal stability of enzymes,33,48-49 and prevented dissociation of the oxygen-evolving complex of photosystem II. 50 One argument often raised against these studies is that the effective concentration necessary for protection in vitro is very high, approximately 500 mM. Such high concentrations are rarely found in vivo. However, when we consider the high concentration of proteins in cells, the concentration necessary

for protection can, we think, be much lower than that required for protection in in vitro assays. In addition, it may not be the solute concentration in solution that is important. Glycinebetaine (which may be present in high or low amounts), for example, protects thylakoid membranes and plasma membranes against freezing damage or heat destabilization,51-53 indicating that the local concentration on membranes or protein surfaces may be more important than the absolute concentration. Two theoretical models have been proposed to explain protective or stabilizing effects of these solutes on protein structure and function. The first is termed the “preferential exclusion model” 54 in which compatible solutes are largely excluded from the hydration shell of proteins that stabilizes protein structure or promotes or maintains protein/ protein interactions. Compatible solutes in this model would not disturb the native hydration water of proteins, but they would interact with the bulk water phase in the cytosol. The second model, the “preferential interaction model”, in contrast, emphasizes interactions between solute and proteins.55 The protein’s hydration shell is crucial for structural stability. During water deficit, these solutes may interact directly with hydrophobic domains of proteins and prevent their destabilization, or they may substitute for water molecules in the vicinity of such regions. While the two models seem to be mutually exclusive at first sight, the actual function of compatible solutes may in fact be explained by both models. The structures of different compatible solutes could accomodate both hydrophobic, van de Waals interactions and charged interactions, but further experiments will be necessary to gain a better insight into the stabilizing effects of compatible solutes that have been documented in in vitro experiments.

Radical Oxygen Species are Unavoidable and Increase During Stress Episodes Compatible solutes may also function as oxygen radical scavengers. Evidence for such a function comes from studies on fungal pathogen interactions where the pathogens are protected by the synthesis and secretion of mannitol. Plants and animals produce oxygen

Stress Tolerance in Crops—How Many and Which Genes? radicals in response to pathogen attack. The rapid production and local accumulation of reactive oxygen species leads to localized cell death in the host, which then may limit the spread of the pathogen.56 In response, some pathogens seem to have evolved mechanisms which detoxify the reactive oxygen species produced by the host. Cryptococcus neoformans, a yeast which opportunistically infects humans with a compromised immune system, produces and secretes mannitol. A mutant strain that does not produce mannitol is less virulent.57 Similarly, during the infection process the tomato pathogen Cladosporium fulvum produces mannitol, which seems to protect the fungi from damage by reactive oxygen species produced by the plants.58 Mannitol has been shown in vitro to function as a scavenger of reactive oxygen species, ROS.59-60 ROS is a generic term which is used to include not only free radicals such as superoxide and hydroxyl radicals, but also singlet oxygen and H 2 O 2 . Smirnoff and Cumbes26 designed experiments that compared the radical scavenging capabilities of different compatible solutes. They reported that mannitol, sorbitol, glycerol, proline, ononitol and pinitol were active scavengers at different concentrations in vitro, while glycinebetaine was not.26,28 The relative radical scavenging efficiency of these compounds seemed dependent on their rate constants for reactions with hydroxyl radicals. For example, the rate constant of mannitol is four-fold higher than that of proline,61 and thus it was more effective than proline as a hydroxyl radical scavenger. Under water deficit conditions, radical production increases in plants,62 and it may be that the accumulation of polyols provides some protective effect against oxidative damage of proteins. Recently, additional experiments shed light on the radical scavenging capacity of mannitol in in vivo experiments.6-7 Mannitol 1-phosphate dehydrogenase was modified such that the protein was imported into chloroplasts, and the gene construct was expressed in transgenic tobacco. We argued that a potential function in radical scavenging in vivo might best be demonstrated with chloroplasts which abundantly produce a variety of ROS when stressed by water deficit (for a

211

recent review, see Noctor and Foyer 63 ). Mannitol was present in concentrations of approximately 100 mM in the chloroplasts.6 Using different conditions, such as illumination with high light, paraquat treatment, enhanced H2O2 generation and DMSO infiltration, it could be shown that plants containing mannitol in their chloroplasts were better able to maintain high carbon fixation rates and showed less chlorophyll bleaching.6 Further experiments indicated that mannitol was active specifically against hydroxyl radicals and not against hydrogen peroxide or radical oxygen. This is important information, considering that chloroplast detoxification systems exist that can deal with H2O2 and radical oxygen, while there is no enzyme system described that could deal with the extremely short lived and highly reactive hydroxyl radicals. Further experiments7 indicated that even under high light conditions the major effect of increased hydroxyl radical production was on the dark reactions of photosynthesis, while the photosystems themselves functioned normally. It could be demonstrated that some enzymes of the Calvin cycle were predominantly affected by hydroxyl radicals. Phosphoribulokinase, PRK, and likely other SH enzymes of the Calvin cycle, showed sensitivity to hydroxyl radicals, and the activity of PRK was protected by the presence of mannitol.7 It appears that mannitol in the chloroplast interferes with the so-called Fenton reaction through which Fe2+ and H2O2 react, producing hydroxyl radicals and Fe3+.6-7 Iron increases have been found to induce the expression of ascorbate peroxidase genes, possibly also for the purpose of prevention the Fenton reaction.64 It would be overextending the compatible solute concept if we assigned a main function in hydroxyl radical scavenging to these solutes. Plants, especially in their chloroplasts and mitochondria, are endowed with an efficient non-enzymatic (α-tocopherol, carotenoids, flavonoids, etc.) and enzymatic (e.g., SOD, catalase, ascorbate/glutathione cycle enzymes) array of armaments to counteract radical species. The unexpected effect and likely function of mannitol, however, highlight the general importance of radical scavenging mechanisms in stress tolerance, especially in

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

plants for which the generation of excited states of chromophores and electron transport is essential to photosynthesis. Several groups have now manipulated resident scavenging enzyme systems for reactive oxygen species (Table 19.2). Protective effects have been observed, for example, by altering catalase, SOD, or the glutathione cycle enzymes.5,65-67 How far engineering of these pathways can go is unclear, because it must be understood that such manipulations might interfere with signaling processes which include the production and recognition of reactive oxygen species (see Bohnert and Sheveleva).68

Manipulation of Reducing Power In this previous discussion of pathways to stress tolerance we have chiefly placed emphasis on the type of protein or product that is induced. In the following, we discuss experiments indicating that the process and not so much the product may equally be important for protection. This experiment tested the long held concept that the function of accumulating metabolites is mainly or exclusively osmotic. The concept was tested in yeast, in which salinity stress leads to a dramatic increase of the simplest acyclic polyol, glycerol, to over 500 mM in the cells. Deleting the two genes encoding glycerol-1phosphate dehydrogenase renders the cells extremely salt sensitive. 69-70 We used the deletion strain lacking these two genes and incorporated genes leading to sorbitol or mannitol synthesis into the line. With sorbitol -6P DH, we achieved polyol amounts (~400 mM) nearly as high as glycerol amounts (~450 mM) in wild type. However, the sorbitol producing yeast cells were not as resistant to salt stress as the wild type, and only marginally more tolerant than the deletion line (Shen et al, in preparation). We do not know why this is the case, but we offer one explanation. The intracellular concentration of glycerol in yeast increases from approximately 25 mM to 450 mM during stress, but much more glycerol is excreted into the medium than is retained by the cells. We estimated that approximately 95% of the glycerol synthesized is being excreted during the experiment. In contrast, the sorbitol-producing cells retained most of

the sorbitol. Approximately only 20% of the sorbitol was excreted or lost to the medium. If this discrepancy is expressed in terms of reducing equivalents, yeast expends about 15 times more energy on synthesizing the glycerol that is in the medium than on sorbitol which mostly stays in the cells.71 As a hypothesis, we suggest that oxidation of NADPH—which would allow respiration to continue—may be more important than generating high amounts of osmolytes inside the cell. Possibly, the term “osmotic adjustment” does not describe the functions of osmolytes adequately.

Controlled Ion and Water Uptake How sodium is taken up during salt stress is not clear. Possibly, monovalent cation channels and transporters serving potassium nutrition could be entry routes for sodium as well (Table 19.1). However, channels of the AKT/KAT type are typically highly selective.72 In contrast, significant influx of sodium through a potassium transporter, HKT in wheat, has been reported.73-74 The HKT transporter from wheat has been described as a K+/ Na+ co-transporter, which mainly transports sodium when the Na + /K + ratio is high. Preliminary data suggest that the homolog of HKT in rice is predominantly a sodium transporter,75 but it is still unclear whether sodium uptake in planta is accomplished by the HKT type system. Equally unresolved is the cell type in which HKT is present. Based on in situ hybridizations, HKT transcripts have been reported in cortical cells of the root in wheat,73 and expressed in cells of the epidermis and endodermis in rice.75 Definite information will have to wait until we have a better understanding about channel distribution in different cells of the tissues where those transport systems are expressed, as well as detailed analyses of their specificity and contribution to overall potassium and sodium transport. As we approach the time when all genes or putative coding regions are known in several eukaryotes, the necessity for additional information becomes paramount. To completely describe function, detailed knowledge will be essential about cellular localization; tissue, developmental and environmental specificity; and bio-

Enzyme Mn-SOD

Mannitol 1-P DH

HVA1-LEA

myo-inositol O-methyltransferase levansucrase

trehalose synthase

choline oxidase

Gene

MnSOD (N. plumbaginifolia)

MtlD (E. coli)

Hva1 (H. vulgare)

Imt1 (ice plant)

SacB (B. subtilis)

Tps1 (S. cerevisiae)

CodA (A. globiformis) A. thaliana

N. tabacum

N. tabacum

N. tabacum

O. sativa

Glycinebetaine accumulation; enhanced tolerance

Low conc. trehalose; increased drought tolerance

Fructan accumulation; higher growth during drought stress

Stress-induced accumulation of D-ononitol

Maintenance of higher growth rate by stressed plants

Sodium tolerance at early growth Enhanced germination in NaCl Chloroplast location, ROS scavenging; Calvin-cycle protected

Organelle targeted expression for reduced damage by ROS

N. tabacum M. sativa N. tabacum A. thaliana N. tabacum

Notes

Host Species

Table 19.2. Transferred genes affecting plant stress tolerance

105

44

104

42

89

101,102 103 6,7

98 99,100

Reference

Stress Tolerance in Crops—How Many and Which Genes? 213

P5CS

Fe-SOD

GST/GPX

P5CS (V. aconitifolia)

FeSOD (A. thaliana)

Gst/Gpx (N. tabacum) N. tabacum

N. tabacum

N. tabacum

Host Species

Increase of oxidized glutathione (GSSG); enhanced seedling growth

PSII/plasma membrane protection/methyl viologen

Proline accumulation leading to lowering of osmotic potential

Notes

5

3

106

Reference

While the effects of overexpression indicate protection, the mechanisms leading to enhanced tolerance under controlled growth conditions are largely not understood. High accumulation of mannitol,107 or sorbitol108 in transgenic tobacco lines reduces growth.

Enzyme

Gene

Table 19.2., (cont'd) Transferred genes affecting plant stress tolerance

214 Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Stress Tolerance in Crops—How Many and Which Genes? physical and biochemical characteristics of the proteins. Water channels, aquaporins (AQP), are found in all organisms as members of a superfamily of membrane proteins, 26-30 kDa in size, termed MIP (major intrinsic protein). 76-77 The presence of aquaporins increases membrane permeability to water in both directions, depending on osmotic pressure differences across the membrane. Some family members encode glycerol facilitators. Other MIPs in microorganisms, animals and plants may mediate ion transport and/or the movement of small non-electrolytes, such as urea.77-78 In vertebrate animals it seems that at most 10 MIP genes are present. They are expressed in different tissues, most highly in erythrocytes, kidney cells and the brain. In contrast, Arabidopsis contains at least 23 MIP-like coding regions,79 and in corn the number of sequences homologous to Mip transcripts is at least 31.80 Sequence signatures of the Arabidopsis MIP indicate two large subfamilies of 10 to 12 proteins each, whose members are either plasma membrane-located (PIP) or tonoplast-located (TIP) and one MIP diverging from the others which has not been characterized in detail.79 While some of the genes might encode facilitators for diverse small metabolites or ions, eight MIP proteins have already been identified as aquaporins. Why are there so many plant aquaporins? There are several possibilities which might explain the high number. We will discuss three possibilities only briefly and the fourth in more detail. First, MIP-intrinsic functional variations might allow for AQP to be active at different membrane osmotic potentials. Second, func- tional differences could have evolved for fine tuning water flux through the plant—with high conductance AQP located in the root cortex and vascular tissues which accommodate bulk fluxes and low conductance channels between mesophyll cells, for example, or even within the cytosol and organelles and the vacuole. Third, cell-specific differences in accommodating water flux, not water transport per se, would determine gene number— requiring different promoters, alterations in RNA stability and translation and protein half life regulation.

215

This explanation is similar to the following, and both find precedence in the presence of, for example, a large number of genes encoding plasma membrane H+-ATPases, AHA, which are differentially expressed throughout the plant.81-82 Last, MIP duplication and diversification could have been dictated by the need for a flexible response to environmental changes in water availability, demanding the presence of several sets of AQP. This assumes evolution of one set of AQP genes for stress responses, and that this set is different from others. It is conceivable that Mip genes exist (set 1) that take care of cell expansion following meristematic activity—and this function (missing from animals) might require regulatory circuits separate from those necessary in genes that perform housekeeping (set 2, also found in animals) and stress-response function (set 3). Alignments of sequences indicate that within the PIP and TIP classes, subfamilies of two to four closely related sequences exist, 78-79,83 which might represent the three sets of genes. These subfamilies can be inferred because of sequence clustering within the alignment tree that unites sequences from plant orders and genera widely separated during evolution. Although we do not have enough data with respect to AQP protein expression, cell specificity and regulation during stress, it may eventually become possible to predict function by position in an evolutionary tree. Several MIP associated with cell expansion, developmental specificity and stress functions have been described. One example from our own work is presented in Figure 19.1. The promoter for the ice plant plasma membrane aquaporin MIPB has previously been expressed in tobacco. Its activity has been monitored by GUS expression.84 The MIPB promoter was also inserted into Arabidopsis to control the expression of luciferase. In homozygous lines, luciferase expression has been monitored in response to various stresses and chemical agents that interfere with signal transduction. In Arabidopsis, this ice plant promoter is induced only by drought conditions and by elevated calcium.85 Sodium stress and osmotic stress by mannitol had no effect. Figure 19.1 compares the luciferase signal under normal growth conditions (left panel) in Arabidopsis seedlings with

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

the signal after a 20 minute drought period (after the seedlings had been removed from the agar plate). Within the 20 minutes, signal strength increased more than 10-fold. Such experiments provide useful information about stress responses and shed light on signaling cascades. In addition, the heterologous promoter could become useful as an expression control element in transgenic engineering projects. Most important for our discussion is how MIP gene expression, protein amount and aquaporin activity are controlled during development and under environmental stress. Regulation is by gene expression and protein amount, and possibly also by posttranslational modification; but as yet we have little information on mechanistic details in plants. Weig et al79 used quantitative PCR amplification for the 23 Arabidopsis MIP and found differences in mRNA amounts spanning several orders of magnitude. Differences in RNA amounts for each MIP in roots, leaves, bolts, flowers and siliques were equally pronounced. No signals were detected for at least three MIP, suggesting that these might be expressed under conditions not found during normal growth or that they are expressed in only a few cells or at very low levels. For several MIP in the ice plant, stress-altered mRNA amounts have been observed (see Maurel77 for a review).83,86 AQP expression also responds to drought and low temperature, hormone treatment (ABA, cytokinine, GA), light and pathogen infection.77-78,87-88 Once all genes are known, the analysis of such a large gene family can best be done by in situ hybridization, immunocytology with specific antibodies and analysis in ordered DNA microarrays. Employing these techniques together, the amount, location and regulation of the genes during development and under different environmental conditions can be known. The discovery and preliminary characterization of AQP in plants has provided more questions than answers. Their existence cannot be questioned, and many MIP are water channels. It is then intuitively obvious that control over their action is important under stress conditions. Although there are few data available, it is equally clear that regulation during stress is complex, involving transcrip-

tional and posttranscriptional controls which seem to involve synthesis, membrane traffic and reversible insertion into membranes, complex assembly and MIP protein half life.

How Many and Which Genes for Stress Tolerance? Experimentation must continue that elucidates mechanisms by which plants achieve stress tolerance. Especially instructive is the comparison of mechanisms gleaned from model systems, yeast and the ice plant, for example, with stress responses present in stress sensitive plants. Additionally, information from various genome analysis projects, genomic sequences and sequences of expressed sequence tags (ESTs) for stress responsive genes will have to be collected and integrated. The cell-, tissue-, and developmental stage-specific expression of stress relevant transcripts and proteins needs to be analyzed in more detail. Finally, technological breakthroughs are imperative regarding targeted gene elimination and gene replacement as well as techniques for the insertion of multiple genes into chromosomes, either through further development of the established vector and gene delivery systems, or by novel techniques such as the construction of artificial, additional chromosomes. Metabolic engineering projects with a focus on stress tolerance have resulted in partially stress protected transgenic models, mainly tobacco and Arabidopsis (Table 19.2, summarized in Nelson et al12 and Bohnert and Sheveleva68). Some dicot crop species have been transformed with the objective of learning about stress tolerance, but very few transgenic analyses have targeted monot crops such as rice or corn. For example, rice has been transformed to achieve the overexpression of an LEA (late embryogenesis abundant) protein. 89 The transgenic plants indeed showed higher osmotic stress tolerance. One must view all generated lines as tester lines; to our knowledge none of these lines has been sufficiently tested under field conditions. In fact, we think field testing would be premature for two reasons. The single gene transfers carried out in the past are unlikely to be sufficient for effective stress protection, and the transgenes are constitutively expressed. Salt tolerant yeast, the ice plant and celery (which

Stress Tolerance in Crops—How Many and Which Genes?

217

Fig. 19.1. Activity of the ice plant MipB promoter in transgenic Arabidopsis. A promoter-luciferase fusion gene was introduced into Arabidopsis thaliana (Columbia) and homozygous lines were selected. Individual seedlings were germinated on agar, sprayed with luciferase substrate and photographed. The activity of the promoter under optimal growth conditions (panel A) is compared to the activity after seedlings experienced 20 minutes of drought (panel B), i.e., after the seedlings had been placed on the laboratory bench for 20 minutes. Quantitation of the signal indicated that luciferease activity increased by 10 to 12-fold under drought conditions. A less pronounced increase, by 3-fold, was obtained when seedlings were incubated with 10 mM calcium chloride. Sodium chloride (150 mM), ABA (100 µM) and mannitol (300 mM) had no effect.

naturally accumulates mannitol), for example, induce responses following stress. Stress defense mechanisms are not constitutively present. Also, it seems unlikely that single gene transfers, which have been carried out in the past, will be sufficient. The brief discussion provided earlier seems to indicate that a large number of genes might have to be transferred to achieve osmotic stress tolerance for the protection of crop plants. Our discussion has concentrated on the biochemical and physiological mechanisms that constitute tolerance to stress. Alternatively, it is possible to focus on stress perception and signal transduction pathways, leading to the induction or enhancement of stress tolerance proteins or pathways that are present in all plants.16,90-93 Mutants of Arabidopsis which reveal stress related signal transduction chains have recently become available,91,94 and their biochemical dissection is under way.95-97 It is therefore conceivable that within a very short time we will have genes available that are high in the hierarchy of regulators. Enhancement of a few genes might then be sufficient for

the induction of resident stress tolerance mechanisms. The best strategy may be a combination of genes to strengthen both stress signal transduction and to add new downstream biochemical protective pathways. One argument favoring proceeding in this way is that crop plants do not possess a complete repertoire of biochemical mechanisms; it seems that some pathways and their genes are missing. Also, we argue that there may have been inadvertent, breeding-associated selection against the induction of stress response mechanisms—or example, by selection for ABA insensitivity which would tend to lead to stomatal opening even under low water conditions. Signal transduction chains could have become inoperative and would not induce stress responses that might have been present before domestication. The combination of genes would first contain genes that enhance the plant equivalent genes of the yeast HOG and calcineurin-related signal transduction pathways (see Nelson et al12). Genes for pathways that enhance the scavenging of radical

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

oxygen species, osmotic adjustment, and controlled uptake of water and ions should be included in any transgenic stress engineering strategy. It is difficult to gauge the usefulness of the LEA genes, which certainly have resulted in increased tolerance in a few model experiments, because there is no mechanistic understanding of their action. Thus, the next generation of transgenic tester lines might include on the order of 20 genes, the transfer of which can be accommodated even by present technologies.

Acknowledgments Our work is or has been supported by grants from the US Department of Energy, the National Science Foundation, the US Department of Agriculture, NEDO Japan, private industry, and the Arizona Agricultural Experiment Station.

References 1. Flowers TJ, Yeo AR. Breeding for salinity tolerance in crop plants: Where next? Aust J Plant Physiol 1995; 22:875-884. 2. Bohnert HJ, Jensen RG. Metabolic engineering for increased salt tolerance—The next step. Aust J Plant Physiol 1996; 23:661-667. 3. Van Camp W, Capiau K, van Montagu M et al. Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe- superoxide dismutase in chloroplasts. Plant Physiol 1996; 112:1703-1714. 4. Palatnik JF, Valle EM, Carrillo N. Oxidative stress causes ferredoxin-NADP + reductase solubilization from the thylakoid membranes in methyl viologentreated plants. Plant Physiol 1997; 115:1721-1727. 5. Roxas VP, Smith RK Jr, Allen ER et al. Overexpression of glutathione S-transferase/ glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress. Nature Biotech 1997; 15:988-991. 6. Shen B, Jensen RG, Bohnert HJ. Increased resistance to oxidative stress in transgenic plants by targeting mannitol biosynthesis to chloroplasts. Plant Physiol 1997a; 113:1177-1183.

7. Shen B, Jensen RG, Bohnert HJ. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiol 1997b; 115:527-532. 8. Downs CA, Heckathorn SA. The mitochondrial small heat-shock protein protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in plants. FEBS Lett 1998; 430:246-50. 9. Willekens H, Chamnongpol S, Davey M, et al. Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. EMBO J 1997; 16:4806-4816. 10. Schwartz A, Wu WH, Tucker EB et al. Inhibition of inward K+ channels and stomatal response by abscisic acid: An intracellular locus of phytochrome action. Proc Natl Acad Sci USA 1994; 91:4019-4023. 11. Ding L, Zhu JK. Reduced Na+ uptake in the NaCl- hypersensitive sos1 mutant of Arabidopsis thaliana. Plant Physiol 1997; 113:795-799. 12. Nelson D, Shen B, Bohnert HB. Salinity tolerance—Mechanisms, models, and the metabolic engineering of complex traits. In: Genetic Engineering, Principles and Methods, Vol. 20. Setlow JK, ed. New York, NY: Plenum Press, 1998a:153-176. 13. Vincent R, Fraisier V, Chaillou S et al. Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants trigger changes in ammonium assimilation and plant development. Planta 1997; 201:424-433. 14. Jones TL, Tucker DE, Ort DR. Chilling delays circadian pattern of sucrose phosphate synthase and nitrate reductase activity in tomato. Plant Physiol 1998; 118:149-158. 15. Jain RK, Selvaraj G. Molecular genetic improvement of salt tolerance in plants. Biotech Annu Rev 1997; 3:245-267. 16. Shinozaki K, ed. Cold, Drought, Heat and Salt Stress: Molecular Responses in Higher Plants. Austin: RG Landes Co, 1998. 17. Yancey PH, Clark, ME, Hand SC et al. Living with water stress: Evolution of osmolyte system. Science 1982; 217: 1214-1222. 18. Le Rudulier D, Strom AR, Dandekar AM et al. Molecular biology of osmoregulation. Science 1984; 224:1064-1068. 19. McCue KF, Hanson AD. Drought and salt tolerance: Towards understanding

Stress Tolerance in Crops—How Many and Which Genes? and application. Biotechnol 1990; 8:358-362. 20. Delauney AJ, Verma DPS. Proline biosynthesis and osmoregulation in plants. Plant J 1993; 4:215-223. 21. Brown AD, Simpson JR. Water relations of sugar-tolerant yeasts: The role of intracellular polyols. J Gen Microbiol 1972; 72:589-591. 22. Bieleski RL. Sugar alcohols. In: Loewus FA, Tanner W, eds. Encyclopedia of Plant Physiology Vol 13A, Plant Carbohydrates I. Berlin: Springer Verlag, 1982:158-192. 23. Blomberg A, Adler L. Physiology of osmotolerance in fungi. Adv Microbiol Physiol 1992; 33:145-212. 24. Bohnert HJ, Nelson DE, Jensen RG. Adaptations to environmental stresses. Plant Cell 1995; 7:1099-1111. 25. Niu X, Bressan RA, Hasegawa PM et al. Ion homeostasis in NaCl stress environments. Plant Physiol 1995; 109:735-742. 26. Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes. Phytochem 1989; 28:1057-1060. 27. Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease: An overview. Meth Enzymol 1990; 186:1-85. 28. Orthen B, Popp M, Smirnoff N. Hydroxyl radical scavenging properties of cyclitols. Proc Royal Soc Edinburgh 1994; 102B:269-272. 29. Borowitzka LJ, Brown AD. The salt relations of marine and halophilic species of the unicellular green alga, Dunaliella. The role of glycerol as a compatible solute. Arch Mikrobiol 1974; 96:37-52. 30. Levitt J. Responses of Plant to Environmental Stress Chilling, Freezing, and High Temperature Stresses, 2nd ed. New York: Academic Press, 1980. 31. Bartels D, Nelson DE. Approaches to improve stress tolerance using molecular genetics. Cell Environ 1994; 17:659-667. 32. Bohnert HJ, Jensen RG. Strategies for engineering water-stress tolerance in plants. Trends Biotech 1996b; 14:89-97. 33. Galinski EA. Compatible solutes of halophilic eubacteria: Molecular principles, water-solute interaction, stress protection. Experientia 1993; 49:487-496. 34. Louis P, Galinski EA. Characterization of genes for the biosynthesis of the compatible solute ecoine from Marinococcus halophilus and osmoregulated expression

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in Escherichia coli. Microbiol 1997; 143: 1141-1149. 35. Vernon DM, Bohnert HJ. A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum. EMBO J 1992; 11:2077-2085. 36. Ishitani M, Majumder AL, Bornhouser A et al. Coordinate transcriptional induction of myo-inositol metabolism during envrinmental stress. Plant J 1996; 9:537-548. 37. Nelson DE, Rammesmayer G, Bohnert HJ. The regulation of cell-specific inositol metabolism and transport in plant salinity tolerance. Plant Cell 1998b; 10:753-764. 38. Nelson DE, Koukoumanos M, Bohnert HJ. Myo-inositol dependent sodium uptake in ice plant. Plant Physiol 1999; 119:165-172. 39. Nikoloff DM, Henry SA. Genetic analysis of yeast phospholipid biosynthesis. Annu Rev Genet 1991; 25:559-583. 40. Anderson AB. Pinitol from sugar pine stump wood. Indus Chem Engin 1953; 45:593-596. 41. Vernon DM, Tarczynski MC, Jensen RG et al. Cyclitol production in transgenic tobacco. Plant J 1993; 4:199-205. 42. Sheveleva E, Chmara W, Bohnert HJ et al. Increased salt and drought tolerance by D-ononitol production in transgenic Nicotiana tabacum L. Plant Physiol 1997; 115:1211-1219. 43. Mackenzie KF, Singh KK, Brown AD. Water stress hypersensitivity of yeast: Protective role of trehalose in Saccharomyces cerevisiae. J Gen Microbiol 1988; 134:1661-1666. 44. Holmström KO, Mäntylä E, Welin B et al. Drought tolerance in tobacco. Nature 1996; 379:683-684. 45. Polland A, Wyn Jones RG. Enzyme activities in concentrated solutes of glycine betaine and other solutes. Planta 1979; 144: 291-298. 46. Brown AD. Microbial Water Stress Physiology, Principles and Perspectives. New York: John Wiley & Sons, 1990. 47. Solomon A, Beer S, Waisel Y et al. Effects of NaCl on the carboxylating activity of Rubisco from Tamarix jordanis in the presence and absence of proline-related

220

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

compatible solutes. Plant Physiol 1994; 90:198-204. 48. Back JF, Oakenfull D, Smith MB. Increased thermal stability of proteins in the presence of sugars and polyols. Biochem 1979; 18:5191-5196. 49. Paleg LG, Douglas TJ, van Daal A et al. Proline, betaine and other organic solutes protect enzymes against heat inactivation. Aust J Plant Physiol 1981; 8:107-114. 50. Papageorgiou G, Murata N. The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving photosystem II complex. Photosyn Res 1995; 44:243-252. 51. Coughlan SJ, Heber U. The role of glycine betaine in the protection of spinach thylakoids against freezing stress. Planta 1982; 156:62-69. 52. Jolivet Y, Larher F, Hamelin J. Osmoregulation in halophytic higher plants: The protective effect of glycine betaine against the heat destabilization of membrane. Plant Sci Lett 1982; 25:193-201. 53. Zhao Y, Aspinall D, Paleg LG. Protection of membrane integrity in Medicago sativa L. by glycine betaine against effects of freezing. J Plant Physiol 1992; 140:541-543. 54. Arakawa T, Timasheff SN. The stabilization of proteins by osmolytes. Biophys J 1985; 47:411-414. 55. Schobert B. Is there an osmotic regulatory mechanism in algae and higher plants? J Theor Biol 1977; 68:17-26. 56. Tenhaken R, Levine A, Brisson LF et al. Function of the oxidative burst in hypersensitive disease resistance. Proc Natl Acad Sci USA 1995; 92:4158-4163. 57. Niehaus WG, Flynn T. Regulation of mannitol biosynthesis and degradation by Crytococcus neoformans. J Bacteriol 1994; 176:651-655. 58. Joosten MHAJ, Hendrickx LJM, de Wit PJGM. Carbohydrate composition of apoplastic fluids isolated from tomato leaves inoculated with virulent or avirulent races of Cladosorium fulvum. Neth J Plant Pathol 1990; 96:103-112. 59. Elstner EF. Metabolism of activated oxygen species. In Davies DD, ed. Biochemistry of Metabolism, Vol.11. San Diego: Academic Press, 1987:253-315.

60. Halliwell B, Grootveld M, Gutteridge JMC. Methods for the measurement of hydroxyl radicals in biochemical systems: Deoxyribose degradation and aromatic hydroxylation. Meth Biochem Analysis 1988; 33:59-90. 61. Buxton GV, Greenstock CL, Helman WP et al. Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J Phys Chem Ref Data 1988; 17:512-579. 62. Moran JF, Becana M, Iturbe-Ormaetxe I et al. Drought induces oxidative stress in pea plants. Planta 1994; 194:346-352. 63. Noctor G, Foyer CH. Ascorbate and glutathione: Keeping active oxygen under control. Annu Rev Plant Physiol Plant Molec Biol 1998; 49:249-280. 64. Vansuyt G, Lopez F, Inzé D et al. Iron triggers a rapid induction of ascorbate peroxidase gene expression in Brassica napus. FEBS Lett 1997; 410:195-200. 65. Foyer CH, Souriau N, Perret S et al. Overexpression of glutathione reductase not glutathione synthetase leads to increase in antioxidant capacity and resistance to photoinhibition in poplar trees. Plant Physiol 1995; 109:1047-1057. 66. Creissen G, Broadbent P, Stevens R et al. Manipulation of glutathione metabolism in transgenic plants. Biochem Soc Trans 1996; 24:465-469. 67. Brisson LF, Zelitch I, Havir EA. Manipulation of catalase levels produces altered photosynthesis in transgenic tobacco plants. Plant Physiol 1998; 116:259-269. 68. Bohnert HJ, Sheveleva E. Plant stress adaptations—making metabolism move. Curr Opin Plant Biol 1998; 1:267-274. 69. Albertyn N, Hohmann S, Thevelein JM et al. GPD1, which encodes glycerol-3- phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Molec Cell Biol 1994; 14:4135-4144. 70. Ansell R, Granath K, Hohmann S et al. The two isoenzymes for yeast NAD dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J 1997; 16:2179-2187.

Stress Tolerance in Crops—How Many and Which Genes? 71. Shen B, Hohmann S, Bohnert HJ. Roles of sugar alcohols in osmotic stress adaptation–replacement of glycerol in yeast by mannitol and sorbitol. Plant Physiol 1999; 121:45-52. 72. Maathuis FJM, Ichida AM, Sanders D et al. Roles of higher plants K+ channels. Plant Physiol 1997; 114:1141-1149. 73. Schachtman DP, Schroeder JI. Structure and transport mechanism of a highaffinity potassium uptake transporter from higher plants. Nature 1994; 370:655-658. 74. Rubio F, Gassman W, Schroeder JI. Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance. Science 1995; 2701660-1663. 75. Golldack D, Su H, Quigley F et al. Differential expression of HKT1-type K+ transporters in salt-sensitive and salttolerant rice lines. 1999; to be submitted. 76. Chrispeels AJ, Agre P. Aquaporins: Water channel proteins of plant and animal cells. Trends Biochem. Sci 1994; 19: 421-425. 77. Maurel C. Aquaporins and water permeability of plant membranes. Annu Rev Plant Physiol Plant Mol Biol 1997; 48:399-430. 78. Tyerman SD, Bohnert HJ, Maurel C et al. Plant aquaporins: Their molecular biology, biophysics and significance for plant water relations molecular. J Exp Botany 1999; in press. 79. Weig A, Deswarte C, Chrispeels MJ. The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group. Plant Physiol 1997; 114: 1347-1357. 80. Barrieu F, Jung R, Pioneer Hibred, personal communication. 81. Sussman MR. Molecular analysis of proteins in the plant plasma membrane. Annu Rev Plant Physiol Plant Mol Biol 1994; 45:211-234. 82. DeWitt ND, Hong B, Sussman MR et al. Targeting of two Arabidopsis H(+)-ATPase isoforms to the plasma membrane. Plant Physiol 1996; 112:833-844. 83. Yamada S, Katsuhara M, Kelly WB et al. A family of transcripts encoding water channel proteins: Tissue-specific expression in the common ice plant. Plant Cell 1995; 7:1129-1142.

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84. Yamada S, Nelson DE, Ley E et al. The expression of an aquaporin promoter from Mesembryanthemum crystallinum in tobacco. Plant Cell Physiol 1997; 38:1326-1332. 85. Li H-X. Water channel expression in transgenic plants. M.S. thesis 1999; University of Arizona. 86. Kirch HH, Golldack, Vera-Estrella R et al. Anti-MIP antibodies recognize distinct cell types in roots and leaves of the ice plant. Plant Physiol 1999; submitted. 87. Phillips AL, Huttly AK. Cloning of two gibberellin-regulated cDNAs from Arabidopsis thaliana by subtactive hybridization: expression of the tonoplast water channel, -TIP, is increased by GA3. Plant Mol Biol 1994; 24:603-615. 88. Kaldenhoff R, Kalling A, Richter G. Regulation of the Arabidopsis thaliana aquaporin gene AthH2 (PIP1b). J Photochem Photobiol B 1996; 36:351-354. 89. Xu D, Duan X, Wang B et al. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 1996; 110:249-257. 90. Serrano R. Salt tolerance in plants and microorganisms: Toxicity targets and defense responses. Int Rev Cytol. 1996; 165:1-52. 91. Ishitani M, Xiong L, Stevenson B et al. Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: Interactions and convergence of abscisic acid-dependent and -independent pathways. Plant Cell 1997; 9:1935-1949. 92. Zhu J-K, Hasegawa PM, Bressan RA. Molecular aspects of osmotic stress in plants. Crit Rev Plant Sci 1997; 16:253-277. 93. Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-stress response. Plant Physiol 1997; 115:327-334. 94. Liu J, Zhu J-K. A calcium sensor homolog required for plant salt tolerance. Science 1998; 280:1942-1945. 95. Hirayama T, Ohto C, Mizoguchi T et al. A gene encoding a phosphatidylinositolspecific phospholipase C is induced by dehydration and slat stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 1995; 92:3903-3907.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

96. Sheen J. Ca2+-dependent protein kinases and stress signal transduction in plants. Science 1996; 274:1900-1902. 97. Urao T, Yakubov B, Yamaguchi-Shinozaki K et al. Stress-responsive expression of genes for two-component response regulator- like proteins from Arabidopsis thaliana. FEBS Lett 1998; 427:175-178. 98. Bowler C, Slooten L, Vandenbranden S, et al. Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J 1991; 10:1723-1732. 99. McKersie BD, Chen Y, de Beus M, et al. Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiol 1993; 103:1155-1163. 100. McKersie BD, Bowley SR, Harjanto E, et al. Water-deficit tolerance and field performance of transgenic alfalfa over expressing superoxide dismutase. Plant Physiol 1996; 111:1177-1181. 101. Tarczynski MC, Jensen RG, Bohnert HJ. Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol. Proc Natl Acad Sci USA 1992; 89: 2600-2604. 102. Tarczynski MC, Jensen RG, Bohnert HJ. Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 1993; 259:508-510.

103. Thomas JC, Sepahi M, Arendall B et al. Plant Cell Environ 1995; 18:801-806. 104. Pilon-Smits EAH, Ebskamp MJM, Paul MJ et al. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol 1995; 107:125-130 105. Hayashi H, Alia, Mustardy L et al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase: Accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J 1997; 12:133-142. 106. Kishor PBK, Hong Z, Miao G, et al. Over-expression of pyrroline-5-carboxylase synthase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 1995; 108:1387-1394. 107. Karakas B, Ozias-Akins P, Stushnoff C et al. Salinity and drought tolerance of mannitol-accumulating transgenic tobacco. Plant Cell Environ 1997; 20:609-616. 108. Sheveleva E, Marquez S, Bohnert HJ et al. Sorbitol-6-phosphate dehydrogenase expression in transgenic tobacco: High amounts of sorbitol lead to necrotic lesions. Plant Physiol 1998; 117:831-839.

CHAPTER 20

Improving Drought, Salt and Freezing Stress Tolerance in Transgenic Plants K. Yamaguchi-Shinozaki, M. Kasuga, Q. Liu, Y. Sakuma, H. Abe, S. Miura and K. Shinozaki

Introduction

D

rought, salt loading, and freezing are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plants respond to these stresses at molecular and cellular levels as well as physiological levels. Expression of a variety of genes has been demonstrated to be induced by these stresses.1,2 The products of these genes are thought to function not only in stress tolerance but also in the regulation of gene expression and signal transduction in stress response.3 Thus, these gene products can be classified into two groups. The first group includes proteins that probably function in protecting cells from dehydration. The second group of gene products contains protein factors that are involved in further regulation of gene expression and signal transduction, and that function in stress response.3 Genetic engineering is thought to be useful for improving the stress tolerance of plants. Recently, several different approaches were attempted to improve the stress tolerance of plants by gene transfer. 4 The genes selected for transformation were those involved in encoding enzymes required for the biosynthesis of various osmoprotectants.5-7 Other genes that have been selected for transformation include those that encoded enzymes for modifying membrane lipids, LEA protein, and detoxification enzyme.8-11 In all these experiments, a single gene for a protective protein or an enzyme was overexpressed under the control of the 35S cauliflower mosaic virus

(CaMV) constitutive promoter in transgenic plants, although a number of genes have been shown to function in environmental stress tolerance and response. The genes encoding protein factors that are involved in regulation of gene expression and signal transduction and function in stress response seem to be useful in improving the tolerance of plants to stresses by gene transfer, as they can regulate many stress inducible genes involved in stress tolerance. Drought is one of the most severe environmental stresses, and affects almost all the plant functions. Abscisic acid (ABA) is produced under water deficit conditions and plays important roles in tolerance against drought. Most of the drought inducible genes that have been studied to date are also induced by ABA. 3 It appears that dehydration triggers the production of ABA, which, in turn, induces various genes. Several reports have described genes that are induced by dehydration but are not responsive to exogenous ABA treatments. 3 These findings suggest the existence of ABA independent, as well as ABA dependent, signal transduction cascades between the initial signal of drought stress and the expression of specific genes. 3 To understand the molecular mechanisms of gene expression in response to drought stress, cis- and trans-acting elements that function in ABA-independent and ABA-responsive gene expression by drought stress have been precisely analyzed. 3 In this article, we summarize recent progress of our

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research on cis- and trans-acting factors involved in ABA-independent gene expression in drought stress response. We also report stress tolerance of transgenic plants that overexpress a single gene for a stress inducible transcription factor, using Arabidopsis as a model.

Function of Water StressInducible Genes A variety of genes are induced by drought stress, and functions of their gene products have been predicted from sequence homology with known proteins. Genes induced during drought stress conditions are thought to function not only in protecting cells from dehydration by the production of important metabolic proteins but also in the regulation of genes for signal transduction in the drought stress response.1, 3 Thus, these gene products are classified into two groups (Fig. 20.1). The first group includes proteins that probably function in stress tolerance:3 water channel proteins involved in the movement of water through membranes, the enzymes required for the biosynthesis of various osmoprotectants (sugars, proline, and betaine), proteins that may protect macromolecules and membranes (LEA protein, osmotin, antifreeze protein, chaperon, and mRNA binding proteins), proteases for protein turnover (thiol proteases, Clp protease, and ubiquitin) and the detoxification enzymes (glutathione S-transferase, soluble epoxide hydrolase, catalase, superoxide dismutase, and ascorbate peroxidase). Some of the stress inducible genes that encode proteins, such as a key enzyme for proline biosynthesis, were overexpressed in transgenic plants to produce a stress tolerant phenotype of the plants.6 The second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response: protein kinases, transcription factors and enzymes in phospholipid metabolism.3 Now it becomes more important to elucidate the role of these regulatory proteins for further understanding of plant responses to water stress and for improving the tolerance of plants by gene transfer. The existence of a variety of drought inducible genes suggests complex responses of

plants to drought stress. Their gene products are involved in drought stress tolerance and stress responses.

Expression of DehydrationInduced Genes in Response to Envitonmental Stresses and ABA The expression patterns of genes induced by drought were analyzed by RNA gel blot analysis. Results indicated broad variations in the timing of induction of these genes under drought conditions. Most of the drought inducible genes respond to treatment with exogenous ABA, whereas some others do not.1,3 Therefore, there are not only ABAdependent but also ABA-independent regulatory systems of gene expression under drought stress. Analysis of the expression of ABA inducible genes revealed that several genes require protein biosynthesis for their induction by ABA, suggesting that at least two independent pathways exist between the production of endogenous ABA and gene expression during stress. As shown in Figure 20.2, we identified at least four independent signal pathways which function under drought conditions: Two are ABA-dependent (pathways I and II) and two are ABA-independent (pathways III and IV). One of the ABA-independent pathways overlaps with that of the cold response (pathway IV). One of the ABA-dependent pathways requires protein biosynthesis (pathway I).1,3 The existence of complex signal transduction pathways in drought response gives a molecular basis for the complex physiological responses of plants to drought stress.

Identification of Cis-Acting Element, DRE, Involved in Drought Responsive Expression A number of genes are induced by drought, salt and cold in aba (ABA deficient) or abi (ABA insensitive) Arabidopsis mutants. This suggests that these genes do not require ABA for their expression under cold or drought conditions. Among these genes, the expression of a drought inducible gene for rd29A/lti78/cor78 was extensively analyzed. 12 At least two separate regulatory systems function in gene expression during drought and

Improving Drought, Salt and Freezing Stress Tolerance in Transgenic Plants

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Fig. 20.1. Drought stress inducible genes and their possible functions in stress tolerance and response. Gene products are classified into two groups. The first group includes proteins that probably function in stress tolerance (Functional Proteins), and the second group contains protein factors involved in further regulation of signal transduction and gene expression that probably function in stress response (Regulatory Proteins). cold stress; one is ABA independent (Fig. 20.2, pathway IV) and the other is ABA dependent (pathway II). To analyze the cis-acting elements involved in the ABA-independent gene expression of rd29A, we constructed chimeric genes with the rd29A promoter fused to the β-glucuronidase (GUS) reporter gene and transformed Arabidopsis and tobacco plants with these constructs. The GUS reporter gene driven by the rd29A promoter was induced at significant levels in transgenic plants by conditions of dehydration, low temperature, or high salt or by treatment with ABA.13 The deletion, the gain of function and the base substitution analysis of the promoter region of rd29A gene revealed that a 9 bp conserved sequence, TACCGACAT (DRE, Dehydration Responsive

Element), is essential for the regulation of the expression of rd29A under drought conditions. Moreover, DRE has been demonstrated to function as a cis-acting element involved in the induction of rd29A by either low temperature or high salt stress. 12 Therefore, DRE seems to be a cis-acting element involved in gene induction by dehydration, high salt or low temperature, but does not function as an ABA responsive element in the induction of rd29A.

Important Roles of the DRE Binding Proteins During Drought and Cold Stresses Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one hybrid screening technique.14 The deduced amino acid sequences

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Fig. 20.2. Signal transduction pathways between initial dehydration stress signal and gene expression. There are at least four signal transduction pathways: two are ABA dependent (I and II) and two are ABA independent (III and IV). Protein synthesis is necessary for one of the ABA-dependent signal pathways (I). ABRE is involved in one of the ABA-dependent pathways (II). In one of the ABA-independent pathways, DRE is involved in the regulation of genes not only by drought and salt but also by cold stress (IV). Another ABA-independent pathway is controlled by drought and salt, but not by cold (III). of DREB1A and DREB2A showed significant sequence similarity with the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethyleneresponsive expression and floral morphogenesis, respectively.15,16 Each DREB protein contained a basic region in its N-terminal region that might function as a nuclear localization signal and an acidic C-terminal region that might act as an activation domain for transcription. These data suggest that each DREB cDNA encodes a DNA binding protein that might function as a transcriptional activator in plants. The ability of the DREB1A and DREB2A proteins expressed in Escherichia coli to bind the wild type or mutated DRE sequences was examined using the gel retardation method.14 The results indicate that the binding of these

two proteins to the DRE sequence is highly specific. To determine whether the DREB1A and DREB2A proteins are capable of transactivating DRE-dependent transcription in plant cells, we performed transactivation experiments using protoplasts prepared from Arabidopsis leaves. Coexpression of the DREB1A or DREB2A proteins in protoplasts transactivated the expression of the GUS reporter gene. These results suggest that DREB1A and DREB2A proteins function as transcription activators involved in the coldand dehydration-responsive expression, respectively, of the rd29A gene (Fig. 20.3).14 We isolated cDNA clones encoding two DREB1A homologs (named DREB1B and DREB1C). The DREB1B clone was identical to CBF1.17 We also isolated cDNA clones encoding a DREB2A homolog and named it

Improving Drought, Salt and Freezing Stress Tolerance in Transgenic Plants DREB2B. Expression of the DREB1A gene and its two homologs was induced by low temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration.14,18 These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low temperature and dehydration conditions, respectively (Fig. 20.3).14

Analysis of the In Vivo Roles of DREB1A and DREB2A by Using Transgenic Plants We generated transgenic plants in which DREB1A or DREB2A cDNAs were introduced to overproduce DREB proteins to analyze the effects of overproduction of DREB1A and DREB2A proteins on the expression of the target rd29A gene. Arabidopsis plants were transformed with vectors carrying fusions of the enhanced CaMV 35S promoter and the DREB1A (35S:DREB1A) or DREB2A (35S: DREB2A) cDNAs in the sense orientation.14,19 All of the transgenic plants carrying the 35S:DREB1A transgene (the 35S:DREB1A plants) showed growth retardation phenotypes under normal growth conditions. The 35S: DREB1A plants showed variations in phenotypic changes in growth retardation that may have been due to the different levels of expression of the DREB1A transgenes for the position effect.14 To analyze whether overproduction of the DREB1A protein caused the expression of the target gene in unstressed plants, we compared the expression of the rd29A gene in control plants carrying the pBI121 vector. Transcription of the rd29A gene was low in the unstressed wild type plants, but high in the unstressed 35S:DREB1A plants.14 The level of the rd29A transcripts under the unstressed control condition was found to depend on the level of the DREB1A transcripts.14 To analyze whether overproduction of the DREB1A protein caused the expression of other target genes, we evaluated the expression of its target stress inducible genes. In the 35S:DREB1A plants the kin1, cor6.6/kin2, cor15a, cor47/rd17 and erd10 genes were expressed strongly under unstressed control conditions, as was the rd29A gene.3,20 In contrast, the transgenic plants carrying the 35S:DREB2A transgene (the 35S:DREB2A

227

plants) showed little phenotypic change. In 35S:DREB2A transgenic plants, the rd29A mRNA did not accumulate significantly, although the DREB2A mRNA accumulated even under unstressed conditions.14 Expression of the DREB2A protein is not sufficient for the induction of the target stress inducible gene. Modification, such as phosphorylation of the DREB2A protein, seems to be necessary for its function in response to dehydration (Fig. 20.3). However, DREB1 proteins can function without modification.

Drought, Salt and Freezing Stress Tolerance in Transgenic Plants The tolerance to freezing and dehydration of the transgenic plant was analyzed using the 35S:DREB1A plants grown in pots at 22°C for 3 weeks. When plants were exposed to a temperature of -6°C for 2 days, returned to 22°C and grown for 5 days, all of the wild type plants died, whereas the 35S:DREB1A plants survived at high frequency. 14,21 Freezing tolerance was correlated with the level of expression of the stress inducible genes under unstressed control conditions (Fig. 20.4; between 80 and 30% survival). 14, 21 To test whether the introduction of the DREB1A gene enhances tolerance to dehydration stress, we did not water the plants for 2 weeks. Although all of the wild type plants died within 2 weeks, between 40 and 20% of the 35S:DREB1A plants survived and continued to grow after rewatering. Drought tolerance was also dependent on the level of expression of the target genes in the 35S:DREB1A plants under unstressed conditions (Fig. 20.4).14,21 Overexpression of the DREB1A cDNA, driven by the constitutive 35S CaMV promoter in transgenic plants, activated strong expression of the target stress inducible genes under unstressed conditions, which, in turn, increased tolerance of freezing, salt and drought stresses. 14,21 Jaglo-Ottosen et al reported that CBF1 overexpression also enhances freezing tolerance.22 However, the overexpression of stress inducible genes controlled by the DREB1A protein caused severe growth retardation under normal growth conditions.14,21

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 20.3. A model of the induction of the rd29A gene and cis- and trans-acting elements involved in stress-responsive gene expression. Two cis-acting elements, DRE and ABRE, are involved in the ABA-independent and ABA-responsive induction of rd29A, respectively. Two types of different DRE binding proteins, DREB1 and DREB2, separate two different signal transduction pathways in response to cold and drought stresses, respectively. DREB1s/CBF1 are transcriptionally regulated whereas DREB2s are controlled posttranslationally as well as transcriptionally. ABRE binding proteins encode bZIP transcription factors. To resolve the problem of growth retardation, we used the stress inducible rd29A promoter to cause overexpression of DREB1A in transgenic plants (rd29A:DREB1A plants).21 Because the rd29A promoter was stress inducible and contained binding sites for the DREB1A protein, it did not cause expression of the DREB1A transgene at high levels under unstressed conditions; instead, it rapidly amplified expression of the DREB1A transgene only under dehydration, salt, and low temperature stress. The rd29A:DREB1A plants revealed strong stress tolerance even though their growth retardation under normal growing conditions was not significant. Moreover, the growth and the productivity of these plants were almost the same as those of the wild type plants under normal growing conditions.21 On the contrary, the rd29A:DREB1A transgenic plants are more tolerant to the stresses than the 35S:DREB1A plants that exhibited growth retardation under normal

growing conditions (Fig. 20.4).21 As the rd29A gene is one of the target genes of the DREB1A protein, the rd29A promoter is more suitable for the tissue specific expression of the DREB1A gene in plants than the 35S CaMV promoter. In the rd29A:DREB1A plants, the target gene products seem to be strongly accumulated in the same tissues that express the products under stress conditions. These results indicate that combination of the DREB1A cDNA with the rd29A promoter would be quite useful for improving drought, salt and freezing stress tolerance in transgenic plants. In a previous studies, we showed that DRE also functions in gene expression in response to stress in tobacco plants,2,13 which suggests the existence of similar regulatory systems in tobacco and other crop plants. DRE-related motifs have been reported in the promoter region of cold inducible Brassica

Improving Drought, Salt and Freezing Stress Tolerance in Transgenic Plants

229

Fig. 20.4. Freezing and drought stress tolerance of the 35S:DREB1Ab, 35S:DREB1Ac and rd29A:DREB1Aa transgenic plants. The stress treatments were conducted as described in the text. Control: 3 week old plants growing under normal conditions; Freezing: plants exposed to a temperature of -6°C for 2 days and returned to 22°C for 5 days; Drought: water withheld for 2 weeks. Percentages of surviving plants and numbers of surviving plants per total tested plants are shown under the pictures. napus and wheat genes.23,24 These observations suggest that both the DREB1A cDNA and the rd29A promoter can be used to improve the dehydration, salt and freezing tolerance of crops by gene transfer.

References 1. Shinozaki K, Yamaguchi-Shinozaki K. Molecular responses to drought and cold stress. Curr Opin Biotech 1996; 7:161-167. 2. Thomashow MF. Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. Arabidopsis. In: Meyro- witz E, Somerville C, eds. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1994:807-834.

3. Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-stress response, Plant Physiol 1997; 115:327-334. 4. Holmberg N, Bulow L. Improving stress tolerance in plants by gene transfer. Trends Plant Sci 1998; 3:61-66. 5. Tarczynski M, Bohnert H. Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 1993; 259:508-510. 6. Kavi Kishor PB, Hong Z, Miao G-U et al. Overexpression of D1-pyrroline-5- carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 1995; 108:1387-1394.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

7. Hayashi H, Mustardy L, Deshnium P et al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J 1997; 12:1334-142. 8. Kodama H, Hamada T, Horiguchi G et al. Genetic enhancement of cold tolerance by expression of a gene for chloroplast w-3 fatty acid desaturase in transgenic tobacco. Plant Physiol. 1994; 105:601-605. 9. Ishizaki-Nishizawa O, Fujii T, Azuma M et al. Low-temperature resistance of higher plants is significantly enhanced by a nonspecific cyanobacterial desaturase. Nature Biotechnol 1996; 14:1003-1006. 10. Xu D, Duan X, Wang B et al. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 1996; 110:249-257. 11. McKersie BD, Bowley SR, Harjanto E et al. Water-deficit tolerance and field performance of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiol 1996; 111:1177-1181. 12. Yamaguchi-Shinozaki K, Shinozaki K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 1994; 6:251-264. 13. Yamaguchi-Shinozaki K, Shinozaki K. Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 1993; 236:331-340. 14. Liu Q, Kasuga, M, Sakuma Y et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 1998; 10:1391-1406. 15. Okamuro JK, Caster B, Villarroel R et al. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci USA 1997; 94:7076-7081.

16. Ohme-Takagi M, Shinshi H. Ethyleneinducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 1995; 7:173-182. 17. Stockinger EJ, Gilmour SJ, Thomashow MF. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA, 1997; 94:1035-1040. 18. Shinwari ZK, Nakashima K, Miura S et al. An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low-temperature-responsive gene expression. Biochem Biophys Res Cmmu 1998; 250:161-170. 19. Mituhara I, Ugaki M, Hirochika H et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 1996; 37:49-59. 20. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K. Characterization of two cDNAs (ERD10 and ERD14) corresponding to genes that respond rapidly to dehydration stress in Arabidopsis thaliana. Plant Cell Physiol 1994; 35:225-231. 21. Kasuga M, Liu Q, Miura S et al. Improving drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnol 1999; 17:287-291. 22. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG et al. Arabidopsis CBF1 overexpression induces cor genes and enhances freezing tolerance. Science 1998; 280:104-106. 23. Jiang C, Iu B, Singh J. Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus. Plant Mol Biol 1996; 30:679-684. 24. Ouellet F, Vazquez-Tello A, Sarhan F. The wheat wcs120 promoter is cold-inducible in both monocotyledonous and dicotyledonous species. FEBS Lett 1998; 423:324-328.

CHAPTER 21

Characterization of Salt Inducible Genes from Barley Plants T. Takabe, T. Nakamura, Y. Muramoto and S. Kishitani

Introduction

T

o understand the gene expression that enables the survival of barley under high salt conditions, we have performed differential display RT-PCR (DDRT-PCR) with the mRNAs expressed in leaves of barley plants subjected to high salt in a stepwise manner. The expression profile of genes often tells us eloquently about what is taking place in a particular cell under a particular situation. We expected that the mRNAs expressed in such salt-stressed leaves include the genes whose products are important in conferring salt tolerance to the plant. DDRT-PCR using various single primers actually enabled us to obtain several DNA fragments, which were amplified preferentially from mRNA expressed in the salt-stressed leaves (Table 21.1). Subsequent Northern blot analysis, using them as probes, confirmed that most of the obtained fragments derived from genes actually expressed in the stressed leaves specifically or preferentially. One of the PCR fragments appeared to encode ascorbate peroxidase, according to the partial sequence. Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme in plant cells.1 It was reported that the expression and activity of ascorbate peroxidase is increased during drought stress. Environmental stress, such as drought, salt, high light and low temperature, causes the evolution of toxic active-oxygen species in chloroplasts, which is called oxidative stress. Therefore, this result validates our approach to isolate salt-induced genes by

differential display, in order to unveil mechanisms of salt tolerance.

Nuclease 12 By PCR using a RAPD primer (5'GCCTGCCTCACG-3'), we detected a DNA fragment which was preferentially amplified from mRNAs prepared from the salt-stressed leaves. We subsequently isolated the full length cDNA clone from a cDNA library prepared from the leaves of salt-stressed barley. The clone was 1283 bp in length and contained an open reading frame predicted to encode a polypeptide of 290 amino acids with the estimated molecular mass of 32.4 kDa. The protein has a sequence which is identical, except for one residue, to the N-terminal region of barley nuclease, expressed at the stage of seed germination (Fig. 21.1).3 Therefore, we concluded that the clone is the gene equivalent to barley nuclease (Nuclease I) and designated this gene as Bnuc1. Bnuc1 protein has a putative signal sequence of twenty-three amino acids at its N-terminus, but no other organelle targeting sequence. This may imply that the protein is secreted to the extracellular space. The transcript of Bnuc1 gene in nonstressed leaves was difficult to detect by Northern blot analysis. However, the expression level was increased markedly under salt stress. It has been reported that barley nuclease is produced in the aleurone layer of seeds and is secreted from the layer in response to GA.4 These

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine ©2000 Eurekah.com.

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Table 21.1. Summary of the clones isolated by differential display Clone

Homology

BD1

RNA helicase

BD8

nuclease

BD9

yeast SSF

BD24

ascorbate peroxidase

observations suggest that barley nuclease degrades the nucleic acids stored in the endosperm to provide the embryo with nucleotides or phosphates for heterotrophic growth. The Bnuc1 gene was not expressed in germinating seeds and the expression did not respond to GA. These result suggest that the Bnuc1 gene does not encode the previously reported barley nuclease, but encodes another member of the family. Actually, in Southern blot analysis using Bnuc1 cDNA as a probe, several bands were detected, suggesting that nuclease genes constitute a gene family on the barley genome. It has been considered that nuclease I enzymes have two major roles, alteration of gene expression and remobilization of nutrients.5 Since Bnuc1 protein has a signal sequence at its N-terminus (Fig. 21.1), different from any nuclear-targeting sequence, Bnuc1 protein does not seem to be a nuclease associated with chromatin.6,7 So we postulate that Bnuc1 protein plays a role in remobilization of nutrients, such as nucleotides and phosphates, for the benefit of young fresh organs under salt stress. Accumulation of salt ions during salt stress is harmful to leaves and causes premature senescence. The aged leaves certainly yellowed first in this situation. Previously, it was reported that sodium and chloride ions accumulate preferentially in older leaves in barley8 and rice9,10 during salt stress, while glycinebetaine, one of the compatible solutes and osmoprotectants, accumu-

lates preferentially in the younger leaves in barley.8 These observations suggest that, in order to survive under high salinity conditions, barley plants have a preference for growth of the fresh tissues which have more sufficient metabolic and physiological activity, and limit the damage by salt to the older tissues which no longer have optimal cellular functions. To examine the spatial expression of Bnuc1 gene, we followed the kinetics of its transcript accumulation in individual leaves during salt treatment. Bnuc1 transcript started accumulating in the older leaves within 24 h. By contrast, the signal was hardly detectable in young leaves. Nuclease activity corresponding to the estimated molecular mass of Bnuc1 protein actually increased more in the older leaves under salt stress. Gan and Amasino11 hypothesized that premature leaf senescence caused by salt stress has an obvious adaptive value, allowing the plant to complete its life cycle even under stressful conditions. They further claimed that plants have evolved mechanisms by which leaf senescence can be induced by limited water and nutrient availability to reallocate nutrients to reproductive organs and to eliminate water consumption by older, less productive leaves. Quite recently, it was also reported that nuclease activity with approximately the same molecular mass as the one we have found increases during leaf aging in barley.12 These observations suggest that during adaptation to salt stress, a nutrient recycling system similar to that occurring during natural leaf senescence is induced, and the barley nuclease I, Bnuc1, likely functions in the system.

ATP-Dependent RNA Helicase We cloned and characterized a saltresponsive transcript, HVD1 (Hordeum vulgare DEAD box protein) (Fig. 21.2), encoding a putative ATP-dependent RNA helicase belonging to a group known as the DEAD box family.14 The DEAD box family was defined first by Linder et al14 and shares several highly conserved motifs among the different organisms ranging from E. coli to human . Most of the members of the DEAD box family have determined or putative ATP-dependent RNA helicase

Characterization of Salt Inducible Genes from Barley Plants

233

Fig. 21.1. Alignment of N-terminal regions of the deduced amino acid sequence of Bnuc1 with barley nuclease3 and Zinnia nuclease.13 X represents ambiguous residues. Black boxes show identical amino acids. The unknown sequence is represented by dashes. activity, modulating RNA secondary and tertiary structure. They are involved in diverse biological functions such as translation initiation, RNA splicing, ribosome assembly and spermatogenesis. In plants, the DEAD box genes encoding eukaryotic initiation factor 4A (eIF-4A) were cloned from tobacco,15 wheat16 and rice.17 The other type of DEAD box gene was cloned from tobacco;18 it ts similar to human p68 of a DEAD box RNA helicase family, but its function is unknown. HVD1 mRNA was detected at low levels in leaves and roots of non-stressed plants, whereas its accumulation was induced 8-fold higher under salt stress. HVD1 cDNA encoded a 764 amino acid polypeptide with molecular mass 81.8 kDa. The deduced HVD1 protein has the eight consensus motifs of the DEAD box family. In addition, the protein contained five repeats of “RGG” known as an RNA-binding motif at its hydrophilic C-terminus, showing a novel DEAD box protein induced by salt stress in plants. The level of mRNA was increased not only by salt but also by cold treatment. It is anticipated that HVD1 protein regulates expression of genes concerned with salt tolerance or important metabolisms such as photosynthesis.

Betaine Aldehyde Dehydrogenase In some higher plants, glycinebetaine is synthesized and accumulated in cells in response to salt stress. Glycinebetaine is a compatible solute and acts as an excellent osmoprotectant. Glycinebetaine of high levels is present in leaves of diverse families of dicotyledons19

and of some monocotyledons.20 The pathway of glycinebetaine biosynthesis in higher plants is as follows: Choline
betaine aldehyde
glycinebetaine. The enzyme which catalyzes the first step is choline monooxygenase (CMO), recently purified and partially characterized.21 The final step is catalyzed by betaine aldehyde dehydrogenase (BADH), which has been well characterized.22-24 Existence of a BADH isozyme was suggested in spinach and sorghum,25,26 and we reported that all monocotyledonous BADHs have a C-terminal tripeptide SKL that is known to be a signal for targeting preproteins to microbodies.27 We cloned two types of BADH cDNA (BBADH1 and BBADH2) and determined their nucleotide sequence. On the basis of an analysis of nucleotide sequence of cDNAs which were cloned into a plasmid, the insert was judged to encode BADH. BBADH1 had the signal sequence (SKL) targeting to microbodies. However, BBADH2 did not have a SKL signal. Predicted amino acid sequence identity between BBADH1 and BBADH2 was 70%. The BBADH2 gene was more similar to the BADH gene of dicotyledons (spinach, sugar beet) than those of monocotyledons (barley, sorghum, rice) previously reported (Table 21.2) A small amount of the 1.9 kb BBADH1 mRNA was expressed under normal growth conditions. Under salinity conditions, BBADH1 gene is expressed, still at a low level. On the other hand, BBADH2 gene expressed at a very low level under non-stress conditions. However, the quantitative analysis revealed that the level of BBADH2 mRNA

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 21.2. Structure of deduced HVD1 protein. under salinity conditions was approximate ten times higher than that under normal growth conditions. These results suggest that BBADH1 gene was expressed constitutively, and BBADH2 gene was induced by salt stress, in barley leaves. Barley had two copies of the BBADH1 gene. Only a single gene, BBADH2, appeared to be present in the genome of barley. In our previous study, it was found that all monocotyledonous BADHs known at that time have a C-terminal tripeptide SKL.27,28 It was thought that monocotyledonous BADHs are localized in microbodies (peroxisomes). In the present study, we confirmed the existence of a BADH isozyme (BBADH2) gene in barley plants. However, we could not find a SKL signal at the C-terminal of the BBADH2 amino acid sequence. Furthermore, BBADH2 was more similar to dicotyledonous BADHs. While the BBADH1 gene was expressed constitutively, BBADH2 gene was salt inducible in barley leaves. Therefore, BBADH2 mainly functions in salt-stressed barley leaves for glycinebetaine synthesis. It was reported that BADH catalyzes oxidation of not only betaine aldehyde but also other aldehydes.29 So BBADH1 may have other functions or catalyze oxidation of other aldehydes.

Production of Transgenic Plants with Increased Salt Tolerance Rice plants are known not to accumulate glycinebetaine as an osmoprotectant and are very sensitive to salt stress. In order to increase salt tolerance of rice plants, they were

transformed with the E. coli betA gene, encoding a bifunctional enzyme (choline dehydrogenase and betaine aldehyde dehydrogenase), using a mitochondrial targeting sequence under the control of CaMV 35S promoter. However, the transformants did not accumulate glycinebetaine. The original betA gene was found to have unfavorable sequences. Accordingly the betA gene was largely modified and then used successfully for transformation of rice plants. The transformants had an increased salt tolerance in terms of both number of seedlings that retained green color after salt treatment (150 mM) for 7 d and that recovered without salt 7 d after the salt treatment, compared with those of control plants. The transformants also acquired an increased drought tolerance. The rice transformed with untargeted versions of either original betA or modified betA did not accumulate glycinebetaine. To evaluate the effects of glycinebetaine on tolerance against cold and heat, transgenic rice plants overexpressing BADH were produced by the intoduction of a BADH (BBADH1) cDNA from barley. The transgenic BADH rice plants converted exogenously applied betaine aldehyde to glycinebetaine of high levels up to 5 µmoles/g FW more efficiently than wild type plants.30 High levels of glycinebetaine accumulation in the transgenic BADH rice plants conferred significant tolerance against cold and heat stresses as well as salt and drought tolerance. The benefit of glycinebetaine accumulation was much higher under a higher

Characterization of Salt Inducible Genes from Barley Plants

235

Table 21.2. Sequence comparison of deduced BADH protein in higher plants

BBADH1 BBADH2 Rice

BBADH2

Rice

Sorghum Atriplex

70

85

73

70

71

69

71

72

63

71

71

71

73

77

70

71

69

71

63

63

61

63

89

87

82

89

83

Sorghum Atriplex Spinach Sugar beet

radiation in rice. The increased stress tolerance against salt, drought, cold or heat was also observed in the rice plants exogenously treated with glycinebetaine.30,31 To produce highly salt-tolerant plants we are planning to use salt-inducible genes obtained by differential display as described above, after each gene is examined for ability to contribute to salt (or other) tolerance, by evaluation using transgenic plants expressing the gene product.

References 1. Mittler R, Zilinskas BB. Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant J 1994; 5:397-405. 2. Muramoto Y, Watanabe A, Nakamura T et al. Enhanced expression of a nuclease gene in leaves of barley plants under salt stress. Gene 1999; 234:315-321. 3. Brown PH, Ho T-HD. Biochemical properties and hormonal regulation of barley nuclease. Eur J Biochem 1987; 168:357-364.

Spinach

Sugar beet

Amaranthus

83

4. Brown PH, Ho T-HD. Barley aleurone layers secrete a nuclease in response to gibberellic acid. Plant Physiol 1986; 82:801-806. 5. Blank A, McKeon TA. Single-strandpreferring nuclease activity in wheat leaves is increased in senescence and is negatively photoregulated. Proc Natl Acad Sci USA 1989; 86:3169-3173. 6. Srivastava BIS. Increase in chromatin associated nuclease activity of excised barley leaves during senescence and its suppression by kinetin. Biochem Biophys Res Commun 1968; 32:533-538. 7. Kalinski A, Chandra GR, Muthukrishnan S. Study of barley endonuclease and α-amylase genes. J Biol Chem 1986; 261:11393-11397. 8. Nakamura T, Ishitani M, Harinasut P et al. Distribution of glycinebetaine in old and young leaf blades of salt-stressed barley plants. Plant Cell Phisiol 1996; 37:873-877. 9. Yeo AR, Flowers TJ. Accumulation and localization of sodium ions within the shoots of rice (Oryza sativa) varieties differing in salinity resistance. Physiol Plant 1982; 56:343-348.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

10. Yeo AR, Flowers TJ. Salinity resistance in rice (Oryza sativa L.) and a pyramiding approach to breeding varieties for saline soils. Aust J Plant Physiol 1986; 13:161-173. 11. Gan S, Amasino RM. Making sense of senescence. Plant Physiol 1997; 113:313-319. 12. Wood M, Power JB, Davey MR et al. Factors affecting single strand-preferring nuclease acivity during leaf aging and dark-induced senescence in barley (Hordeum vulgare L.). Plant Sci 1998; 131:149-159. 13. Thelen MP, Northcote DH. Identification and purification of a nuclease from Zinnia elegans L.: A potential molecular marker for xylogenesis. Planta 1989; 179:181-195. 14. Linder P, Lasko PF, Ashburner M et al. Birth of the D-E-A-D box. Nature 1989; 337:121-122. 15. Owttrim GW, Hofmann S, Kuhlemeier C. Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco. Nucl Acids Res 1991; 19:5491-5496. 16. Metz AM, Browning KS. Sequence of a cDNA encoding wheat eukaryotic protein synthesis initiation factor 4A. Gene 1993; 131:299-300. 17. Nishi R, Kidou S, Uchimiya H et al. Isolation and characterization of a rice cDNA which encodes the eukaryotic initiation factor 4A. Biochim Biophys Acta 1993; 1174:293-294. 18. Itadani H, Sugita M, Sugiura M. Structure and expression of a cDNA encoding an RNA helicase-like protein in tobacco. Plant Mol Biol 1994; 24:249-252. 19. Weretilnyk EA, Bednarek S, McCue KF et al. Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons. Planta 1989; 178:342-352. 20. Ishitani M, Arakawa K, Mizuno K et al. Betaine aldehyde dehydrogenase in the Gramineae: Levels in leaves of both betaine-accumulating and nonaccumulating cereal plants. Plant Cell Physiol 1993; 34:493-495. 21. Burnet M, Lafontaine PJ, Hanson AD. Assay, purification, and partial characterization of choline monooxygenase from spinach. Plant Physiol 1995; 108:581-588.

22. Arakawa K, Takabe T, Sugiyama T et al. Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody. J Biochem 1987; 101:1485-1488. 23. Weretilnyk EA, Hanson AD. Betaine aldehyde dehydrogenase from spinach leaves: Purification, in vitro translation of the mRNA, and regulation by salinity. Arch Biochem Biophys 1989; 271:56-63. 24. Arakawa K, Mizuno K, Kishitani S et al. Immunological studies of betaine aldehyde dehydrogenase in barley. Plant Cell Physiol 1992; 33:833-840. 25. Weretilnyk EA, Hanson AD. Betaine aldehyde dehydrogenase polymorphism in spinach: Genetic and biochemical characterization. Biochem Genet 1988; 26:143-151. 26. Wood AJ, Saneoka H, Rhodes D et al. Betaine aldehyde dehydrogenase in sorghum. Plant Physiol 1996; 110:1301-1308. 27. Nakamura T, Yokota S., Muramoto Y et al. Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes. The Plant J 1997; 11:1115-1120. 28. Ishitani M, Nakamura T, Han SY et al. Expression of the betaine aldehyde dehydrogenase gene in barley in response to osmotic stress and abscisic acid. Plant Mol Biol 1995; 27:307-315. 29. Trossat C, Rathinasabapathi B, Hanson AD. Transgenically expressed betaine aldehyde dehydrogenase efficiently catalyzes oxidation of dimethylsulfoniopropionaldehyde and w-aminoaldehydes. Plant Physiol 1997; 113:1457-1461. 30. Kishitani S, Suzuki M, Takanami T et al. Compatibility of glycinebetaine in rice plants that do not accumulate it; evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase gene from barley. Plant Cell Environ 1999. in press. 31. Harinasut P, Tsutsui K, Takabe T et al. Exogenous glycinebetaine accumulation and increased salt-tolerance in rice seedlings. Biosci Biotec Biochem 1996; 60:366-368.

CHAPTER 22

Transgenic Rice: Development and Products for Environmentally Friendly Sustainable Agriculture S.K. Datta

Introduction

R

ice is one of the most important food crops of the world, feeding nearly 2.5 billion people. By the year 2020, the number of rice consumers will be almost double. It is estimated that 60% more rice needs to be produced with less land, less water, and less labor. Attacks by insect pests, sheath blight and bacterial blight, and abiotic stresses can cause yield losses in rice equivalent to 200 million tons (Table 22.1).1,2 Crop protection plays a vital and integral role in sustainable rice production. Pesticide applications worldwide are now estimated to cost approximately US $8.1 billion per annum and Japan tops the list of pesticide users (Tables 22.2 and 22.3).3 This tremendous use of pesticides has reduced the effective life span of some compounds. It has also led to serious environmental consequences and concerns for human health.4 In addition to integrated pest management (IPM), crop rotation and resistant crops through genetically engineered rice varieties would appear to be the best options for environmentally friendly sustainable agriculture (Tables 22.4 and 22.5).5 Transgenic Bt rice conferring resistance to stem borer,5-8 bacterial blight resistance (Fig. 22.1),9 sheath blight resistance,10,11 and stripe virus resistance12 have been developed. Transgenic resistant plants would produce more yield while growing with fewer agrochemicals, particularly pesticides.

Case Study of Transgenic Rice Transgenic Bt Rice for Resistance to Stem Borer Stem borer damage is a serious problem in rice, causing estimated losses of 10-30% of the total yield. Scirpophaga incertulas (yellow stem borer) and Chilo suppressalis (striped stem borer) are the major stem borers, and are widely distributed from Japan to India. Stem borer larvae start their attack by boring through the inner portion of the leaf sheath. The subsequent boring through the stem by caterpillars causes considerable damage, resulting in “deadheart” symptoms, and the affected tillers do not bear panicles. Panicles often emerge with empty grains, called “whiteheads” (Fig. 22.2a). Bacillus thuringiensis (Bt), the common soil bacterium, produces crystals containing insecticidal proteins. These toxins kill insects by binding to and creating pores in the midgut membranes. Bt toxins are highly specific and therefore are not toxic to beneficial insects, birds, and mammals, including humans.13 But, Bt toxins are insecticides and, like conventional chemical insecticides, insects may quickly adapt to them unless Bt plants are carefully designed and deployed. A greater assurance of durable resistance can be achieved if a Bt toxin is combined with a second unrelated type of toxin.14 Tissue-specific promoters, particularly the green-tissue specific

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine ©2000 Eurekah.com.

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Table 22.1. Estimated yield losses by groups of constraints (kg ha-1) in the main rice seasons

Region

Insects

Diseases

Drought

Soils

Other abiotic stresses Others

China

92

88

n.a.

635

557

91

Eastern India

104

93

177

74

81

167 (weeds, lodging, birds

West Bengal

43

213

n.a.

n.a.

772

-

Southern India

215

137

44

72

7

87 (weeds, lodging, nutrients)

Bangladesh

135

111

172

27

106

22 (weeds)

Indonesia

399

24

n.a.

n.a.

n.a.

189 (rats)

Thailand

78

12

n.a.

n.a.

n.a.

5 (rats)

Nepal

810

560

210

150

10

150 (rats, weeds, lodging)

Philippines

250

198

n.a.

407

90 (variety, others)

Source: Herdt, 1996.2

Table 22.2. Insect control costs (US $ million)

Table 22.3. Use (%) of pesticides by countries and regions

Crop

Control cost

Substitution value

Country

Rice

1190

422

Japan

45.7

Korea

27.9

Cotton

1870

1161

Corn/maize

620

158

Fruits and vegetables

2465

891

Other

1965

Total

8110

Source: Krattiger, 1998.3

2632

Percentage

Europe

8.8

USA

6.3

Thailand

5.9

Indonesia

2.5

India

1.3

Latin America

0.9

Africa

0.6

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Table 22.4. Development and use of transgenic rice for biotic stresses Rice

Method used

Genes transferred

Traits

Japonica

Protoplast

nptII*

Resistant to kanamycin

21

Japonica

Protoplast (electroporation)

hpt*

Resistant to kanamycin

22

Indica

Protoplast (PEG)

hpt*

Resistant to kanamycin

17

Japonica

Protoplast (PEG)

nptII

Resistant to kanamycin

23

Indica (IR72) Protoplast (PEG)

bar*

Resistant to herbicide

24

Japonica

Protoplast (electroporation)

cp-stripe virus

Resistant to stripe virus

12

Japonica

Protoplast (electroporation)

Bt

Resistant to insects

25

Japonica

Agrobacterium

hpt*

Resistant to hygromycin

26

Indica

Protoplast (PEG)

chi11

Resistant to sheath blight

10

Japonica

Protoplast

cc

Resistant to insects

27

Japonica

Biolistic

Xa-21

Resistant to bacterial blight

28

Japonica

Biolistic

cpTi

Resistant to insects

29

Indica

Protoplast and biolistic

Bt/(DWR)

Resistant to stem borer

7

Japonica/ Indica

Agrobacterium

hpt*

Resistant to hygromycin

30

Japonica

Biolistic and protoplast

pinII

Resistant to insects

31

Indica

Biolistic

Bt

Resistant to stem borer

32

Indica/ Japonica

Biolistic and protoplast

early nodulin

Biological N2 fixation

33

Indica

Biolistic

Bt

Resistant to stem borer

6

Indica/ Japonica

Biolistic and protoplast

Bt Resistant to (tissue-specific) stem borer

5

Indica (IR72) Biolistic

Xa-21

Resistant to bacterial blight

9

Indica

Biolistic

Bt ML for hybrid rice

Resistant to stem borer

8

Indica

Biolistic

PR genes

Resistant to sheath blight

11

*Used as selectable marker gene; ML, maintainer line; DWR, deepwater rice.

Reference

Challenge to the Crisis of the Earth's Biosphere in the 21st Century

240

Table 22.5. Production of transgenic rice for field testing

Cultivars

Methoda

Genesb

Plants in greenhouse (total #)

Southern+ enzyme assay

Fertility status (%)

Transgenic lines selected

IR72

B

Bt, chi, Xa-21

32

20

60

12

IR64

B, P

Bt, chi

20

3

20

1

CBII

P

Bt, chi

1800

160

80

18

IR58

P, B

Bt, chi others

210

2

60

1

IR51500

P, B, A

Bt, chi

60

3

40

2

Basmati 370

P, B

Bt, chi, others

72

16

20

3

Basmati 122

P, B, A

Bt, chi

66

18

30

4

New plant others

B, P

Bt, chi, others

120

66

30

15

Maintainer line for hybrid rice

B, P

Bt, chi, others

140

22

60

12

MH63

B

Bt, chi

62

16

70

11

Vaidehi-1

B

Bt, chi

26

6

80

2

aB, biolistic; P, protoplast-mediated; A, Agrobacterium bBt, encoding resistance for stem borer; chi, chitinase gene for sheath blight resistance cPlants chosen based on Southern, Western, and bioassay data

promoter (PEPCP) used in Bt gene expression in rice, were allowed to express preferentially in green tissue and significantly reduced expression in grain. Thus, Bt rice plants with PEPCP or pith promoter, either alone or in combination, should provide a better strategy for providing rice plants with protection against insect pests, thus minimizing the expression of the CryIA(b) protein in seeds and other tissue.5

Transgenic Rice for Fungal Resistance In response to pathogen invasion, most plants synthesize an assortment of pathogenesis-related proteins (PR) as a defense mechanism (Table 22.6). Among PR proteins, chitinases have been studied most intensively and have been shown to have antifungal activity.15 In addition to sheath blight resistance, other fungal resistance has also been observed.10,11,16 Constitutive overexpression of

Transgenic Rice: Environmentally Friendly Sustainable Agriculture

241

Fig. 22.1. Schematic presentation of transfer of Xa-21 from original source to cultivated rice by conventional and molecular breeding. From Datta, 1998.5 PR genes in intra- and extracellular tissue provides enhanced fungal resistance, which allows a reduced application of fungicides. We have introduced a PR-5 gene, D34 (thaumatin-like protein gene), in rice, which showed enhanced resistance to sheath blight (Fig. 22.2b).11 We are now aiming to develop transgenic rice with multiple genes, and assume that this is a better strategy for durable resistance because of gene pyramiding (Table 22.5). Transgenic Rice With Xa-21 Conferring Resistance To Bacterial Blight Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases of rice throughout the world. A schematic presentation compares conventional breeding and the relative advantage of molecular breeding with Xa-21 (Figs. 22.1 and 22.2c). However, durable resistance can be achieved only with gene pyramiding, as was done in transgenic IR72. This is a very efficient way to improve BB resistance in a

desirable cultivar without the negative impact of genetic linkage dragging.

Environmentally Friendly Selectable Marker Genes The production of transgenic plants involves a suitable transformation vector carrying a selectable marker gene driven by a promoter to select the transformed tissue/ regenerants. Hygromycin is an excellent selectable agent for rice transformation when hph is driven by a constitutive promoter using all three transformation systems.17-19 However, though there has been no report of any toxic effect of HPH on the human system and it is not used in preparing any antibiotic drug for human use, public concerns still remain about having HPH in the final product. Alternatively, a new selection strategy involves the use of mannose, which cannot be metabolized by many plant species. A schematic presentation shows the conversion of mannose to mannose- 6-phosphate (M-6-P) by a hexokinase,

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 22.2. a) Symptoms of "whiteheads" showing panicles without grain caused by stem borer (left), and rice plants with stem borer resistance (right). b) Control plants showing the symptoms and damage due to sheath blight (left) versus transgenic plants showing enhanced resistance (right; from Datta et al, 1999a). c) Rice plants cv. IR72 with Xa-21 showing resistance to bacterial blight (race 6) versus control plants showing disease symptoms (right, from Tu et al, 1998b). T=transgenic; C=control.

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243

Table 22.6. Pathogenesis-related protein families in plants (recognized and proposed)

Family

Representative plants

Molecular weight (kDa)

Biochemical properties

PR-1

Tobacco

14, 15, 17

Antifungal, unknown

PR-2

Tobacco

31, 33, 35

b-1, 3-glucanase

PR-3

Tobacco

27, 28, 32, 34

Chitinase

PR-4

Tobacco

13, 15, 20

Similar to potato win proteins

PR-5

Tobacco Rice

24, 26 23

Thaumatin-like

PR-6

Tomato

8, 13

Proteinase inhibitor

PR-7

Tomato

69

Endo-proteinase

PR-8

Cucumber

28

Chitinase

PR-9

Tobacco

39, 40

Lignin-forming peroxidase

PR-10

Parsley

17-19

Ribonuclease-like

PR-11

Tobacco

41, 43

Class V chitinase

a. Thionins

Barley

5

Antimicrobial

b. Plant defensins

Radish

5

Antifungal

Others

resulting in severe growth inhibition (Fig. 22.3). The gene product phosphomannose isomerase converts M-6-P to fructose 6-phosphate, which is readily metabolized, giving the transformed cells a metabolic advantage. Mannose-6-phosphate is toxic to nontransgenic cells/tissue, which supports the mechanism of mannose selection. We are now working on this positive selection, aiming at an environmentally friendly and publicly

acceptable marker gene in rice. Similar work has been reported in the selection of transgenic sugar beet.20

Acknowledgments I thank BMZ (Germany), the Rockefeller Foundation (USA), and several collaborators who provided us with the gene constructs. A special mention is due to Novartis for providing us with the phosphomannose isomerase

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 22.3. Selection system using phsophomannose isomerase gene and mannose as a selectable agent. gene and to Bill Hardy for valuable comments on the manuscript. I sincerely thank my laboratory colleagues Karabi Datta, Jumin Tu, Mohammad F. Alam, Celsa Quimio, Lina Torrizo, Editha Abrigo, and Norman Oliva for their generous help and contributions.

References 1. Herdt RW. Research priorities for rice biotechnology. In: Khush GS, Toenniessen GH, eds. Rice Biotechnology. Wallingford: CAB International and Manila, International Rice Research Institute, 1991:19-54. 2. Herdt RW. Summary, conclusions and implications. In: Evenson RE, Herdt RW, Hossain M, eds. Rice Research in Asia: Progress and Priorities. Wallingford: CAB International and Manila, International Rice Research Institute. 1996:393-405. 3. Krattiger A. The importance of agbiotech to global prosperity. Ithaca: The International Service for the Acquisition of Agri-biotech Applications, 1998:1-11. 4. Dirham B. The Pesticides Hazard. London: The Pesticides Trust, 1993.

5. Datta K, Vasquez A, Tu J et al. Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest. Theor Appl Genet, 1998: 97:20-30. 6. Tu J, Datta K, Alam MF et al. Expression and function of a hybrid Bt toxin gene in transgenic rice conferring resistance to insect pests. Plant Biotech 1998a; 15:183-191. 7. Alam MF, Datta K, Abrigo E et al. Production of transgenic deepwater indica rice plants expressing a synthetic Bacillus thuringiensis cryIA(b) gene with enhanced resistance to yellow stem borer. Plant Sci 1998; 135:25-30. 8. Alam MF, Datta K, Alrigo E et al. Transgenic insect resistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Rep 1999; 18:572-575. 9. Tu J, Ona I, Zhang Q et al. Transgenic rice variety IR72 with Xa-21 is resistant to bacterial blight. Theor Appl Genet 1998b: 7:31-36. 10. Lin W, Anuratha CS, Datta K et al. Genetic engineering of rice for resistance to sheath blight. Bio/Technology 1995; 13:686-691.

Transgenic Rice: Environmentally Friendly Sustainable Agriculture 11. Datta K, Velazhahan R, Oliva N et al. Overexpression of cloned rice thaumatinlike protein (PR-5) in transgenic rice plants enhances environmental-friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet 1999a; 98:1138-1145. 12. Hayakawa T, Zhu Y, Itoh K et al. Genetically engineered rice resistant to rice stripe virus, an insect-transmitted virus. Proc Natl Acad Sci, 1992: 89:9865-9869. 13. Koziel MG, Beland GL, Bowman C et al. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 1993; 11:194-200. 14. Cohen MB, Savary S, Huang N et al. Importance of rice pests and challenges to their management. In: Dowling NG, Greenfield SM, Fischer KS, eds. Sustainability of Rice in the Global Food System. Manila: Pacific Basin Study Center and IRRI, 1998:145-164. 15. Boller T. Induction of hydrolases as a defense reaction against pathogens. In: Key JL, Kosuge T, eds. Cellular and Molecular Biology of Plant Stress. New York: Alan R. Liss, 1985: 247-262. 16. Datta K, Muthukrishnan S, Datta SK. Expression and function of PR-protein genes in transgenic plants. In: Datta SK and Muthukrishnan S, eds. PathogenesisRelated Proteins in Plants. CRC Press, 1999b: 261-277. 17. Datta SK, Peterhans A, Datta K et al. Genetically engineered fertile Indica-rice recovered from protoplasts. Bio/Technology 1990; 8:736-740. 18. Datta K, Torrizo L, Oliva N et al. Production of transgenic rice by protoplast, biolistic and Agrobacterium systems. Proceedings of the Fifth International Symposium on Rice Molecular Biology. 14-15 October 1996. Taipei, Taiwan, 1996. 19. Datta SK, Torrizo L, Tu J et al. Production and molecular evaluation of transgenic rice plants. IRRI Discussion Paper Series No. 21. Manila: International Rice Research Institute, 1997. 20. Joersbo M, Donaldson I, Kreiberg J et al. Analysis of mannose selection used for transformation of sugar beet. Mol Breed 1998; 4:111-117.

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21. Toriyama K, Arimoto Y, Uchimiya H et al. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technology 1988; 6:1072-1074. 22. Shimamoto K, Terada R, Izawa T et al. Fertile transgenic rice plants regenerated from transformed protoplast. Nature 1989; 337:274-276. 23. Peterhans A, Datta SK, Datta K et al. Recognition efficiency of Dicotyledoneaespecific promoter and RNA processing signals in rice. Mol Gen Genet, 1990; 222:361-368. 24. Datta SK, Datta K, Soltanifar N et al. Herbicide-resistant indica rice plants from IRRI breeding line IR72 after PEGmediated transformation of protoplasts. Plant Mol Biol 1992; 20:619-629. 25. Fujimoto H, Itoh K, Yamamoto M et al. Insect resistant rice generated by introduction of a modified δ-endotoxin gene of Bacillus thuringiensis. Bio/Technology 1993; 11:1151-1155. 26. Hiei Y, Ohta S, Komari T et al. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis ofEnhe boundaries of the T-DNA. Plant J 1994; 6:271-282. 27. Irie K, Hosoyama H, Takeuchi T et al. Transgenic rice established to express corn cystatin exhibits strong inhibitor activity against insect gut proteinases. Plant Mol Biol 1996; 30:149-157. 28. Song WY, Wang GL, Chen LL et al. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 1996; 270:1804-1806. 29. Xu D, Xue Q, McElroy D et al. Constitutive expression of a cowpea trypsin inhibitor gene cpTi in transgenic rice plants confers resistance to two major rice insect pests. Mol Breed 1996; 2:167-173. 30. Datta K, Oliva N, Torrizo L et al. Genetic transformation of indica and japonica rice by Agrobacterium tumefaciens. Rice Genet Newsl 1996; 13:136-139. 31. Duan X, Li X, Xue Q et al. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotech 1996; 14:494-498.

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32. Nayak P, Basu D, Das S et al. Transgenic elite indica rice plants expressing cryIAc δ-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas). Proc Natl Acad Sci 1997; 94:2111-2116. 33. Reddy PM, Ladha JK, Ramos MC et al. Rhizobial lipooligosaccharide nodulation factors activateexpression of the legume early nodulin gene ENOD12 in rice. Plant J 1998; 14:693-702.

34. Datta SK. Transgenic cereals: Oryza sativa (rice). In: Vasil IK, ed. Molecular Improvement of Cereal Crops. Vol 5, Dordrecht: Kluwer Academic Publishers, 1999:149-187.

CHAPTER 23

Plant Programmed Cell Death and Environmental Constraints— Adenylate Homeostasis and Aerenchyma Formation H. Uchimiya, P. K. Samarajeewa and M. Kawai

Introduction

A

denylate kinase (ATP:AMP phosphotransferase) is known to supply ADP in energyproducing systems. Adenylate metabolism is directly associated with energy-producing metabolism under conditions of O2 deficiency. The enzymatic activity of adenylate kinase was stimulated in submerged rice seedlings. This activity was enhanced in every organ of the submerged plants. Treatment with N2 gas had the same effect as submergence, suggesting that O2 depletion is a major cause of the stimulation of adenylate kinase activity. Furthermore, different induction patterns of enzymatic activity were seen in two rice varieties, FR13A and IR42, which are, respectively, tolerant and intolerant of complete submergence. NaCl-induced adenylate kinase activation was confirmed in the indica rice cultivar IR28 susceptible to salinity stress, but not in the NaCl-tolerant indica cultivar Nona Bokra. In salt sensitive rice plants, adenylate kinase may play some role in adenylate homeostasis for the early stages of rice seedlings subjected to salt stress. Cellular events which occur before cell collapse were examined in the root cortex of rice during aerenchyma formation. The cell collapse started at a specific position in the mid-cortex. These cells were distinct in shape

from those located to the periphery. Cells destined to collapse expanded more radially than in an anti-radial (tangential) direction before death. Furthermore, cell collapse was preceded by acidification and loss of plasma membrane integrity in cells of the mid-cortex. Sequential death of neighboring cells followed a radial path. Further analysis of the role of NaCl in suppression of cortical cell death was confined to the delay of early stages of cell collapse, which was caused by tonoplast disruption and plasma membrane destruction.

Stimulation of Adenylate Kinase in Rice Seedlings under Submergence Stress Introduction Higher plants are exposed to numerous environmental pressures, such as changes in temperature, dehydration, salinity stress and other adverse soil conditions.1 To survive such stressful conditions, plants have acquired mechanisms by which they adjust their gene expression to adapt to these various environmental constraints. Unlike other graminaceous monocots, rice (Oryza sativa L.) is extremely tolerant of submergence stress, which is one form of abiotic stress to which land plants are exposed. The biochemical mechanisms which

Proceedings of the 12th Toyota Conference: Challenge of Plant and Agricultural Sciences to the Crisis of Biosphere on the Earth in the 21st Century, edited by Kazuo Watanabe and Atsushi Komamine ©2000 Eurekah.com.

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

underlie the adaptation of plants to O 2 deficiency are still not completely understood, but high fermentative metabolism is important for tolerance of this condition.2-4 Adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) is known to supply ADP in energy-producing systems. This is a monomeric enzyme that catalyzes the interconversion of adenine nucleotides according to the equation ATP + AMP2ADP.5 It has been postulated that adenylate metabolism is directly associated with energy-producing metabolism under conditions of O2 deficrtant role in maintaining the energy charge of the adenylate pool.5-6 It would be interesting to examine whether any changes occur in this system in response to submergence stress. We demonstrated that rice adenylate kinase complemented the mutation in a temperature-sensitive adenylate kinasedeficient mutant strain of Escherichia coli.7 Anti-adenylate kinase antibody reacted with a 27 kDa protein in callus, root, and leaf tissues. A cellular fractionation analysis showed that these proteins were mainly located in the cytosol and mitochondrial fractions. Tissue printing immunoblot analysis revealed that adenylate kinase protein was strongly expressed in vascular tissues.8,9 Furthermore, we proposed that sodium chloride stimulated adenylate kinase in several salt-sensitive rice varieties.10 Due to the role of this enzyme in the generation of energy, it is important to investigate the enzymatic activity of adenylate kinase in plants that are known to adapt to O2 deficiency. We examined adenylate kinase activity in rice seedlings that had been subjected to submergence.11

in unsubmerged control plants. The concentration of soluble protein in submerged plants showed a tendency to be decreased compared to controls, and both values slightly declined with incubation. Estimated total adenylate kinase activity in each shoot also indicated enhanced activity in submerged seedlings at 72 h. Adenylate kinase activity in each organ was measured after submergence for 72 h, and relative induction against control plants was calculated. Adenylate kinase specific activity was enhanced in every organ by submergence. The comparison of total activity in each organ indicated that strong enhancement was shown in coleoptile and endosperm. Effects of Partial Submergence and N2 Gas on Adenylate Kinase Activity To determine whether the induction of adenylate kinase activity by submergence was due to oxygen deficiency or to some other aspect of submergence, rice seedlings were exposed to partial submergence or to nitrogen gas. The adenylate kinase activity in partially submerged seedlings was similar to that in aerobic seedlings (control). However, nitrogen gas alone stimulated adenylate kinase activity, which reached a maximum 72 h after the start of treatment. The changes in protein concentrations were similar in all seedlings.

Results

Adenylate Kinase mRNA Accumulation in Rice Seedlings by Submergence To examine the level of adenylate kinase mRNA in response to submergence, Northern blot analysis was carried out using adenylate kinase cDNA as a probe. The relative amount of transcripts under submergence stress reached a peak at 24 h of treatment.

Effects of Submergence on Rice Seedlings When 4 day old rice seedlings were entirely submerged in water, a substantial reduction in the rate of root growth, with shrinking and curling, became apparent within 24 h. Prolonged exposure to submergence (72 h) suppressed the growth of roots and shoots, but stimulated elongation of coleoptiles. The adenylate kinase activity in shoots of submerged plants was elevated at 72 h compared to that

Adenylate Kinase Activity in Submergence Tolerant and Intolerant Rice Varieties FR13A and IR42 are, respectively, tolerant and intolerant of submergence.12 A substantial reduction in shoot growth was observed when 4 day old IR42 seedlings were entirely submerged. Shoots of the two varieties were used for enzyme assay to determine whether different patterns of adenylate kinase activity could be seen. The changes in adenylate kinase

Plant Programmed Cell Death and Environmental Constraints activity were different; the maximum adenylate kinase activity in FR13A and IR42 occurred at 24 h and 96 h, respectively. The estimated total activity in a shoot also demonstrated similar results; a tremendously high amount of adenylate kinase activity, 14.6 m units/shoot, was shown in submerged FR13A at 24 h. The other samples, control IR42, submerged IR42 and control FR13A, indicated 6.6, 5.6, and 5.2 m units/shoot at 24 h, respectively. The changes in protein concentration between control and submerged seedlings were similar in each variety.

Discussion Rice plants adapt well to submergence. Among graminaceous monocots, rice plants possess extremely well developed aerenchyma tissues in their leaves, as well as in their roots. These air spaces allow rice plants to survive under heavily flooded conditions.13,14 However, severe flooding leads to complete submergence of plants. Under such conditions, oxidative phosphorylation in mitochondria is inhibited.15,16 Thus, plants are limited to the production of ATP via the glycolytic pathway.3,17,18 The effects of anaerobiosis on gene expression have been studied extensively. Transcriptional induction under anaerobic conditions has been investigated for enzymes involved in alcoholic fermentation and glycolysis such as alcohol dehydrogenase, 9,20 glyceraldehyde phosphate dehydrogenase21 and α-amylase.22 Umeda and Uchimiya 23 analyzed the expression levels of genes associated with glycolysis and alcohol fermentation in rice plants under submergence stress. They reported that two types of gene (type I and type II) with respect to the accumulation of mRNA were expressed in response to submergence stress. Transcripts of type I genes, such as the genes for glucosephosphate isomerase, phosphofructokinase, glyceraldehydephosphate dehydrogenase and enolase, reached a maximum level after 24 h of submergence. In contrast, transcripts of type II genes, genes such as those for aldolase and pyruvate kinase, reached a maximum level after 10 h of submergence. This latter pattern coincides with the expression pattern of a gene for ribosomal protein. In

249

the case of adenylate kinase, the accumulation of mRNA was similar to that of type I genes, and reached a maximum at 24 h after the start of submergence. When rice seedlings were only partially submerged, the activity of adenylate kinase was not induced, which is similar to the expression patterns of type I genes. The difference between the times of the maximum enzyme activity (72 h) and mRNA accumulation (24 h) in adenylate kinase may be due to the stabilities of protein or mRNA, or to some other factors. Treatment with N2 gas was as effective as submersion in inducing adenylate kinase activity, which suggests that oxygen deficiency might be a major factor in the induction of adenylate kinase activity by submergence stress. It seems likely that an oxygen deficit increases the transcription of genes for adenylate kinase and several other proteins associated with the anaerobic production of energy. Adenylate kinase activity was highly induced in coleoptile and endosperm under submergence conditions. In rice plant, the elongation of coleoptile is a well known characteristic of submerged seedlings. In young seedlings, energy supply for growth is dependent on starch breakdown in endosperm. Among several cereal seeds, only rice is able to degrade the starchy endosperm under anaerobic conditions.24 It is interesting that stimulation of adenylate kinase activity was shown in such organs in submerged conditions. Enzyme assay using the submergence tolerant rice FR13A and submergence intolerant IR42 showed induction in FR13A after 24 h of submergence. In IR42, adenylate kinase activity continued to increase steadily to 96 h. Umeda and Uchimiya23 analyzed the mRNA level of glycolysis and alcohol fermentation genes, and considered that the transcript levels of type I genes (glucose phosphate isomerase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase and enolase) may change in FR13A. These results suggest that the expression of adenylate kinase may be coregulated with several glycolysis and alcoholic fermentation genes.

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Sodium Chloride Stimulates Adenylate Kinase Level in Seedlings of Salt-Sensitive Rice Varieties

of adenylate kinase activities by NaCl treatment was observed in roots and shoots of IR28, but not of Nona Bokra.

Discussion Introduction Adenylate kinase (AK) has been reported to occur as a monomeric protein in both prokaryotes and eukaryotes.6 The enzyme possesses a biologically important housekeeping function in the regulation of energy metabolism. Thus, it is abundant in metabolically active tissues. Adenylate kinase is important to maintain adenylate nucleotide homeostasis in plant cells that are being subjected to various abiotic stresses. Analysis was conducted on the level of AK protein in rice seedlings under salinity stress.10

Results Growth Responses and Adenylate Kinase Activity of Seedlings Salinity stress suppressed root elongation more than shoot growth. Adenylate kinase activities were also compared in seedlings grown under the same conditions. There were no significant differences in adenylate kinase activities in shoots, whereas in roots a salt-induced enhancement of the enzyme activity was apparent. The procedure that was followed to obtain root segments yielded a uniform root growth enabling the cutting of even root segments. Comparison of enzyme activities in such root segments of seedlings grown in the absence or presence of 1.0 % NaCl for 5 d indicated that adenylate kinase activities in NaCl-treated roots were enhanced. This was observed in every root segment, with the most distal segment showing the highest activity. Adenylate Kinase Activity in Different Rice Varieties In order to know whether or not the activation of adenylate kinase was related to salinity-sensitive characteristics, enzyme activities were compared between two indica rice varieties, namely Nona Bokra (salt tolerant) and IR28 (salt sensitive). The stimulation

In the present study, the salt concentrations (0.5% under submerged conditions or 1.0% on paper towel) maintained inhibited growth of seedlings. It was evident that root growth was more inhibitory to the effects of salt. When adenylate kinase activity was measured in the japonica variety, root tissues exhibited an enhanced activity while shoot tissues did not do so. These observations were further confirmed by Western blot. Increased activity of the enzyme was not confined to a specific region of roots. Results from tissue print analysis suggested that though adenylate kinase is mainly localized to vascular tissues and other peripheral tissues under saline conditions, induced expression of the protein can occur even in cortical tissues. Adenylate kinase is found in several subcellular localizations. In rice, localization of the enzyme at tissue and cellular levels has been done previously . 9 It is distributed mainly to the cytosol fraction. Therefore, in the absence of chloroplastic fractions, the increased enzyme activity in root tissues, shown here in several experiments, can possibly be attributed to the increase in cytosol fraction. However, no remark can be addressed to the observed difference between roots and shoots in their adenylate kinase activity, though root growth was shown to be more sensitive to salinity. It remains to be known whether there is any correlation between high sensitivity of root growth and increased response of adenylate kinase in roots to salinity stress. In order to see whether there is any varietal difference for the relationship between salt stress and adenylate kinase activity, a salt tolerant variety and a salt sensitive variety were used. Induction of enzyme activity was seen in the salt sensitive variety. This may imply that in salt sensitive rice plants adenylate kinase may play some role in adenylate homeostasis in response to

Plant Programmed Cell Death and Environmental Constraints salt stress. The response of the adenylate kinase level to salinity stress is an interesting phenomenon.

Dissection of Programmed Cell Death in Root Cortex in Rice

251

lecular trafficking through plasmodesmal connections are also described. Most importantly, a degradation pattern of cortical cells occurred in the cell file, where molecular movement to radial direction was dominant.29

Results Introduction Programmed cell death has been observed in various developmental processes in higher plants. Such events are characteristic of xylem development,25 tapetum cell degradation to sustain pollen development,26 sexual organ formation27 and other processes. The presence of air-filled spaces known as aerenchyma in numerous plant species is considered to be an important anatomical adaptive feature necessary for their survival under flooded conditions. Ample evidence has been presented to show that this system provides a diffusion path of low resistance for the transportation of oxygen from aerial plant parts, roots or rhizomes, thus enabling the plants to grow in a waterlogged, O2-deficient environment.13 Thus, plants with highly developed root porosity show better oxygen transport, which contributes to their survival under flooded conditions. The formation of porosity in roots under flooded conditions is common in a number of plant species well adapted to waterlogged conditions.16 Under waterlogged conditions, many plant species develop aerenchyma in their roots. For instance, in maize roots air space development was caused by endogenous as well as exogenously applied ethylene.28 Gas-filled spaces in rice arise from the death of certain cortical cells, and from separation of cell walls from adjacent cells so that the radial walls from the collapsing cells aggregate, forming “forks” leaving a large gas-filled or lacunal space between them. The cell collapse in rice roots thus occurs selectively and is regulated by a naturally programmed degenerative processes.14 We demonstrate here that the initiation of cell death in cortical cells of rice roots is characterized by spatial acidification and membrane leakage in specific cell positions. Anatomical characterization of rice cortical cells and the possible existence of macromo-

Cell Collapse Occurred in Specific Locations in the Root Cortex Aerenchyma formation was examined in primary seminal roots from seeds either presoaked or not in water for 24 h before germinating on wet paper towels. Roots were divided into different sections. The soaking treatment stimulated the rate of cavity formation in roots. Little porosity was observed toward the tip. Cell collapse occurred in the central cortical cells, and this collapse then expanded radially to include peripheral cells. Rice root has synchronous and distinctively oriented cortical cell files. Furthermore, the number of cortical cells of rice is less than in species such as maize and Phragmites australis, which also show cortical cell death. In roots of 70 day old rice plants, the highly developed aerenchyma is present in the cortex. However, not all the cortical cells collapse; the outermost or the innermost cells remain intact. The remains of the collapsed cortical cells form “forks” or “spokes of a wheel”. To determine where cells first collapsed, the cortical cells were numbered radially, 1 to 7 from the inside. Using young seminal roots (20 mm long, derived from unsoaked seeds), the position of the first cells which underwent lysis in the radial files was determined in 80 different cross sections. Cells at position five located near the center of the cortex showed the highest incidence of early lysis. Observation of Cell Death Using Dye Probes We analyzed the frequency and location of cells staining with neutral red, which reddens visibly at low pH. The oldest regions of the root contained the most red cells. The first cells to stain with neutral red (section II) were at position 5 in the cortex. Thus, acidification was apparent in the mid-region of the cortex.

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It is assumed that Evans blue can only be taken up by cells where plasma membranes were damaged. Thus, tissues excised from section III were treated with 2.5% Evans blue. Cells staining with this dye appeared most frequently in the central cell cortex. Cells stained with Evans blue in older tissue extended radially away from the mid-region of the cortex. The cell positions that stained most readily with Evans blue were also those staining strongly with neutral red. Although there were differences in the numbers of cortical cells and patterns of root growth, similar results using neutral red and Evans blue were obtained with seminal roots of maize. Rice roots, however, have some advantages over maize because of distinctive cell positions allowing accurate observation of position-related cell death. Cell to Cell Movement in Cortex Once cell collapse has started, neighboring cells become stained with Evans blue, suggesting, perhaps, radial movement of a solute associated with cell death. To understand the cell to cell plasmodesmal connection in root cortical parenchyma, we injected Lucifer yellow (LY) and FITC conjugated dextran (F-dextran) of different molecular weights into root cortical cells. We used cortical tissues which had been treated with Evans blue for such microinjection experiments. In cells which did not stain with Evans blue, there was a distinctive molecular exclusion limit between 9.3-19.6 kDa in the cortical cells. Regardless of the root position, intercellular movement of 4.4 kDa F-dextran was also observed. In every case tested, we saw no evidence of leakage of injected probes from unstained cells. We found that about 50% of the cells that stained with Evans blue showed leakage of injected substances, which may have been caused by the loss of membrane integrity. Amounts of molecules in neighboring cells declined compared with those in injected cells. More cells (67%) showed radial transfer of F-dextran of 9.3 kDa than with 4.4 kDa (27%) in unstained cells. These results indicated that movement of 9.3 kDa molecules

was predominantly in the radial direction, similar to the radial pattern of cell death.

Discussion The objective of this investigation was to dissect the events leading to cortical cell death, which eventually resulted in gas-space formation in plant roots. Investigation focused upon the spatial and temporal development of cortical cell death in rice roots. The most significant finding is that cell death is initiated at a specific cell position. The first cells to collapse were located at the center of the cortical tissues surrounding the stele. Cells in this position were characterized by shorter length, and a larger radial diameter, than other cortical cells. It appears that cells destined to collapse expand in a radial direction before dying. It has been reported that anoxia causes cytoplasmic acidosis in maize root tips30 and hypoxia stimulates the formation of aerenchyma in plant roots.31,32 However, the roots in our study were neither hypoxic or anoxic; staining with neutral red in the central cell position (position 5 and neighboring cells) may indicate cytoplasmic acidification resulting from loss of tonoplast integrity and diffusion of H+ from the vacuole. Once cell collapse occurred, neighboring cells were systematically destroyed in a radial direction in cortical parenchyma tissues. As was noted, before cell death occurred, expansion of the cell in the radial direction was apparent. This may be due to relaxation of cell wall matrix and development of plasmodesmal connections in the radial direction to enable the transmission of molecules, which may be related to cell death. The results of microinjection indicate that cells that were not stained with Evans blue showed a molecular exclusion limit between 9.3-19.6 kDa. We did not observe any leakage of fluorescent substances in such cells. With regard to the direction of movement, 67% of cells showed radial transfer with F-dextran 9.3 kDa. These results indicate that the direction of cell to cell movement of large molecules (9.3 kDa) in cortical cells is mainly radial, which coincided with the direction of cell

Plant Programmed Cell Death and Environmental Constraints death. The leakage of substrate in half of the cells stained with Evans blue may indicate that these cells are in a late stage of cell death. In mesophyll cells, the exclusion limit for movement has been shown to be below 1 kDa.33 We noted that cortical cells of roots permit plasmodesmal movement up to 9.3 kDa, suggesting the possible existence of macromolecular trafficking through plasmodesmata in root cortical cells. Cell-specific acidosis and membrane breakdown appear to coincide with cortical cell death. Since high doses of H2O2 induce cell death in higher plants,34 it is interesting to speculate that systemic spread of cell death may be due to H2O2 produced by oxidative burst. It would be interesting to further investigate the interaction of these signals in conjunction with ethylene, which has been known to stimulate aerenchyma formation.

Effects of NaCl on Cortical Cell Death Introduction Mechanisms underlying salt stress in plants are complex not only physiologically but also genetically. Responses to salinity stress at the whole plant level are very variable and depend on a number of factors such as plant age, tissue organization and distribution of ions throughout the plant body, and the number of salt ions in the external environment.35 In a saline environment, the first reaction of plants to the adverse effects of low external water potential is uptake of salt ions and their accumulation to high intracellular concentrations.36 Entry into part of the root can occur along apoplastic pathways. The salt tolerance of the plant depends decisively on the tolerance of the roots.37 The mechanisms underlying root growth inhibition and other cellular responses need to be examined in detail in order to understand whole plant response. As yet there have been no attempts to examine the internal characteristics of rice roots when challenged by NaCl. The effects of NaCl on cell proliferation, cell enlargement and cell collapse were analyzed.38

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Results Effects of NaCl on Aerenchyma Development in Roots The basic anatomical structures appeared unchanged by salt stress. Control roots showed some cell collapse at 5-10 mm from the root tip in the mid-cortex. More extensive cortical cavities were evident in older parts further from the root tip. The percentage aerenchyma was measured for cross sections of 20 mm and 30 mm long roots. Maximum gas spaces of about 25% for 20 mm long roots and about 30% for 30 mm roots were recorded in control roots. Under salt stress, significantly less aerenchyma was observed throughout the roots irrespective of growth stage. No more than 12% gas space was found in the middle portions (15-20 mm from root tip) of 30 mm roots. To observe the progress of cell death with time, aerenchyma formation in roots at different intervals was analyzed. Only a limited amount of aerenchyma was observed under salinity stress from 60 h after germination, while in controls gas spaces were clearly developed after only 36 h. Effects of NaCl on Cell Number and Cell Size in Cortical Cells of Roots Cell number, cell length and cell diameter measurement in cortical tissues of 20 and 30 mm roots are given, respectively. The cell number was substantially limited in almost every portion of the root tip (0-1.2 mm) under saline conditions. The cell length increased earlier and more closer to the root tip in salt-stressed roots, shortening the phase between cell division and cell elongation. Final cell length (~80 µm) in root portions proximal to the root tip was similar in control and salt-stressed roots. With the exception of cells in the apical 1 mm, there was no significant difference in cell diameter between treatments. The average diameter for mature cells in the middle cell position was approximately 25 µm.

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Discussion The present studies indicate that cell death can be delayed by salt stress. Where the functional properties of aerenchyma (i.e., aeration) are concerned, this could be an important issue, since some rice plants are confronted both by salt and waterlogged conditions in the field. In mature rice plants, reduced aeration would lead to physiological distress. Responses to salinity stress are often expressed as anatomical and cellular changes.39 In the present work, compared to control roots the basic anatomical structure was retained in roots subjected to NaCl. However, higher concentrations of salt may be expected to cause irregularities such as lessening of root diameter and delaying of differentiation.40 This notion is in agreement with present observations. We examined changes in the dimensions of cortical cells from the cell division phase to the cell death phase. The measurements were confined to central cortical cells in the root zone, because cell death initiation occurred in this central cell portion.29 Hence, no significant difference was seen in final cell length between longitudinal cell files. As observed in other roots41 no clear demarcation was observed between the two phases (i.e., cell division and elongation); both cell division and elongation occur in the same zone. The limited number of cells observed in the cell proliferation phase under salt stress suggests that the zone of cell division was shortened. The zone of cell elongation began closer to the root tip, implying that there was a more rapid transition from cell division to cell elongation. Ishikawa and Evans41 named the region between the zones of cell division and cell elongation the “distal elongation zone.” The cells in this zone react differently from cells in other zones in response to stimuli. Thus, salt stress seems to stimulate the transition phase from cell division to elongation, suggesting a shift in the elongation zone toward the root tip.

Acknowledgments This research was supported by Grants-inAid for Scientific Research from the Ministry of Education, Culture and Science, Japan; by a

grant from the Rockefeller Foundation; and by a grant from the Ministry of Agriculture, Forestry and Fisheries, Japan.

References 1. Dhariwal HS, Kawai M, Uchimiya H. Genetic engineering for abiotic stress tolerance in plants. Plant Biotech 1998; 15:1-10. 2. Bertani A, Brambilla I, Menegus F. Effect of anaerobiosis on rice seedlings: Growth, metabolic rate, and fate of fermentation products. J Exp Bot 1980; 31:325-331. 3. Raymond P, Al-Ani A, Pradet A. ATP production by respiration and fermentation, and energy charge during aerobiosis and anaerobiosis in twelve fatty acid starch germinating seeds. Plant Phys 1985; 79:879-884. 4. Setter TL, Ella ES. Relationship between coleoptile elongation and alcoholic fermentation in rice exposed to anoxia. I. Importance of treatment conditions and different tissues. Ann Bot 1994; 74:265-271. 5. Atkinson DE. The energy charge of adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochem 1968; 7:4030-4034. 6. Noda LH. Adenylate kinase. In: Boyer PD, ed. The Enzymes. New York: Academic Press, 1973:279-305. 7. Kawai M, Kidou S, Kato A, Uchimiya H. Molecular characterization of cDNA encoding for adenylate kinase of rice (Oryza sativa L.). Plant J 1992; 2:845-854. 8. Kawai M, Uchimiya H. Biochemical properties of rice adenylate kinase and subcellular location in plant cells. Plant Mol Biol 1995; 27:943-951. 9. Kawai M, Uchimiya H. Tissue-specific localization of adenylate kinase in rice (Oryza sativa L.) plants. J Plant Phys 1995; 146:239-243. 10. Samarajeewa PK, Kawai M, Anai T et al. Sodium chloride stimulates adenylate kinase level in seedlings of salt-sensitive rice varieties. J Plant Phys 1995; 147:277-280. 11. Kawai M, Umeda M, Uchimiya H. Stimulation of adenylate kinase in rice seedlings under submergence stress. J Plant Phys 1998; 152:533-539.

Plant Programmed Cell Death and Environmental Constraints 12. Jackson MB, Waters I, Setter T et al. Injury to rice plants caused by complete submergence; a contribution by ethylene (ethene). J Exp Bot 1987; 38:1826-1838. 13. Armstrong W. Radial oxygen losses from rice roots as affected by distance from the apex, respiration and water logging. Physiol Plant 1971; 25:192-197. 14. Webb T, Armstrong W. The effects of anoxia and carbohydrates on the growth and viability of rice, pea and pumpkin roots. J Exp Bot 1983; 34:579-603. 15. Sachs MM, Ho THD. Alteration of gene expression during environmental stress in plants. Ann Rev Plant Phys 1986; 7:363-376. 16. Justin SHFW, Armstrong W. The anatomical characteristics of roots and plant response to soil flooding. New Phytol 1987; 106:465-495. 17. Davis DD. Anaerobic metabolism and the production of organic acids. In: Biochemistry of Plants, London: Academic Press 1980; 2:581-611. 18. Dolferus R, deBruxelles G, Denis E et al. Regulation of the Arabidopsis Adh gene by anaerobic and other environmental stress. Ann Bot 1994; 74:301-308. 19. Cobb BG, Kennedy RA. Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings. Plant Cell Environ 1987; 10:633-638. 20. Xie Y, Wu R. Rice alcohol dehydrogenase genes: Anaerobic induction, organspecific regulation and characterization of cDNA clones. Plant Mol Biol 1989; 13:53-68. 21. Ricard B, Rivoal J, Pradet A. Rice cytosolic glyceraldehyde 3-phosphate dehydrogenase contains two subunits differentially regulated by anaerobiosis. Plant Mol Biol 1989; 12:131-139. 22. Perata P, Guglielminetti L, Alpi A. Mobilization of endosperm reserves in cereal seeds under anoxia. Annals Bot 1997; 79:49-56. 23. Umeda M, Uchimiya H. Differential transcript levels of genes associated with glycolysis and alcohol fermentation in rice plants (Oryza sativa L.) under submergence stress. Plant Phys 1994; 106:1015-1022.

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24. Perata P, Geshi N, Yamaguchi J et al. Effect of anoxia on the induction of α-amylase in cereal seeds. Planta 1993; 191:402-408. 25. Mittler R, Lam E. In situ detection of nDNA fragmentation during the differentiation of tracheary elements in higher plants. Plant Phys 1995; 108:489-493. 26. Davies RA, Singh MB, Knox RB. Identification and in situ localization of pollenspecific genes. Int Rew Cytol 1992; 140:19-34. 27. Delong A, Calderon-Urrea A, Dellaporta SL. Sex determination gene TASSELSEED2 of maize encodes a short- chain alcohol dehydrogenase required for stage-specific floral organ abortion. Cell 1993; 74:757-768. 28. Drew MC, Jackson MB, Giffard S. Ethylene-promoted adventitious rooting and development of cortical air spaces (aerenchyma) in roots may be adaptive responses to flooding in Zea mays L. Planta 1979; 147:83-88. 29. Kawai M, Samarajeewa PK, Barrero RA et al. Cellular dissection of the degradation pattern of cortical cell death during aerenchyma formation of rice roots. Planta 1998; 204:277-287. 30. Roberts JKM, Callis J, Wemmer D et al. Mechanism of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia. Proc Natl Acad Sci USA 1984; 81:3379-3383. 31. Drew MC, Jackson MB, Giffard SC et al. Inhibition by silver ions of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to exogenous ethylene or to oxygen deficiency. Planta 1981; 153:217-224. 32. Jackson MB, Fenning TM, Drew MC et al. Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen. Planta 1985; 165:486-492. 33. Lucas WJ (1995) Plasmodesmata: Intercellular channels for macromolecular transport in plants. Curr Opin Cell Biol 1995; 7:673-680. 34. Levine A, Tenhaken R, Dixon R et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 1994; 79:583-593.

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35. Yeo AR, Flowers TJ. Accumulation and localization of sodium ions within the shoots of rice (Oryza sativa L.) varieties differing in salinity resistance. Physiol Planta 1982; 56:343-348. 36. Greenway H, Munns R. Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant Phys 1980; 31:149-190. 37. Jeschke WD, Wolf O. External potassium supply is not required for root growth in saline conditions: Experiments with Ricinus communis L. grown in a reciprocal split-root system. J Exp Bot 1988; 39:1149-1167. 38. Samarajeewa PK, Barrero RA, UmedaHara C et al. Cortical cell death, cell proliferation, macromolecular movement and rTipl expression pattern in roots of rice (Oryza sativa L.) under NaCl stress. Planta 1999; 207:354-361.

39. Hajibagheri MA, Yeo AR, Flowers TJ. Salt tolerance in Suaeda maritima (L.) Dum. fine structure and ion concentrations in the apical region of roots. New Phytol 1985; 99:331-343. 40. Serrato-Valenti G, Ferro M, Ferraro D, Riveros F. Anatomical changes in Prosopis tamarugo Phil. seedlings growing at different levels of NaCl salinity. Ann Bot 1991; 68:47-53. 41. Ishikawa H, Evans ML. Specialized zones of development in roots. Plant Physiol 1995; 109:725-727.

SECTION VII INTRODUCTION

Biotechnology of Woody Plants S. Kitani

R

apidly increasing population is enhancing the expansion of slash-and-burn agriculture in tropical areas. Cultivation in the arable land obtained by slash-and-burn agriculture becomes impossible several years later. This is the cause of desertification in the tropical area. Seventeen million hectares of the forests have been deforested or destroyed per year. Of the tropical forest it is known that extremely variant plants, animals and microbes are alive there. By destruction of the tropical forest, the treasury of such various genetic resources has been disappearing rapidly, without contributing to future human beings. Under these conditions, we must stop the decrease of tropical forests and try to reforest, but the area of reforestation per year is only equal to 10% of the decreased area. The forest area in temperate and cold climate regions has also been decreasing. On the other hand, the world demand for wood products is predicted to rise sharply in the next decade, and shortages have been forecasted. We must increase the productivity of woody plants to improve the outlook for these problems. It is expected that “biotechnology of woody plants” is useful to the analysis and preservation of a gene resource, and can also contribute to the reproduction of the destroyed forest. “Biotechnology of woody plants” includes micropropagation, genetic engineering and marker-aided selection. The possibilities of the “biotechnology of woody plants” are described below.

Micropropagation Micropropagation is an advanced technology for producing a large number of genetically superior and pathogen-free transplants in a limited time and space. Professor Kozai has reported on photoautotrophic or sugar-free micropropagation. Moreover, Dr. Gupta has presented the possibility of micropropagation by asexual embryogenesis.

Genetic Engineering By recent progress in genetic engineering, it would be possible to introduce expected traits for a comparatively short period of time. Genetic engineering is expected to be useful in woody plant breeding. Dr. Ebinuma has reported a new transformation system using oncogenes of Agrobacterium as a selectable marker. And, Professor Morohoshi has presented the possibility of transgenic plants having lower lignin content. Moreover, the possibility of acid soil-tolerant trees was also mentioned in this session.

Molecular Tools Progress in molecular genetics and biotechnology is bringing us the tools necessary for the production of valuable compounds out of these natural resources, without harming the ecosystems of origin. Professor Montagu has reported molecular tools for capturing the value of the tropical rain forest.

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CHAPTER 24

Molecular Tools for Capturing the Value of the Tropical Rain Forest M. Van Montagu

Introduction

T

he ongoing destruction of tropical rain for ests, estimated now to be proceeding at a rate of 154,000 km2 per year, poses serious concerns not only for ecologists, but also for all responsible inhabitants of this planet. The rain forest harbors the highest diversity in plants, insects and microorganisms of all habitats. The irreversible loss of this biodiversity represents a tremendous setback for both the “classical” biologists as well as for molecular geneticists and biotechnologists. How can the scientist combat this? Governments of many developing countries have great difficulty in recognizing the value of these forests in light of more pressing economic problems. Hence, they permit the slash-and-burn agriculture of landless farmers, or they collect immediate revenue by licensing destructive logging or, worse still, by authorizing the highly polluting and poisoning action of the “garimpeiros” (gold diggers). The only value recuperated from such indiscriminate logging or burning is additional acreage for agriculture. Sometimes the land surface that is thus recuperated is used for certain monocultures, such as rubber or palm tree plantations, but often, as in Brazil, the soil is so poor that within only a few years the area has to be abandoned. Traditionally, biologists consider that biodiversity is of intrinsic value for humanity in itself. It is, after all, a monument to life heritage. I would propose, however, that it is of paramount importance to demonstrate the social and economic values of biodiversity. Otherwise, it will be difficult to convince the

developing countries to preserve the rain forests and representative freshwater and marine ecoregions that contain the majority of this biodiversity. This is the challenge scientists are facing today. The tools that are presently being developed by molecular biologists and plant biotechnologists are bringing a unique opportunity to all biologists. The impressive progress that has been made in molecular sciences is enabling identification, stock-taking and preservation of this biodiversity. It is the urgent task of the scientific community to introduce ecologists, soil scientists, agronomists, taxonomists and physiologists to the molecular approaches available and to establish together the necessary networks that can demonstrate the economic value of this biodiversity for all countries. Only then can we expect serious efforts to be made toward saving the remaining rain forests.

The Model Plant Arabidopsis Understanding plant growth and development in molecular terms is a prerequisite if one wishes to understand, rather than to just observe, how an ecological community is established, functions and deteriorates. It is also a prerequisite for engineering economically improved plants that are better adapted to a human-dominated environment. Collecting and collating this knowledge has been and still remains a huge task. Up until ten years ago, very few plant genes had been cloned. As a result, the molecular mechanisms underlying physiological processes

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could only be advanced in a few instances, and the concepts of plant signal transduction pathways or plant cell cycle had not even been considered. The basic molecular research into such processes has been carried out by science faculties and in some far-sighted companies, but rarely in Schools of Agriculture or public or private Plant Breeding Stations. Indeed, when molecular biologists proposed to concentrate their research efforts on a model plant, such as Arabidopsis thaliana (L.) Heynh, many remained skeptical, pointing out that it was a weed, not a crop plant. However, Arabidopsis thaliana has particular advantages: It possesses a small genome (only 120,000 kb), a small size, a fast growth cycle and a large seed set (many thousands). In retrospect, the choice of this small crucifer, a close relative of some Brassica species, was an excellent one. Nearly all plant molecular biology laboratories have joined the Arabidopsis effort and results have been generated at an ever increasing pace. Today, it is possible to buy “filters” carrying 12,000 different expressed sequences of Arabidopsis genes. As a result, several steps of some important events in plant development, such as flower formation, are becoming understood at the level of molecular interactions.1 Although directed gene displacement is still not possible due to constraints in homologous recombination, efficient seed mutagenesis allows the construction of many mutant alleles. Even banks of temperaturesensitive mutants are now becoming available. Map-based cloning technology, which again was principally elaborated with Arabidopsis, enables rapid cloning of the mutated alleles and is the start of a future understanding of aspects of plant biochemistry.

The Genome Research The remarkable progress in automated DNA sequencing, and the medical and industrial interest in the human genome, have stimulated a variety of genome studies, including the systematic sequencing of some plant genomes. Again, the results obtained with the model plant Arabidopsis are very encouraging. Several industrial concerns are even rumored to have completely sequenced the genomic clone banks, which probably represent the entire Arabidopsis genome. A

concerted effort among several European, American and Japanese laboratories has already obtained nearly half of the Arabidopsis genome in an ordered and annotated sequence (http://www.tigr.org). A first analysis indicates that plants probably function with a mere 20,000 genes. Of these sequenced genes, half are not found in the databases of sequences from other organisms. A parallel effort, mainly by Japanese researchers, is taking place for rice, and the United States has also started a sequence program for corn. Taken together, all these data confirm the limited gene complexity of plant genomes, and this in turn will stimulate efforts toward the identification of the function of plant genes. In the medical field, this has already become an important priority and has even resulted in a new name for this research: functional genomics.2 Here too, new technology and instrumentation is rapidly evolving and many start-up companies specialize in supplying these on a contract basis.

Functional Genomics In a first set of attempts to visualize the levels of gene expression in individual tissues, micro-array hybridization technologies have been developed. Here, a library of clones representing as many as possible of the expressed sequences of a genome is distributed by a robot as individual microspots on 1 x 1 cm filters, called microchips. In the case of the yeast (Saccharomyces cerevisiae) genome, such filters contain approximately 6,000 spots. For larger genomes, 10 to 100-fold more clones need to be spotted, but robots capable of applying 50,000 spots per square cm of filter are already available. Another approach (Argonne National Laboratories US together with Packard Motorola and the Engelhardt Institute Moscow) based on polyacrylamide gel pads seems also very promising. Hybridization of these filters with a labeled copy of the total mRNA population of a single cell type reveals the genes that are expressed in these cells. Comparing the variation in expression under different physiological conditions, stress situations, or during development will help us understand how a complex multicellular organism functions. Or, as is presently being explored for yeast, how the crosstalk

Molecular Tools for Capturing the Value of the Tropical Rain Forest between various biochemical pathways is functioning.3 Nevertheless, an alternative approach will be needed for ecological studies in the tropical forest because such an approach depends on the availability of a complete gene library and because such libraries do not exist for most of the species under study. In these cases, amplified fragment length polymorphism4 (AFLP)-based mRNA profiling techniques are being developed. AFLP is a very efficient method for the comparison of genomes in taxonomic studies or for the analysis of the outcome of crosses in markerassisted breeding. As such, the method has been used preferentially for studying plant and microbial genomes, although work with other organisms is currently in progress. mRNA profiling that is based on AFLP allows us to detect rare messengers (down to a level of one in five million). The method is also highly reproducible and can be used to distinguish the expression of different alleles in the case of heterozygotic organisms. The rapid progress of these technologies should stimulate us to apply them now to forestry studies. Attempting to correlate on the one hand growth characteristics, pathogen resistance, wood quality, seed set and beneficial characteristics in general with the presence of characteristic messengers, on the other hand, is a great challenge.5 However, this information is urgently required because it lies at the basis of future tree selection programs.

Proteomics Functional genomics is not just restricted to mRNA identification and dosage. Progress in two-dimensional gel electrophoresis makes it possible to prepare an inventory, or fingerprint, of the protein composition of individual tissues. Often, the amount of protein detected is high enough to obtain amino acid sequences from some of the peptides of these proteins and in this way it is possible to identify the enzyme or protein present in such a gel spot. The new classes of tabletop mass spectrometers now available permits detection sensitivities down to the femtomole level as well as simultaneous N- and C-terminal sequencing of protein-derived peptides within minutes, rather than hours, as is the

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case with the traditional Edman-based N-terminal sequencing.

Metabolite Profiling It is now becoming possible to measure the fluctuations of key metabolites in some tissues. Characterization of the metabolic profiles of plants will help us to identify the biochemical perturbations underlying altered gene expression, which may not be straightforward in the absence of a clear phenotype. For a wide variety of solutes, the further development of capillary electrophoresis (CE) has enormous potential in this direction because of its high peak capacities, resolution and sensitivity of detection. In combination with mass spectrometry (CE-MS), this raises the possibility of the rapid and unequivocal identification of the analyzed compounds. Already, analysis of the contents of a single cell of a citrus fruit has been achieved, and studies on the release of biogenic amines from individual nerve cells have been published. There is no doubt that in the future CE-MS will become an essential tool in helping to identify the biosynthetic pathways of plant secondary metabolites. This identification is essential if one wants to explore and secure the value of the compounds extracted from tropical forest species. Indeed, a combination of characterization (by CE-MS) of the intermediates in a biosynthesis and the identification of the enzymes present in the biosynthetic tissues must allow us to unravel the pathway and to clone the genes encoding it. Further recombinant technology work can then allow the engineering of the pathway in more economically favorable plants. Another aspect of metabolite profiling is the analysis of the volatile molecules of a plant. Often such volatiles play an important role in plant signaling. An extensive literature is already available on the ecological significance of plant volatiles, for instance during pathogen interactions, as attractant of pollinators or in plant-plant communication. There are even indications that the capacity of some plants to conquer a given territory and to form an ecological niche depends on their ability to release volatile molecules. To really substantiate these data, we urgently need the molecular proof through the identification

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of the genes encoding the information for the regulated synthesis of these compounds, as well as to identify the receptor molecules and the signal transduction pathways involved. This ambitious project is feasible thanks to the high separation capacities and sensitivity of gas chromatography, when coupled to mass spectrometry (GC-MS), and particularly thanks to new approaches in head-space analysis to detect minimum amounts of chemicals in the gas phase surrounding plant organs. Traditionally, these compounds were adsorbed and concentrated on the surface of, for instance, active carbon. A temperature chase transported them to the GC-MS analyzer. Such a procedure functions well for hydrophobic compounds that can be released from the adsorbent at nondestructive temperatures. More recently, gum phase columns are being used for the absorption of gaseous compounds, and this requires only a mild temperature increase to release polar and hydrophobic compounds with a minimum of degradation. One may safely predict that many new compounds, other than the already well studied terpenoids, aromatic compounds or esters, will be identified. Coupling this compound identification with judicious field work and with the introduction of the molecular biologist’s tools will open a completely new area of molecular ecology.

Biosynthetic Pathways for Secondary Metabolites The importance of secondary metabolites of plants is still very much underestimated. Organic chemists have often expressed an interest in these “natural compounds,” but mainly as a means to sharpen their capacity for structure determination, the reward being the elucidation of an as yet not described structural skeleton. Because an important fraction of our pharmaceuticals are still derived from plant origins, it was also often rewarding to chemically synthesize these compounds, particularly when the active component was isolated from a rare or difficult to cultivate species. Frequently, therefore, an enormous amount of effort has gone into the identification and synthesis of minute amounts of a new compound that only ended

up being an addition to the ever growing encyclopedia of known organic compounds. As discussed above, the fascinating progress in plant molecular biology has created a huge potential for the characterization of biosynthetic pathways and the physiological activities of compounds. This imparts an entirely new importance and significance to plant secondary metabolite studies. These molecules are synthesized through interactions with enzymes and have evolved in nature through their capacity to bind target macromolecules, mostly proteins. They are thus ideal starting products for the development of drugs or agrochemicals that interact tightly with a given target protein, be it a receptor, an enzyme, a signaling or a regulatory protein. Until now, although more than 8,000 proteins have been crystallized and their three-dimensional structure determined, it turns out that only 650 different protein folds have been identified.6 This means that all proteins, whether from microbial, plant, invertebrate or vertebrate origin, are built up using a limited number of three-dimensional structures. Hence, by applying a high throughput screening for compounds interacting with a given receptor protein, specific molecules can be isolated that interact with a characteristic protein fold. Such a compound will be an ideal starting molecule for further drug design. Plant molecular biologists now have the tools to isolate the genes involved in its biosynthesis and engineer the pathway in a fast growing plant. In this way, an abundant or continuous production can be assured. The compound can then be the starting material for combinatorial chemists who will synthesize variants that better fit the fold in the target protein against which a drug needs to be developed.

Tropical Diversity Studies Sustainable exploitation and efficient management of a tropical rain forest requires a thorough insight into the structure and dynamics of this ecosystem. The stability of an ecosystem is largely determined by the intraspecific genetic diversity of the different interacting species, because this diversity holds a reservoir of potential adaptations to changing environmental conditions. Although the measurement of visual traits

Molecular Tools for Capturing the Value of the Tropical Rain Forest reveals the existence of genetic variation, it does not give a good indication of the structure of diversity within populations or how this diversity is maintained.7 Progress in molecular biology has recently offered new, more powerful tools based on the polymerase chain reaction. DNA analysis visualizes the genetic information directly, independently of environmental factors, tissue or developmental stage. For this reason, molecular techniques are used more and more for the study of genetic structures of tropical ecosystems. Applications include the use of molecular markers for species identification and for the development of general sets of molecular markers with which to assess genetic diversity. The Department of Plant Genetics (University of Gent, Gent, Belgium) has initiated a collaboration project with Silvolab (French Guyana) and INRA-Bordeaux (France) in order to introduce and adapt DNA marker technology for tropical forest studies. The main objective of the project is to describe the levels and distribution of genetic diversity in a selected number of tropical tree species that display contrasting life history traits and to analyze the dynamics of this diversity. The marker methods used are AFLP and microsatellites which, combined, can reveal almost all aspects of genetic diversity and gene flow. The further development and use of sets of molecular markers will greatly facilitate future diversity studies on a large scale. Microorganisms are another crucial component of tropical forest ecosystems. Indeed, trees of tropical forests are usually dependent on obligate root symbioses, such as mycorrhizae, for water and nutrient acquisition, and for protection against soil-borne pathogens. In a recent study, the conclusion was that the mycorrhizal fungal diversity determines the plant biodiversity, as well as the ecosystem variability and productivity.8 The same molecular techniques, as applied to trees, can be used for microorganisms. This study will lead to a deeper insight into the dynamics and stability of soil beneficial organisms in their interaction with trees of tropical forests. This is essential for sustainable forest management, in protected reserves and sanctuaries or for a sustainable use of diversity.

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It will be also very useful for reforestation and agroforestry.

Forestry Research Despite their high economic and environmental values, molecular geneticists have, until recently, largely neglected trees as a research area. This is understandable, since genetic analysis of organisms with such long reproductive cycles is frustratingly slow. Nonetheless, many biochemical reactions that are important for primary and secondary cell wall formation, and thus are determinants of wood quality, should attract the attention of molecular biologists. Indeed, certain fast growing trees, such as eucalyptus and poplar, are now being studied. Many thousands of expressed sequence tags have been sequenced and as a result, a wealth of new genes has been discovered. Transgenic trees that are downregulated in enzymes involved in lignin biosynthesis have been generated. In this way, it has been possible to obtain poplar wood from which lignin can be more easily extracted. When applied, this might lead to a decreased use of chemicals during cellulose production for the paper industry. By altering the degree of lignin methylation or by altering the levels of appropriate peroxidase enzymes, transgenic poplars with different lignin composition have been obtained. This pioneering work will hopefully attract additional researchers to molecular tree research, whose input will be required if work on tropical trees is to be initiated. Selection of putatively superior trees, even within the genus Populus, is, however, a very long term process. Many genotypes flower only once every five or ten years. This means that screening the progeny for certain important economic traits involves a waiting period of 15 years until the tree has matured. Powerful molecular marker technologies, such as AFLP, can circumvent this problem by providing tree breeders early on with the necessary information to increase the efficiency of the analysis of the outcome of crosses. In our own work with fungal disease resistance (Melampsora larici-populina), bulk segregant analysis has identified rather efficiently a DNA fragment that is present in resistant trees but absent in sensitive trees.9 This

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marker is now being used in poplar breeding schemes, demonstrating the power of AFLP in breeding of trees in general.

Stronger Plants Through Plant Engineering Rather recently, the first transgenic plants reached the market place. These concern crops such as corn, soybean, cotton and rapeseed. They are transgenic through engineering of a bacterial gene. Their new properties, such as insect resistance, herbicide tolerance or yield increase through engineered hybrid vigor, are in the first place important for large scale, industrial agriculture. The increased research on molecular plant physiology is now preparing the second wave of plant products. Satisfactory results have been obtained for engineering delayed ripening of tomatoes and melons by interfering with ethylene biosynthesis.10,11 Such an approach might be attempted with some tropical fruits. Many interesting types of fruit are locally collected and consumed by people living in or near the tropical forest. These fruits are often fast ripening and sensitive to bruising, so that they cannot be transported or marketed. Initiating marker-assisted breeding and engineering firmness and delayed ripening might result in a higher value fruit. It could be the beginning of propagation in the tropics of a new type of orchard derived from fruit trees identified in the tropical forest. A completely different class of transgenic products consists in plants producing antigens or antibodies.12 At present, Medicare, as practiced in Europe, the United States or Japan, is prohibitively expensive for developing countries. Since, in the next century, 85% to 90% of the world population will be living in these areas, less expensive alternatives should be urgently pursued. Expression of antibodies has been successful. Such constructs can in principle be used for altering a biochemical reaction in the plant, for instance when the antibody is reacting with a key enzyme of the pathway. Antiviral antibodies can also be expressed, or antibodies can be produced that are needed in clinical or biochemical laboratories or even industrial productions (antibody columns). Antigens expressed in fruits

or vegetables, which are eaten uncooked, can be used for oral vaccination. For the tropics, it would be important if such developments could be done with help from the developed world.

The Changing World of Industry The major chemical and agrochemical industries now agree that they should give absolute priority to nonpolluting production processes and to the development of less polluting agrochemicals. Trying to evolve into a more sustainable industry, these industries give priority to biotechnological approaches and have declared themselves “life sciences” companies. All show great interest in plant biotechnology and in the possibility to make new products through plants. Also the science management organizations of the United States, such as the National Institutes of Health or the National Science Foundation, watch this new trend closely. These agencies just gave six grants of $500,000 US to major American companies, as well as start-up companies, for prospecting biodiversity worldwide for new active compounds and natural products. How will these multinational concerns make the transition into life science companies and achieve the necessary know-how? Monsanto and Dupont prepared for this change long since and built up in house research capacities. Other companies acquired biotechnology start-up companies to gain access to technology and bought up seed companies to allow the production of novel plants. However, the first wave of transgenic plants focused on improved biotic stress resistance, high yielding crops and engineering that allows the use of less polluting agriculture through the possibility of using new environmentally acceptable herbicides. At present, 20 million hectares of transgenic plants are already cultivated in the United States. All predictions are that in the next years this will substantially increase so that half of the large scale agriculture will be using transgenic plants.

Molecular Tools for Capturing the Value of the Tropical Rain Forest

Conclusion Today, it must be possible to demonstrate the economic value of the endangered ecosystems, such as tropical rain forest, to society. Molecular biologists have now created the tools to achieve this. To successfully reach this goal we should, however, closely note how the first plant biotechnology products were developed. It is likely that the same road will have to be followed. This involves three main stages, the discovery, the prototype development and the commercialization of the product. The innovative fundamental research will be performed at universities and public or private research institutes. In view of the immense size and the urgency of the task, close interaction and networking between different specialized teams will have to be established. Prototype development is better suited to a semi-industrial environment. This will ensure that the progress and findings of the scientists are efficiently translated into candidate prototype products. For this reason we should encourage the creation of start-up companies also for these “forest products”. Only such spin offs from research laboratories will have the necessary focus and financial strength to reach this goal. If, at the outcome, the product is a plant, a lot of breeding will then still be needed before a competitive commercial product is obtained. If it is a chemical, costly environmental toxicity tests will be required. The validation costs of such plants or chemicals are currently so high that only major “life sciences” industries can pursue this path to the end. Therefore, to really capture the potential of the tropical rain forests we urgently need to create all three stages of this process and secure a cooperative link between them.

Acknowledgments Chris Simoens, Mark Davey and Dominique Van Der Straeten are acknowledged for their help in preparing the manuscript and Martine De Cock for final layout.

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References 1. Meyerowitz EM, Somerville CR, eds. Arabidopsis; Cold Spring Harbor Monograph Series, Vol.27. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1994. 2. Bouchez D, Höfte H. Functional genomics in plants. Plant Physiol 1998; 118:725-732. 3. DeRisi JL, Iyer VR, Brown PO. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 1997; 278:680-686. 4. Vos P, Hogers R, Bleeker M et al. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res 1995; 23:4407-4414. 5. Vos P, Simons G, Jesse T, Wijbrandi J et al. The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids. Nature Biotechnol 1998; 16:1365-1369. 6. Wang Z-X. A re-estimation for the total numbers of protein folds and superfamilies. Protein Engineering 1998; 11:621-626. 7. Loveless M. Isozyme variation in tropical trees: Patterns of genetic organization. New Forests 1992; 6:67-94. 8. Van der Heijden MGA, Klironomos JN, Ursic M et al. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 1998; 396:69-72. 9. Guis M, Botondi R, Ben-Amor M, Ayub R et al. Ripening associated biochemical traits of cantaloupe charentais melons expressing an antisense ACC oxidase transgene. J Am Soc Horticult Sci 1997; 122:748-751. 10. Cervera M-T, Gusman J, Steenackers M et al. Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus. Theor Appl Genet 1996; 93:733-737. 11. Oeller PW, Min-Wong L, Taylor LP et al. Reversible inhibition of tomato fruit senescence by antisense RNA. Science 1991; 254:437-439. 12. Artnzen C. High-tech herbal medicine: Plant-based vaccines. Nature Biotechnology 1997; 15:221-222.

CHAPTER 25

Improvement of a New Transformation Method: MAT Vector System H.Ebinuma

Introduction

Transformation Procedure

W

We summarize transformation procedures of two prototype MAT vectors (pNPI132, 702) (Figs. 25.1 and 25.3). Plasmid pNPI132 has a “hit & run” cassette in which both the chimerical ipt (isopentenyl transferase)7-9 and R (recombinase) genes fused with 35S promoters are located between two directly oriented RS (recombination site) sequences (Fig. 25.1). pNPI132 transformation and selection were as follows (Fig. 25.3A): 1. Tobacco leaf segments were infected with A. tumefaciens containing a MAT vector (pNPI132), and cultured on a hormone-free MS medium without kanamycin (nonselective medium). When adventitious shoots were regenerated, they were separated from the leaf segments and transferred to the same medium; 2. After one month of cultivation, we visually identified and selected for further cultivation abnormal shoots (called ESP for extreme shooty phenotype) that lost apical dominance; 3. Normal shoots exhibiting normal apical dominance appeared from ESP shoots. We visually identified, selected and transferred these normal shoots to the same medium. These shoots grew normally and rooted. In PCR analysis, the chimerical ipt gene was present in the chromosomal DNA of the ESP shoots, but was excised from that of the “normal” shoots along with the “hit and run” cassette. These results indicate that these nomally grown

e introduce our improvement of a new transformation method (MATVS: MultiAuto-Transformation Vector System) for a practical use. In current systems, selective agents (antibiotics, herbicides etc.) and the corresponding resistance genes (selectable marker genes) are used for the selection of transgenic cells. However, these systems have three problems: 1. The selective agents have negative effects on proliferation and differentiation of plant cells; 2. There is uncertainty regarding the environmental impact of many selectable marker genes; 3. It is difficult to repeat transformation using the same selectable marker in order to pyramid desirable genes.1 The MATVS is designed to overcome these difficulties.2-4

Principle of MAT Vectors The MATVS is based on a novel concept that oncogenes (ipt or rol genes etc.) of Agrobacterium are used as selectable markers to regenerate transgenic cells and select marker-free transgenic plants.4 Commonly, oncogenes have not been used as the selectable marker gene to transform plants because the resulting transgenic plants exhibit a seriously abnormal phenotype. The MATVS is designed to remove the oncogenes from transgenic plants after a transformation by inserting them into removal elements: maize transposable element Ac;5 yeast site-specific recombination system R/ RS (Figs. 25.1 and 25.2).6

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Fig. 25.1. Construction of MAT vectors. Plasmid pNPI106 has a “hit and run” cassette in which the chimeric ipt gene with 35S promoter is inserted into Ac as a selectable marker. The pNPI132 and 702 vectors use the excision system of R/RS to remove the chimeric ipt gene or rol genes and has a “hit and run” cassette in which these marker genes and R (recombinase) genes fused with a 35S promoter are located between two directly oriented RS (recombination site) sequences. The gusA and nptII genes are unselected markers in these experiments. shoots are marker-free transgenic plants containing only desired genes. Using the pNPI106 vector, we could obtain marker-free transgenic tobacco plants from only 3 of 63 ESP lines (4.8%) by 8 months after infection of Agrobacterium.2 Also, we obtained marker-free transgenic tobacco plants from 10 of 48 ESP lines by 4 months after infection using the pNPI132 vector. Finally, marker-free transgenic tobacco plants appeared from 32 of 48 ESP lines (67%) by 8 months after infection.3 These results indicate that the MATVS could produce marker-free transgenic plants without sexual crossing and that the R/RS-type MATVS is the more practical of the two (Table 25.1). Plasmid pNPI702 has a “hit and run” cassette in which the 7.6 kb DNA fragment containing rolA, B, C, D 9-11 and the R genes with a 35S promoter are located between two directly oriented RS sequences. pNPI170 transformation and selection were as follows (Fig. 25.3B):

1. Tobacco leaf segments were infected with A. tumefaciens containing a MAT vector (pNPI702) and cultured on a hormone-free MS medium without kanamycin (nonselective medium). Hairy roots were regenerated two weeks after infection. After one month, they were separated from the leaf segments and transferred to a shootinducing medium with hormones; 2. After two months half of cultivation, transgenic shoots were regenerated from such roots. We separated these shoots and transferred to a nonselective medium for further cultivation. 3. We visually identified and selected normal plants from abnormal plants with wrinkled leaves, reduced apical dominance or shortened internodes. Commonly, crossing of chimeric plants segregates out non-chimerical progenies. Therefore, we subjected the seedlings of chimerical plants to PCR analysis and observed

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A

B

Figure 25.2. Principle of MATVS. Hormone synthetic genes ipt used for selection are removed after transformation. (A) Recombination between two directly repeated sites (RS) results in deletion of the ipt gene lying between the sites. (B) Marker-free transgenic plants are identified as normal shoots which recover apical dominance. (a): Regeneration of adventitious shoots; (b): Differentiation of abnormal shoots which have lost apical dominance; (c): Appearance of normal shoots which have recovered apical dominance.

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a

b

c

d

Fig. 25.3.A. Transformation procedure for tobacco plants using MAT vectors. Cytokinin type (pNPI132): (a): Regeneration of adventitious shoots from leaf segments on a nonselective medium (hormone-free MS medium containing 500 mg/l carbenicillin); (b): Differentiation of extreme shooty phenotype (ESP) from adventitious shoots; (c): Appearance of “normal” shoots which have recovered apical dominance; (d): “Normal” rooted plant. the segregation of non-chimerical marker-free transgenic plants with a high frequency.4 These results indicate that the MATVS (pNPI702) also could generate non-chimerical marker-free transgenics without sexual crossing, and produce them more efficiently through crossing. In addition to tobacco, regenerated shoots have been obtained in several recalcitrant species (sweet potato, snapdragon) using

MATVS. A. rhizogenes are susceptible by a wide variety of dicotyledonous plants (116 species). 12 Transgenic plants have been regenerated from hairy roots of 53 plant species. Therefore A. rhizogenes has been used to transform many recalcitrant plant species, including fruit trees and forest trees. However, the characteristic altered phenotype caused by the integrated Ri T-DNA limits their use for

Improvement of a New Transformation Method: MAT Vector System

a

b

c

d

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Fig 25.3.B. Transformation procedure for tobacco plants using MAT vectors, cont. Auxin type (pNPI702): (a): Differentiation of hairy roots from leaf segments on nonselective medium (hormone-free MS medium containing 500 mg/l carbenicillin); (b): Regeneration of adventitious shoots from hairy roots; (c): Normal shoots; (d): Abnormal shoots (wrinkled leaf). commercial genetic engineering. The MATVS enables us to overcome the disadvantage of altered phenotype and improve transformation efficiency in a wide variety of plant species.

Improvement of MAT Vectors In our evaluations of two prototype MAT vectors, we found several problems with practical use and developed new MAT vectors (pMAT21, 22, 101, pTL7, pIPT53, 54, prol103,

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Table 25.1. Transformation efficiency of tobacco plants ESP lines which formed normal shoots ESP Vector

Number

4 months after infection Number

(%)

8 months after infection Number

(%)

pNPI 106

63

2

3.2

3

4.8

pNPI 132

48

10

20.8

32

66.6

Transformation of tobacco plants using pNPI 106 and 132 vectors, and appearance of marker-free transgenic plants at hormone-free MS medium.

108) (Figure 25.4). Our improvement is as follows. 1. Cloning sites: We constructed two kinds of vectors. One is a binary cloning vector pTL7 in which multicloning sites except for an SseI site are available for desired genes, and an SseI site outside the multicloning sites, for a “hit & run” cassette of MAT vectors. The other type is MAT cassette vectors (pIPT53, 54, prol103, 108). After cloning valuable genes into multi cloning sites, a MAT cassette is put into an Sse site. 2. Second markers: A selectable marker gene (nptII) is inserted into a “hit & run” cassette (pMAT22, pIPT53, prol108) and a reporter gene (GUS) into a cassette (pMAT21, pIPT54, prol103). Now we are evaluating MAT vectors (rol genes type) containing GFP genes which enable us to easily distinguish transgenic roots from non-transgenic ones. 3. Control of recombinase: We have detected dropouts of a MAT cassette by excision events during cultivation of Agrobacterium. Therefore, we inserted an intron of Eucalyptus histone genes into the R genes to inactivate in Agrobacterium. In addition to the 35S promoter (constitutive type), rbcS,13 parB 14 and GST II15 promoters (inducible type) are joined

to the R genes for control of its expression. rbcS and parB promoters are under evaluation. Our evaluation work shows that the GST II promoter is preferable for controlling excision events during transformation. 4. Control of ipt genes: When we used the ipt genes fused with a 35S promoter to regenerate shoots at tobacco transformation, we detected inactivation of GUS genes in several plants. At transformation of hybrid aspen, we could get a lot of regenerated shoots, but most shoots were nontransgenic. Therefore, we examined native ipt and rbcS promoters to compare with a 35S promoter. A native ipt promoter has been isolated from A. tumefaciens PO22. On the other hand, a rbcS promoter came from tomato. In hybrid aspen, rbcS is absolutely greater than others in ESP formation. In tobacco, rbcS and native ipt are better than 35S. These results might depend on plant species. 5. Combination with iaaM/H genes: Endogenous hormonal levels are very different across plant species and plant tissues. The ipt genes are combined with iaaM/H genes16 to manipulate the auxin/cytokinin ratio according to the physiological state of plant tissues. Now the vector is in the process of evaluation.

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A

B

Fig. 25.4. Construction of new MAT vectors. The ipt genes are jointed to a native or rbcS promoter. The R (recombinase) genes fused with a 35S promoter have an intron sequence of Eucalyptus histone genes. The gusA and nptII genes are second selectable marker genes. Unique SmaI and BamHI sites (A) binary type; or MCS, multicloning sites (B), two component type; of lacZ’ DP: Dumb quoteare available for cloning of desirable genes.

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In current transformation methods, we have utilized only the gene transfer system of Agrobacteria, but not a plant hormone regulation system of oncogenes. Agrobacteria are able to induce “crown gall” or “hairy root” in a wide variety of plant species. Such wide adaptation is achieved by oncogenes, which highly regulate endogenous hormone balance. However, oncogenes have not been commonly used for transformation because the resulting transgenic plants show abnormal morphology. Our study shows that oncogenes can be used for transformation by combining with removal elements.

Plasmid Release The plasmid materials and manuals of MATVS have been released for researchers through the MAT Vector Association (President: A. Komamine; Correspondent: H. Ebinuma).

References 1. Yoder JI, Goldsbrough AP. Transformation systems for generating marker-free transgenic plants. Bio/Technol 1994; 12:263-267. 2. Ebinuma H, Sugita K, Matsunaga E, Yamakado M. Selection of marker-free transgenic plants using the isopentenyl transferase gene as a selectable marker. Proc Natl Acad Sci USA 1997; 94:2117-2121. 3. Sugita K, Matsunaga E, Ebinuma H. Effective selection system for generating marker-free transgenic plants independently of sexual crossings, Plant Cell Rep 1999; 18:941-947. 4. Ebinuma H, Sugita K, Matsunaga E, Yamakado M. Principle of MAT vector. Plant Biotechnol 1997; 14:133-139. 5. Müller-Neumann M, Yoder JI, Starlinger P. The DNA sequence of the transposable elements Ac of Zea mays L. Mol Gen Genet 1984; 198:19-24. 6. Araki H, Jearnpipatkulpatkkul A, Tatsumi H et al. Molecular and functional organization of yeast plasmid pSR1. J Mol Biol 1987; 182:191-203. 7. Akiyoshi D E, Klee H, Amasino RM et al. T-DNA of Agrobacterium tumefaciens encode an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci USA 1984; 81:5994-5998.

8. Barry GF, Rogers SG, Fraley RT, Brand L. Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci USA 1984; 81:4776-4780. 9. Gaudin V, Vrain T, Jouanin L. Bacterial genes modifying hormonal balances in plants. Plant Physiol Biochem 1994; 32:11-29. 10. White FF, Taylor BH, Huffman GA, Gordon MP. Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J Bacteriol 1985; 164:33-44. 11. Slightom JL, Durand-Tardif M, Jouanin L, Tepfer D. Nucleotide sequence analysis of the TL-DNA of Agrobacterium rhizogenes type plasmid. J Biol 1986; 261:108-121. 12. Christey MC. Transgenic crop plants using Agrobacterium rhizogenes-mediated transformation. In: Doran PM, ed. Hairy Roots: Culture and Using Agrobacterium Rhizogenes-Mediated Transformation. Hairy Roots: Culture and Application. Amsterdam: Hardwood Academic Publishers, 1997:99-111. 13. Sugita M, Gruissem W. Developmental, organ-specific, and light-dependent expression of the tomato ribulose-1,5bisphosphate carboxylase small subunit gene family. Proc Natl Acad Sci USA 1987; 84: 7104-7108. 14. Takahashi Y, Sakai T, Ishida S, Nagata T. Identification of auxin-responsive elements of parB and their expression in apices of shoot and root. Proc Natl Acad Sci USA 1995; 92: 6359-6363. 15. Holt DC, Lay VJ, Clarke ED et al. Characterization of the safener-induced glutathionne S-trransferase isoform II from maize. Planta 1995; 196: 295-302. 16. Thomashow LS, Reeves S, Thomashow MF. Crown gall oncogenesis: Evidence that a T-DNA gene from the Agrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyses synthesis of indoleacetic acid. Proc Natl Acad Sci USA 1984; 81: 5051-5075.

CHAPTER 26

Formation and Characterization of Transformed Woody Plants Inhibiting Lignin Biosynthesis Noriyuki Morohoshi Abstract We have tried to form a super-tree by new biotechnological and genetic engineering techniques. The first object of our research is to form a tree with less lignin content,by controlling the lignin biosynthesis genes using antisense RNA. We have succeeded in isolating and sequencing the phenylalanine ammonia-lyase (PAL), O-methyltransferase (OMT) and peroxidase (PO) genes from hybrid aspen (Populus kitakamiensis), and also isolating the promoter regions of these genes. These results show that the genes of PAL, OMT and PO involved in lignification are palg2a, homt1 and prxA3a, respectively. We have been able to construct a system which includes transducing a foreign gene to the hybrid aspen by use of the Ti plasmid and infecting with Agrobacterium tumefaciens by the leaf desk method.

In this paper, we focus on the peroxidase gene. First, transgenic poplars were made using prxA1 of a peroxidase gene and the CaMV 35S promoter by the antisense RNA method. The transgenics could not grow to young plants, because the promoter cannot control its expression in situ and temporally. Therefore, a new vector having the original peroxidase (prxA3a) promoter and the antisense prxA3a gene, involved in lignification, was constructed. The transformants with this vector can grow as well as non-transformants. The transgenic poplars have lower total peroxidase activity (10-25%) than that of the control. From the result of peroxidase isozyme analysis by isoelectric focusing, a peroxidase band (pI 3.8) involved in lignification disappeared in the transgenic plants. Lignin content in transgenic plants decreases 40-80% compared with control (100%), on the basis of potassium permanganate oxidation results. On the other hand, the amount of glucose determined by the alditol acetate method in transformants increases 5-10% compared with non-transformants. These results show that it is possible to form transgenic poplars having lower lignin content and higher glucose content, which indicates the cellulose content.

Introduction

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e have had several serious problems up to the present time: for example, the exhaustion of energy and resources, as well as aggravation of the environment situation on earth. This situation will continue into the near future. One means to solve these problems is to achieve an increased yield of biomass and to develop useful techniques of biomass

utilization, able to take the place of present energy and resource power. As trees are a major biomass, it is very important to aim at improving trees by use of genetic engineering techniques and to establish some useful conversion systems of biomass in the near future. First of all, we focused on the lignin synthetic pathway, occupying a huge second

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metabolism system in trees, and tried to inhibit lignin biosynthesis and to form a useful tree with of less lignin content yet able to use polysaccharide materials effectively. Populus kitakamiensis is used as the plant material, and the target gene of the lignin biosynthesis pathway is the peroxidase involved in lignification. The gene inhibited by antisense RNA.

Development of the Technical Requisites to Inhibit Lignin Biosynthesis There are three technical requisites to form an improved plant by genetic engineering. These three requisites are as follows: 1. To establish developmental and regenerative techniques from callus to mature plant;1 2. To establish a stable transformation system to integrate a foreign gene into plants;2 and 3. To isolate and analyze some target genes and their promoters.

Development and Regeneration System of Hybrid Aspen The callus of hybrid aspen grows in Murashige-Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D: 0.5 mg/l). Formation of adventitious buds from the callus is carried out in MS medium containing benzyladenine (BA: 0.1 mg/l) and zeatin (1.0 mg/l). The root developing from the shoot is formed in MS medium containing 1-naphthaleneacetic acid (NAA: 1.5 mg/l).

Integration with Foreign Genes The vector used for the transduction is pBI121, which has several target genes. The transformation system is the Ti plasmid method; the infection of samples by Agrobacterium tumefaciens is carried out by the leaf disk method.

Isolation and Analysis of Target Genes Many enzymes and genes are involved in the pathway of lignin biosynthesis in plants. We focused on phenylalanine ammonia-lyase (PAL), which is a first step enzyme in the general phenypropanoid synthesis in plants; O-methyltransferase (OMT), an enzyme

deciding the characteristics of lignin structure between those of soft and hard woods, and peroxidase (PO), only one of the enzymes involved in polymerization from monomer units to a macromolecule. In PAL genes, palg1, palg2a, palg2b and palg4 were isolated from P. kitakamiensis (Fig. 26.1)3 and palg2a was chosen as a PAL gene involved in lignin biosynthesis on the basis of Northern hybridization analysis results and expression analysis of the promoter. OMT and PO genes involved in lignin biosynthesis were also chosen: homt1 of homt1 and homt2 (Fig. 26.2)4 and prxA3a (Fig. 26.3)5,6 from among prxA1, prxA2a, prxA2b, prxA3a, prxA4a and HPOX14 by use of the same analysis methods.

Identification of a Peroxidase Enzyme Involved in Lignification There are many isozymes of peroxidase in plants; therefore it is very important to identify the enzyme involved in lignification in order to inhibit lignin biosynthesis only. Crude enzyme fractions were extracted from one year old hybrid aspen samples prepared every month and analyzed by isoelectric focusing. It was found that the acidic peroxidase isozyme clearly expresses in July, August and September, when lignin is synthesized vigorously, and that the lignification occurs in regenerated shoot and one year old trunk, but not in the callus, green callus and leaf. This isozyme band has a pI value of 3.8-4.2. Ultimately, we assumed that the acidic peroxidase enzyme is involved in lignification. Furthermore, in order to verify this assumption by histochemical localization analysis of this enzyme, we isolated the peroxidase isozyme and made a monoclonal antibody. We can find that immunogold-silver staining is detected in the xylem part near the cambial zone where lignin biosynthesis occurs in July. The lignification in July was also ascertained by ultraviolet microscopic observation. Conclusively, this result indicates that this acidic peroxidase isozyme is involved in the lignification of hybrid poplar.

Formation and Characterization of Transformed Woody Plants Inhibiting Lignin Biosynthesis

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Fig. 26.1. Restriction enzyme maps of PAL genes from P. kitakamiensis. Bars indicate the probes used for Southern and Northern blot analyses. E: EcoRI, B: BamHI, P: Pstl, RV: EcoRV, Sl: Sall, Sc: Sacl, X: Xhol.

Fig. 26.2. Restriction enzyme maps of OMT genes from P. kitakamiensis. Black and white boxes indicate exons and introns, respectively. Arrows indicate positions of primerse for PCR. E: EcoRI, B:BamHI, H: HindIII, Bg: BglII, P: PstI, X: XbaI.

Fig. 26.3. Restriction enzyme maps of PO genes (prxA3a and prxA4a) from P. kitakamiensis.

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Properties of the Poplar Controlled by the Peroxidase Gene Peroxidase is a final step enzyme in the pathway of lignin biosynthesis. This enzymatic step has no other bypass and is a rate limiting step for the dehydropolymerization of lignin. Therefore, we thought that it would be possible to inhibit lignin biosynthesis effectively using the peroxidase gene for the control of lignin content. A vector having the CaMV 35S promoter, followed by a reversed prxA1, was constructed and introduced into the hybrid poplar. Transformants obtained with POX1 have died because the preoxidase enzyme activity of the POX1 transformants was strongly inhibited in all parts of the transformants. This fact shows that the CaMV 35S promoter, which though it can express everywhere in plants, yet cannot be used for inhibition by antisense RNA in the case of the peroxidase gene. Therefore, we constructed an antisense vector having the original promoter of prxA3a (Fig. 26.4) and transformants were formed. These transformants have grown similarly to the control without death. Some transformants have changed characteristics, for example, more branches and delayed root growth. Figure 26.5 shows the peroxidase activity of the transformants. Obtained results indicate that peroxidase activity in the stem of transformants decreases 75-90% compared with control, and in the leaf decreases 20-80%. This result suggests that the prxA3a gene specifically expresses in the stem. From the results of isoelectric focusing analysis, we found that the transformants lack an acidic peroxidase band, pI 3.8, which mainly expresses in the wild stem. In order to analyze the lignin chemical structure of transformants in detail, the samples are subjected to potassium permanganate oxidation7 and thioacidolysis analyses.8 These results show that the lignin content of transformants decreases 20-60% in comparison with the control (Fig. 26.6) and the characteristic chemical structure of transformants is a decrease of p-hydroxy, guaiacyl and syringyl units evenly. From this result, it seems that there is no effect for the process of polymerization

among aromatic units when the peroxidse is inhibited by antisense RNA. As the amount of uncondensed units is almost similar between transformants and control, it is concluded that the the main effect of inhibition of peroxidase in transformants is to decrease the lignin content in the plant. To clarify the amount and quality of polysaccharides in transformed aspens, the composition of monosaccharide degraded by the alditol acetate method9 was analyzed. This result indicates an increasing tendency for glucose, which forms cellulose, and a decreasing tendency for xylose that forms the hemicellulose. This result might mean that transformed aspens increase in cellulose content and decrease hemicellulose fractions in comparison with the wild.

Conclusion In order to make a lower lignin content tree, the aspen, we tried to isolate the peroxidase gene involved in lignin biosynthesis from the hybrid aspen. prxA3a was isolated and the DNA sequence and promoter of it were analyzed. As the promoter of prxA3a expressed in the stem specifically, it was assumed that prxA3a is involved in lignin biosynthesis. By using the antisense RNA method, some transformed aspens inhibitied in peroxidase activity and lignification were formed. These transformants have lower peroxidase activity (70-90% in stem) and lower lignin content (15-60%), and an increasing tendency for glucose than the wild type. Especially, the characteristic lignin chemical structure in the transformants is lower contents of p-hydroxy, guaiacyl and syringyl units, an increasing degree of tendency toward the condensed type and less toward the uncondensed than in the wild. These results give us hope that we may succeed in forming useful trees by means of genetic engineering techniques.

References 1. Ebinuma H, Sugita K, Matunaga E et al. Selection of marker-free trnsgenic plant using the isopentenyl transferase gene. Proc Natl Acad Sci USA 1997; 94:2117-2121

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279

Fig 26.4. Structure of a plasmid containing anti-prxA3a.

Fig. 26.5. Peroxidase activity of transgenic aspen in stem and leaf. POX43, POX44, POX49, POX53: TransformantS.

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Fig. 26.6. Lignin content of transformed hybrid aspen estimated by permanganate oxidation. POX43, POX44, POX49, POX53: Transformants. 2. Kajita S, Osakabe K, Katayama Y et al. Agrobacterium-mediated transformation of poplar using a disarmed binary vector and the overpression of a specific member of a family of poplar peroxidase gene in transgenic poplar cell. Plant Science 1994; 103:231-239 3. Osakabe Y, Ohtsubo Y, Kawai S et al. Structure and tissue-specific expression of genes for phenylalanine ammonia-lyase from a hybrid aspen, Populus kitakamiensis. Plant Science 1995; 105:217-226 4. Hayakawa T, Nanto K, Kawai S et al. Molecular cloning and tissue-specific expression of two genes that encode caffeic acid and O-methyltransferases from Populus kit- akamiensis. Plant Science 1996; 113:157-165 5. Osakabe K, Koyama H, Kawai S et al. Molecular cloning and the nucleotide sequences of two novel cDNAs that encode anionic peroxidases of Populus kitaka- miensis. Plant Science 1994; 103:167-175

6. Osakabe K, Koyama H, Kawai S et al. Molecular cloning of two tandemly arranged peroxidase genes from Populus kitaka- miensis and their differential regulation in the stem. Plant Molecular Biology 1995; 28:677-689 7. Erickson M, Larsson S, Miksche GE. Gaschromatographische Analyse von Ligninoxydationsprodukten. VII. Ein verbessertes Verfahren zur Charakterisierung von Ligninen durch Methylierung und oxydativen Abbau. Acta Chemica Scand 1973; 27:127-140 8. Lapierre C, Monties B, Rolando C. Thioacidolysis of poplar lignin—Identification of monomeric syringyl products and characterization of guaiacyl-syringyl lignin fractions. Holzforschung 1986; 40:113-118 9. Blakeney AB, Harris PJ, Henry RJ et al. A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr Res 1983; 113:291-299

CHAPTER 27

Tolerance of Acacia Mangium to Acid Soil S. Kitani, N. Higuchi and I. Yasutani

Introduction

R

ecently, environmental problems have been increasing; especially, the destruction of tropical forests is one of the most serious problems in the world. Tropical and subtropical forests are estimated to comprise twenty billion hectares (ha). However, it is reported that 10% of it has already been destroyed. In addition, seventeen million ha of the forest has been deforested or destroyed per year. Under these conditions, we must stop the decrease of tropical forests and try to reforest, but the area of reforestation per year is only equal to 10% of the decreased area. The efficiency of reforestation is very poor. We have started research on improvement technologies of reforestation by molecular breeding. Of many types of environmental stress, acid stress was chosen as the target because it is one of the most serious environmental problems in tropical areas. Acacia mangium was chosen as the model plant for molecular breeding of acid-tolerant plants because it is an important tropical tree for reforestation and can resist acid.

Materials and Methods Sulfuric Acid Treatment of Acacia Mangium Seedlings The seeds of Acacia mangium were purchased from Hikariryokuchisangyo. The A. mangium seedlings of were grown in vermiculite for four weeks. These seedling roots were exposed to acidic water, pH adjusted to 1.0 or pH 2.0 with sulfuric acid for an hour per day. After 14 days of acid treatment, fresh weights

of shoots, roots and maximum root lengths were measured, and the morphological changes were observed by naked eye.

Identification of Acid Responsive Proteins in Acacia Mangium Preparation of Proteins of Acacia Mangium We extracted proteins of Acacia mangium according to an altered Hurkman and Tanaka’s method.1 Tissue (100 mg) was homogenized with liquid nitrogen and transferred into 200 µl of preparation buffer (0.7 M sucrose, 0.5 M Trizma base, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2%-mercaptoethanol). The homogenate was incubated at 4°C for 10 min and had added 50 µl of phenol solution saturated with water. After ten minutes of incubation at room temperature, it was centrifuged at 8,000 rpm for 10 min at room temperature. The phenol phase was collected and added to 50 µl of Hurkman and Tanaka’s preparation buffer. Following a second 10 min incubation, the extract was again centrifuged at 8,000 rpm for 10 min at room temperature. The phenol phase was collected and added to a five-fold volume of 0.1 M ammonium acetate-methanol. The extract was stored -20°C overnight and centrifuged at 30,000 rpm for 10 min at 4°C. The protein precipitate was washed with 0.1M ammonium acetate-methanol. Two-Dimensional Electrophoresis Method The precipitate of the extracted protein was dissolved with the sample buffer described

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below. After addition of 80 mg of urea, the protein solution was centrifuged at 10,000 rpm for 10 min at room temperature. Two-dimensional electrophoresis was performed. The Identification of Acid Responsive Proteins The pattern of two-dimensional electrophoresis was detected by Coomassie brilliant blue staining. To identify the responsive protein, we compared the electrophoresis pattern of the protein fraction extracted from acid treated Acacia mangium root with that of non-acid treated roots. Using Image Master (Pharmacia), the electrophoresis pattern was analyzed, and the amount of each protein was estimated.

Blotting and Protein Sequence The proteins were blotted on PVDF membrane at 30 V overnight with blotting buffer containing 10 mM CAPS, 10% methanol, pH 11.0. The membranes were stained with CBB for 1 h. After washing with methanol and distilled water, positive spots were excised from the membrane and sequenced by 473A protein sequencer (Perkin Elmer).

Construction of cDNA Library in Acacia Mangium Etiolated two week old seedlings were used. These were grown at 25°C in the dark. Before extraction of all RNA, seedlings were treated with 1 mM of Fe-EDTA solution for 12 h. The extraction of total RNA was performed as described by Brawermam2 and Watanabe and Price.3 Poly (A)+ RNA was purified by an oligo-(dT)-cellulose column. The cDNA was synthesized by using a great length TM cDNA Synthesis Kit (Clontech). The cDNA library was constructed with λgt10, EcoRI, and CIAP-treated Vector Kit (Stratagene).

Cloning and Sequence of cDNA of Ferritin in Acacia mangium Construction Of DNA Probe DNA fragments specific to the cDNA of the ferritin of Acacia mangium were amplified by the polymerase chain reaction (PCR). The two primers were as described below:

1. 5'-GTG ATW TTT GAA CCS TTT GAR GAG GTT-3' 2. 5'-CAA ACA CCR TGA CCC TTT CCA ACC CTT-3' The cDNA for the template was synthesized with an RT-PCR Kit (Stratagene). The amplified DNA fragment was subcloned into pT7 Blue Vector (Stratagene) and used as a probe to screen for the cDNA of ferritin. Screening of Ferritin cDNA DIG DNA labeling and Detection System Kit was used to screen for ferittin cDNA. Plaques were screened by plaque hybridization according to kit instructions. After a second screening, the inserted DNA fragment of positive plaques was subcloned into pUC118 and sequenced by a PRISM Dye Terminator Cycle Sequencing Kit (Perkin Elmer) with an automated DNA sequencer (model 373; Perkin Elmer).

Introduction of Ferritin Gene to Tobacco Tobacco (Nicotiana tabacum L., cv SR1) was transformed with the ferritin (AMFE1-1) cDNA expressed under control of a cauliflower mosaic virus 35S promoter/enhancer to produce the precursor of ferritin. Seeds of the T3 generation after repeated selfing were used. Ninety transformed strains were prepared.

Analysis of Ferritin Expression Polyclonal antibody to ferritin of Acacia mangium was prepared. We synthesized a peptide (20 amino acid residues) of N-terminal sequence. After conjugating the peptide to BSA, we immunized rabbits. Immunoblots were performed for analyzing the expression of ferritin.

Growth Tests Against Excessive Iron Plants of wild type tobacco and the transformed lines were germinated and grown in vermiculite for approximately 3 weeks. They were transferred to hydroponic culture containing different Fe-EDTA concentrations. One week later the morphological changes were observed by naked eye.

Tolerance of Acacia mangium to Acid Soil

Results Acid Treatment of Acacia mangium Seedlings After treatment with pH 1.0 sulfuric acid, all seedlings died. But following treatment with pH 2.0 sulfuric acid, the leaves and stems of the seedlings became pale green or yellow. There were not any remarkable morphological changes. Following pH 2.0 treatment, a 15% decrease in growth was observed in both shoots and roots. According to these results, the acid threshold was determined as pH 2.0.

Identification of Acid Induced Proteins The two-dimensional electrophoresis pattern of the root proteins derived from acid treated four week old seedlings is shown in Figure 27.1. There were some spots showing a changed amount of expressed protein; in a result from Image Master analysis, the most remarkably increased spots were 3.3-fold greater than that of the control. One was a protein of molecular mass of about 28 kDa. As a result of protein sequence analysis, its N-terminal amino acid sequence was determined.

Construction of cDNA Libraries in Acacia mangium By comparison of N-terminal amino acid sequence, the sequence of this protein was similar to ferritin protein derived from some legumes (Fig. 27.2). Based on this result, we estimated that this 28 kDa acid responsive protein might be the ferritin protein of Acacia mangium and cloning of the cDNA of this protein was performed. We tried the amplification of a fragment DNA from it by PCR. As a result, there were two kinds of DNA fragments. Both the sequences of these two DNA fragments were similar to the sequence of ferritin cDNA of Phaseolus vulgaris. The ratio of these two DNA fragments was 9:1; we named the major fragment AMFE1-p, and the minor fragment AMFE2-p (Fig. 27.3). Part of the amino acid sequence, which was estimated from the DNA sequence of AMFE1-p, was completely the same as the amino acid sequence of acid responsive protein.

283

Screening of Ferritin cDNA At the first screening, thirty-five thousand plaques were screened. As a result, 22 positive plaques were detected. Finally, 16 positive plaques were isolated after the second screening. Two kinds of cDNA were subcloned, and each clone was named either pAMFE1-1 or pAMFE1-2. Following subcloning, the sequence of these two clones was analyzed. Both clones had full length ferritin cDNA. The DNA sequence and the amino acid sequence are shown in Figure 27.4. In Figure 27.4, N-terminal amino acid sequences of acid responsive protein are completely in accord with the sequences of the underlined parts (b). It was confirmed that this cDNA clone encoded the acid responsive protein. Alignment of the amino acid sequence of acid responsive protein with the plant ferritin sequences showed 77% identity, and the putative ferric ion biding site was also conserved. These results indicated that this cDNA was the ferritin cDNA of Acacia mangium.

The Specific Antibody Against Ferritin of Acacia mangium We performed immunoblot analysis using the prepared antibody. Figure 27.5 shows that the prepared antibody reacts with ferritin of Acacia mangium but never reacts with ferritin of tobacco. Using this antibody, we selected 47 strains expressing Acacia mangium ferritin from 90 transformed strains.

Growth Tests Against Excessive Iron We tested the tolerance of the transformed plants expressing Acacia mangium ferritin against excessive iron. The results show that some strains acquired tolerance of excessive iron (Fig. 27.6).

Discussion Generally the factors which inhibit growth in acid soil may be summarized as follows: 1. The effects of toxic ions such as Al or Mn; 2. The effects of shortage of macro elements such as P, Ca, Mg, K etc.; 3. The effects of low pH on the soil microorganisms which are important to plant growth.

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Fig 27.1. Identification of sulfuric acid responsive protein. (a) Sulfuric acid treatment (pH2.0), (b) Control.

Fig. 27.2. Amino acid sequence comparison of sulfuric acid responsive protein 28kDa and those of some legumes. (a) Sulfuric acid responsive protein 28kDa, (b) Pisum sativum,2 (c) Phaseolus vulgaris.3

Fig. 27.3. Comparison of amplified cDNA with PCR and known ferritin cDNA. Box: Amino acid sequence identical to that of 28kDA sulfuric acid responsive protein.

Tolerance of Acacia mangium to Acid Soil

285

Fig. 27.4. Comparison of amino acid sequence between sulfuric acid responsive protein and ferritin precursors of some legumes. (a) AMFE1-1, (b) Pisum sativum,4 (c) Phaseolus vulgaris.5 Underline: Transit peptide part; Iron-binding residues.

Fig. 27.5. Antibody against ferritin of Acacia mangium. (a) Total protein of Acacia mangium. (b) Total protein of tobacco.

From the results of the previous studies, the mechanism producing acid resistance was considered to be somewhat as follows: for example, absorption of toxic ions by organic acid secreted from roots, absorption of toxic substances by mucus secreted from roots, increase in soil pH by substances secreted from roots. Recently, the effect of organic acid on acid resistance was vigorously analyzed. Kojima reported that an Al tolerant carrot cell line secretes a similar amount of citric acid as that from roots, and it forms a chelate compound with Al ions to decrease Al toxicity.7 There have been many studies on isolating stress resistance genes. For example, the heat shook proteins, the proteins induced by drought and the specific proteins for infection have been analyzed. But, there are few studies on the acid soil resistance. As described before, the mechanism that gives acid resistance is very complicated. Factors cooperate with each other, and might produce acid resistance in the plants. In this report, it has been revealed that ferritin was induced by sulfuric acid treatment of the roots of Acacia mangium. Its full length cDNA was isolated; its size was about 1.1 kbp. The putative molecular weight is 24 kDa, but the actual molecular weight calculated from electrophoresis was 28 kDa. This difference of molecular weight might be caused by modification of the protein after translation. The iron storage protein ferritin is widely distributed in living organisms. Plant ferritin exists mostly in chloroplasts; it forms large molecular subunits of about 440-550 kDa. Its

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Fig. 27.6. Tolerance of the transformed plants against excessive iron. 1: Non-transformed tobacco, 2-10: Transformed strains of tobacco. function is the storage of iron, and the supply of iron to heme proteins. The ferritin cDNA of Acacia mangium contains an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting, as reported for other plant ferritin cDNAs. In this experiment, ferritin cDNA was isolated from roots. Ferritin might be accumulated in plastids. In previous studies in suspension cells of Phaseolus vulgaris4 and Glycin max,8 ferritin was increased by the addition of iron. It was reported that ferritin might act for the detoxification of excessive iron.9 Further, researchers working within the electric power industry have shown that plants acquired tolerance to excessive iron by inducing the ferritin gene. Our results also support these results. Considering the relationship between ferritin and treatment with sulfuric acid, it was supposed that ferritin might be induced by the increase in free iron caused by soil acidification. In this case, through the detoxification of iron, ferritin played a part in acid resistance in Acacia mangium. In fact, in a tropical soil with acid sulfate, excessive iron is included. It has been reported that there are some isoforms of ferritin in maize, and that their expression pattern was changed by the kinds of stress.10 By RT-PCR, it was revealed that two

kinds of ferritin also exist in Acacia mangium. It is guessed that the expression of ferritin isoforms is changed by the kinds of stress, as in maize. Therefore, the ferritin induced by a stress may be involved in the resistance to each stress. So, we are considering that the ferritin proteins might play an important role in the detoxification of acid stress. Further, experiments will be made in the future to reveal the relationship between ferritin and acid resistance by analyzing the expression pattern of ferritin.

References 1. Hurkman WJ, Tanaka GK. Plant Physiol 1986; 81:802-806. 2. Brawerman: Anal. Biochem. 1974; 72: 413-427. 3. Watanabe A, Price CA. Proc Natl Acad Sci USA. 1982; 79:6034-6038. 4. Spence MJ, Henzl MT, Lammers PJ. Plant Molecular Biology 1991; 17:499-504. 5. Lobreaux S, Yewdall SJ, Briat JF et al. Biochem J. 1992; 288:931-939. 6. Tanaka A, Hayakawa Y. Japanese Journal of Soil Science and Plant Nutrition. 1974; 45:561-570. 7. Ojima K, Ohira K. Proc. 5th Intl. Plant Tissue and Cell culture, Plant Tissue Culture. 1982:475-476 .

Tolerance of Acacia mangium to Acid Soil 8. Lescure AM, Proudhon D, Pesey H et al. Proc Nat Acad Sci USA. 1991; 88: 8222-8226.

287 9. Laulhere JP, Briat JF. Biochem J. 1993; 290:693-699. 10. Fobis-Loisy et al. Eur J Biochem. 1995; 231:609-619.

CHAPTER 28

Developing a Mass Propagation System for Woody Plants T. Kozai, C. Kubota, S. Zobayed QT Nguyen, F. Afreen-Zobayed and J. Heo

Introduction

T

he population in tropical countries has been increasing at an annual rate of 2.8% during the last ten years, and it will continue to increase at a similar rate for the next 10-20 years. On the other hand, the total area under forest in those countries has been decreasing at an annual rate of 0.8% according to a survey by FAO. It was 1,910 million hectares in 1981, but it decreased to 1,756 million hectares in 1990, i.e., it decreased by 15.4 million hectares per year. An annual afforestation and reafforestation area in those countries is estimated to be 1.8 million hectares during the period 1981-1990, resulting in a yearly net decrease in forest area of 13.6 (15.4 - 1.8) million hectares. The forest area in temperate and cold climate regions has also been decreasing. In addition, the decrease in biomass of woody plants due to desertification in arid regions is significant. Such local and global decrease of the forest areas, and consequently the plant biomass, are the factors causing recent climate changes on different geographical scales. It has been predicted that, in future decades, demands for woody transplants will rise considerably in the pulp, paper, timber, plantation, horticulture and furniture industries, for re-afforestation, afforestation and desert rehabilitation, for environment conservation and for energy/food production.1-3 Use of plant biomass in the industries mentioned above is essential to reduce the consumption of fossil fuels and to lower atmospheric CO2 concentration, and to stabilize local and global climate changes.

Transplant production based upon micropropagation has advantages over transplant production using seeds or cuttings, with respect to genetic and phenotypic uniformity and scheduled year round production of disease-free or pathogen-free transplants.4 We have been developing a system for producing a large number of quality transplants at low production cost, based on the idea of photoautotrophic (no sugar in the culture medium) or photosynthetic micropropagation under high CO2 concentration or CO2 enrichment.5-7 In this chapter we demonstrate experimental results of photoautotrophic microprop agation for woody plant species such as eucalyptus (Eucalyptus camaldulensis), coffee (Coffea arabusta), acacia (Acacia mangium) and mangosteen (Garcinia Mangostana), and we discuss the advantages and disadvantages of photoautotrophic micropropagation over the conventional, heterotrophic and photomixotrophic micropropagation (with use of culture medium containing sugar). In addition, we discuss advantages of air porous supporting materials such as vermiculite or cellulose fibers and large culture vessels (5-1000 liter in air volume) over gelled supporting materials such as agar and small or conventional culture vessels (0.1-1 liter in air volume), respectively. We also emphasize the importance of in vitro acclimatization over ex vitro acclimatization. Finally, we discuss possibilities of factory style micropropagation systems.

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Reasons for High Production Costs and Their Reduction by Photoautotrophic Micropropagation4-7 Micropropagation is an advanced vegetative propagation technology for producing a large number of genetically superior and pathogen-free transplants in a limited time and space. However, widespread use of micropropagation for woody plant species is still limited, mainly due to its high production costs.4 High production costs of micropropagated plants are mostly attributed to their low growth rate, a significant loss of plants in vitro by microbial contamination, poor rooting, low percent survival at the ex vitro acclimatization stage and high labor costs due to intensive manual operations of small plants and small culture vessels. Most of the above factors, which bring about the high production costs, are directly or indirectly related to the heterotrophic or photomixotrophic nature of plant growth in conventional micropropagation, where sugar is supplied to the culture medium as a sole or a main carbon and energy source for plant growth in vitro. Our recent research achievements 5-7 have revealed that most chlorophyllous plants in vitro have photosynthetic ability to grow photoautotrophically, and that the low CO2 concentration in the airtight culture vessel during the photoperiod is a main cause of the low net photosynthetic rate of plants in vitro. Also, the net photosynthetic rate of plants in vitro is considerably lower when cultured on sugar-containing medium than when cultured on sugar-free medium. Furthermore, we have shown that the photoautotrophic growth of chlorophyllous plants in vitro can be significantly promoted by increasing CO2 concentration and light intensity or photosynthetic photon flux (PPF), by decreasing the relative humidity in the culture vessel, and by the use of fibrous and/or porous supporting materials with high air porosity. By using a culture medium containing no sugar, the loss of plants in vitro due to microbial contamination can be significantly reduced. When a culture vessel with high ventilation rate or high number of air exchanges is used, the relative humidity in the vessel is reduced. This reduction in relative

humidity results in enhanced rooting and high percent survival at the ex vitro acclimatization stage, especially when the porous supporting materials are used in vitro. In the following sections, we show the beneficial effects of in vitro environment control under photoautotrophic and photomixotrophic conditions on growth promotion of some woody plant species in small culture vessels with natural ventilation, and large culture vessels with forced ventilation.

Growth Promotion and Quality Improvement Using Small Culture Vessels Experimental results on photoautotrophic micropropagation using relatively small (or conventional) culture vessels like Magenta culture vessels with an air volume of 300-400 ml are shown below. For photoautotrophic micropropagation, gas permeable filter discs are attached to the lid or sides of the culture vessel. These filter discs are effective for enhancing the natural ventilation of the culture vessel, and thus keeping the CO2 concentration in the culture vessel during the photoperiod higher to increase the net photosynthetic rate, and keeping the relative humidity comparatively lower to increase the transpiration rate, than those in the culture vessels without the gas permeable filter discs.

Eucalyptus8-11 Eucalyptus camaldulensis shoots (2.2 cm long) were cultured in vitro photoautotrophically in Magenta type culture vessels (air volume: 370 ml) containing different types of supporting materials (agar, Gelrite, plastic net or vermiculite) for six weeks under CO 2 non-enrichment (400 µmol mol -1 in the culture room) or CO2 enriched (1200 µmol mol-1 in the culture room) conditions. The gas permeable filter discs (Milli Seal, Millipore Seal, Millipore Japan, Tokyo) were attached to side walls of the vessel. CO 2 enrichment significantly increased growth (dry mass and number of primary roots) of plants in vitro regardless of the type of supporting materials (Tables 28.1). The growth in vitro was greatest in the vermiculite, followed by the plastic net, Gelrite, and agar (in descending order) under either CO 2-nonenriched or CO2-enriched

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291

Table 28.1. Effects of CO2 enrichment and supporting material in vitro dry mass, leaf area, number of primary roots, and length of primary roots of Eucalyptus plants on 6 weeks in vitro8 CO2 condition

Supporting material

Dry mass mass (mg)

Leaf area (cm2)

No. of primary roots

Length of primary roots(mm)

45

8

1

27

Gelrite

49

9

1

30

Plastic net

64

11

4

39

Vermiculite

82

12

5

42

54

9

2

32

Gelrite

62

10

2

37

Plastic net

76

12

5

45

Vermiculite

103

13

6

49

8

3

1

11

CO2 condition (C)

**

NS

*

*

Supporting material (S)

**

*

**

**

CxS

NS

NS

NS

NS

Non-enriched Agar (400 µmol mol-1)

Enriched Agar (1200 µmol mol-1)

LSDP=0.05

Analysis of variance

*, ** Significant at P=0.05 and 0.01, respectively. NS, Nonsignificant at P=0.05.

conditions (Fig. 28.1). The growth of plants ex vitro was highest, and percent damaged leaves/roots was lowest, in the vermiculite under the CO2-enriched condition (Table 28.2). An extensive root system with many secondary roots was produced in vitro in the vermiculite (Fig. 28.2)

Coffee12,13 Growth of Coffea arabusta plants cultured in vitro as affected by sucrose concentration, type of supporting materials and vessel ventilation rate per plantlet was investigated at PPF (photosynthetic photon flux) of 100-200 µmol

m-2 s-1. Single nodal cuttings of in vitro coffee plants were cultured on medium without sugar and with 20 g l -1 sucrose. Two types of supporting materials, agar and Florialite (porous solid cubes consisting of a mixture of vermiculite and cellulose fibers, Nisshinbo Ind. Inc., Japan), and two levels of ventilaton rate per plantlet, 27 and 310 ml h-1, were examined (Table 28.3). Fresh mass, shoot length, root length and leaf area of plants cultured on Florialite with sugar-free nutrient solution under high vessel ventilation rate per plantlet were greater than those cultured on sugarcontaining agar or Florialite (Fig. 28.3 and

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Challenge to the Crisis of the Earth's Biosphere in the 21st Century

Fig. 28.1. Eucalyptus camaldulensis plantlets cultured for six weeks on sugar-free MS medium under CO2 non-enriched and CO2 enriched conditions, using four different supporting materials (By courtesy of C. Kirdmanee).8 Upper: CO2 non-enriched, Lower CO2 enriched. From left to right: Agar, Gelrite, platic net and vermiculite. Table 28.4). At the end of a 40 day culture period, callus were observed at the shoot base of plants grown on agar containing sugar. The net photosynthetic rate of coffee plants in vitro was significantly increased when cultured on Florialite with sugar-free nutrient solution under high vessel ventilation rate per plantlet (data not shown).

Acacia14,15 Effects of presence (30 g l-1) and absence of sugar and growth regulator (IBA, 1 mg l-1) in the medium, number of air exchanges of the culture vessel (0.6 or 6.7 h -1 ), CO 2 concentration in the culture room (400 or 1500 µmol mol-1) and types of supporting materials (agar and Florialite) on the growth in vitro and survival percentage ex vitro of Acacia mangium plants were examined. The combination of high CO2 concentration (CO2 enrichment) and high number of air exchanges of the culture vessel increased the fresh and dry mass of the plants, while presence/absence of sugar and growth regulator did not have any significant effects (Table 28.5). The plants cultured in a conventional way (with sugar

and IBA containing agar medium in the culture vessel and with low number of air exchanges under low CO2 concentration in the culture room) gave the lowest fresh and dry mass, and no root formation, while rooting of the plants was enhanced in other treatments (Fig. 28.4). Net photosynthetic rate was higher throughout the culture period in the treatment with sugar-free Florialite supporting material in the culture vessel with high number of air exchanges under high CO2 concentration (data not shown).

Mangosteen14,16 Mangosteen (Garcinia mangostana) shoots excised from aseptically germinated seedlings were cultured on vermiculite or agar medium with presence (30 g l-1)/absence of sugar and growth regulator (10 ml l-1 of 2-ip and 1 mg l-1 of IBA) in a culture vessel with low (0.1 h-1) or high (4.4 h-1) number of air exchanges at a PPF of 110 µmol m-2 s-1 under high CO2 concentration (1300 µmol mol-1 in the culture room). Fresh and dry mass of the plants on day 30 were not significantly different among the treatments. Addition of the

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293

Table 28.2. Effects of CO2 enhancement and supporting material in vitro on damaged leaf percentage and damaged root percentage 1 week after transplanting ex vitro, as well as effect on shoot length, leaf area, and number of primary roots of Eucalyptus plantlets 4 weeks after transplanting ex vitro8

CO2 condition

Supporting material

Damaged leaves(%)

Damaged roots(%)

Shoot length (mm)

Leaf area (cm2)

No. of primary roots

Non-enriched (400 mol mol-1)

Agar

33

50

46

27

2

Gelrite

29

50

49

29

2

Plastic net

50

33

69

25

6

Vermiculite

10

14

90

40

7

Agar

20

40

53

29

2

Gelrite

18

35

57

40

3

Plastic net

45

28

91

26

7

Vermiculite

5

0

121

49

5

8

12

6

1

CO2 condition (C)

**

**

**

**

*

Supporting material (S)

**

**

**

**

**

CxS

NS

NS

**

NS

NS

Enriched (1200 mol mol-1)

LSDP=0.05

Analysis of variance

*, ** Significant at P=0.05 and 0.01, respectively. NS, Nonsignificant at P=0.05

regulator in the medium increased the number of leaves. Twenty to forty percent of shoots exhibited root induction in the treatments with high number of air exchanges and vermiculite as supporting material, either with or without sugar/growth regulator (Fig. 28.5). On the other hand, in the control treatment with low number of air exchanges and sugar-containing agar medium, no rooting was observed (Table 28.6). The CO2 concentration was the lowest, and the net photosynthetic rate per leaf area in the control treatment was about 10 % of that in the treatments with high number of air exchanges (data not shown).

Interpretation of Growth Promotion and Quality Improvement Growth of chlorophyllous explants (shoots and leafy nodal cuttings) of most woody plant species examined above was greater under photoautotrophic than under photomixotrophic conditions, when CO 2 concentration in the culture vessel was raised by using the gas permeable filter discs and by increasing the CO 2 concentration in the culture room. Increase in PPF was also required in most cases. Increase in CO 2 concentration and PPF promoted photosynthesis and thus dry matter accumulation.4-6,17

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Fig. 28.2. Roots of Eucalyptus camaldulensis plants cultured for six weeks on sugar-free MS medium under CO2 enriched conditions, using four different supporting materials (By courtesy of C. Kirdmanee).8 Upper left: Agar; Upper right: Gelrite; Lower left: Plastic net; Lower right: Vermiculute. In addition, an increase in the number of air exchanges of the culture vessel enhanced the air movement or air current speed around the plants in the culture vessel and promoted diffusion of CO2 and water vapor around the plants, resulting in the promotion of photosynthesis and transpiration of in vitro plants.18 The increase in the number of air exchanges decreased the relative humidity in the culture vessel from nearly 100% to 85-90%.4 This decrease in relative humidity, in combination with the enhancement of air movement in the culture vessel, increased the transpiration rate of in vitro plants significantly, and thus increased water and nutrient uptake of in vitro plants.4 The decrease in relative humidity also enhanced cuticular wax formation of leaves and stomatal functioning, and it improved the water stress tolerance of plants. 19 The decrease in relative humidity, in combination with the use of air porous supporting materials such as vermiculite and Florialite, improved root formation and functioning,

especially formation of secondary roots and normal vascular systems. Increase in the number of air exchanges also decreased ethylene concentration in the culture vessel. Ethylene is a gaseous phytohormone produced by plants, which, in turn, affects the differentiation, development, morphology and growth of plants. Plants in the culture vessel with high number of air exchanges did not show any physiological or morphological disorder such as hyperhydration, which is often considered to be caused by a combination of high ethylene concentration and high relative humidity in the culture vessel.

In Vitro Acclimatization and Rooting20 The term acclimatization is defined in this chapter as the environmental adaptation of a tissue-cultured or micropropagated plant that has been moved to a new environment, i.e., a greenhouse or a field environment. During the acclimatization, the plant’s environment is changed gradually with time,

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295

Table 28.3. Experimental description of Coffea arabusta Sucrose concentration (g l-1)

Supporting material

No. of air exchanges (h-1)

Ventilation rate (ml h-1/ platelet)

SAL

20

Agar

0.2

27

SAH

20

Agar

2.3

310

SFL

20

Florialite

0.2

27

SFH

20

Florialite

2.3

310

FAL

0

Agar

0.2

27

FAH

0

Agar

2.3

310

FFL

0

Florialite

0.2

27

FFH

0

Florialite

2.3

310

Treatment code*

* S and F at the left represent sugar-containing and sugar-free medium; A and F at the middle represent agar and Florialite; L and H at the right represent low and high number of air exchanges per hour of the vessel, N, or vessel ventilation rate = N. vessel air volume/ No. of plantlets in the vessel).

starting with the 'near' in vitro environment and finishing with the ‘near’ greenhouse or field environment. Acclimatization conducted in a greenhouse or in a field under shade is called ‘ex vitro acclimatization’. In photoautotrophic micropropagation, acclimatization can be almost completed in the tissue culture vessel, which is called ‘in vitro acclimatization’. Rooting in vitro of many woody plant species is known to be difficult, and application of plant growth regulators or other chemicals to the medium has been tried for promoting rooting, without success in many cases. Thus, rooting is often conducted during the ex vitro acclimatization, which is often called ‘direct (ex vitro) rooting’. Under photoautotrophic micropropagation, root formation, development and growth in vitro of physiologically and morphologically normal plants are common. The plants are able to control the transpiration and thus control water loss when exposed to the

ex vitro environment; thus, wilting is not obvious even without the necessity of any specialized ex vitro acclimatization. This is probably due to: 1. A higher net photosynthetic rate and thus a greater accumulation of carbohydrates; 2. A higher production rate of phytohormone by the in vitro plant itself; 3. A higher transpiration rate and thus a higher uptake rate of minerals in the medium during the in vitro culture; and 4. A higher air porosity of the supporting material, which gives a higher dissolved oxygen concentration around the shoot base. In photoautotrophic micropropagation, plants with supporting material can be transplanted to the soil in the greenhouse or field, with little bacterial and fungal contamination,

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Fig 28.3. Coffea arabusta plantelets in vitro on day 40 as affected by presence/absence of sugar in the culture media, types of supporting materials (agar and Florialite) and high/low number of air exchanges of the vessel.12-14 For treatment codes, see Table 28.1. provided that the sugar-free supporting material consists of inorganic components and its surface is dry. Transplanting of plants with supporting material reduces root damage, saves labor costs, and gives a possibility of automatic transplanting.

Limitation of Use of Small Culture Vessels with High Number of Air Exchanges As described above, the CO2 concentration inside the culture vessel can be increased and the relative humidity and ethylene concentration can be decreased by attaching gas permeable filter discs to the lid or the wall of the culture vessels, or by changing the gaseous concentrations in the culture room. However, the CO2 concentration and other gaseous concentrations in the culture vessel with natural ventilation are interrelated with a number of factors such as the metabolic activity of the plants in vitro, the size and leaf area of the plants, the number of air exchanges of the culture vessel and the culture room environment. Thus, the gaseous concentrations in the culture vessel with natural ventilation are often unpredictable and uncontrollable. It is also difficult to measure the gaseous concentrations in the vessel continuously. Furthermore, it is difficult to provide a high number of air exchanges for a large culture vessel.

Forced Ventilation Micropropagation Systems and Their Application Forced ventilation is a method of ventilation which involves the use of mechanical

force such as an air pump and an air compressor to flush in a particular gas mixture directly through the culture vessel. With this system, the gaseous composition (CO2, water vapor, etc.) of incoming air and forced ventilation rate and/or air current speed in the culture vessel can be controlled relatively precisely by use of a needle valve, mass flow controller or an air pump with an inverter.4-6 One of the key advantages of photoautotrophic micropropagation is that it makes it possible to use large culture vessels with minimum risk of microbial contamination. In photoautotrophic micropropagation using a large culture vessel, forced ventilation has several advantages over natural ventilation. The use of larger culture vessels with forced ventilation is expected to reduce labor costs by nearly 50%, as compared with those in conventional micropropagation. By appropriately controlling the gaseous composition, the growth and development of plants can be promoted significantly or controlled properly. Another application of a large culture vessel with a nutrient supply system can also make it possible to measure and control the pH, composition and volume of nutrient solution in the culture vessel. Fujiwara et al.21 developed a large culture vessel (58 cm long, 28 cm wide and 12 cm high) with a forced ventilation system for enhancing the photoautotrophic growth of strawberry (Fragaria x ananassa Duch.) explants and/or plants during the rooting and acclimatization stages (Fig. 28.6). This was a kind of aseptic microhydroponic system with a nutrient solution control system.

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Table 28.4. Effects of sucrose concentration, supporting material and number of air exchanges on increased fresh mass (FM), % dry matter (DM), shoot length (SL), leaf area (LA), number of unfolded leaves (NUFL) and root length (RL) of coffee (C. arabusta) plantlets on day 40 of culture Treatment code SAL SAH SFL

FM (mg)

DM (%)

SL (mm)

LA (cm2)

NUFL

RL (mm)

130

19

4.6b

9.7

8.2a

0.0d

23

d

4.5

c

0.0d

bc

0.0d

bc

27 62

31

2.1

b

4.6

6.1

5.3

SFH

59

34

3.9

5.3

4.6

1.7c

FAL

23

15

2.7cd

5.1

4.1c

0.0d

4.5

b

0.0d

bc

11.0b

bc

5.1

24.1a

1.5

2.6

FAH FFL FFH

39 61 277

18 18 15

LSDP=0.05

bc

4.3

bcd

3.2

bc

4.2

13.0

a

5.9 13.0

1.6

6.1 5.1

Analysis of variance Sucrose conc. (A)

*

**

**

NS

NS

**

Supporting material (B)

**

**

**

**

NS

**

Ventilation rate (C)

*

NS

**

**

NS

**

AxBxC

NS

NS

**

NS

**

**

NS, *, **: Nonsignificant or significant at P=0.05* and 0.01**, respectively. For symbols of treatment, see Table 28.3.

Kubota and Kozai22 showed that the net photosynthetic rate and photoautotrophic growth of potato (Solanum tuberosum L.) plants cultured using a large culture vessel with forced ventilation, containing a multicell tray with rock-wool cubes, were significantly greater than those cultured using a conventional (small) culture vessel with natural ventilation. Heo and Kozai23 developed a forced ventilation micropropagation system with a culture vessel containing a multicell tray widely used for plug seedling production. The cells were filled with sterilized vermiculite or

cellulose plugs. The photoautotrophic growth of sweet potato plants cultured with this system was several times greater than the photomixotrophic growth of plants cultured with conventional or small culture vessels containing sugar and with natural ventilation. However, the growth in the culture vessel was not uniform, with larger plants near the air inlet and comparatively small plants near the air outlet. Zobayed et al.24 developed large culture vessels with air distribution pipes for forced ventilation. The major aim of the system was to provide an air current pattern which enables

No Yes

No

Yes

No

No

0 30

30

0

0

30 (control

155 ± 21

**

NS

NS

14

w

20 ± 4

NS

NS

NS

0

46

82

75

82 38

100

81

94

rooting

%

zMedium contained 1 mg l-1 IBA. yCO concentration inside the culture room with or without CO enrichment was 1500 or 400 µmol mol-1, 2 2 respectively. xVermiculite and cellulose fiber mixture. wOnly one replication for treatment with 30 g l-1 sucrose, without growth regulator and no CO2 enrichment, and control treatments. vANOVA (Analysis of variance) was applied for 9 treatments (except for the control treatment). NS, nonsignificant; **, significant at P<0.01.

**

100

w

139 ± 38

18 ± 4

17w

116w 110 ± 33

41 ± 14 22 ± 2

62 ± 24

32 ± 1

40 ± 21

(mg/plantlet)

DM

298 ± 4 152 ± 15

410 ± 145

CO2 enrichment and number of air exchanges(h-1)

Agar

V-CF mix

V-CF mix

V-CF mix

V-CF mix V-CF mix

V-CF mix

V-CF mix

222 ± 89

NS

100

150

150

150

150 150

150

150

V-CFmixx

Substrate

(mg/plantlet)

NS

0.7

0.7

0.7

0.7

6.7 0.7

6.7

6.7

150

PPF (µmol m-2s-1)

Sucrose conc.

No

No

No

No

Yes No

Yes

Yes

6.7

No. of air exchanges (h-1)

FM

Growth regulator

Analysis of variancev

No

Yes

30

Yesy

Yesz

30

0

CO2 enrichment

Growth regulator

Sucrose Conc. (g l-1)

Treatment

Table 28.5. Fresh (FM) and dry mass (DM), and percent rooting of Acacia mangium plants cultured for 28 days.14 Means ± SD are shown

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299

Fig. 28.4. Acacia mangium plants on day 28.14,15 S0: Sugar-free medium; CE: CO2 enriched; NE: CO2 non-enriched, GR: presence of growth regulator in the medium; NR: absence of growth regulator; C: control (sugar and growth regulator containing medium, CO2 non-enrichment, airtight vessel).

Fig. 28.5. Garcinia mangostana plants on day 30.14,16 S0: sugar-free medium, S30: sugar containing (30 g l-1 sucrose) medium; GR and NoGR: Presence and absence of growth regulator in the medium, respectively; Control: sugar and growth regulator containing medium. uniform distributions of CO2 concentration and relative humidity as well as those of air current speeds, and thus uniform plant

growth. Their aims have been achieved successfully. Applications of those systems for eucalyptus and coffee plants are now underway.

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Scaled-Up Micropropagation System by Use of an Aseptic Culture Room25,26 The idea of the forced ventilation micropropagation system can be further extended to an idea of an aseptic culture room, considering the aseptic culture room itself as a large culture vessel containing many small sterile trays with plants on the culture shelves. This kind of micropropagation system can be also considered as a transplant production system using small cuttings under disease-free conditions or a closed vegetative propagation and transplant production system with artificial light. Research and development of such a photoautotrophic micropropagation system with automation facility are now underway. For commercialization of such a system, considerable reduction in electricity consumption for lighting and air conditioning is essential. Currently, electric energy consumption per transplant has been estimated to be 0.1 MJ and its cost in Japan to be 2-3 yen (1.5-2 US cents) per transplant.27 Remarkable reductions in electricity consumption can be expected in the near future by improving the lighting and air conditioning systems. In this system, no person is permitted to enter the culture room for handling the trays with plants or for environment control in normal production modes. Thus, tray transportation and environmental control in the culture room must be automated.

Conclusion In the near future, photoautotrophic or sugar-free micropropagation will increase its importance for producing a large number of genetically superior and pathogen-free transplants at low production costs, to help solve the global problems of environmental conservation, food production and bioresource production in the 21st century. We have been conducting research on a large scale mass- propagation system based on photoautotrophic micropropagation and have obtained successful results.

References 1. Kurata K, Kozai T. Transplant Production Systems. Dordrecht: Kluwer Academic Publishers, 1992:299. 2. Kozai T, Kubota C, Kitaya Y. Greenhouse technology for saving the earth in the 21st century. In: Goto T, Kurata K, Hayashi M et al., eds. Plant Production in Closed Ecosystems. Dordrecht: Kluwer Academic Publishers, 1997:139-152. 3. Kozai T, Kubota C, Fujiwara K et al, eds. Proc. of ISHS Symposium on Plant Production in Closed Ecosystems, Acta Horticulturae, 1996; 440:674. 4. Aitken-Christie J, Kozai T, Smith MAL. Automation and Environmental Control in Plant Tissue Culture. Dordrecht: Kluwer Academic Publishers, 1995:574. 5. Kozai T. Micropropagation under photoautotrophic conditions. In: Debergh PC, Zimmerman RH, eds. Micropropagation-Technology and Applications. Dordrecht: Kluwer Academic Publishers, 1991:447-469. 6. Jeong BR, Fujiwara K, Kozai T. Environmental control and photoautotrophic micropropagation. Horticultural Reviews 1995; 17:125-172. 7. Nguyen QT, Kozai T. Environmental effects on the growth of plantlets in micropropagation. Environment Control in Biology 1998; 36(2):59-75. 8. Kirdmanee C, Kitaya Y, Kozai T. Effects of CO 2 enrichment and supporting material in vitro on photoautotrophic growth of Eucalyptus plantlets in vitro and ex vitro. In Vitro Cell Dev Biol-Plant 1995; 31:144-149. 9. Kirdmanee C. Environmental control in micropropagation of woody species. Abstract book of 9th International Congress on Plant Tissue and Cell Culture. Jerusalem, Israel: Kluwer Academic Publishers , 1999:651-654. 10. Kirdmanee C, Kitaya Y, Kozai T. Effects of CO2 enrichment and supporting material on growth, photosynthesis and water potential of Eucalyptus shoots/plantlets cultured photoautotrophically in vitro. Environment Control in Biology 1995; 33(2):133-141. 11. Kirdmanee C. Environmental control and its effect in Eucalyptus micropropagation under photoautotrophic conditions. Ph.D. Dissertation, Chiba University, 1995:172.

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Fig. 28.6. Configuration and schematic diagram of hte photoautotrophic micropropagation system with forced ventilation and nutrient solution control system.21 Upper: Components. Gas and nutrient solution flow lines are represented by solid lines and electrical lines by dash-and-dotted lines. Lower: the culture box assembly.

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12. Kozai T, Nguyen QT, Kubota C et al. Growth promotion of coffee (Coffea arabusta) plantlets in vitro by use of fibrous supports containing no sugar and culture vessels with high number of air exchanges. Jpn J Trop Agr 1998; 42(Extra issue 1):27-28. 13. Niu G, Nguyen QT, Kozai T et al. Net photosynthetic rates of sweetpotato and coffee plantlets cultured photoautotrophically and photomixotrophically in vitro. Jpn J Trop Agr 1998;42 (Extra issue 1):25-26. 14. Ermayanti TM, Imelda M, Tajuddin T et al. Growth promotion by controlling in vitro environment in micropropagation of tropical plant species. Proc of International Symposium on Access and Benefit Sharing of Bioresources, Tokyo, 1999:10-25. 15. Imelda M, Kubota C, Kozai T et al. Photoautotrophic micropropagation of Acacia Plantlets. Jpn J Trop Agr 1998; 42(Extra issue 1):29-30. 16. Kubota C, Tajuddin T, Kozai T. Photoautotrophic micropropagation of Mangosteen plantlets. Jpn J Trop Agr 1998; 42(Extra issue 1):31-32. 17. Kozai T, Zimmerman RH, Kitaya Y et al. eds. Environmental effects and their control in plant tissue culture. Acta Horticulturae 1995; 393:230 18. Kitaya Y, Ohmura Y, Kozai T et al. Visualization and analysis of air currents on plant tissue culture vessels. Environment Control in Biology. 1997; 35(2):139-141. 19. Ziv M. In vitro acclimatization. In: Aitken-Christie J, Kozai T, Smith MAL eds. Automation and Environmental Control in Plant Tissue Culture. Dordrecht: Kluwer Academic Publishers, 1997:493-516. 20. Kozai T, Zobayed SMA. Acclimatization. In: Spier R, ed. Encyclopedia of Cell Technology, The Wiley Biological Series. 1999 (in press).

21. Fujiwara K, Kozai T, Watanabe I. Development of a photoautotrophic tissue culture system for shoots and/or plantlets at rooting and acclimatization stages. Acta Horticulturae 1988; 230:153-158. 22. Kubota C, Kozai T. Growth and net photosynthetic rate of Solanum tuberosum in vitro under forced and natural ventilation. Hort Science 1992; 27(12):1312-1314. 23. Heo J and Kozai T. Development of a forced ventilation micropropagation system for enhancement of the photoautotrophic growth of sweetpotato plug plantlets cultured in vitro. Jpn J Trop Agr. 42(Extra issue 1) 1998:21-22. 24. Zobayed SMA, Kubota C, Kozai T. Development of a forced ventilation micropropagation system for large-scale photoautotrophic culture and its utilization in sweetpotato. In Vitro Cell and Dev. biol Plant 1999; 354:350-355. 25. Kozai T, Kubota C, Heo, J et al. Towards efficient vegetative propagation and transplant production of sweetpotato (Ipomoea batatas L. Lam.) under artificial light in closed ecosystems. In: LaBonte D, Yamashita M, Mochida H eds. Proceedings of International Workshop on Sweetpotato Production towards the 21st Century. Miyakonojyo, Japan: Kyusyu Agric Exp Stn, 1998:201-214. 26. Kozai T et al. Transplant production under artifical light in closed systems. Proc. of the 3rd Asian Crop Science Conference, Taichung, Taiwan, 1999:296-308. 27. Ohyama K, Kozai T. Estimating electric enegy consumption and its cost in a transplant production factory with artificial lighting: A case study. J Soc High Technology in Agriculture 1997; 10(2):96-107.

CHAPTER 29

Advances in Conifer Tree Improvement Through Somatic Embryogenesis P. K. Gupta, R. Timmis, K. Timmis, J. Grob, W. Carlson, E. Welty and C. Carpentar

Introduction

T

he world demand for wood products is expected to rise sharply over the coming decade, and shortage has been forecast by the 21st century. To meet this burgeoning demand, the forest productivity of our limited lands will have to be increased. There is an urgent need for mass production of improved quality planting stock for reforestation. The conventional methods of tree improvement and selection offer only a limited possibility of meeting this demand. Over the past few years, biotechnological methods have been developed which are predicted to have significant impacts on commercial forestry in the 21st century. These include micropropagation, genetic engineering, marker-aided selection etc. Micropropagation has been approached in two ways: via somatic (asexual) embryogenesis and via organogenesis (enhancement of axillary bud break or adventitious budding). Micropropagation via somatic embryogenesis offers an inexpensive and efficient way to produce an unlimited supply of genetically uniform superior clones for reforestation. The most important advantage of somatic embryogenesis is that the embryogenic tissue can be cryopreserved indefinitely without any genetic change and can be bulked for mass production at any time. In forestry, the production of somatic embryos throughout the year provides

a complementary technology to reduce the risk where seed production is limited or uncertain. Considerable progress has been made over the last decade in the development of somatic embryogenesis systems for large scale clonal propagation of conifers.1,2 Since the first report in 1985, several hundred papers have been published on this subject.3 Here we discuss the current status of the technology and its implementation for clonal propagation of conifer trees for reforestation.

Culture Establishment Induction of embryo suspensor masses (ESMs) has been reported from immature embryos, mature embryos, hypocotyls, cotyledons, and explants of somatic and zygotic seedlings of Norway spruce (Picea abies).4 Recently, ESM induction has also been reported from explants of 10-20 year old trees of Pinus radiata,5 Pinus pinaster and Picea abies.4 At Weyerhaeuser, we have initiated ESM from immature embryos (pre-dome and dome stages before development of cotyledons) of Douglas fir (Pseudotsuga menziesii) and loblolly pine (Pinus taeda). ESM cultures were initiated onto solid medium with auxin and cytokinin, details of which have been published earlier.6 There is no callus stage in ESM induction from immature embryos. ESM form directly from heads of early stage embryos

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through cleavage polyembryony. Cleavage polyembryony is natural in several conifer species, and multiple embryos develop inside the megagametophyte through this process. Cleavage polyembryony continues in vitro in the presence of auxin and cytokinin in the medium. After initiation, ESM cultures are transferred to maintenance medium for continuation of true-to-conifer type cleavage polyembryony under lower concentrations of auxin and cytokinin.6 At this time, osmolality is increased from 90-100 to 190-200 mM/ kg, with 5 g/l myo-inositol and 30 g/l maltose (compared with 0.1 g/l myo-inositol and 15 g/l sucrose in the initiation medium). We have found that the type of sugar is very important for subsequent embryo development on plates. Early stage embryos were able to fully mature only when grown in the maltose maintenance medium.7 Without increased osmolality treatment, which leads to larger embryonal heads of early stage embryos, many genotypes of Douglas fir and loblolly pine did not develop good quality cotyledonary embryos.8 ESM multiplication is faster in liquid than gelled medium. We have established ESM cultures of Douglas fir and loblolly pine in liquid suspension in 1 liter Erlenmeyer flasks and maintained them by weekly subculture. Each flask contains over 10,000 early stage embryos which may double or triple in number weekly.

Embryo Development, Maturation and Germination Abcisic acid (ABA) and osmoticum play important roles in cotyledonary embryo development. ABA inhibits cleavage polyembryony and allows embryos to separate and develop further.9 However, ABA alone could not inhibit precocious germination of cotyledonary embryos; increased osmolality of the medium was found to be necessary for embryos to mature. ESM cultures were settled in a measuring cylinder for 30 minutes. Supernatant was discarded and 1.0 ml settled ESM culture was transferred onto pads imbibed in liquid medium. Good quality cotyledonary embryos were produced by combining activated

charcoal, ABA, GA4/7, and osmoticum.10 Polyethylene glycol (PEG), MW 4,000-8,000, proved to be the best osmoticant for embryo maturation.11 Fifty to one hundred cotyledonary embryos were produced per ml of settled ESM suspension culture on development medium (Fig. 29.1). The number and quality of cotyledonary embryos produced varied considerably among genotypes. For germination, good quality cotyledonary embryos were selected individually (as looking similar to zygotic embryos) by hand from development medium under the stereo microscope. Selected embryos were transferred onto semi-solid medium for germination. Culture plates were incubated the first 5-7 days in the dark, followed by transfer to light. Percentage germination varied considerably among genotypes. Germinated embryos bearing an epicotyl were then selected by hand and transplanted into 10 cubic inch Supercell pots containing a mixture of peat, vermiculite and perlite, and incubated in the greenhouse with frequent misting for acclimatization and growth. After establishment in soil in the greenhouse, somatic seedlings were grown in the nursery for one year before transplanting to forest regeneration sites.

Cryopreservation Cryopreservation is at present considered essential for the large scale commercial use of somatic embryogenesis technology for clonal forestry, because it permits genetic material to be saved at low cost until its genetic value can be measured in field tests (see implementation section below). We and others have found that almost 100% of embryogenic culture lines, frozen under controlled conditions and stored in liquid nitrogen, can be recovered after 5 years.6 Trees produced from them are visually indistinguishable from the products of unstored cultures, and there is no evidence of genetic change.6 Space requirements for cryogenic storage are minimal, since even a 45 liter storage unit typically can accommodate 8-10 thousand vials. A completely reliable system for maintenance of liquid nitrogen service, complete with alarm systems and backups, is essential. We have cryostored ESM of over 750

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Fig. 29.1. Cotyledonary embryo development of Douglas fir on the pad in a bioreactor: Temporary immersion of the lower pad surface. clones of Douglas fir from superior full-sib families.

Field Performance Somatic seedlings have now been growing for four years on a typical forest regeneration site in Washington State for clonal demonstration purposes. Strikingly uniform growth is apparent for somatic seedlings within a clone (Figure 29.2) compared with the less uniform zygotic seedlings (Figure 29.3). Somatic seedlings from ESM of 20-25 year old trees of Pinus radiata5 and Picea abies 4 have also been grown in the field. Morphology of these somatic seedlings, too, was reported to be similar to zygotic seedlings. Recently, we have produced over 30,000 somatic seedlings of Douglas fir from a large number of genotypes for a clonal field test.

Large Scale Production For large scale production, suspension cultures were grown in 1 to 2 liter Erlenmeyer

flasks. One liter nipple flasks have also been used to bulk up embryogenic suspension cultures of conifers.9 Large scale production of somatic embryos can be achieved in a bioreactor. 12 Bioreactors provide several advantages for the growth of embryogenic suspension cultures, including larger volume (than shake flasks), maintenance of more homogeneous cultures, and control of the cultural and physical environment for optimum growth.12 Two to ten liter bioreactors have been used to scale up embryogenic cultures of several conifer species, different types of bioreactors being used for different conifer species.13,14 Maturation has not been achieved for embryos immersed in liquid medium. Recently, cotyledonary embryo development of Sitka spruce has been reported in an airlift bioreactor,15 but their low germination shows that biochemical maturity was not attained. At Weyerhaeuser, ESM cultures of Douglas fir were grown in low speed, winged, magnetically driven stirred bioreactors.16 Their

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Fig. 29.2. One replicate plot of clones (from control crossed genetically improved family) of Douglas fir somatic seedlings growing in the field. Photo shows the uniformity of somatic seedlings. yields of cotyledonary embryos, after plating on pads soaked with development medium, did not differ significantly from flask yields, but their germination was improved.16 Cotyledonary embryos have been produced in a bioreactor using an absorbent pad above the surface of liquid medium.17,18 At Weyerhaeuser, we have produced cotyledonary embryos of Douglas fir in a bioreactor by temporary immersion of the lower pad surface.14 Liquid medium was pumped in and out of the bioreactor from a reservoir at different intervals. Higher yields of good quality cotyledonary embryos were produced using this method.14

Embryo Sorting Somatic embryo development is not synchronized. Embryos produced on development medium vary in shape, size and morphology. Separating the high quality (zygotic-like)

embryos from non-embryo tissue and embryos of lower quality by hand is very labor-intensive, and automation is essential to render these steps economically feasible. Machine vision and image analysis systems are being developed currently to automatically count, sort, size and grade somatic embryos of several herbaceous plants such as carrot and sweet potato. Harrell and Cantiffe19 developed a noninvasive machine vision system that could automatically measure the quality of sweet potato somatic embryos produced in an airlift bioreactor. A computer vision system has also been developed for the classification of Norway spruce somatic embryos using algorithm recognition.20 The somatic embryos from development plates were moved with constant speed, and embryos were recognized as they passed a line-scan camera. We have research in progress for sorting of somatic embryos of Douglas fir and loblolly pine using image analysis.21

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Fig. 29.3. One replicate plot of zygotic seedlings (from seeds as control) of Douglas fir growing in the field. Photo shows the lesser degree of uniformity as compared to somatic seedlings (Fig. 29.2).

Manufactured Seed

Redenbaugh22 defined a synthetic seed as a somatic embryo inside a coating, and as being directly analogous to natural seed. Several names have been used for such a “seed”, including artificial seed, seed analog and somatic embryo seed. At Weyerhaeuser Company, we use the term “manufactured” seed, to reflect the nature of the construct more accurately.23 Several patents and papers have been published on synthetic seed design exhibiting low percentage germination.17,22 Germination is limited by lack of oxygen, nutrition, tolerance to desiccation and mechanical damage. Carlson et al24 published a seed design which overcomes many of these problems. This was accomplished by providing oxygen carrier emulsions, a hard seed coat (wax impregnated paper) to prevent mechanical damage and desiccation, and a cotyledon restraint system to ensure emergence of the germinating embryo without trapping in the gel. Manufactured seed

research is moving rapidly and will make it possible to deliver somatic embryos to the field economically.

Clonal Field Tests The implementation of somatic embryogenesis in forestry has already begun at Weyerhaeuser, and several other forestry organizations worldwide have begun the establishment of clonal field tests and corresponding clone banks of cryogenically stored ESM (Figure 29.4). Performance of the individual genotypes in these tests will be carefully measured to identify outstanding genotypes as soon as possible. During this evaluation period (5+ years), continuing R&D is expected to bring the costs of somatic embryogenesis and manufactured seed technologies within acceptable limits. The selected genetic material will be retrieved from the clone bank, then multiplied and treated to produce embryos in

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Fig. 29.4. A vision of 21st century forest biotechnology. Somatic embryos are regenerated from ESM of control crossed seeds from elite families (top left). A sample of ESM from each genotype is stored cryogenically (center) and the rest are used to produce somatic seedlings for clonal performance (left). After 5-15 years, field performance data is used to select clones, which are then scaled up in a bioreactor from cryostored ESM (center). Mature embryos are selected by machine vision and transported for manufactured seed production, which are machine sown in forest nurseries (right). Genes conferring specifically desired traits may be inserted into the ESM of selected clones before their multiplication, for the production of genetically improved forests. larger vessels or bioreactors. Mature embryos will be harvested with the aid of a machine vision system, and fed to a manufactured seed assembly line to produce the quantities needed for reforestation. Manufactured seed will be storable, and sown through standard container facilities or nurseries.

Conclusion Clonal testing is viewed as an ongoing endeavor linked to the ongoing advancement of the conventional breeding program, and leading to continuous improvement of the reforestation clonal mix through time. We anticipate that in the future genetic transformation of selected clones at the ESM stage, using gene constructs controlling, e.g., wood properties or disease resistance, will further enhance genetic value. This integrated approach to

improvement of forest crops, if sustained through prudent investment on existing commercial forest lands, will have a major impact on the industry’s ability to meet demand for forest products in the next century.

References 1. Gupta PK, Pullman G, Timmis R et al. Forestry in the 21st century: The biotechnology of somatic embryogenesis. Bio/ Technology 1993; 11:254-459. 2. Timmis R. Bioprocessing for tree production in the forest industry: Conifer somatic embryogenesis. Biotechnology Progress 1998; 14(1):156-166. 3. Gupta PK, Grob JA. Somatic embryogenesis in conifers. In: Jain SM, Gupta PK, Newton RJ eds. Somatic Embryogenesis in Woody Plant Vol.1. Dordrecht:Kluwer Academic Publishers, 1995:81-98.

Advances in Conifer Tree Improvement Through Somatic Embryogenesis 4. Paques M. Somatic embryogenesis: A technology underway to value old selected Norway spruce. In Proceedings abstracts of 8th conifer biotechnology working group. Ruthgers: Rutgers University Press, 1998:5. 5. Smith DR. The role of in vitro methods in pine plantation establishment: The lesson from New Zealand. Plant Tissue Culture and Biotechnology 1997; 3(2):63-73. 6. Gupta PK, Timmis R, Timmis K et al. Clonal propagation of conifers via somatic embryogenesis. In: Ahuja MR, Boerjan W, Neale DB eds. Somatic Cell Genetics and Molecular Genetics of Trees. Dordrecht : Kluwer Academic Publishers, 1996:3-10. 7. Gupta PK. Methods for reproducing coniferous plants by somatic embryogenesis using maltose enriched maintenance medium. 1996. U.S. Patent. No.5,563,061. 8. Gupta PK, Pullman GS. Methods for reproducing coniferous plants by somatic embryogenesis. 1990. U.S. Patent No. 4,957,866 9. Durzan DJ, Gupta PK. Somatic embryogenesis and polyembryogenesis in Douglas fir cell suspension cultures. Plant Sci 1987; 52:229-235. 10. Pullman GS, Gupta PK. Method for reproducing coniferous plants by ssomatic embryogenesis using mixed growth hormones for embryo culture. 1994. U.S. Patent No. 5,294,549. 11. Gupta PK, Pullman GS. Methods for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation. 1991. U.S. Patent No. 5,036,007. 12. Ammirato PV, Styer DJ. Strategies for large scale manipulation of somatic embryos in suspension culture. In: Zaitlin M, Day P, Hollaender A eds. New York: Academic Press, 1985:161-178. 13. Tautorus TE, Lulsdrof SL, Kikcio SI et al. Nutrient utilization during bioreactor cultures, and maturation of somatic embryos of black spruce and interior spruce somatic embryos. In Vitro Cell Dev Bio Plant 1994; 308:58-63. 14. Gupta PK, Timmis R, Timmis K et al. Conifer somatic embryo production from liquid culture. In proceedings abstracts of IX International Congress on Plant Tissue and Cell Culture. Jerusalem, Israel, 1998:50.

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15. Ingram B, Mavituna F. Picea sitchensis somatic embryogenesis: Proliferation and maturation in bioreactor. In proceedings abstracts of IX International Congress on Plant Tissue and Cell Culture. Jerusalem, Israel, 1998:83 16. Timmis R, Surerus-Lopez HA, Barton BM et al. Preliminary experiments on Douglas fir somatic embryo yield and quality from stirred bioreactors. Abstract. In Vitro Cell Dev Bio Plant 1998; 34:21A. 17. Attree SM, Pomoroy MK, Fowke LC. Production of vigorous, desiccation tolerant white spruce synthetic seeds in bioreactor. Plant Cell Rept 1994; 12:601-606. 18. Paques M, Bercetche J, Palada M. Prospects and limits of somatic embryogenesis of Picea abies. In: Jain M, Gupta PK, Newton RJ, eds. Somatic Embryogenesis in Woody Plant Vol.1. Dordrecht: Kluwer Academic Publishers, 1995:399-414. 19. Harrel RC, Cantliffe DJ. Automated evaluation of somatic embryogenesis in sweet potato by machine vision. In: Vasil IK, ed. Scale-up and automation in plant propagation. New York: Academic Press, 1991;179-195. 20. Hamalainen JJ, Jokinen KJ. Selection of Norway spruce somatic embryos by computer vision. Optics in Agriculture and Forestry 1992;1836:195-204. 21. Timmis R, Ghermay T. Liquid culture morphology related to embryo yield in Douglas fir. In: Abstract Proceedings of joint meeting of the IUFRO working parties of Somatic Cell Genetics and Molecular Genetics of Trees, Quebec City, Canada, 1997:74. 22. Redenbaugh K, Viss P, Slade D et al. Scale-up: Artificial seeds. In: Green CE, Sommers, DA, Hackett WP et al. eds. Plant Tissue and Cell Culture. New York: Alan R. Liss,1986:473-494. 23. Carlson WC, Hartle JE. Manufactured seeds of woody plants. In: Jain M, Gupta PK, Newton RJ, eds. Somatic Embryogenesis in Woody Plant Vol.1. Dordrecht: Kluwer Academic Publishers, 1995:253-264. 24. Carlson WC, Hartle JE, Bower BK. Oxygenated analogs of botanic seed. 1993. U.S. Patent No. 5,236,469

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