Genetically Modified Organisms in Food: Production, Safety, Regulation and Public Health 9780128022597, 2232242242, 2602602612, 0128022590, 9780128025307, 0128025301

Table of contents :
Front Cover......Page 1
Genetically Modified Organisms in Food......Page 4
Copyright......Page 5
Contents......Page 6
Contributors......Page 16
SECTION III: GOVERNMENT REGULATION AND LITIGATION FOR GMO FOODS......Page 20
SECTION VII: DEMAND AND USES OF NON-GENETICALLY MODIFIED FOODS, AND GMO’S FOR HUMANS AND ANIMALS......Page 21
Acknowledgments......Page 22
Section I - Development, Testingand Safety of Plant and Animal GMO foods......Page 24
INTRODUCTION......Page 26
SOME COMMENTS ABOUT TRANSGENIC SOYBEANS......Page 27
COMPARATIVE STUDIES INVOLVING TRANSGENIC AND NONTRANSGENIC SOYBEAN SEEDS OR PLANTS......Page 28
CONCLUSIONS......Page 31
REFERENCES......Page 32
BIOSAFETY MEASUREMENT......Page 34
BT GENE AND STACKED TRAITS......Page 36
PCR AND REAL-TIME PCR......Page 37
IMMUNO-PCR......Page 38
REFERENCES......Page 39
WEBSITE REFERENCES......Page 41
GENETICALLY MODIFIED ORGANISMS......Page 42
APPLICATIONS OF TRANSGENIC ANIMALS......Page 45
REFERENCES......Page 47
BIOCHEMICAL AND NUTRITIONAL PROPERTIES......Page 50
INSECT PESTS OF BRINJAL......Page 51
DEVELOPMENT OF INSECT-RESISTANT BT-BRINJAL......Page 52
FRUIT AND SHOOT BORER MANAGEMENT IN BT-BRINJAL......Page 53
DETECTION OF BT-BRINJAL......Page 54
FOOD SAFETY ASSESSMENT OF BT-BRINJAL......Page 55
ENVIRONMENTAL RISK ASSESSMENT OF BT-BRINJAL......Page 57
REFERENCES......Page 58
CROP COMPOSITION, NUTRITIONAL CONTEXT, AND THE SUITABILITY OF ANIMAL STUDIES......Page 62
HISTORY OF ANIMAL STUDIES FOR NUTRITIONAL ASSESSMENT OF GENETICALLY MODIFIED CROPS......Page 66
REGULATORY ASSESSMENTS......Page 67
REFERENCES......Page 71
VARIOUS NCRNA-BASED SILENCING PLATFORMS......Page 74
APPREHENSIONS OF NONCODING RNA-BASED GENETICALLY MODIFIED CROPS......Page 77
PREDICTIVE ENVIRONMENTAL RISK ASSESSMENT......Page 79
IMPACT ON PLANT PROTECTION MEASURES......Page 82
REFERENCES......Page 83
AGROBACTERIUM-MEDIATED T-DNA TRANSFER PROCESS......Page 86
FACTORS INFLUENCING AGROBACTERIUM-MEDIATED TRANSFORMATION......Page 89
ADVANCES IN AGROBACTERIUM-MEDIATED TRANSFORMATION......Page 90
APPLICATIONS......Page 92
SUMMARY AND FUTURE PROSPECTS......Page 93
REFERENCES......Page 94
INTRODUCTION......Page 98
THEORETICAL FRAMEWORK AND HYPOTHESES DEVELOPMENT......Page 99
RESEARCH METHODOLOGY......Page 102
RESULTS......Page 103
REFERENCES......Page 106
INTRODUCTION......Page 110
SCREENING STRATEGIES FOR BT EGGPLANT......Page 112
QUALITATIVE ANALYSIS OF EE1 EVENT......Page 115
DIAGNOSTICS FOR EE1 EVENT: TO ENSURE GM-FREE CONSERVATION OF GERMPLASM......Page 116
REFERENCES......Page 117
INTRODUCTION......Page 120
GMO BIOSENSORS......Page 122
REFERENCES......Page 131
INTRODUCTION......Page 134
DNA QUALITY AND PURITY: FROM EXTRACTION TO PCR ANALYSIS......Page 135
EFFECT OF FOOD PROCESSING ON GMO DETECTION......Page 136
ACKNOWLEDGMENT......Page 139
REFERENCES......Page 140
BIOSENSORS......Page 142
ALTERNATIVE DNA AMPLIFICATION METHODS......Page 148
FINAL REMARKS......Page 150
REFERENCES......Page 151
DIFFERENT TYPES OF SDNS: FROM MEGANUCLEASES TO CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)......Page 156
THE DIFFERENT USES OF SDNS......Page 157
WHAT HAS ALREADY BEEN DONE IN PLANTS?......Page 158
HOW THE NUCLEASE IS DELIVERED IN PLANT CELLS......Page 161
SDNS TO OBTAIN NEW PLANTS FOR COMMERCIAL USES: LEGAL FRAMEWORK......Page 163
CONCLUSIONS......Page 164
REFERENCES......Page 165
Section II - Social and Economic Contextof GMO Foods......Page 170
OVERVIEW ON PERCEPTION, POLICIES, AND POLITICS ON GM CROPS IN EUROPE, USA, AND THE REST OF THE WORLD......Page 172
PUBLIC ATTITUDES AND TRUST IN INSTITUTIONS......Page 175
THE FRAMING OF THE PUBLIC DEBATE......Page 176
METHODOLOGIES TO ASSESS PUBLIC AND CONSUMER ATTITUDES......Page 177
CONCLUDING REMARKS......Page 178
REFERENCES......Page 179
SALMON DECLINE: FROM WILD TO FARMED FISH......Page 182
THE SOCIAL AND ECONOMIC CONTEXT OF GENETICALLY MODIFIED SALMON......Page 184
GENETICALLY MODIFIED SALMON: A SMALLER ECOLOGICAL FOOTPRINT?......Page 185
CONCLUSION......Page 186
REFERENCES......Page 187
THEORETICAL FRAMEWORK: A MEDIATOR MODEL......Page 190
RESEARCH METHODS......Page 192
RESULTS: THE MEDIATOR MODEL......Page 195
ACKNOWLEDGMENT......Page 198
REFERENCES......Page 201
INTRODUCTION......Page 204
MATERIALS AND METHODS......Page 205
RESULTS......Page 206
DISCUSSION......Page 210
REFERENCES......Page 212
Section III - Government Regulation and Litigation for GMO foods......Page 216
METHODS......Page 218
RESULTS......Page 219
CASE STUDY: INFORMATION EFFECTS ON WTP FOR FOLATE BIOFORTIFIED RICE......Page 225
CONCLUSIONS......Page 226
REFERENCES......Page 228
GMO FEEDS: GENERAL CHARACTERISTICS, REGULATIONS, AND HEALTH ISSUES......Page 230
DETECTION METHODS OF GENETICALLY MODIFIED ORGANISMS IN FEED......Page 233
MOLECULAR ANALYSIS......Page 234
CONCLUSIONS......Page 240
REFERENCES......Page 241
THE CURRENT MARKET SITUATION OF GENETICALLY MODIFIED ORGANISMS......Page 246
CONCERNS ABOUT THE POTENTIAL PROBLEMS OF GMOS......Page 247
GENE CONTAINMENT......Page 248
SMG AND SMG PROTEIN FREE IN GMOS......Page 252
REFERENCES......Page 255
BIOGENESIS AND MECHANISMS OF NEGATIVE REGULATORS OF MRNA......Page 260
ROLES OF MIRNA IN PLANT GENETIC MODIFICATION......Page 262
REFERENCES......Page 271
INTRODUCTION......Page 280
THE EUROPEAN CONTEXT AND GMOS......Page 281
GMOS AND COMPETING JOURNALISTIC TRADITIONS OF JOURNALISTIC OBJECTIVITY AND INTERPRETATION......Page 283
THE CHALLENGE OF REPORTING ON GMOS: FACTS AND SCIENTIFIC DISPUTES......Page 285
REFERENCES......Page 286
LITIGATION OVER BIOTECH CROPS......Page 290
REFERENCES......Page 295
THE “FROM FARM TO FORK” EUROPEAN APPROACH......Page 298
GMOMETHODS DATABASE: DATA SOURCE AND SELECTION CRITERIA......Page 299
THE GMOMETHODS DATABASE CONTENT......Page 300
JOINT RESEARCH CENTER (JRC) GMO-MATRIX......Page 301
PRACTICAL CONSIDERATIONS......Page 305
NEXT GENERATION SEQUENCING......Page 306
FUTURE PROSPECTIVE FOR GMO ANALYSIS......Page 307
NGS......Page 308
REFERENCES......Page 309
Section IV - Role of GMC (Genetically Modified Crops) in Increasing the Food Supply in the Developing and Developed Countries......Page 312
HISTORICAL BACKGROUND OF GM CROPS......Page 314
FEEDING GM CROPS IN LIVESTOCK......Page 315
CONCLUSIONS AND FUTURE PROSPECTIVES......Page 316
REFERENCES......Page 317
INTRODUCTION......Page 320
REFERENCES......Page 325
INTRODUCTION......Page 328
APPROACHES TO STUDYING STRESS TOLERANCE IN PEANUT......Page 329
ISSUES WITH THE TRANSGENIC/GMO......Page 331
REFERENCES......Page 332
POPULATION VERSUS AGRICULTURAL PRODUCTIVITY......Page 336
NUTRITION SECURITY......Page 337
COTTON......Page 339
MARKER ASSISTED SELECTION......Page 340
HORTICULTURE......Page 341
ENHANCEMENT OF NUTRITION CONTENT......Page 342
OILSEEDS......Page 343
PULSES......Page 344
REFERENCES......Page 345
INTRODUCTION......Page 348
REFERENCES......Page 355
Section V - Potential Health Benefits, Acceptance and Risks due to Incorporation of Novel Plant Gene Products into the Food Supply......Page 358
THE TRADITIONAL METHODS OF GENOME WALKING......Page 360
THE NOVEL METHODS: A-T LINKER PCR......Page 361
RANDOMLY BROKEN FRAGMENT PCR WITH 5′ END-DIRECTED ADAPTOR FOR GENOME WALKING......Page 362
NEXT-GENERATION SEQUENCING IN OBTAINING THE MOLECULAR CHARACTERIZATION OF GENETICALLY MODIFIED ORGANISMS......Page 363
REFERENCES......Page 364
QUALITATIVE PCR DETECTION TECHNIQUES......Page 366
ENZYME-LINKED IMMUNOSORBENT ASSAY DETECTION TECHNIQUE......Page 367
HIGH-THROUGHPUT DETECTION TECHNIQUES......Page 368
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION......Page 371
REFERENCES......Page 372
STRATEGIES FOR METABOLIC ENGINEERING IN THE CAROTENOID PATHWAY......Page 376
ASSESSING THE IMPACT OF VITAMIN A BIOFORTIFICATION ON HUMAN NUTRITION......Page 377
GENETIC ENGINEERING OF CAROTENOID ACCUMULATION IN CEREAL GRAINS......Page 378
GENETIC ENGINEERING OF CAROTENOID ACCUMULATION IN STEM AND TUBER CROPS......Page 379
FUTURE PERSPECTIVES......Page 380
REFERENCES......Page 381
INTRODUCTION......Page 384
GENETICALLY ENGINEERED FOOD FROM TOMATO......Page 387
CAROTENOIDS METABOLIC ENGINEERING IN TOMATO......Page 393
REFERENCES......Page 399
PLANT DEFENSINS: ORIGIN AND DISTRIBUTION IN PLANTS......Page 404
PLANT DEFENSINS: STRUCTURE......Page 407
PLANT DEFENSINS: MECHANISM OF ACTION......Page 408
ENGINEERING FUNGAL RESISTANCE IN TRANSGENIC PLANTS USING PLANT-DERIVED DEFENSINS......Page 409
COMBINATORIAL STRATEGY APPROACH USING PLANT DEFENSINS......Page 412
TARGETED MODIFICATION OF NATIVE DEFENSINS FOR IMPROVED ANTIMICROBIAL ACTIVITY......Page 413
FUTURE PROSPECTS......Page 414
REFERENCES......Page 415
INTRODUCTION......Page 420
ALLERGEN DATABASES......Page 421
BIOINFORMATICS METHODS TO PREDICT PROTEIN ALLERGENICITY......Page 424
BIOINFORMATICS METHODS USED IN REGULATORY ASSESSMENT OF PROTEIN ALLERGENICITY......Page 428
REFERENCES......Page 431
MATERIALS AND METHOD......Page 436
RESULTS......Page 437
DISCUSSION......Page 441
REFERENCES......Page 444
INTRODUCTION......Page 446
INTROGRESSION......Page 447
EVOLUTIONARY AND AGRICULTURAL CONSEQUENCES OF INTROGRESSION OF INSECT-RESISTANCE GENES......Page 449
CONCLUSION......Page 450
REFERENCES......Page 451
Section VI - Safety of Genetically Modified Foods for Humans and Animals......Page 454
THE CURRENT BIOTECHNOLOGY DEBATE......Page 456
THE REGULATORY ENVIRONMENT OF GM FOOD LABELING POLICIES IN THE UNITED STATES......Page 457
CONCLUSIONS......Page 462
REFERENCES......Page 463
INTRODUCTION......Page 466
INSECTICIDAL ACTIVITY OF BT CRY PROTEINS......Page 467
HISTORY OF SAFE USE AND HUMAN DIETARY EXPOSURE ASSESSMENT OF CRY PROTEINS......Page 468
ASSESSMENT OF FOOD PROCESSING ON CRY PROTEIN INSECTICIDAL ACTIVITY......Page 469
ALLERGY AND IMMUNOGENICITY ASSESSMENT OF CRY PROTEINS......Page 470
TOXICOLOGY TESTING OF CRY PROTEINS......Page 471
TOXICOLOGY FEEDING STUDIES IN RODENTS FED BT CROPS......Page 472
REFERENCES......Page 473
INTRODUCTION......Page 478
MOLECULAR PROPERTIES OF FOOD ALLERGENS......Page 479
PROTEIN FAMILIES OF FOOD ALLERGENS AND ALLERGEN DATABASES......Page 480
APPLICATIONS OF ALLERGENOMICS FOR ASSESSMENT OF ALLERGENICITY IN GM PLANTS......Page 481
CONCLUSIONS......Page 483
REFERENCES......Page 484
Section VII - Demand and Uses ofNon-Genetically Modified Foods, and GMO’s for Humans and Animals......Page 488
INTRODUCTION......Page 490
MATERIALS AND METHODS......Page 491
RESULTS AND DISCUSSION......Page 495
REFERENCES......Page 500
INTRODUCTION......Page 504
REASON ANALYSIS......Page 505
REFERENCES......Page 509
Index......Page 512

Citation preview

Genetically Modified Organisms in Food

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Genetically Modified Organisms in Food

Production, Safety, Regulation and Public Health

Ronald Ross Watson

Mel and Enid Zuckerman College of Public Health Health Promotion Sciences Division University of Arizona, Tucson, AZ, USA

Victor R. Preedy

Department of Nutrition and Dietetics King’s College London, London, UK

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD • PARIS SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Copyright © 2016 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-802259-7 British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress For information on all Academic Press publications visit our website at http://store.elsevier.com/

Publisher: Nikki Levy Acquisition Editor: Patricia Osborn Editorial Project Manager: Jaclyn Truesdell Production Project Manager: Caroline Johnson Designer: Greg Harris Typeset by TNQ Books and Journals www.tnq.co.in Printed and bound in the United States of America

Contents Contributorsxv Prefacexix Acknowledgmentsxxi

Section I Development, Testing and Safety of Plant and Animal GMO foods 1. Soybean as a Food Source: Comparative Studies Focusing on Transgenic and Nontransgenic Soybean M.A.Z. Arruda, R.M. Galazzi, B.K. de Campos, M.A. Herrera-Agudelo, S.C.C. Arruda and R.A. Azevedo Introduction3 Some Comments about Transgenic Soybeans4 The Necessity of Using an Herbicide 4 Genetic Modification of Soybean–Glyphosate Resistance4 A Brief History of the Transgenic Soybean in Brazil4 Comparative Studies Involving Transgenic and Nontransgenic Soybean Seeds or Plants 5 Bioaccessibility Studies 5 Enzymes Involved in the Oxidative Stress 6 Metabolites and (Metallo)proteins 7 Trends8 Conclusions8 References9

2. Genetically Modified Crops: Biosafety Regulations and Detection Strategies Suchitra Kamle and Dawei Li Introduction11 Biosafety Measurement 11 Labeling Issues 13 Bt Gene and Stacked Traits 13

Detection Strategies 14 PCR and Real-Time PCR 14 Biosensors15 Protein-Based Detection 15 Immunoassays15 Immunostrip15 Immuno-PCR15 Future Prospects 16 References16

3. Genetically Modified Food Animals: An Overview Renu Pandey, Meenakshi Dwivedi, Shishir Kumar Gupta and Daman Saluja Introduction19 Genetically Modified Organisms 19 Definition19 Advent of GMOs 19 Methods for Introduction of Transgenesis 20 Applications of Transgenic Animals 22 Biological Research 22 Xenotransplantation23 Biopharming23 Environmental Sustainability 23 Food23 Concerns24 Future Directions 24 References24

4. Genetically Modified Aubergine (Also Called Brinjal or Solanum melongena) Lalitha R. Gowda General Description of Brinjal 27 Biochemical and Nutritional Properties 27 Insect Pests of Brinjal 28 Development of Insect-Resistant Bt-Brinjal 29 Fruit and Shoot Borer Management in Bt-Brinjal30 Fungal-Resistant Dm-AMP1-Aubergine Plants 31 Detection of Bt-Brinjal 31 Current Regulatory Framework of India for Recombinant DNA Technology 32 v

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Food Safety Assessment of Bt-Brinjal 32 Environmental Risk Assessment of Bt-Brinjal 34 Commercialization of Bt-Brinjal 35 References35

5. Nutritional Assessment of Genetically Modified Crops Using Animal Models R.D. Ekmay, S. Papineni and R.A. Herman Crop Composition, Nutritional Context, and the Suitability of Animal Studies 39 Processed Products 43 History of Animal Studies for Nutritional Assessment of Genetically Modified Crops 43 Regulatory Assessments 44 42-Day Broiler Study 45 90-Day Rodent Study 47 Other Animal Models 47 Conclusion48 References48

6. Noncoding RNA-Based Genetically Modified Crops: Concepts and Challenges S.V. Ramesh and Shelly Praveen Introduction51 Various ncRNA-Based Silencing Platforms 51 Apprehensions of Noncoding RNA-Based Genetically Modified Crops 54 Persistence of ncRNAs 56 Predictive Environmental Risk Assessment 56 Impact on Plant Protection Measures 59 Food and Feed Safety 60 Nutritional Composition and Equivalency 60 Conclusions and Future Directions 60 References60

7.  Agrobacterium-Mediated Alien Gene Transfer Biofabricates Designer Plants Shweta Mehrotra and Vinod Goyal The Biology of Agrobacterium63 Agrobacterium-Mediated T-DNA Transfer Process63 Signal Recognition 64 Bacterial Colonization 64 Induction of Bacterial Virulence System 64 Generation of the T-DNA Transfer Complex 65 Transfer of T-DNA 65 Integration of T-DNA into the Plant Genome66 Comparison of T-DNA Transfer to Conjugative DNA Transfer 66 Factors Influencing Agrobacterium-Mediated

Transformation66 Osmotic Treatment of Explants 66 Desiccation of Explants 66 Culture Medium 66 Antinecrotic Treatments 67 Temperature67 Surfactants67 Antibiotics67 Promoters67 Selectable Markers 67 Reporter Genes 67 Advances in Agrobacterium-Mediated Transformation67 Agrobacterium-Mediated Transformation of Dicotyledonous Plants 68 Agrobacterium-Mediated Transformation of Monocotyledonous Plants 68 Agrobacterium-Mediated In Planta Transformation68 Agrobacterium-Mediated Transformation of Nonplant Organisms 69 Challenges in Agrobacterium-Mediated Transformation69 Applications69 Biosafety Aspects 70 Summary and Future Prospects 70 References71

8. Understanding the Factors Influencing Attitudes toward Genetically Modified Rice Latifah Amin and Hasrizul Hashim Introduction75 Theoretical Framework and Hypotheses Development76 Engagement76 Confidence in Key Players 77 Attitudes to Technology 77 Attitudes to Nature 78 Religiosity78 Perceived Moral Concerns 78 Perceived Risks and Perceived Benefits 79 Religious Acceptance 79 Research Methodology 79 Survey Data Collection 79 Instrument79 Statistical Analysis 80 Results80 Measurement Model (Confirmatory Factor Analysis)80 Structural Equation Modeling 80 Construct Reliability and Validity 80 Relationships among the Variables 80 Conclusion83

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Acknowledgment83 References83

9. Qualitative and Quantitative Diagnostics for EE1 Event of Bt Eggplant Gurinder J. Randhawa and Monika Singh Introduction87 Screening Strategies for Bt Eggplant 89 GMO Matrix 89 LAMP-Based Screening 90 Multiplex PCR-Based Screening 92 Qualitative Analysis of EE1 Event 92 Construct- and Event-Specific PCR Assays 92 Ready-to-Use TaqMan® Real-Time PCR-Based Multitarget System 92 Diagnostics for EE1 Event: To Ensure GM-Free Conservation of Germplasm 93 Conclusion94 References94

10. Biosensors for Detection of Genetically Modified Organisms in Food and Feed Mary A. Arugula and Alex L. Simonian Introduction97 Standard Approaches for Detection of GMO 99 GMO Biosensors 99 Optical Biosensors 100 Piezoelectric Biosensors 103 Electrochemical Biosensors 106 References108

11. Genetically Modified Organism Analysis as Affected by DNA Degradation Telmo J.R. Fernandes, Joana Costa, Alexandra Plácido, Caterina Villa, Liliana Grazina, Liliana Meira, Maria Beatriz P.P. Oliveira and Isabel Mafra Introduction111 DNA Quality and Purity: From Extraction to PCR Analysis 112 Effect of Food Processing on GMO Detection113 Mechanical Processing 113 Effect of Temperature 114 Oil Extraction and Refining 115 Final Remarks 116 Acknowledgment116 References117

12. Novel Strategies for Genetically Modified Organism Detection Alexandra Plácido, Joana S. Amaral, Joana Costa, Telmo J.R. Fernandes, M. Beatriz P.P. Oliveira, Cristina Delerue-Matos and Isabel Mafra Introduction119 Biosensors119 Electrochemical Biosensors 120 Optical Biosensors 124 Piezoelectric Biosensors 125 Microarrays125 Alternative DNA Amplification Methods 125 Final Remarks 127 Acknowledgment128 References128

13. Targeted Genetic Modification in Crops Using Site-Directed Nucleases Cécile Collonnier, Fabien Nogué and Josep M. Casacuberta Introduction133 Different Types of SDNs: From Meganucleases to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 133 The Different Uses of SDNs 134 What Has Already Been Done in Plants? 135 How the Nuclease is Delivered in Plant Cells 138 Comparison of SDN-Based Approaches to Currently Used Techniques in Plant Breeding 140 SDNs to Obtain New Plants for Commercial Uses: Legal Framework 140 Conclusions141 References142

Section II Social and Economic Context of GMO Foods 14. Agricultural Biotechnology and Public Attitudes: An Attempt to Explain the Mismatch between Experience and Perception Philipp Aerni Introduction149 Overview on Perception, Policies, and Politics on GM Crops in Europe, USA, and the Rest of the World 149 Europe150 USA151 Rest of the World 152

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Public Attitudes and Trust in Institutions 152 The Framing of the Public Debate 153 Methodologies to Assess Public and Consumer Attitudes 154 The Problem with Surveys that Measure Stated Consumer Preferences 154 The Problem with Surveys that Measure State Political Preferences 154 Why Concrete Experience with the Technology Matters 155 Concluding Remarks 155 References156

15. Fishy Business: Genetic Engineering and Salmon Aquaculture Rebecca Clausen, Stefano B. Longo and Brett Clark Introduction159 Salmon Decline: From Wild to Farmed Fish 159 The Social and Economic Context of Genetically Modified Salmon 161 Genetically Modified Salmon: A Smaller Ecological Footprint? 162 Conclusion163 References164

16. Consumer Behavior Regarding Genetically Modified Foods: A Mediator Model Macario Rodríguez-Entrena and Melania Salazar-Ordóñez Introduction167 Theoretical Framework: A Mediator Model 167 Research Methods 169 Sample169 Methodological Issues 169 Results: The Mediator Model 172 Conclusions175 Acknowledgment175 References178

17. Are Ready for Market Genetically Modified, Conventional and Organic Soybeans Substantially Equivalent as Food and Feed? T. Bøhn, M. Cuhra, T. Traavik and J. Fagan Introduction181 Long-Term Studies of Food and Feed Products and Agrochemicals are Missing 181 Soy Production is Dominated by HerbicideTolerant (HT) RR GM Soy 181

The Daphnia magna Model 182 Previous Studies in D. magna182 Materials and Methods 182 Soy Samples and Characterization 182 Feeding Studies in D. magna182 Results183 Glyphosate and AMPA Residues in the Soybeans 183 Main Constituents of the Soy—Individual Samples183 Discriminant Analysis 185 Life-Time Feeding Studies in D. magna with the Different Soy Types 185 Discussion187 Residues of Pesticides in the Soy 187 Increases in MRL of Glyphosate in Food and Feed 187 Toxicity and Health Relevance of Pesticide/ Glyphosate Residues 188 Nutritional Components 188 Conclusion189 Acknowledgment189 References189

Section III Government Regulation and Litigation for GMO foods 18. Consumer Acceptance and Willingnessto-Pay for Genetically Modified Foods with Enhanced Vitamin Levels Hans De Steur, Dieter Blancquaert, Simon Strobbe, Shuyi Feng, Jeroen Buysse, Christophe Stove, Willy Lambert, Dominique Van Der Straeten and Xavier Gellynck Introduction195 Methods195 Search Strategy 195 Study Selection 196 Data Extraction 196 Results196 Acceptance of GM Foods with Enhanced Vitamin Levels 196 Willingness-to-Pay for GM Foods with Enhanced Vitamin Levels 197 Determinants of Acceptance and Willingness-to-Pay200 Case Study: Information Effects on WTP for Folate Biofortified Rice 202 Information Effects 203 Conclusions203 References205

Contents  ix

19. Detection of Genetically Modified Organisms in Feed Özgür Çakır, Sinan Meriç and Şule Arı Introduction207 GMO Feeds: General Characteristics, Regulations, and Health Issues 207 Detection Methods of Genetically Modified Organisms in Feed 210 Molecular Analysis 211 Genomic DNA Extraction 211 Conventional PCR Analysis for Identifying Plant Species 213 Conventional PCR Analysis for Detection of GMOs 214 Nested PCR Analysis for Determining GMO Events in Feed 214 Real-Time PCR Analysis for Quantifying GMO in Feed 215 Interpreting Results 216 Conclusions217 References218

20. Development of Molecular Strategies for Gene Containment and MarkerFree Genetically Modified Organisms Ning Yuan, Steve Cogill and Hong Luo The Current Market Situation of Genetically Modified Organisms 223 Concerns about the Potential Problems of GMOs 224 Gene Flow and Introgression from GMOs 224 Unnecessary Foreign DNA and Protein in the Final Product 225 Gene Containment 225 Male Sterility 225 Maternal Inheritance 226 GURT-Based Technology Protection System 227 Gene Self-Deleting System 229 SMG and SMG Protein Free in GMOs 229 Site-Specific Recombination 229 Homologous Recombination 230 Cotransformation231 Conclusion232 Acknowledgment232 References232

21. Negative Regulators of Messenger RNA and the Role of microRNA for Plant Genetic Engineering Shuangrong Yuan and Hong Luo Introduction237 Biogenesis and Mechanisms of Negative Regulators of mRNA 237

Biogenesis and Mechanisms of miRNA 237 Biogenesis and Mechanisms of siRNA 238 Roles of miRNA in Plant Genetic Modification 239 MiRNA-Mediated Plant Organ Development for Plant Genetic Engineering 239 MiRNA-Mediated Plant Response to Abiotic Stress for Plant Genetic Engineering 243 MiRNA-Mediated Plant Response to Biotic Stress for Plant Genetic Engineering 246 MiRNA-Based Gene Silencing Strategies in Plant Genetic Modification 248 Concluding Remarks 248 Acknowledgment248 References248

22. Genetically Modified Organisms and European Journalism Anita Howarth Introduction257 The European Context and GMOs 258 The Politicization of Food and the Sensitizing of Journalists 258 Arguments and Counter-Arguments 259 GMOs: Campaigns and Counter-Campaigns 260 GMOs and Competing Journalistic Traditions of Journalistic Objectivity and Interpretation260 The American Tradition of an “Objectivity” Ritual260 The “Anglo–American Model” and British Newspapers261 Continental European Traditions of Journalism261 The Challenge of Reporting on GMOs: Facts and Scientific Disputes 262 Conclusion263 References263

23. Agricultural Biotechnology Regulation and Litigation: Preventing “Contamination” Thomas P. Redick, Theodore A. Feitshans and Megan R. Galey Regulatory Background 267 Litigation Over Biotech Crops 267 Syngenta Litigation Regarding Major Market Approval in Federal and State Courts 268 Monsanto’s Wheat Woes 269 Inadvertent Reproduction of Patented Seed 269 Bayer’s Billion-Dollar LibertyLink Rice Settlement269 Aventis and the StarLink Corn Recall 270 GMOs Are Not “Natural” 270 The National Environmental Policy Act 271

x Contents

Litigation Involving Ordinances Banning or Restricting Biotech Crops in States with Biotech Crop Opponents 272 References272

24. The European Union Reference Methods Database and Decision Supporting Tool for the Analysis of Genetically Modified Organisms: GMOMETHODS and JRC GMO-Matrix Laura Bonfini, Alexandre Angers-Loustau, Mauro Petrillo, Ilaria Ciabatti, Francesco Gatto, Sabrina Rosa, Antoon Lievens and Joachim Kreysa The “From Farm to Fork” European Approach 275 Harmonized Analytical Approaches for GMO Detection: Role of EU-RL GMFF and the European Network of GMO Laboratories (ENGL)276 GMOMETHODS Database: Data Source and Selection Criteria 276 The GMOMETHODS Database Content 277 Joint Research Center (JRC) GMO-Matrix 278 The Central Core Sequence Information System282 The in silico Pipeline Supporting the JRC GMO-Matrix282 Practical Considerations 282 Next Generation Sequencing 283 Challenges284 Future Prospective for GMO Analysis 284 Prespotted Plates 284 Digital PCR 284 NGS285 Conclusions286 References286

Section IV Role of GMC (Genetically Modified Crops) in Increasing the Food Supply in the Developing and Developed Countries 25. Genetically Modified Crops: An Alternative Source of Livestock Feeding Rajib Deb, T.V. Raja, Sandip Chakraborty, Shishir K. Gupta and Umesh Singh Introduction291 Historical Background of GM Crops 291 What is a GM Crop? 292

Risks and Concerns of GM Crops 292 Feeding GM Crops in Livestock 292 Large Ruminants 292 Small Ruminants 292 Poultry293 Fish293 Conclusions and Future Prospectives 293 References294

26. Transgenic Food: Uncertainty, Trust, and Responsibility Lucia Martinelli, Małgorzata Karbarz and Vincenzo Pavone Introduction297 Promises and Concerns over GMOs: A Reorienting Strategy? 297 Conclusion302 Acknowledgment302 References302

27. Engineering Stress Tolerance in Peanut (Arachis hypogaea L.) Bhavanath Jha, Avinash Mishra and Amit Kumar Chaturvedi Introduction305 Factors Affecting Peanut Production 306 Approaches to Studying Stress Tolerance in Peanut 306 Molecular Breeding 306 Functional Genomics 307 Genetic Engineering 307 Enhanced Stress Tolerance in Peanut: A Case Study 308 Issues with the Transgenic/GMO 308 Conclusion and Future Prospective 309 Acknowledgment309 References309

28. Why India Needs Biotechnology to Ensure Food and Nutrition Security? Chenna R. Aswath, P.U. Krisnaraj and Govindarajan Padmanaban Introduction313 Population versus Agricultural Productivity 313 Nutrition Security 314 Cotton316 Rice317 Hybrid Rice 317 Marker Assisted Selection 317 Transgenic Rice 318

Contents  xi

Rice Genomics 318 Horticulture318 Enhancement of Nutrition Content 319 Oilseeds320 Pulses321 Conclusions322 Acknowledgment322 References322

29. Wheat Storage Proteins in Transgenic Rice Endosperm Maria Oszvald, Ferenc Békés and László Tamás Introduction325 The Unique Feature of Wheat Proteins 325 Characterization of Rice Proteins and Rice Dough 326 Production and Analysis of Transgenic Rice Plants 327 Identification and Characterization of 1Dx5 HMW Glutenin in Rice Endosperm 328 Functional Properties of Rice Dough Made from Transgenic Rice 330 Trafficking of Wheat Proteins in Transgenic Rice Endosperm 330 Acknowledgment332 References332

Section V Potential Health Benefits, Acceptance and Risks due to Incorporation of Novel Plant Gene Products into the Food Supply 30. Event-Specific Identification Technology of Genetically Modified Organisms Wentao Xu and Ying Shang Introduction337 The Traditional Methods of Genome Walking337 The Novel Methods: A-T Linker PCR 338 Loop-Linker PCR 339 Randomly Broken Fragment PCR with 5′ End-Directed Adaptor for Genome Walking 339 Next-Generation Sequencing in Obtaining the Molecular Characterization of Genetically Modified Organisms 340 Prospect341 References341

31. The Detection Techniques of Genetically Modified Organisms Wentao Xu and Ying Shang Introduction343 Qualitative PCR Detection Techniques 343 Quantitative PCR 344 Enzyme-Linked Immunosorbent Assay Detection Technique 344 High-Throughput Detection Techniques 345 Multiplex PCR 345 Universal Primer Multiplex PCR (UP-M-PCR) 345 Multiplex Ligation-Dependent Probe Amplification346 Single Universal Primer Multiplex LigationDependent Probe Amplification 346 Multiplex PCR with Pyrosequencing for GMO Detection 347 Loop-Mediated Isothermal Amplification 348 Prospect349 References349

32. Carotenoids, Genetically Modified Foods, and Vitamin A Nutrition Li Tian Introduction353 Strategies for Metabolic Engineering in the Carotenoid Pathway 353 Assessing the Impact of Vitamin A Biofortification on Human Nutrition 354 Genetic Engineering of Carotenoid Accumulation in Cereal Grains 355 Genetic Engineering of Carotenoid Accumulation in Oilseeds and Legumes 356 Genetic Engineering of Carotenoid Accumulation in Stem and Tuber Crops 356 Genetic Engineering of Carotenoid Accumulation in Vegetables and Fruits 357 Future Perspectives 357 References358

33. Food from Genetically Engineered Plants: Tomato with Increased β-Carotene, Lutein, and Xanthophylls Contents Caterina D’Ambrosio, Adriana L. Stigliani and Giovanni Giorio Introduction361 Legislation in the United States of America and the European Union 361 Genetically Engineered Crop Cultivation in the World 362

xii Contents

Genetically Engineered Food and Feed in the EU 363 The Pros and Cons of Extant and Future Genetically Engineered Plants 364 Genetically Engineered Food from Tomato 364 Genetics, Breeding, and Nutritional Aspects of Tomato 364 Carotenoid Metabolism in Tomato 367 Carotenoids Metabolic Engineering in Tomato 370 Overcoming the Developmental Block of Lycopene Cyclases 371 Stacking of Cyclase Transgenes: The CD Line (HighCaroxHighDelta) 372 Boosting the Hydroxylation of β-Ionone Rings374 Carotenoid Composition and Nutritional Values of Transgenic Tomatoes 375 Final Comments 376 References376

34. Plant Defensins for the Development of Fungal Pathogen Resistance in Transgenic Crops Siddhesh B. Ghag, Upendra K. Singh Shekhawat and Thumballi R. Ganapathi Introduction381 Plant Defensins: Origin and Distribution in Plants 381 Plant Defensins: Structure 384 Plant Defensins: Mechanism of Action 385 Plant Defensins: Role in Normal Growth and Development 386 Engineering Fungal Resistance in Transgenic Plants Using Plant-Derived Defensins 386 Combinatorial Strategy Approach Using Plant Defensins 389 Tissue Specific Overexpression of Plant Defensins390 Targeted Modification of Native Defensins for Improved Antimicrobial Activity 390 Biosafety Issues Regarding the Use of Plant Defensins391 Future Prospects 391 References392

35. Bioinformatics Application in Regulatory Assessment for Potential Allergenicity of Transgenic Proteins in Food Crops Ping Song Introduction397 Allergen Databases 398

Bioinformatics Methods to Predict Protein Allergenicity401 Sequence Homology Identification Using Local Alignment Search Tools 403 Matches of Short Segments of Contiguous Amino Acids Based on the Estimated Minimum Length for IgE Epitope Binding 403 Epitopic Prediction Using the Physicochemical Properties of a Query Protein Calculated Using Epitopic Information of Known Allergens403 Prediction by Searching Motifs Contained in Known Allergens 404 A Combination of Sequence Homology Searches and Motif Identification 404 Machine Learning Model 404 Prediction based on Tertiary Structure 405 Bioinformatics Methods Used in Regulatory Assessment of Protein Allergenicity 405 Conclusion408 Acknowledgment408 References408

36. Attitudes of Polish Adolescents toward Genetically Modified Organisms and Genetically Modified Food Anna Jurkiewicz Introduction413 Objective413 Materials and Method 413 Characteristics of the Study Group 413 Results414 Discussion418 Conclusion421 References421

37. Consequences of Gene Flow between Transgenic, Insect-Resistant Crops and their Wild Relatives Henri Darmency Introduction423 Introgression424 Introgression among Cultivated and Wild Forms of the Same Biological Species 424 Introgression among Distantly Related Species425 Evolutionary and Agricultural Consequences of Introgression of Insect-Resistance Genes 426 General Considerations 426 The Case of Oilseed Rape 427 Conclusion427 References428

Contents  xiii

Section VI Safety of Genetically Modified Foods for Humans and Animals 38. Labeling of Genetically Modified Food in the United States Anton E. Wohlers Introduction433 The Current Biotechnology Debate 433 The Regulatory Environment of GM Food Labeling Policies in the United States 434 Conclusions439 References440

39. The Food Safety Assessment of Bt Crops Bruce Hammond Introduction443 History of Safe Use of Bt Microbial Pesticides444 Insecticidal Activity of Bt Cry Proteins 444 History of Safe Use and Human Dietary Exposure Assessment of Cry Proteins 445 Regulatory Guidance for the Safety Assessment of Cry Proteins Introduced into GM Crops 446 Digestibility of Cry Proteins When Consumed in the Diet 446 Assessment of Food Processing on Cry Protein Insecticidal Activity 446 Allergy and Immunogenicity Assessment of Cry Proteins 447 Toxicology Testing of Cry Proteins 448 Acute Toxicity Testing 448 Toxicology Feeding Studies in Rodents Fed Bt Crops 449 Food and Feed Safety Benefits of Bt Crops 450 Reduced Insecticide Application 450 Reduced Mycotoxin Contamination of Grain 450 Conclusions450 Acknowledgment450 References450

40. Allergen Analysis in Plants and Use in the Assessment of Genetically Modified Plants Rie Satoh and Reiko Teshima Introduction455 Molecular Properties of Food Allergens 456

Protein Families of Food Allergens and Allergen Databases 457 Applications of Allergenomics for Assessment of Allergenicity in GM Plants 458 Conclusions460 References461

Section VII Demand and Uses of Non-Genetically Modified Foods, and GMO’s for Humans and Animals 41. Usage of Genetically Modified Foods: The Extent of Genetically Modified Rice, Maize, and Soy Consumption in Saudi Arabia Mohamed Fawzy Ramadan, Rafaat M. Elsanhoty and A.I. Al-Turki Introduction467 Materials and Methods 468 Materials468 Sampling468 Sample Pretreatment 468 Extraction of Genomic DNA 468 Extraction of DNA from Processed Food Samples469 DNA Yield and Quality 469 PCR Detection of Lectin Gene and Invertase Gene 469 GMOScreen 35S/NOS 471 Results and Discussion 472 DNA Extraction 472 Detection of Lectin and Invertase Genes 473 Detection of GMO Specific Genetic Elements (35S Promoter or NOS Terminator) 473 Specific Detection of RR Soybean 474 Specific Detection of GM Maize Lines 474 Quantitative Detection of GM Soybean and Maize Lines 476 Conclusion477 References477

42. Reasons Analysis of Chinese Urban Consumers Opposing Genetically Modified Food—An Overview Yue Ma and Shaoping Gan Introduction481 Background481 Statement of the Problem 482

xiv Contents

Reason Analysis 482 Health Issues 483 Economic Issues 484 Environmental Issues 484 Other Issues 485 Discussion486

Recommendations486 References486

Index489

Contributors Philipp Aerni Center for Corporate Responsibility and Sustainability (CCRS), University of Zurich, Switzerland

T. Bøhn  GenØk – Centre for Biosafety, Tromsø, Norway; UiT The Arctic University of Norway, Faculty of Health Sciences, Tromsø, Norway

A.I. Al-Turki  Al-Qassim University, Department of Plant Production and Protection, Qassim, Kingdom of Saudi Arabia

Laura Bonfini Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Joana S. Amaral  REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; ESTiG, Instituto Politécnico de Bragança, Bragança, Portugal

Jeroen Buysse  Ghent University, Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent, Belgium

Latifah Amin  Pusat Citra Universiti, Kebangsaan Malaysia, Selangor, Malaysia

Universiti

Alexandre Angers-Loustau Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy Şule Arı  Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey; Research and Application Center for Biotechnology and Genetic Engineering, Istanbul, Turkey

Özgür Çakır  Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey Josep M. Casacuberta  Center for Research in Agricultural Genomics, CRAG (CSIC-IRTA-UAB-UB), Campus UAB, Cerdanyola del Vallès Barcelona, Spain Sandip Chakraborty Animal Resources Development Department, Agartala, Tripura, India Amit Kumar Chaturvedi  Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India

M.A.Z. Arruda  University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil; University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil

Ilaria Ciabatti Biotechnologies Unit, DG SANTE, European Commission, Bruxelles, Belgium

S.C.C. Arruda University of São Paulo, Department of Genetics, Laboratory of Genetics Biochemistry of Plants, ESALQ, Piracicaba, Brazil

Rebecca Clausen Fort Lewis College, Department of Sociology, Durango, CO, USA

Mary A. Arugula Auburn University, Department of Materials Engineering, Auburn, AL, USA Chenna R. Aswath Division of Biotechnology, Indian Institute of Horticultural Research, Bangalore, India R.A. Azevedo University of São Paulo, Department of Genetics, Laboratory of Genetics Biochemistry of Plants, ESALQ, Piracicaba, Brazil Ferenc Békés  FBFD Pty. Ltd., Beecroft, NSW, Australia Dieter Blancquaert Ghent University, Laboratory of Functional Plant Biology, Department of Physiology, Ghent, Belgium

Brett Clark  University of Utah, Department of Sociology, Salt Lake City, UT, USA

Steve Cogill  Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA Cécile Collonnier INRA AgroParisTech, IJPB, UMR 1318, INRA Centre de Versailles, Versailles, France Joana Costa REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal M. Cuhra  GenØk – Centre for Biosafety, Tromsø, Norway; UiT The Arctic University of Norway, Faculty of Health Sciences, Tromsø, Norway Caterina D’Ambrosio Centro Ricerche Metapontum Agrobios, ALSIA, Metaponto (MT), Italy

xv

xvi Contributors

Henri Darmency Institut National de la Recherche Agronomique, UMR1347 Agroécologie, Dijon, France B.K. de Campos University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil; University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil Hans De Steur  Ghent University, Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent, Belgium Rajib Deb  ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India Cristina Delerue-Matos  REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal Meenakshi Dwivedi Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India R.D. Ekmay  Dow AgroSciences LLC., Indianapolis, IN, USA Rafaat M. Elsanhoty El-Sadat City University, Institute of Genetic Engineering and Biotechnology, Department of Industrial Biotechnology, Branch of Food and Dairy Biotechnology, Sadat City, Egypt

Xavier Gellynck  Ghent University, Department of Agricultural Economics, Faculty of Bioscience Engineering, Ghent, Belgium Siddhesh B. Ghag  Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India Giovanni Giorio  Centro Ricerche Metapontum Agrobios, ALSIA, Metaponto (MT), Italy Lalitha R. Gowda  Chief Scientist (Former), CSIR-Central Food Technological Research Institute, Department of Protein Chemistry and Technology, Mysore, Karnataka, India Vinod Goyal  University of Delhi, Delhi, India Liliana Grazina  REQUIMTE, Departamento de Ciências Químicas, Universidade do Porto, Porto, Portugal Shishir K. Gupta Indian Veterinary Research Institute, Izatnagar, Bareilley, Uttar Pradesh, India Shishir Kumar Gupta Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India; Indian Veterinary Research Institute, Izatnagar, Bareilley, Uttar Pradesh, India

J. Fagan  Earth Open Source Institute, Fairfield, IA, USA

Bruce Hammond Private Consultant, St Charles, MO, USA

Theodore A. Feitshans  North Carolina State University, Raleigh, NC, USA

Hasrizul Hashim Pusat Citra Universiti, Universiti Kebangsaan Malaysia, Selangor, Malaysia

Shuyi Feng Nanjing Agricultural University, College of Public Administration, Weigang, Nanjing, China

R.A. Herman  Dow AgroSciences LLC., Indianapolis, IN, USA

Telmo J.R. Fernandes  REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

M.A. Herrera-Agudelo  University of Campinas– Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil; University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil

R.M. Galazzi  University of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil; University of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil Megan R. Galey Husch Blackwell LLP, St. Louis, MO, USA

Anita Howarth Brunel University London, Kingston Lane, Uxbridge, UK Bhavanath Jha  Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India

Thumballi R. Ganapathi  Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India

Anna Jurkiewicz  Institute of Rural Health, Department of Public Health, Lublin, Poland

Shaoping Gan  Philosophy Institution of Chinese Academy of Social Science, Beijing, China

Małgorzata Karbarz University of Rzeszów, Institute of Applied Biotechnology and Basic Sciences, Kolbuszowa, Poland

Francesco Gatto Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Suchitra Kamle  Shanghai Jiao Tong University, School of Pharmacy, Shanghai, China

Joachim Kreysa  Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy

Contributors  xvii

P.U. Krisnaraj College of Agriculture, Department of Agricultural Microbiology, Bijapur, India Willy Lambert  Ghent University, Laboratory of Toxicology, Department of Bioanalysis, Ghent, Belgium Dawei Li Shanghai Jiao Tong University, School of Pharmacy, Shanghai, China Antoon Lievens  Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy Stefano B. Longo North Carolina State University, Department of Sociology and Anthropology, Raleigh, NC, USA Hong Luo Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA

Mauro Petrillo Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy Alexandra Plácido  REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Porto, Portugal Shelly Praveen Indian Agricultural Research Institute, Advanced Centre for Plant Virology, Division of Plant Pathology, New Delhi, India T.V. Raja  ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

Lucia Martinelli  MUSE – Science Museum, Trento, Italy

Mohamed Fawzy Ramadan  Zagazig University, Agricultural Biochemistry Department, Zagazig, Egypt; Umm Al-Qura University, Institute of Scientific Research and Revival of Islamic Heritage, Makkah, Kingdom of Saudi Arabia

Yue Ma  Graduate School of Chinese Academy of Social Science, Beijing, China

S.V. Ramesh Indian Council of Agricultural ResearchICAR, Directorate of Soybean Research, Indore, India

Shweta Mehrotra  University of Delhi, Delhi, India

Gurinder J. Randhawa  Division of Genomic Resources, Indian Council of Agricultural Research (ICAR)National Bureau of Plant Genetic Resources, New Delhi, India

Isabel Mafra REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal

Liliana Meira REQUIMTE, Departamento de Ciências Químicas, Universidade do Porto, Porto, Portugal Sinan Meriç Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Istanbul, Turkey; Kultur University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, Istanbul, Turkey Avinash Mishra  Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat, India Fabien Nogué INRA AgroParisTech, IJPB, UMR 1318, INRA Centre de Versailles, Versailles, France Maria Beatriz P.P. Oliveira  REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal Maria Oszvald Eotvos Lorand University, Department of Plant Physiology and Molecular Plant Biology, Budapest, Hungary Govindarajan Padmanaban  Indian Institute of Science, Department of Biochemistry, Bangalore, India

Thomas P. Redick  Global Environmental Ethics Counsel LLC, Clayton, MO, USA Macario Rodríguez-Entrena  IFAPA-Institute of Agricultural Research and Training, Department of Agricultural Economics and Rural Studies, Alameda del Obispo Center, Córdoba, Spain Sabrina Rosa  Molecular Biology and Genomics Unit, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy Melania Salazar-Ordóñez  Universidad Loyola Andalucía, Department of Economics, Córdoba, Spain Daman Saluja  Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India Rie Satoh Analytical Science Division, National Food Research Institute, National Agriculture and Food Research Organization Tsukuba, Ibaraki, Japan

S. Papineni Dow AgroSciences LLC., Indianapolis, IN, USA

Ying Shang China Agricultural University, Laboratory of Food Safety, College of Food Science and Nutri­ tional Engineering, Beijing, China; The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing, China

Vincenzo Pavone  Institute of Public Goods and Policies (IPP-CSIC), Madrid, Spain

Alex L. Simonian Auburn University, Department of Materials Engineering, Auburn, AL, USA

Renu Pandey  Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India

xviii Contributors

Upendra K. Singh Shekhawat Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, India Monika Singh Division of Genomic Resources, Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources, New Delhi, India Umesh Singh ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India Ping Song Dow AgroSciences LLC, Indianapolis, IN, USA Adriana L. Stigliani Centro Ricerche Metapontum Agrobios, ALSIA, Metaponto (MT), Italy Christophe Stove  Ghent University, Laboratory of Toxicology, Department of Bioanalysis, Ghent, Belgium Simon Strobbe  Ghent University, Laboratory of Functional Plant Biology, Department of Physiology, Ghent, Belgium László Tamás Eotvos Lorand University, Department of Plant Physiology and Molecular Plant Biology, Budapest, Hungary Reiko Teshima Division of Foods, National Institute of Health Sciences, Setagaya, Tokyo, Japan

Li Tian University of California, Department of Plant Sciences, Davis, CA, USA T. Traavik GenØk – Centre for Biosafety, Tromsø, Norway; UiT The Arctic University of Norway, Faculty of Health Sciences, Tromsø, Norway Dominique Van Der Straeten  Ghent University, Laboratory of Functional Plant Biology, Department of Physiology, Ghent, Belgium Caterina Villa REQUIMTE, Departamento de Ciências Químicas, Universidade do Porto, Porto, Portugal Anton E. Wohlers Cameron University, Academic Enrichment, Lawton, OK, USA Wentao Xu  China Agricultural University, Laboratory of Food Safety, College of Food Science and Nutritional Engineering, Beijing, China; The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing, China Ning Yuan  Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA Shuangrong Yuan Clemson University, Department of Genetics and Biochemistry, Clemson, SC, USA

Preface Recent media reports suggest that 70% of production in USA is by GMO (genetically modified organisms) plants and animals, principally corn.

SECTION I: DEVELOPMENT, TESTING AND SAFETY OF PLANT AND ANIMAL GENETICALLY MODIFIED CROPS AS FOODS This book focuses on scientific evaluation of published research relating to general responses by public health agencies to new GMO food products to assert their safety as well as potential health risks. The initial section discusses production and safety of GMO. Therefore comparative studies on transgenic and standard soybeans are evaluated followed by discussions of biosafety regulations and detection strategies. GMO plants as food have been evaluated for nutrition using animal models. GMO plants based upon noncoding RNAs help understand the concepts and challenges of GMO. It is clear that some aspects of GMO affect people’s use so the factors influencing use of genetically modified rice are discussed. New and improved methods to detect GMO in food and feed are needed including biosensors, DNA degradation, and other novel strategies in three chapters.

SECTION II: SOCIAL AND ECONOMIC CONTEXT OF GMO FOODS GMO technology has doubled the yields of some crops which is huge in its effects on food availability and practice of agriculture with public health implications for areas with many inadequately fed peoples. However concerns about newly introduced nonfood genes into foods have resulted in General Mills, a major breakfast cereal food producer in the USA announcing in 2014 that its classic Cheerios breakfast cereal will be GMO free. The market could be the 20% of Americans with concerns about GMO foods, double what it was a decade ago. Half of the American states had bills introduced to require GMO labeling of foods, but all failed. The non-GMO breakfast cereal is possible because most of Cheerios is oats and noncorn sources will be used for sweetening. Yet the rest of General Mills other foods will contain GMO products due to economic and availability reason. Therefore what are the economic, nutrition, food availability, and risks/benefits of using these GMO crops as foods? Therefore Phillip Aerni tests agricultural biotechnology to explain mismatch between public attitudes and perceptions about GMO foods. Food animals, fish are appearing and affecting the salmon aquaculture whose sociological effects are described by Rebecca Clausen and others. A mediator model is used by Macario RodrigueaEntrena to understand consumer behavior with GMO. A group in Norway asks and answers the important question whether GMO soybeans are equivalent as food and feed to organic ones.

SECTION III: GOVERNMENT REGULATION AND LITIGATION FOR GMO FOODS The United States Agriculture Department proposed eliminating restrictions on the use of corn and soybean seeds genetically engineered to resist common weed killers including Roundup recently. This move was welcomed by many farmers but concerning to some scientists and environmentalists who worry it could invite growers to use more chemicals. The herbicide, known as 2, 4-D, has had limited use in corn and soybean farming because it is toxic to the plants early in their growth. While the American farmers and public have been generally welcoming of GMO crops European governments and public has been stronger in concerns about safety and health. To help understand the different views a group in Belgium review consumer acceptance and pay for high nutrient GMO in Europe. Ozgur Caku reviews detection of GMO in animal feeds. Then gene containment and marker free GMO are described with Shuangrong Yuan also discussing microRNA for plant engineering. A British author, A. Howard, discusses the effects of European journalism on GMO use. This is timely as in 2014 an insect-resistant corn is on the verge of being approved by the European Union. The majority of EU nations opposed this action but failed to block it. In the USA litigation is frequent for many issues. An attorney, Thomas Redick, xix

xx Preface

reviews agricultural litigation and regulation. Finally in this section the European Union Reference methods database and decision supporting tool for the analysis of GMO is reviewed by a large group of authors in Italy.

SECTION IV: ROLE OF GENETICALLY MODIFIED CROPS IN INCREASING THE FOOD SUPPLY IN THE DEVELOPING AND DEVELOPED COUNTRIES Supporters of GMO crops argue that they offer an unrivaled opportunity to increase yields, but opponents say they pose unknown health and environmental risks. American companies pushing this product note “a legal obligation to [DuPont Pioneer], to their farmers and scientists and its trade partners”. It has taken 13 years after submission to reach approval, suggesting strongly the need for compilation of reviews on many aspects of GMO preparation and public health effects. A group in India reviews the benefits of GMO in livestock feeding. Then Lucia Martinelle describes the role of transgenic food’s uncertainty as well as the producers’ responsibilities. Groups in developing countries review stress tolerance in peanuts to make it easier to grow under challenging environmental conditions while another describes why India needs biotechnology to ensure food and nutrition security. Then Maria Oszvald discusses an example in detail where key genes for protein in wheat are transferred to rice.

SECTION V: POTENTIAL HEALTH BENEFITS, ACCEPTANCE AND RISKS DUE TO INCORPORATION OF NOVEL PLANT GENE PRODUCTS INTO THE FOOD SUPPLY Acceptance of genetically modified foods is their identification and especially safety. Wentao Xu discusses event-specific identification technology as well as detection techniques. Several groups discuss putting into GMO different genes for precursors to nutrients like carotenoids for increased vitamin A, important particularly in developing countries where childhood vitamin A levels are often low. It is possible to reduce allergenicity of some crops as reviewed by Siddhesh B Ghag while plant defensins for development of fungal pathogen resistance can improve yield. Finally Henri Darmency identifies gene flow from GMC to wild or original crops.

SECTION VI: SAFETY OF GENETICALLY MODIFIED FOODS FOR HUMANS AND ANIMALS Safety concerns persist while frequently scientific studies overwhelmingly show safety of GMO foods. Therefore Anton E. Wohlers discussed labeling of GMO foods in USA as part of safety concerns. Bruce Hammond also reviews aspects of GMO food safety. Rie Satoh has a chapter on allergen analysis in assessment of GMO foods.

SECTION VII: DEMAND AND USES OF NON-GENETICALLY MODIFIED FOODS, AND GMO’S FOR HUMANS AND ANIMALS Mohamed Fawzy Ramadan genetically modified rice, maize, and soy consumption in Saudi Arabia. Yue Ma concludes the book with reasons for Chinese urban consumers opposing GMO.

Acknowledgments The work of Dr Ronald Watson’s assistant, Bethany L. Stevens, in communicating and working with authors on the manuscripts was critical to the successful completion of this book. It is very much appreciated. Support for Ms Stevens’ and Dr Watson’s work was graciously provided by the Natural Health Research Institute (www.naturalhealthresearch.org) and Southwest Scientific Editing & Consulting, LLC. Appreciation for suggesting the topic by Dr Michael Lelah is noted. Finally, the work of the librarian at the Arizona Health Sciences Library, Mari Stoddard, was vital and very helpful in identifying the key researchers who participated in the book.

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Section I

Development, Testing and Safety of Plant and Animal GMO foods

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Chapter 1

Soybean as a Food Source: Comparative Studies Focusing on Transgenic and Nontransgenic Soybean M.A.Z. Arruda1,2, R.M. Galazzi1,2, B.K. de Campos1,2, M.A. Herrera-Agudelo1,2, S.C.C. Arruda3, R.A. Azevedo3 1University

of Campinas–Unicamp, Institute of Chemistry, National Institute of Science and Technology for Bioanalytics, Campinas, Brazil; of Campinas–Unicamp, Department of Analytical Chemistry, Campinas, Brazil; 3University of São Paulo, Department of Genetics, Laboratory of Genetics Biochemistry of Plants, ESALQ, Piracicaba, Brazil 2University

INTRODUCTION Food, especially food derived from plant sources, has always played a vital role in human nutrition and development. Besides supplying nutrients, its importance is justified by its functional properties and capacity to protect human organisms against a diversity of diseases (Suliburska and Krejpcio, 2014). Hunger and malnutrition are among the most devastating problems affecting a large part of the world’s population. In this perspective, soybean (Glycine max (L.) Merril) is one of the most important crops because it is an inexpensive source of protein and oil in the human and animal diet. Typically, soybean seeds contain about 40% protein and 20% oil (Natarajan et al., 2013; Yamada et al., 2012), and they are rich in several elements important to body functions (Mataveli et al., 2010). In addition, soybean has physiologically active metabolites such as isoflavones, lecithins, tocopherols, and saponins, which are important for maintaining good health (Yamada et al., 2012). According to some studies, the regular consumption of soybean and soy productions reduces the risks of cancer and others illnesses (Natarajan et al., 2013; Cassidy and Faughnan, 2000). However, soy foods may exhibit allergenic symptoms due to antigenic proteins present in soybeans, causing allergic reactions in sensitive consumers (Natarajan et al., 2013; Berneder et al., 2013). Moreover, it exhibits a low content of methionine, an important and essential amino acid for animal nutrition (Azevedo et al., 1997). Considering the significance of soy production in different countries and areas, its high production and productivity are imperative. One alternative to attain these necessities is the production of genetically modified organisms (GMOs). Over the last 20 years, transgenic crops have accounted for much of the world’s seed production. Soybean is one of the most important crops due to its commercial importance for a diversity of countries, including Brazil and the USA. The genetic modification (GM) is carried out by the insertion, elimination, altered expression, and/or the replacement of exogenous genes in order to (1) synthesize new substances, (2) promote the absence of proteins, which were currently synthesized before the GM, or (3) enhance the synthesis of substances already present in the organism. In addition, GM improves the production and the nutritional quality of crops, and is therefore a possible solution for reducing or eliminating some problems associated with allergens and antinutrients present in nontransgenic (NT) soybeans. In 2013, the global planted area of GMOs in 27 countries was more than 175 M ha, corresponding to an increase of 3% of cultivated area when compared to 2012. Since 1996, the total cultivated area of transgenic crops increased over 100 times (James, 2013). Soybean is one of the most cultivated crops in the world. The USA is the largest producer, with c. 90 M tons in 2013 (USDA, 2014), and Brazil is the second largest producer, with c. 87 M tons in 2013/2014 (CONAB, 2014; EMBRAPA, 2014a). As noted, the production of GMOs has intensified. This, due to a set of nonconventional tools, in rapid development, enables the transfer of genetic information from one organism, being a microorganism, a plant, or an animal, to another. Additionally, multiple genes or large amounts of transfers have also been reported in the literature, such as the application of rice, canola, and corn as well as genes conferring the production of polyunsaturated fatty acids and vitamin E in soybean and Arabidopsis (Shetty et al., 2007; Porfirova et al., 2002). In this context, plants are the main targets of the studies. The advancement of techniques for the genetic improvement of these organisms allows for biotechnological goals such as increasing productivity, reducing postharvest losses, obtaining Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00001-4 Copyright © 2016 Elsevier Inc. All rights reserved.

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crops that are more tolerant to environmental stress, and/or obtaining crops that use nitrogen and phosphorus more efficiently, increasing the nutritional value of the food, resistance to herbicides, pests, and/or diseases, and the development of alternatives for industries such as the fuel and pharmaceutical enterprises (Herman et al., 2003; Kliebenstein et al., 2001). Since the first report as a GMO, the importance of soybeans to different countries, including Brazil, exponentially increased. Different aspects of this culture, focusing on comparative studies involving transgenic (T) and NT soybean seeds and plants, will be emphasized in this chapter. These studies involve aspects such as bioaccessibility of metals and evaluation of differential enzymes and their activities as well as metabolites, proteins, and metalloproteins.

SOME COMMENTS ABOUT TRANSGENIC SOYBEANS The Necessity of Using an Herbicide Soybean, like many others crops, is subject to the action of several stress factors, such as drought and temperature, actions of pathogens, and fungi and pests that currently reduce the production (Ghosh et al., 2013). Thus, to avoid the action of pests on crops some types of herbicides are used. Glyphosate [N-(phosphomethyl) glycine] is the highest selling herbicide in the world. It is employed to control pests in the cultivars because of its systemic and postemergent action. This herbicide is continually translocated from roots, accumulating in distinct compartments of plants, inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Arruda et al., 2013a; Arango et al., 2014; Zobiole et al., 2010), which participates in the synthesis of aromatic amino acids, whose absence results in plant death (Arruda et al., 2013a; Nakatani et al., 2014; Gomez et al., 2009). The impact of glyphosate in the soil microbiota and rhizosphere communities is still not well understood, and the literature reports antagonisms (Arango et al., 2014; Nakatani et al., 2014). For example, some studies showed that glyphosate application can modify the biomass content in soil microbiota as well as in microbial respiration (Nakatani et al., 2014). On the other hand, no significant effects on the soil microbiota biomass, respiration, and metabolism and CO2 production were observed in other studies after the application of the glyphosate (Arango et al., 2014; Nakatani et al., 2014).

Genetic Modification of Soybean–Glyphosate Resistance In most cases, the GM is done by the introduction of a DNA fragment, usually from a bacterium that is tolerant or resistant to the action of herbicides, pests, and insects, among others (Natarajan et al., 2013; Xue et al., 2012; Li et al., 2013). The first reported GM of the soybean was carried out for its improvement to herbicide tolerance. Although the glyphosate, as the main component of the herbicide, presents excellent characteristics, as already mentioned, it is nonselective. In other words, all plants could die, including those of commercial and nutritional value, such as the soybean. For solving this problem, the insertion of the cp4-EPSPS exogenous gene into the soybean, from a soil bacterium Agrobacterium sp, was carried out, so that its expression resulted in a glyphosate tolerance phenotype. The EPSPS enzyme is now continuously produced, even in the presence of the herbicide, allowing the use of glyphosate on T soybean cultivation. Due to the production of T crops presenting tolerance to the glyphosate, the use of this herbicide as well as the GM crops has been increasing (Arango et al., 2014; Nakatani et al., 2014). The first variety of T soybean was called Roundup Ready® (RR) (Natarajan et al., 2013; Li et al., 2013), and it was the first genetic crop in the world produced by the Monsanto Company. Considering all GM crops, those presenting tolerance to herbicides represent the most planted area, with 25.9 M ha. In addition to GM, which confers tolerance to herbicides, another modification involving the insertion of Bt cry1Ac gene, which confers resistance to insects, corresponds to 16.2% of the total planted area (Céleres, 2013; Kim et al., 2012). More recently, another variety of the T soybean (Intacta RR2®), combining the resistance to pests with herbicide tolerance, was produced, which currently accounts for 7.8 M ha (19.3% of the total cultivated area) (Céleres, 2013; MONSANTO, 2014).

A Brief History of the Transgenic Soybean in Brazil The USA introduced soybeans to Brazil in 1882, and after some tests, the first cultivars were registered in distinct areas of Brazil between 1900 and 1901. In the mid-1950s, the soy crop was encouraged due to its properties and appropriated climatological conditions, especially in the south of Brazil. Since then, soybean production and its importance to the economy are increasing (EMBRAPA, 2014b). Regarding the T soybean in Brazil, the first GM crop was obtained in 1998 (Embrapa Soja, 2003). GM soybean varieties account for 27 M ha (91.1% of the total soybean crop area in 2013/2014), which represents 67.2% of the total GM crop area in Brazil. In terms of production, soybeans also represent the highest crop production in Brazil, with c. 87 M tons, including GM, such as maize and cotton, and non-GM crops (CONAB, 2014; Céleres, 2013). Considering the importance of soybean

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crops in different areas such as economics, food, and the environment, comparative studies involving T and NT soybeans are necessary to evaluate both varieties and their specificities.

COMPARATIVE STUDIES INVOLVING TRANSGENIC AND NONTRANSGENIC SOYBEAN SEEDS OR PLANTS Bioaccessibility Studies Soybean seeds are known to contain elements such as Ca, Cu, Fe, Mg, Mn, P, and Zn, which serve several functions in the human body, such as bone and teeth formation. They are also constituents of a diversity of enzymes responsible for cell metabolism. An accurate determination of these elements in soybeans can provide access to their nutritional quality (Mataveli et al., 2010). A comparative study regarding total element concentrations (Mataveli et al., 2010) revealed that Fe, Co, and Cu are present at a higher concentration in T (variety MSOY 7575 RR) rather than NT soybean seeds (variety MSOY 7501). As a result, the T seeds seem to have the ability to take up and store higher amounts of these elements. Differences in Cu and Fe concentrations between T and NT soybean seeds were also previously established by Sussulini et al., 2007. One typical and valuable use would be the control of iron deficiency anemia in certain populations around the world through the consumption/use of iron-rich transgenic soybeans in food production. Considering other soybean plant compartments (roots, stems, and leaves) as well as other Roundup Ready transgenic varieties (MSOY 7211 RR–T and MSOY 8200–NT soybeans), Oliveira et al. (2014) carried out a tracer experiment to evaluate if GM can influence the uptake and translocation of Fe. This study concluded that T and NT plants present similar profiles on Fe distribution between plant compartments; however, NT plants were able to accumulate higher amounts of Fe than T plants, even when different sources of Fe were provided. For estimating the nutritional soybean contribution, it is of utmost importance to know the amount of nutrients that are delivered (bioaccessible fraction) for the absorption in the organism during human digestion (Fernández-García et al., 2009), since not all of the total content is efficiently absorbed and used. In this way, in vivo and in vitro methods are currently used to determine the bioaccessible fraction encountered in the gastrointestinal tract during the digestion, that is, the fraction really available to enter the bloodstream (Hur et al., 2011). In vivo methods allow the determination of the amount of nutrients absorbed, bioactive compounds, or their metabolites through studies of mass balance and tissue concentration (Fernández-García et al., 2009). The studies of mass balance determine the nutrient amount absorbed by measurements of the amount of ingested and excreted nutrients. In studies of tissue concentrations, the increase of the nutrient concentration into the blood plasma is monitored. Some examples in the literature report the application of these methods in animals or humans, with the aim of determining the absorption of carbohydrates, minerals, and vitamins, among others (Vaisberg et al., 2006; Weber et al., 2006; Scalbert and Williamson, 2000). Most traditional in vitro methods are those considered to be statics, which are proposed to make a sequential exposure of the sample in order to simulate its digestion inside the mouth, stomach, and intestine, simulating human physiological conditions, such as pH, gastric, intestinal, and hepatic enzymes as well as residence time. These conditions have been currently modified/adapted for performing bioaccessibility methods. On the other hand, temperature is the only physiological condition that usually remains at 37 °C (Hur et al., 2011; Intawongse and Dean, 2006; Le et al., 2012). After the simulation, the remaining solution is used for quantifying the nutrient of interest, allowing the evaluation of its bioaccessibility. A wide variety of in vitro methods have been developed with the aim of establishing the best representation of the human physiological system, such as the Relative Bioaccesibility Leaching Procedure method, recommended by the American Agency EPA, In Vitro Gastrointestinal, Physiologically Based Extraction Test, Dutch National Institute for Public Health and the Environment method, and Unified Bioaccessibility Method (Le et al., 2012). In vitro models are an alternative to objectively establish an analysis of the matrix composition in the digestive process. For functional foods, in vitro methods provide a first insight into the behavior of the bioactive ingredient, transport, absorption, and bioaccessibility (Fernández-García et al., 2009). Several studies have been published, with the main objective to assess the bioavailability of selected chemicals in certain foods. Studies of the metal composition of genetically modified food are often performed, but most of them are limited to the determination of total content (Xin et al., 2008; Gayen et al., 2013), providing little information about the amount available to be absorbed and utilized efficiently by the organism. The work carried out by our research team (Mataveli et al., 2010) was, up until now, unique where the effects of GM in the bioaccessiblity of some elements by comparing T and NT soybean seeds were investigated. The analysis was carried out by an inductively couple plasma (ICP)-sector field-mass spectrometry (MS). The bioaccessible fractions obtained through

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a simulation of gastric and intestinal conditions revealed that the contributions of bioaccessible fractions of Cu, Fe, and other elements (Mn, S, Zn) for T soybean seeds appear to be larger than those found in NT soybean seeds. Therefore, the comparative studies involving bioaccessibility between nutritional species of T and NT soybeans are of utmost importance, due to the increase in demand for food worldwide and its quality as a functional food. Research with T soybeans is scarce, since there few studies available allow for the collection of unpublished and important data, which may drive the availability of nutritional information for society.

Enzymes Involved in the Oxidative Stress Earth is the only planet able to support aerobic life as the way that we understand its meaning, and the presence of oxygen is derived from the photosynthetic activities of cyanobacterias and plants (Arruda et al., 2013b). Its presence in its atmosphere led to a diversity of oxygen-based reactions. Under some situations, such as normal metabolic activity or when under environmental disturbance, the oxygen can be switched to an excited state, with the production of free radicals and similar forms, such as H2O2; O2 ·; HO2−; and OH− (Gratão et al., 2005), which can act in different ways in the cellular environment (Foyer and Noctor, 2000). Positive and negative aspects regarding the oxygen-based reactions can occur, and this process is known as oxidative stress. Oxidative stress can be described as a central factor in abiotic and biotic stress that occurs due to imbalances in any cell compartment between the production of reactive oxygen species (ROS) and antioxidant defense (Gratão et al., 2005). Despite the importance of evaluating ROS and the enzymes involved in its combat (i.e., superoxide dismutase–SOD (­Fridovich, 1995), catalase–CAT (Scandalios et al., 1997), glutathione reductase–GR, among others), the few examples found in the literature for soybeans (Arruda et al., 2013a) are not focused in comparative studies involving T and NT soybeans. In fact, there are only two examples that were carried out by our research team and will be briefly reviewed in this section. Barbosa et al. (2012) reported the evaluation of enzyme activity and the abundance of some proteins from T (MSOY 7575 RR) and NT (MSOY 7501) soybean seeds. The analysis of enzymes such as ascorbate peroxidase (APX), GR, and CAT revealed higher levels (30.6, 71.4, and 35.3%, respectively) in T than in NT seeds. Additionally, lipid peroxidation was also evaluated through the malondialdehyde (MDA) concentration, which is an indicator of lipid peroxidation and oxidative stress, showing 29.8% higher concentration in T than in NT seeds. Such results suggest that the T genotype naturally exhibited a level of stress higher than that of the NT genotype. Nevertheless, such higher levels also suggest that this genotype is possibly better prepared than NT when facing adverse conditions. In the referred work, four proteins were differentially found by using 2-D DIGE, as glycine-rich RNA-binding protein, cytosolic glutamine synthetase, actin, and glycinin G1 subunit. Even though there is no information in the literature regarding ROS production and GM, particularly when seeds are concerned, it is well known that plants are highly adapted to respond quickly to biotic or abiotic stresses by altering gene expression and metabolism as a result of cell signaling, which may be mediated by ROS (Schmidt et al., 2010). In the same report, the authors commented that there was a previous GM, conferring on the plant a resistance to glyphosate. In plants, posttranscriptional control mechanisms are growing in importance, and proteins involved in RNA processing and alternative splicing, RNA transport, messenger RNA (mRNA) translation, mRNA stability, and mRNA silencing mechanisms have been shown to be required for normal plant development and the responses of plants to altered environments (Kazan, 2003; Cheng and Chen, 2004; Wang and Li, 2007). In the work by Barbosa et al. (2012) only the glycine-rich RNA-binding protein was differentially found after DIGE analysis, a protein shown to be correlated with ROS production (Schmidt et al., 2010; Kim et al., 2007). The cytosolic glutamine synthetase (another differentially abundant protein found through DIGE analysis) is involved in nitrogen fixation and is a target of herbicides. Pageau et al. (2006) also demonstrated that oxidative stress can control the expression of nitrogen-metabolism genes. It is easy to show that cytosolic glutamine synthetase can be altered because of the oxidative stress observed in the T soybean line, mainly when higher levels of H2O2 are present, which is correlated to higher activities of CAT. There is no report in the literature concerning actin or glycinin G1 subunit and ROS production, and the authors commented that this fact might open up a new possibility for future studies (Barbosa et al., 2012). Barbosa et al. (2012) also evaluated protein species and enzymes, but in T (MSOY 7575) and NT (MSOY 7501) soybean leaves, collected from plants obtained from the seeds used. The enzymes involved in ROS combat, such as APX, CAT, GR, and SOD exhibited higher activities (21, 70, 14, and 31%, respectively) in T leaves when compared to NT leaves. Such a relationship was also observed for MDA and H2O2 concentrations, which increased (c. 44 and 69%, respectively) in T when compared to NT leaves. These results indicated that the GM itself might be a stressful factor to the plant, possibly due to the need for a new equilibrium in its metabolism, since the stress condition is being maintained within the levels that the plant can deal with.

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Metabolites and (Metallo)proteins Techniques based on modern biotechnology known as omics sciences, studying DNA, RNA, proteins, metabolites, and metals, among others, have been used in an attempt to answer some questions about the soybean culture and for evaluating the effects of the T soybean species at the molecular level (Arruda et al., 2013a; Barbosa et al., 2012; Clark et al., 2013). Despite more than a decade of use, no case of T food human consumption was reported as malicious. However, there are some results about food safety in the literature obtained from rats showing some antagonisms, since the total absence of effects to changes in organic functions, such as increase in cholesterol and triglycerides (Brasil et al., 2009; Daleprane et al., 2009a,b, 2010; Battistelli et al., 2010). Additionally, several studies in the literature raise some criticism with regard to side effects that plants have suffered in its metabolism (Arruda et al., 2013b). Recently, the metabolic profile between GM and non-GM soybean was performed by Clark et al., 2013 in order to obtain data on biosafety. For this task, they investigated the soybean metabolic profile from 29 different cultivars cultivated in different seasons. A total of 169 metabolites have been identified, 159 of them corresponding to primary metabolism and 15 to secondary metabolism. The primary metabolites are essential to plants for functions such as growth and fertilization, while the secondary metabolites perform other functions, such as protection and defense (Lin et al., 2014). Most of the literature concerning the content or metabolite profile in biological samples reports the use of the mass spectrometry technique, usually coupled to a separation technique such as liquid or gas chromatography. The metabolites extraction for chemical analysis is well established and it is easy to handle. For example, the method proposed by De Voz et al. (2007) uses 100 mg of a macerated sample with N2 and 1 mL of methanol (75% v/v), acidified with 0.1% formic acid (v/v) for 15 min sonication. Studies involving comparative metabolomics between T and NT soybeans are still scarce in the literature; much more comprehensive research should be on the agenda if a better understanding of the metabolism of the plant and the effects that can cause these genetically modified organisms is to be better understood. For instance, some studies suggest that metabolite levels may have been dramatically altered by combining genetic or environmental impacts (Clark et al., 2013). It is also known that the effects of T soybean plants are becoming evident when it comes to proteomic studies. To the naked eye, the T and NT plants developments are similar, although the T plant has increased productivity and presented a shorter cultivation period; however, it has not yet been possible to correlate these changes with the changes observed in their proteomic profile. Proteomics is defined as the area of science that studies proteins, including not only identification but also quantification, determination, modifications, interactions, activities, and functions thereof. It is known that from the total protein content in soybean seeds, 70–80% is glycinin and β-conglycinin and its subunits, presenting storage function and being responsible for nutritional and physicochemical properties. Much of the studies found in the literature are focused on these proteins (Natarajan et al., 2009). However, other studies involve a more thorough proteomic investigation of soybean proteins (Arruda et al., 2013a; Barbosa et al., 2012; Garcia et al., 2006; Natarajan et al., 2007, 2009) as well as studies involving food safety and the possible effects T organisms (Daleprane et al., 2009; Krzyzowska et al., 2010). Krzyzowska et al. (2010) evaluated the possible effects of T triticale, which is a hybrid between wheat and rye, on animal health in five successive generations of rats. The results indicated that the use of T triticale in rodent diets has increased the concentration of white blood cells and also the expansion of B-cells in secondary lymphoid organs. Similarly, Daleprane et al., 2009 studied the use of GM soybean in the diet of rats. The study was carried out by separating three groups of rats, the first being fed with feed organic soybean, the second with feed T soybean, and third using casein base. The results showed a significant difference in the weight of the rats in both groups (experimental and control) and also that the organic soybean has a better protein utilization in relation to the transgenic soybean line. However, as far as we are aware, there are no case studies yet available on the consequences of a feeding base of transgenic organisms in humans. In order to display differential proteins between two different cultures, for example T and NT soybean, polyacrylamide gel electrophoresis (2D-DIGE) is clearly a good choice. In this context, Barbosa et al. (2012) studied, by means of proteomic comparison, T and NT soybeans. The image of the gels showed four spots with different intensities, indicating that the proteins had different abundances between samples due to GM suffered by T soybean seeds. This fact was confirmed by the identification of the protein cp4-EPSPS, characterizing the GM. Additionally, 192 proteins were able to be identified by 2D-PAGE separation and identification by MALDI-TOF. Another area that studies omics content is metallomics, which is defined as the totality of metal and metalloid species in a cell or tissue, its identity, and location. Most metal biological organisms are bound to proteins, which are called metalloproteins (Arruda and Azevedo, 2009; Mataveli et al., 2012). Our group performed a comparative study of the content of metals present in T and NT soybeans by ICP-MS. It was observed that the T and NT soybeans have statistically different concentrations of some metals such as Cu and Fe (higher in T soybeans). Using a three-dimensional separation technique—the first

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chromatographic separation by size exclusion (SEC), the second using an anion exchange (AEX), and, finally, one dimension by electrophoresis (SDS-PAGE)—fractions containing different metals were identified. These fractions were taken for identification by mass spectrometry with electrospray ionization source (ESI-MS). This approach identified 33 proteins, some of them being metalloproteins such as seed lipoxygenase-1, β-conglycinin, the chain lipoxygenase-3, and beta-amylase, among others (Mataveli et al., 2010, 2012). In this context, the genes encoding genetically modified organisms in different cultivars of plants, added to the plant with the aim of agronomic improvement as herbicide tolerance and insect resistance, should be used in a way that does not affect the performance of the culture and does not produce changes unsafe to food derived from these cultivars. The omics sciences in contribution with the analytical techniques have presented data suggesting an improvement regarding the safety of GM crops; however, more studies are needed for a biochemical vision and extensive vegetable physiology for conclusive results.

TRENDS Several regulatory agencies, such as Food and Agricultural Organization of the United Nations (FAO), The European Food Information Council, Brazilian Health Surveillance Agency (ANVISA), and others regulate the safety levels of determined nutrients and metals in feed that can be consumed by humans. In this way, many studies evaluate the elemental composition of some foods as well as if a GM changes the levels of nutrients and if the transgenic food would be safe to the consumer. In addition to the elements determination in seeds, studies that evaluate the bioaccessibility of the main elements contained in T and NT seeds are still scarce, and more attention should be given to this type of research, such as the one published by Mataveli et al. (2010). In addition to the elemental composition and bioaccessibility studies evaluating the nutrients, an approach focusing on carotenoids, wherein the total concentration as well as the carotenoids composition changes according to different kinds of GM (Rivera et al., 2013), was also carried out. Nonetheless, despite the bioaccessibility studies already performed, which provide preliminary and important information on how each bioaccessible compound could be absorbed by the human body, more elaborate tests, named bioavailability studies, must be carried out because in such studies it is possible to evaluate whether a bioaccessible element is able to pass through membranes that simulate the digestive wall and can thus be absorbed. More than assessing what kind of changes the GM may cause in terms of bioavailability, a chemical speciation study to know what kind of given element/compound would be bioavailable and if these species are toxic to humans would also add interesting new information that is missing from current studies. When GM foods are concerned, a study of the generation of seeds is important to assess whether the effects caused by T intensify or diminish with successive plantings of seeds obtained. Such studies can be performed using various approaches, such as comparing the proteomic profile of generations, studies evaluating the metabolites of each generation, bioaccessibility and bioavailability tests, and also enriched isotope tracer studies to assess the biochemical absorption by the plant, such as the unique study published by Oliveira et al. (2014). Another study which would be interesting is related to the use of glyphosate in T crop planting to assess whether this herbicide changes the composition of seeds obtained as well as if glyphosate is incorporated into the plant and if it has the absorption of some potentially toxic metals of the association with glyphosate and the possible consequences of consumption, by humans, of these contaminated seeds.

CONCLUSIONS Besides a diversity of byproducts (i.e., milk, cakes, flour), there is no doubt that soybean is a major source of proteins and oil, placing this legume species on a special list as a key plant species to be used as a food source. Additionally, the necessity for obtaining more productive cultivars opened some new possibilities in terms of using molecular genetics, creating a generation of GMOs, including T soybean lines. As an organism led by thousands of years in terms of evolutionary adaption, it is easy to realize that the modified species may follow the same path. In this way, some questions may appear. Is the behavior always the same when these species are compared with natural ones? What is, in fact, changed when both are compared? What are the consequences of these changes? Is the nutritional quality of these GMOs similar when it is compared to that of natural organisms? This chapter presented some comparative results involving T and NT soybeans, hoping to answer some of these questions but leaving a door open to future research on T soybean as well as T crops plants as a whole. We can say that there are some differences, indicating that the GMO, that is, the soybean, is searching for a new equilibrium as a living organism in nature. What is needed from now on is the understanding of each difference observed and perhaps making possible the demystification of the GM. Besides being of great opportunity to research, we also will be contributing to making our food safer.

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Talanta 69, 1–15. Gayen, D., Sarkar, S.N., Datta, S.K., Datta, K., 2013. Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart. Food Chem. 138, 835–840. Ghosh, P., Roy, A., Chakraborty, J., Das, S., 2013. Biological safety assessment of mutant variant of Allium sativum leaf agglutinin (mASAL), a novel antifungal protein for future transgenic application. J. Agric. Food Chem. 61, 11858–11864. Gomez, E., Ferreras, L., Lovotti, L., Fernandez, E., 2009. Impact of glyphosate application on microbial biomass and metabolic activity in a Vertic Argiudoll from Argentina. Eur. J. Soil Biol. 45, 163–167. Gratão, P.L., Polle, A., Lea, P.J., Azevedo, R.A., 2005. Making the life of heavy metal-stressed plants a little easier. Funct. Plant Biol. 32, 481–494. Herman, E.M., Helm, R.M., Jung, R., Kinney, A.J., 2003. Genetic modification removes an immunodominant allergen from soybean. Plant Physiol. 132, 36–43. Hur, S.J., Lim, B.O., Decker, E.A., McClements, D.J., 2011. In vitro human digestion models for food applications. Food Chem. 125, 1–12. Intawongse, M., Dean, J.R., 2006. In-vitro testing for assessing oral bioaccessibility of trace metals in soil and food samples. Trends Anal. Chem. 25 (9), 876–886. James, C., 2013. Global Status of Commercialized Biotech/GM Crops: 2013. ISAAA Brief No. 46. ISAAA, Ithaca, NY. Kazan, K., 2003. Alternative splicing and proteome diversity in plants: the tip of the iceberg has just emerged. Trends Plant Sci. 8, 468–471.

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Kim, M.J., Kim, J.K., Kim, H.J., Pak, J.H., Lee, J.H., Kim, D.H., Choi, H.K., Jung, H.W., Lee, J.D., Chung, Y.S., Ha, S.H., 2012. Genetic modification of the soybean to enhance the β-carotene content through seed-specific expression. PLoS One 7, 1–12. Kim, Y.O., Pan, S., Jung, C.H., Kang, H., 2007. A zinc finger containg glycine-rich RNA-binding protein, atRZ-la, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress condition. Plant Cell. Physiol. 48, 1170–1181. Kliebenstein, D.J., Kroymann, J., Brown, P., Figuth, A., Pedersen, D., Gershenzon, J., Mitchell-Olds, T., 2001. Genetic control of natural variation of Arabidopsis thaliana glucosinolate accumulation. Plant Physiol. 126, 811–825. Krzyzowka, M., Wincenciak, M., Winnicka, A., Niemialtowski, M., 2010. The effect of multigenerational diet containing genetically modified triticale on imune system in mice. J. Vet. Sci. 13, 423–430. Le, X.C., Li, X.F., Lee, H.K., 2012. Extraction techniques and applications: biological/medical and environmental/forensics. In: Board, J.E. (Ed.), Comprehensive Sampling and Sample Preparation. Elsevier Science, pp. 487–507. Editor-in-Chief: Janusz Pawliszyn. Li, S.X., Chen, L.H., Zheng, F.Y., Li, Y.C., 2013. Effect of the cp4-epsps gene on metal bioavailability in maize and soybean using bionic gastrointestinal tracts and ICP-MS determination. J. Agric. Food Chem. 61, 1579–1584. Lin, H., Rao, J., Shi, J., Hu, C., Cheng, F., Wilson, Z.A., Zhang, D., Quan, S., 2014. Seed metabolomic study reveals significant metabolite variations and correlations among different soybean cultivars. J. Integr. Plant Biol. 56, 826–836. Mataveli, L.R.V., Pohl, P., Mounicou, S., Arruda, M.A.Z., Szpunar, J., 2010. A comparative study of element concentrations and binding in transgenic and non-transgenic soybean seeds. Metallomics 2, 800–805. Mataveli, L.R.V., Fioramonte, M., Gozzo, F.C., Arruda, M.A.Z., 2012. Improving metallomics information related to transgenic and non-transgenic soybean seeds using 2D-HPLC-ICP-MS and ESI-MS/MS. Metallomics 4, 373–378. MONSANTO. Intacta RR2 PRO®. Available in: http://intactarr2pro.com.br/ (accessed 02.12.14.). Nakatani, A.S., Fernandes, M.F., de Souza, R.A., da Silva, A.P., dos Reis-Junior, F.B., Mendes, I.C., Hungria, M., 2014. Effects of the glyphosate-­ resistance gene and of herbicides applied to the soybean crop on soil microbial biomass and enzymes. Field Crops Res. 162, 20–29. Natarajan, S.S., Xu, C., Bae, H., Bailey, B.A., 2007. Proteomic and genomic characterization of kunitz trypsin inhibiton in wild and cultivated soybean genotypes. J. Plant Physiol. 6, 756–763. Natarajan, S.S., Xu, C., Cregan, P., Caperna, T.J., 2009. Utility of proteomics techniques for assessing protein expression. Regul. Toxicol. Pharmacol. 54, 32–36. Natarajan, S., Luthria, D., Bae, H., Lakshman, D., Mitra, A., 2013. Transgenic soybeans and soybean protein analysis: an overview. J. Agric. Food Chem. 61, 11736–11743. Oliveira, S.R., Menegário, A.A., Arruda, M.A.Z., 2014. Evaluation of Fe uptake and translocation in transgenic and non-transgenic soybean plants using enriched stable 57Fe as a tracer. Metallomics 6, 1832–1840. Pageau, K., Reisdorf-Cren, M., Morot-Gaudry, J.F., Masclaux-Daubresse, C., 2006. The two senescence-related markers, GS1 (cytosolicglutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves. J. Exp. Biol. 57, 547–557. Porfirova, S., Bergmüller, E., Tropf, S., Lemke, R., Dörmann, P., 2002. Isolation of an Arabidopsis mutant tacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis. PNAS 99, 12495–12500. Rivera, S.M., Vilaró, F., Zhu, C., Bai, C., Farré, G., Christou, P., Canela-Garayoa, R., 2013. Fast quantitative method for the analysis of carotenoids in transgenic maize. J. Agric. Food Chem. 61, 5279–5285. Scalbert, A., Williamson, G., 2000. Dietary intake and bioavailability of polyphenols. J. Nutr. 130, 2073–2085. Scandalios, J.G., Guan, L., Polidoros, A.N., 1997. Catalases in plants: gene structure, properties, regulation, and expression. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defenses. New York. Cold Spring Harbor Laboratory Press, Plainview, pp. 343–406. Schmidt, F., Marnef, A., Cheung, M.K., Wilson, I., Hancock, J., Staiger, D., Ladomery, M., 2010. A proteomic analysis of oligo(dT)-bound mRNP containing oxidative stress-induced Arabidopsis thaliana RNA-binding proteins ATGRP7 and ATGRP8. Mol. Biol. Rep. 37, 839–845. Shetty, K., Paliyath, G., Pometto, A., Levin, R.E. (Eds.), 2007. Functional Foods and Biotechnology. 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Chapter 2

Genetically Modified Crops: Biosafety Regulations and Detection Strategies Suchitra Kamle, Dawei Li Shanghai Jiao Tong University, School of Pharmacy, Shanghai, China

INTRODUCTION Genetically modified (GM) crops are gaining acceptance worldwide. From 1996 to 2014, the global area of biotech crops has continued to increase for the 18th year at a sustained growth rate of 3–4% or 6.3 million hectares (∼16 million acres), reaching 181.5 million hectares or 448 million acres. GM crops have set a case in point that the biotech area has grown impressively every single year for the past 18 years, with a striking 100-fold increase since the commercialization began in 1996. A record 175.2 million hectares of biotech crops were grown globally in 2013 at an annual growth rate of 3%, up five million from 170 million hectares in 2012. The year 2013 marked the 18th year of commercialization when growth continued after a remarkable 17 consecutive years of increases; notably 12 of the 17 years were double-digit growth rates. Thus, GM crops are considered to be the fastest adopted crop technology in the history of modern agriculture. GM crops have been successfully grown in accumulated 1.78 billion hectares (4.4 billion acres) (James, 2014). A disputed journey of GM crops was always swinging like a pendulum for their acceptance in between the lawmakers and anti-GM groups. But, successful acceptance of GM crops raises interrogations about consumers’ concerns and human biosafety. Arguably, the anti-GM groups (Greenpeace and Gene Campaign) are voicing their reservations fearing the growth of several nonapproved varieties and the possibility of cross-contamination of GM crops (Paarlberg, 2002; Smythe et al., 2006). To resolve such issues, International Regulatory (IR) bodies, corporate council chambers, lawsuits, and lawmakers become active besides research laboratories. These regulatory bodies, after due deliberation, regulate the release of GM crops accepted world over (Codex, 2003; Stewart et al., 2000). Labeling is mandatory to avoid unintended commingling of GM and non-GM crops, thus providing assurance to the consumer (Gruère and Rao, 2007). Creating acceptability of GM crops similar to non-GM crops will continue to remain as a gainsayer.

BIOSAFETY MEASUREMENT Since the introduction of GM technology in 1996, at the international and national levels, several amendments have been made and finally parliamentary bills were implemented after due debates on the use and release of GM crops. All of this keeps in view the general concern and stringent biosafety issues for the protection of society and the environment. Harmonization and synchronization in society require rigorous verification, just to avoid any unintentional commingling of GM and non-GM crops (Smythe et al., 2006). With the contriving drives and mutual agreements, many countries are accepting GM technology and have started planting GM crops. The commercialization of GM crops is also gaining acceptance despite occasional reverberation of consumer concerns (Gruère and Rao, 2007). The legislative chambers and lawsuit members have critically probed this issue. On January 29, 2000, in Montreal, Canada, the Cartagena Protocol on Biosafety was formulated and finally adopted, which entailed an overnight discussion (Raustalia and Victor, 1996; Kinderlerer, 2008). This was the very first legally approved biosafety protocol on GM organisms having a series of clauses and rules which were de jure bound in an agreement made for crossing transboundaries. Following overnight debates, the protocol was approved during the early hours of the morning (CBED and UNEP, 2003). This Cartagena Protocol was unanimously constituted based on the legal opinions of members of the committee. Thus, the much needed commencement of the biosafety issues was enforced amicably (Paarlberg, 2000; Gupta, 2004). Upon the commencement of the safe use of modern biotechnology, the technology transfer was finally activated after the adoption of Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00002-6 Copyright © 2016 Elsevier Inc. All rights reserved.

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the Cartagena Protocol. However, it was not limited to human biosafety and consumer concerns, as its scope was extended (Kinderlerer, 2008). The developed countries accepted the biosafety protocols to avoid any international sanctions. While the acceptance of biotech crops was steadily gaining momentum the world over, the dispute also continued to simmer at an alarming rate. The politicians, lawmakers, and anti-GM groups made interrogative remarks about the biosafety rules and guidelines, drawing the attention of the masses on the Cartagena Protocol on Biosafety. It was projected that the Cartagena Protocol on Biosafety under the United Nations Convention on Biological Diversity had several potential loopholes and therefore did not ensure a secure future for GM crops (Gupta, 2004). On the other hand, the developing countries also felt the pressing need for regulatory frameworks to deal with GM biosafety issues. Gradually, attention was focused on developing national biosafety regulations both for the safety of the consumers and the environment. Accordingly, countries like India and Brazil implemented the biosafety rules in 1990. The developed world’s policies and safety measurements dominated the developing world, crossing legal boundaries. It was found that a research institute in the United States, without permission from the government of Argentina, started genetically testing the rabies vaccine, which was ethically wrong. Actually, in 1986, the first field test of a recombinant rabies vaccine developed by Wistar Institute of Philadelphia of the United States was conducted in Argentina by the commissioned Pan American Health Organization (PAHO) without the knowledge of Argentina government. This event promoted the Organization for Economic Cooperation and Development (OECD) urged the developing countries to adopt more stringent guidelines over the release of novel recombinant organisms in another country to prevent the exploitation of developing countries’ lax rules (Koprowski, 1988; Connor, 1988). Clearly, a developing country should not have been used as a platform for unethical testing purposes (Krueger, 1999). It is obvious that developed and developing countries have several conflicting views about genetically modified organisms (GMOs) and consequently this has become a world issue. The bottom line behind all of these concerns was only the safety of humans and the environment. In defiance of prevalent resistance, GM crops grow over a billion hectares of land with every passing year, despite the lurking biosafety issues. European countries have their own regional directives and measurements (Regulation (EC) 2001; Alexandrova et al., 2005). According to the Cartagena Protocol on Biosafety, consent for any foreign trade and potential risk assessment are both mandatory for the transfer of GMOs (Hagen and Weiner, 2000). The Cartagena Protocol deals with both the intention to release GMOs into the environment and to GM products consumed by animals and humans as feed and food (Cartagena Protocol, 2000). Often, the GMOs biosafety issue becomes headlines in the broadcasting media where society’s rage and ferocity against GM crops are aired. The media outburst was seen despite the fact that regulatory bodies, legislatives, and lawsuit members all were involved in drafting new agreements, documentation, measurements, and guidelines to address the GM biosafety issue and ensure harmony and synchronization between the developed and developing world (Kamle and Ali, 2013). As the situation stands now, a scientifically sound risk assessment is necessary together with the due safety concern. The “Biosafety Clearing House” has made it mandatory to have advance written official notice related to GM products both from the importer and exporter prior to any business transaction (Gupta, 2004; Hagen and Weiner, 2000). With the results, several countries got involved and imposed biosafety regulations, formulated directives, and ensured measurements to make it a sound scientific practice across the world. Subsequently, worldwide biosafety regulations on genetically modified crops (GMCs) are revised and published, highlighting the safety guidelines. In the process, Agenda 21, emphasizing ecofriendly management, was introduced in the United Nations conference. In addition, the Convention on Biological Biodiversity, the World Trade Organization Technical Barrier to Trade, and the Codex Alimentarius Commission made efforts to regulate biosafety measures accordingly (Codex Alinorm, 2006; Kamle and Ali, 2013). These regulatory bodies and organizations took overall responsibility for monitoring and enforcing biosafety rules, encompassing a global trade system (Haslberger, 2003). By law, these regulatory frameworks ensure comprehensive biosafety assessment of GM crops and administer enforcement, compliance, and accreditation, keeping in view the national and international coordination of the policies. To protect the environment, society, and economic interests, the detection strategies and labeling of GM crops have to be as robust as that of their envisaged acceptance. The international directives were freshly regulated by the biosafety guidelines to mandate the labeling of GMCs (Maltsbarger and Kalaitzandonakes, 2000). For instance, a GMC carrying insecticidal genes needs to be detected both at the DNA and protein levels (Kamle et al., 2011a,b). These regulations require advancement in the detection system. After an extensive improvement in the current detection methods for the transgene, mRNA expression can be easily detected (Morisset et al., 2009). With technical advancements and innovation, modern biotechnology has metamorphosed from the conventional polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA) to real-time PCR, immuno-PCR, and finally to the biosensors and capillary electrophoresis for the detection of GMCs (Kamle and Ali, 2013; Cifuentes, 2012). The regulatory authorities mandate the labeling of GMOs, which varies from country to country. GM crops “precautionary principle,” introduced by the European Union, was also made mandatory for the biosafety of society (Regulation (EC) 2001; Kamle and Ali, 2013).

Biosafety and Detection of GM Crops Chapter | 2  13

The Codex Guidelines 2003, World Trade Organization, and IR bodies and organizations have all made agreements, treaties, and protocols regarding the use and release of GMOs for the purpose of the biosafety of human health, including the safety of biodiversity of flora and fauna (Haslberger, 2003). The risk assessment and safety measures are strictly regulated by the governing bodies throughout the world. Sometimes, owing to insufficient scientific data and evidence, the biosafety issue remains unresolved. The governance directs scientific analysis of GMOs before their commercialization (Hagen and Weiner, 2000; McKay White and Veeman, 2007). These safety guidelines and protective measurements are critically assessed and revised well in time. Countries such as India have a number of regulatory bodies, including the Ministry of Environment and Forest, the Recombinant DNA Advisory Committee, the Institutional Biosafety Committee (IBC), the Genetically Engineered Appraisal Committee (GEAC), and the Biological Diversity Act which legally provide overall control of the GM issues. Now, several nations such as the USA, Argentina, China, Brazil, Japan, South Africa, Australia, New Zealand, Korea, Malaysia, Thailand, Philippines, Uruguay, Paraguay, Romania, Mexico, Spain, and the European Union have accepted GM technology and grow GMCs covering millions of hectares of land areas (James, 2014; Kamle and Ali, 2013; Report of APO Study, 2002; Ladics and Selgrade, 2009). GM technology, despite its acceptance, continues to remain controversial as the issues related to biosafety continue to crop up in one form or another with the introduction of new GM crops. Thus, with each upcoming GM crop, new technology is warranted, fulfilling the requirements of detection strategies, safety, and security. The prime directive therefore should not be to impede the development of GM crops but instead to ensure the overall acceptance and then biosafety to both humans and environment. GM not only offers an alternate source of food but also provides food for thought in the context of ever changing global climates and the status of food security.

LABELING ISSUES The labeling of GM crops is a contentious issue. The international authorities are drafting guidelines for the proper labeling of GM crops and their products. Exact labeling requires an extensive identity preservation system, from granger to the elevator to grain processor to food processing manufacturer and finally to the consumer through the retailer (Maltsbarger and Kalaitzandonakes, 2000). The labeling of GM crops is compulsory to keep consumers informed. Consumers must know that the GM crop has been declared safe by the authorities (Hansen, 2004; McKay White and Veeman, 2007; Streiffer and Rubel, 2003). Moreover, labeling helps to enhance the surveillance and tracing of GM food. Labeling is required when GM crops are substantially different from their conventional counterpart (e.g., a change in composition, nutritional value, or allergenic nature). The Food and Drug Administration’s stance is that GM and non-GM crops are substantially equivalent. It is difficult to label each fruit as it would incur additional prices to the products that would be shifted to the consumer (Bansal and Ramaswami, 2007). The GM labeling requirement for food products as a precautionary measure was introduced by the European Union (Regulation (EC) 258/97) and approved lawfully to provide safety to society. Thus, biosafety measurements and regulations are made to create a “safety net” by testing and labeling GM products. Usually, country-specific labeling policies are made. In many countries, the labeling of grains, feed, and foodstuffs is mandatory if the GMO content exceeds a certain threshold level. The proposed threshold level is 1%, but it has been urged to amend this to as low as 0.01%. The threshold value is based on the percentage of GMO material in a non-GM background (Hansen, 2001). Normally, no GM food labeling would be required if the food contains GM material below the threshold level. Countrywise, the degree of the labeling pattern varies greatly (Bansal and Gruere, 2010; Carter and Gruere, 2003). The Codex Committee on Food Labeling has drafted advanced recommendations on the labeling of biotech products and is directly linked with the World Trade Organization through an agreement. The Codex process for standard development is based on creating an international consensus, to protect consumers and to facilitate trade by developing the best labeling policies for harmonization (Codex, 2003; Haslberger, 2003; Ladics, 2008). As of this writing, there is no authentic global approval and legal registration of GM crops and their processed food products. Therefore, GM testing and its legal registration must be made mandatory and operational world over (Goodman and Tetteh, 2011).

BT GENE AND STACKED TRAITS The modern biotechnological approach allows genes to be introduced into a plant genome. These foreign genes may originate from prokaryotes (unicellular) or eukaryotes (multicellular), either from plants or from animals. The first GM organism was Bt; due to its broad applications it was called Bt technology. In its first application, Bt genes were transferred into tobacco and tomato (Fischhoff et al., 1987) and following this, many other crops were developed (Jouanin et al., 1988). A GM maize (Bt11) has been developed to express the Cry1Ab insecticidal protein. This Cry1Ab was found to be toxic against some lepidopterons

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such as Helicoverpa punctigera, Helicoverpa zea, and Pectinophora gossypiella insects (Bruderer and Leitner, 2003). Various GM crops harboring Bt genes (cry1Ac, cry1Ab, cry2Aa, cry2Ab, cry2Ac, cry1F, epsps, and vip-3A) encoding insecticidal proteins were derived from a ubiquitous soil bacterium Bt. These insecticidal proteins generally have molecular weights between 65 and 88 kDa (Hofte and Whiteley, 1989) and are known to be lethal against dipteran, coleopteran, and lepidopteron insects. Since the commercialization of GM crops, herbicide tolerance (HT) has consistently been the dominant trait and is used in soybeans, followed by insect resistance used in Bt maize, Bt cotton, and Bt canola. Such GM crops tolerate more herbicides, such as glyphosate and ammonium glufosinate, and are resistant to different pests. GM crops expressing insecticidal proteins are steadily gaining acceptance and are grown throughout the world (James, 2014). Stacked events are those which combine in the same plant by conventional breeding or retransformation of one or more existing traits (http://ftp.jrc.es/EURdoc/report_GMOpipeline_online_preprint.pdf). The first-generation GM crop has a single Bt gene (e.g., Bollgard-I: cry1Ac) and now the second and third generations of GM crops are stacked with multiple genes (e.g., Bollgard-II: cry1Ac + cry2Ab), having one copy of each event to achieve long-lasting resistance. GM maize stacked with 13 double, three triple, and one quadruple event is currently under European Union assessment. The stacked GM crops which are likely to be commercialized are soybean, maize, cotton, rapeseed, rice, and potato. A database for GM crops has been established to provide uniform and updated information world over (http://cera-gmc.org/index. php?action=gm_crop_database). GM crops that have been commercialized are Bt cotton in five different countries, roundup ready (RR) soybean in Argentina, Bt maize in Canada and Argentina, and HT maize in Canada. Argentina gave approval to Syngenta to grow fourstack (GA2 × Bt 11 × MIR60 × MIR162) Viptera maize (Que et al., 2010). Japan has approved maize event 4114 (HR + IR). Mexico has given food approvals to a new cotton event, MON88701 × MON88913 × MON15985 (HT + IR), and two herbicide tolerant soybean events, FG72 and DAS44406. Taiwan has granted food approvals to three events: COT102 (cotton), MON89034 × TC1507 × NK603 × DAS40278 (maize), and the new event TC1507 × MON810 × MIR162 (maize). The USA has granted nonregulated status to herbicide tolerant events MON88701 (cotton) and MON87708 (soybean). Additionally, the Russian Federation approved the insect resistant maize event 5307 for food and feed. South Korea has added 10 new approvals: MON88302 × RF3 (food), MON88302 × MS8 × RF3, 3006-210-23 × 281-24-236 × MON88913 × COT102, 4114, GA21 × T25, DAS40278, TC1507 × MON810, MON87427 × MON89034 × NK603, MON87460 × MON89034 × MON88017, and SYHT02 food and feed approvals (http://www.isaaa.org/gmapprovaldatabase/default.asp).

DETECTION STRATEGIES GM content-based verification requires testing of GM products for the presence of foreign DNA or protein. The enforcement of threshold values has created a pressing demand for the development of reliable GM analysis methods of a rapid and inexpensive character. Reliable screening methods are important both for detection of unauthorized GM crops and labeling control (Morisset et al., 2009). Unauthorized GM crops can challenge the present analytical system on the grounds of practical application of detection methods such as regulatory sequences common to all GM crops. Different screening methods based on DNA and proteins are employed for the detection of GM crops and their products.

PCR AND REAL-TIME PCR PCR is the preferred method for the identification and quantification of Bt gene because of its versatility, sensitivity, specificity, and high-throughput applications (Morisset et al., 2009). To detect any Bt gene, it is necessary to know the sequence of the genes used in the GM construct. These may include plasmid vector sequences, selectable markers, promoters, and terminators. As mentioned earlier, commonly used detection methods for GM crops are based on PCR (Stull, 2001). To identify GM crops and products, a primer needs to be designed for the amplification of the inserted gene. This basic requirement is ascertained by restriction endonuclease digestion of the gene followed by hybridization with a specific DNA probe. Alternatively, the PCR product itself may be used for direct sequencing. In addition, a nested PCR in which two sets of standard primers are used that bind specifically to the target sequences may also be employed. Multiplex and transgene construct-specific PCR assays for cry1Ab, cry1Ac, cry2Ab, and vip-3A transgenes have been reported (Kamle et al., 2011a; Randhawa et al., 2010). Real-time PCR is used to quantify a targeted DNA molecule. For the detection of the products, sequence-specific oligonucleotides labeled with a fluorescent reporter are used, which allow the detection of the amplified product as the reaction advances. Real-time PCR has great value in validating and estimating the number of copies of inserted genes into the host genome (Bonfini et al., 2002; Zhang et al., 2003). This has been reported for several GM crops such as maize, cassava, rapeseed, wheat, cotton, and brinjal (Aguilera et al., 2008; Ballari et al., 2013; Beltrán et al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007). Furthermore,

Biosafety and Detection of GM Crops Chapter | 2  15

a sensitive loop-mediated isothermal amplification method employed for the detection of three GM rice events has been reported (Chen et al., 2012b; Kiddle et al., 2012). Besides these techniques, microarray-based detection systems are under development. Bt-176 transgenic maize (cry1Ab) was quantified by ligation detection reaction combined with a universal array approach (Bordoni et al., 2004).

BIOSENSORS A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component. GM detection has encouraged the development of sensitive sensor technology that promises to generate quick results. The biosensor’s prominent attribute is the immobilization of the probe on an electrode surface such as altered cysteamine gold. Currently, different types of biosensors (electrochemical sensors, piezoelectric biosensors, surface plasmon resonance/optical biosensors) are used to detect transgenes in GM crops such as soybean, maize, cotton, rice, tomato, and canola (Bai et al., 2007; Feriotto et al., 2003; Mariotti et al., 2002; Stobiecka et al., 2007; Tichoniuk et al., 2008). Recently, in Surface Enhanced Raman Scattering Spectroscopy, a barcoded nanosensor has been developed to detect cry1Ab and cry1Ac transgenes in GM rice (Chen et al., 2012a; Chen et al., 2012b).

PROTEIN-BASED DETECTION An immunoassay technique based on antibodies is a standard approach for qualitative and quantitative detection of protein of a known target analyte (Brett et al., 1999). Both monoclonal (highly specific) and polyclonal (often more sensitive) antibodies can be used, depending on the specificity of the detection system. On the basis of typical concentrations of a transgenic material in plant tissues (>10 μg per tissue), the limit of detection (LOD) of a protein immunoassay can predict the presence of recombinant protein in up to 1% of GMOs (Stave, 2002).

IMMUNOASSAYS ELISA has a significant advantage of protein analysis in GM crops and their products. A sandwich ELISA is the preferable immunoassay used for the detection of Bt protein, where an analyte is sandwiched in between the two antibodies: a capture antibody and the detector antibody. In sandwich ELISA, protein concentration is directly proportional to the color intensity (the higher the protein concentration, the greater the color intensity). ELISA was successfully used for the detection of protein encoded by cp4-epsps gene in an RR soybean (Rogan, 1999). Also, monoclonal antibodies are being used for the development of sensitive and single-epitope-specific immunoassays for the detection of Bt proteins such as Cry1Ac and Cry1Ab (Vázquez-Padrón et al., 2000). For the detection of Cry1Ab, a capillary electrophoresis competitive immunoassay and a highly sensitive quanti-dot based fluorescence linked immunosorbant assay have been developed (Giovannoli et al., 2008; Zhu et al., 2011). Similarly, a monoclonal antibody-based sandwich immunoassay having a 100 ng/g LOD for Cry1Ac and a 1 pg/g LOD for Cry2Ab in cotton seed/leaf samples has been reported (Shan et al., 2007; Kamle et al., 2011b, 2013).

IMMUNOSTRIP The use of a different format, such as ELISA, with a nitrocellulose strip rather than microtiter wells, led to the development of lateral flow strip/dipstick/immunostrip technology. Immobilized double antibodies, specific to recognize expressed protein, are conjugated to a color reactant (gold nano-particles) and incorporated into a nitrocellulose strip. This nitrocellulose strip, when dipped in the protein extract of plant tissue (e.g., GM cotton leaf) harboring a GM protein, leads to an antibody reaction releasing color. This red colored gold-conjugated complex flows to the other end of the strip through capillary movement to a porous membrane that has two captured antibody zones. One zone is specific for the GM protein and the other one is specific for untreated antibodies coupled to the reagent. The immunostrips can give results as either “Yes” or “No” within 5–10 min. The immunostrip is an economical, easy, and field tractable detection method. These immunostrips are commercially available to detect Cry1Ab, Cry1Ac, Cry2Ab, and CP4-EPSPS (Lipton et al., 2000; Fagan et al., 2001).

IMMUNO-PCR Immuno-PCR potentially offers a sensitive and specific method for detecting the antigen, in which a specific DNA molecule is used as a marker. It combines the specificity of an ELISA with the sensitivity of the assay using PCR (Sano et al.,

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1992). An immuno-PCR assay has been reported for the detection of Cry proteins expressing GM crops such as Cry1Ac (Allen et al., 2006; Zhang and Guo, 2011).

FUTURE PROSPECTS The first generation of Bt crops (MON810) has been extraordinarily successful with a few examples of pest populations evolving resistance. These crops are already being replaced with a second or third generation of GM crop varieties having two or more traits/events. This is not a matter of complacency and still needs more efficacious and potent Bt strains to meet the future requirement (Christou et al., 2006; Crickmore, 2006). With the development of newer transgene crops, detection methods are also likely to be improved. The International Regulations and the Codex guidelines acting together with biosafety issues and the labeling of GMOs seem to be a promising proposition toward the acceptance of GM crops. Currently, new GM crops and traits stacked with different genes, namely herbicide resistance, drought resistance, and salt or osmosis resistance, which could be commercialized in the upcoming years, are going under field testing. Simplot, the developer of the Innate™ potato, gets the United States Department of Agriculture nod for the cancer reducing GM potato. Many other different kinds of GM crops are ready to be commercialized. GM or biotech crops are not a panacea but can potentially make a significant contribution to curving down the graph of poverty by increasing crop productivity, which can be accomplished by the collaboration of the public and private sectors. In conclusion, the productivity of GMCs assures the reduction of hunger and poverty from this planet. Lawfully, ­biosafety measurements and consumer concerns also need to be scrutinized from time to time to maintain synchronization and harmonization in society. Improvement of the GM crop detection strategies is a pressing need to avoid unintentional mixing of GM and non-GM crops.

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Chen, X., Wang, X., Jin, N., Zhou, Y., Huang, S., Miao, Q., Zhu, Q., Xu, J., 2012b. End point visual detection of three genetically modified rice events by loop mediated isothermal amplification. Int. J. Mol. Sci. 13, 14421–14433. Christou, P., Capell, T., Kohli, A., Gatehouse, J.A., Gatehouse, A.M.R., 2006. Recent developments and future prospects in insect pest control in transgenic crops. Trends Plant Sci. 11, 302–308. Cifuentes, A., 2012. Food Analysis: Present, future and foodomics. ISRN Anal. Chem. 2012, 1–16. Codex Alimentarius, 2003. Codex Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarius Commission, Twenty-fifth Session, Rome, Italy 30 June–5 July, 2003. Appendix III, Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants, and Appendix IV, Annex on the Assessment of Possible Allergenicity. Codex Alimentarius Commission, Rome 47–60. Codex Alinorm, 2006. Food standards programme. 06/29/34: Joint FAO/WHO. 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Kamle, S., Ojha, A., Kumar, A., 2011b. Development of an enzyme linked immunosorbant assay for the detection of Cry2Ab protein in transgenic plants. GM Crops Food 2, 125–132. Kamle, S., Ojha, A., Kumar, A., 2013. Development of enzyme-linked immunosorbant (ELISA) assay for the detection of Bt protein in transgenic cotton. In: Baohong, Z. (Ed.), Transgenic Cotton, vol. 958. Humana Press, pp. 131–138. Kiddle, et al., 2012. GMO detection using a bioluminescent real time reporter (BART) of loop mediated amplification (LAMP) suitable for field use. BMC Biotechnol. 12, 15. Kinderlerer, J., 2008. The Cartagena Protocol on Biosafety. Collect. Biosaf. Rev. 4, 12–65. Koprowski, H., 1988. Argentine trials. New Scientist. 18. Krueger, J., 1999. What’s to become of trade on hazardous wastes? The Basel convention on decade later. Environment 41, 11–21. Ladics, G.S., 2008. Current codex guidelines for assessment of potential protein allergenicity. Food Chem. Toxicol. 46, 20–23. Ladics, G.S., Selgrade, M.K., 2009. Identifying food proteins with allergenic potential: evolution of approaches to safety assessment and research to provide additional tools. Regul. Toxicol. Pharmacol. 54, S2–6. Lee, S.H., Kang, S.H., Park, Y.H., Min, D.M., Kim, Y.M., 2006. Quantitative analysis of two genetically modified maize lines by real-time PCR. J. Microbiol. Biotechnol. 16, 205–211. Li, Z., Hansen, J.L., Liu, Y., Zemetra, R.S., Berger, P.H., 2004. Using real-time PCR to determine transgene copy number in wheat. Plant Mol. Biol. Rep. 22, 179–188. Lipton, C.R., Dautlick, J.X., Grothaus, G.D., Hunst, P.L., Magin, K.M., Mihaliak, C.A., 2000. Guidelines for the validation and use of immunoassays for determining of introduced proteins in biotechnology enhanced crops and derived food ingredients. Food Agric. Immunol. 12, 153–164. Maltsbarger, R., Kalaitzandonakes, N., 2000. Direct and hidden costs in identity preserved supply chains. AgBioForum 3, 4. Mariotti, E., Minunni, M., Mascini, M., 2002. Surface plasmon resonance biosensor for genetically modified organisms detection. Anal. Chim. Acta 453, 165–172. McKay White, K., Veeman, M.M., 2007. A Survey of Literature on Genetically Modified Crops: Economics, Ethics, and Society (Staff Paper 07–01) Department of Rural Economy, Faculty of Agriculture, Forestry and Home Economics, University of Alberta, Edmonton, Canada, Alberta, Canada. http://purl.umn.edu/7380 (accessed 01.02.14.).

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Morisset, D., Demsar, T., Gruden, K., Vojvoda, J., Steih, D., Zel, J., 2009. Detection of genetically modified organisms—closing the gaps. Nat. Biotechnol. 27, 700–701. Paarlberg, R.L., 2000. Genetically modified crops in developing countries: promise or peril. Environment 42, 19–27. Paarlberg, R.L., 2002. The real threat to GM crops in poor countries: consumer and policy resistance to GM foods in rich countries. Food Policy 27, 247–250. Que, Q., et al., 2010. Trait stacking in transgenic crops: challenges and opportunities. GM Crops Food 4, 220–229. Randhawa, G.J., Singh, M., Chhabra, R., Sharma, R., 2010. Qualitative and quantitative molecular testing methodologies and traceability systems for commercialized Bt cotton events and other Bt crops under field trials in India. Food Anal. Methods 4, 295–303. Raustalia, K., Victor, D.G., 1996. Biodiversity since Rio: the future of the convention on biological biodiversity. Environment 38, 1–11. Regulation (EC) 258/97 of the European Parliament and of the Council of 27th January 1997 concerning novel foods and novel food ingredients. Off. J. Eur. Union Lt. 043, 14.02.1997, 0001–0006 (accessed 01.02.14.). Regulation (EC), 2001. Deliberate release into the environment of genetically modified organisms. Report of the APO Study, 2002. Meeting on Use and Regulation of Genetically Modified Organisms Held in the Republic of China, 18–23 November. (02-AG-GE-STM-04-B) (accessed 01.02.14.). Rogan, G.J., 1999. Immunodiagnostic methods for selection of 5-enolpyruvyl shikimate-3-phosphate synthase in Roundup Ready soybeans. Food Control 10, 407–414. Sano, T., Smith, C.J., Cantor, C.R., 1992. Immuno-PCR: very sensitive antigen detection by means of specific antibody–DNA conjugates. Science 258, 120. Shan, G., Embrey, S.K., Schaffer, B.W., 2007. A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton. J. Agric. Food Chem. 55, 5974–5979. Smythe, S., Kerr, W.A., Davey, K.A., 2006. Closing markets to biotechnology: does it pose an economic risk if markets are globalised? Int. J. Technol. Glob. 2, 377–389. Stave, J.W., 2002. Protein immunoassay methods for detection of biotech crops: applications, limitations and practical considerations. J. AOAC Int. 85, 780–786. Stewart, C.N., Richards, H.A., Halfhill, M.D., 2000. Transgenic plants and biosafety: science, misconception and public perceptions. Biotechniques 29, 832. Stobiecka, M., Ciesla, J.M., Janowska, B., Tudek, B., Radecka, H., 2007. Piezoelectric sensor for determination of geneticallymodified soybean Roundup Ready in samples not amplified by PCR. Sensors 7, 1462–1479. Streiffer, R., Rubel, A., 2003. Choice versus autonomy in the GM food labeling debate. Stull, D., 2001. A feat of fluorescence. Scientist 15, 20–21. Tichoniuk, M., Ligaj, M., Filipiak, M., 2008. Application of DNA hybridization biosensor as a screening method for the detection of genetically modified food components. Sensors 8, 2118–2135. Vázquez-Padrón, R.I., et al., 2000. Cry1Ac protoxin from Bacillus thuringiensis sp., kurstaki HD-73 binds to surface proteins in the mouse small intestine. Biochem. Biophys. Res. Commun. 271, 54–58. Wu, Y., Wu, G., Xiao, L., Lu, C., 2007. Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1 × RF1 and MS1 × RF2. J. Agric. Food Chem. 55, 8380–8389. Zhang, D., Guo, J., 2011. The development and standardization of testing methods for GMO and their derived products. J. Integr. Plant Biol. 3, 539–551. Zhang, Y., Zhang, D., Li, W., Chen, J., Peng, Y., Cao, W., 2003. A novel real-time quantitative PCR method using attached universal template probe. Nucleic Acids Res. 31, 123. Zhu, X., Chen, L., Shen, P., Jia, J., Zhang, D., Yang, L., 2011. High sensitive detection of Cry1Ab protein using a quanti-dot based fluorescence-linked immunosorbant assay. J. Agric. Food Chem. 23, 2184–2189.

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Chapter 3

Genetically Modified Food Animals: An Overview Renu Pandey1, Meenakshi Dwivedi1, Shishir Kumar Gupta1,2, Daman Saluja1 1Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India; 2Indian Veterinary Research Institute, Izatnagar, Bareilley, Uttar Pradesh, India

INTRODUCTION The population of the world, according to UN estimates, is growing at the rate of 1.14% per annum and is expected to surpass 8 billion by 2024 (United Nations, 2014). With 7.2 billion humans on the earth as of this writing, there is an imminent demand for food to cater to the growing numbers. With limited availability of agricultural land and increasing malnutrition among developing countries, the situation is grim and concerted efforts are needed to address the situation. Age old methods of cropping and livestock rearing will not be able to provide for the growing hunger. Biotechnology, or molecular genetics in particular, is the science that we look forward to with anticipation in this regard to ameliorate the crises of malnutrition and inflated food costs. It also has an added advantage of reduced environmental footprints. Biotechnology is not a novel idea; it has been here since the Neolithic revolution, when man first domesticated wheat and barley through breeding and selection of high-yielding varieties (Zohary et al., 1969). Traditional methods for the improvement of animal production have been in practice for a long time and rely basically on the selection of animals with superior traits. These changes appear over a long period of time due to gradual modification of the genome. Such methods employ the selection of an animal with the desirable trait that results from random mutations in the genome. As such, mutations occur randomly, and the efficiency of the selection method is highly dependent on the uncontrolled mutations. Furthermore, considering the complexity of the genome and the fact that most of the traits are polygenic in nature, and the genes involved are, often, located on the different chromosomes, making a coordinated selection of the involved genes is a complex process. Also, in the standard selection method, there is a transfer of undesirable alleles along with the desired ones, which may result in deleterious side effects. Transgenesis, or modification of an organism’s genome by the introduction of a foreign gene or an altered gene of the same organism through genetic engineering, obliterates the flaws of the conventional breeding system. Sequencing, along with progressive genome mapping data, has put a plethora of information regarding the genomic composition of many animals of interest into the public domain. This vital information can be utilized to selectively hunt or recognize a gene, influencing a particular trait through techniques like reverse or forward genetics and inducing stable and inheritable genetic changes (Carviel et al., 2009).

GENETICALLY MODIFIED ORGANISMS Definition According to the European Union (EU), genetically modified organisms (GMOs) are defined as any organism, except humans, carrying an altered genetic material that does not occur naturally through natural selection or mating (Francescon, 2001) in the Directive 2001/18/EC on the deliberate release into the environment of GMOs issued by the EU and including techniques of recombinant DNA (rDNA) technology along with microinjection techniques as a tool for genetic modifications. All such tinkered organisms are known by synonymous terms like “genetically engineered,” “genetically modified,” “transgenic,” “biologically engineered,” and “biologically modified” organisms.

Advent of GMOs A scientific breakthrough was achieved around three decades ago when the first recombinant mouse was generated through microinjection into the pronuclei of mouse oocyte (Gordon et al., 1980; Jaenisch and Mintz, 1974). Since then, genetically Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00003-8 Copyright © 2016 Elsevier Inc. All rights reserved.

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modified (GM) mice have been an indispensable part of medical research, serving as disease models for various congenital, metabolic disease, cancer, and aging disorders (Bedell et al., 1997; Halford and Shewry, 2000; Heckl et al., 2014). Other animals have also been genetically modified for reasons other than research. Considerable success has been achieved in the introduction of novel traits into different animals. There is now an extended list of cows, swine, sheep, and goats genetically modified or fortified for food (and other applications) generated through various rDNA technologies and epigenetic approaches (reviewed later in the chapter), creating heritable and nonheritable changes in the genome. Transgenesis is an extension of the conventional breeding practices: Being more robust, direct in approach, and effective than conventional breeding; Bringing about heritable changes once a foreign gene is inserted; l  Not restricted by fertility barriers in which only closely related species could be crossed, even heterologous genes have been inserted into animals with substantial success, for example, the Enviropig™, containing a phytase-producing gene from Escherichia coli (Forsberg et al., 2003); and l Inducing both heritable and nonheritable changes with the usage of appropriate methodology. l l

Methods for Introduction of Transgenesis The introduction of transgenesis can be roughly grouped into three categories based on the changes in the genome desired: the introduction of a new gene, the replacement of a gene through homologous recombination, and the alteration of gene expression through epigenetics. Before venturing into the details of the methods the foremost point of consideration is identification of the gene of interest (Houdebine, 2007).

Identification of the Gene of Interest Identification of the gene of interest is the most vital spoke in the wheel of genetic engineering. When generating a GM organism, a transgene is inserted into the host animal’s genome. Among the many available routes for screening and identification of the gene of interest, the exact approach would depend on the organism and the rDNA technology available for that organism, along with other factors like whether the mutation is recessive or dominant, which screens are available for identification of the mutant and the wild-type phenotype, known homology with other genes, etc. The gene of interest can be screened from sequence homology, targeting the RFLP (restriction fragment length polymorphism) markers around the gene of interest, or from the cDNA libraries (utilizing the technique of “primer walking”) available in the public domain or by creating your own libraries (Figure 1).

FIGURE 1  Schematic representation of the methodology used to generate genetically modified organisms.

Genetically Modified Food Animals Chapter | 3  21

Microinjection Microinjection or pronuclear injection was at one time the most successful method for producing GM animals (Chen et al., 2004; Hammer et al., 1985). Around 400–500 copies of the transgene construct are directly injected using a very fine needle into one of the two pronuclei of the fertilized embryo. Transgene gets inserted into the host genome randomly with low efficiency and such in-vitro fertilized embryo thus generated is then cultured in the artificial environment and transplanted into an impregnated surrogate mother. Microinjection at a later stage (after the single celled stage) leads to the appearance of “mosaic” patterns. The microinjection technique has been used most widely for biopharming (Campbell et al., 1994). This method has its own lacunae, too, like a very low degree of efficiency of ∼5%, high screening costs, and being unsuitable for creating GM birds/poultry as it is challenging to access the fertilized egg at the single-celled stage. Therefore, the method of viral transfection is best suited in case of aves. Nonetheless, microinjecting the gene construct into the zygote was reported to produce transgenic birds with some success (Love et al., 1994). Advances in combining pronuclear injection with integrases and recombinases hold greater possibilities for creating targeted mutagenesis (Ohtsuka et al., 2012). The first report of generating transgenic mice with high muscle mass through direct microinjection of the recombinant lentiviral vector containing shRNA against the myostatin gene holds promise for quality food from livestock (Tessanne et al., 2012).

Viral Transfection Lentiviruses—slow growing members of the viral family of retroviridae categorized by long incubation periods—have been the most utilized vectors to deliver the transgene by virtue of their ability to integrate with the host genomic DNA and proliferate with every replication of the host genomic content (Jaenisch, 1976). However, adenoviruses—the nonenveloped doublestranded DNA viruses of the family adenoviridae—are also being used. Recombinant adenoviruses have been used in swine to generate vaccines against swine influenza virus (Wesley et al., 2004). The “replication competent” viruses reintegrate the transgene multiple times and ensure transgene overexpression in the offspring and sometimes even in other animals coming into contact, due to their high infection rates. In contrast, “defective competent” viruses can transfect the cells only once. Retrovirus-mediated transfer has been successfully used to transfer a foreign gene into cows, chickens, monkeys, swine, and sheep (Chan et al., 1998, 2001; Clements et al., 1994; Houdebine, 2000) through mammalian and avian retroviruses like Rous sarcoma virus (RSV), Moloney leukemia virus (MLV), Avian leukosis virus (ALV), Simian virus, etc. Viralmediated genetic modification has some major practical limitations: It limits the size of the transgene to be inserted, making it unviable for larger gene constructs. The long terminal repeats of the virus interfere with the promoter of the transgene. l Viral vectors lack the ability to replicate in early embryo cells, resulting in “chimeras” where the transgene is expressed in only some of the cells of the GM animal (McCluskie et al., 1999). l l

Embryonic Stem Cells Modification Embryonic stem (ES) cells are self-renewing pluripotent cells derived from the inner cell mass of early dividing embryos called blastocysts. The first successful establishment of the embryonic stem cell culture was carried out from a murine embryo in the early 1980s (Evans and Kaufman, 1981) and concerted the effort for the isolation of ES cells from nonhuman primates and finally from humans (Prelle et al., 2002; Thomson et al., 1998). The transgene delivery system, as in the viral delivery system, could be “directed” or “random,” depending on the strategy implied. Directed gene “knock-in” or “knock-out” relies on homologous recombination (Smithies, 2001), a DNA repair mechanism where the nucleotide sequences are exchanged between genomic and exogenous sequences through the event of crossing over between homologous sequences (Li and Heyer, 2008; Te Riele et al., 1992). The transformed ES cells are then transferred into a recipient embryo, implanted into an impregnated surrogate, and “chimeras” or “completely transformed” animals are screened. Unfortunately, this technology is still in its infancy as stable ES cell lines have not been established for livestock (Nowak-Imialek and Niemann, 2012).

Spermatocyte-Mediated Gene Transfer Transgene is incorporated into the spermatocyte through electroporation, polyethylenimine-mediated gene transfection, or viral-mediated delivery. The transfected spermatocyte is used as a vector after in-vitro fertilization, where the attachment of exogenous DNA to the sperm is facilitated by the specific DNA binding proteins (DBPs) present on the postacrosomal surface of the sperm (Wolf et al., 2000). The first transgenic mice were generated through sperm-mediated gene transfer by Lavitrano et al. (1989), mixing plasmid DNA with spermatocytes. But the results of sperm-mediated gene transfers are marred by low

22  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

repeatability and efficiency in the case of mammals (Gandolfi, 2000). Intra-cytoplasmic sperm injection directly into unfertilized oocytes has substantially increased the efficiency of transgene expression in the recovered offspring (Perry et al., 1999).

Cloning: Somatic Cell Nuclear Transfer (SCNT) The emergence of cloning technologies facilitating nuclei transfer from the somatic cells (Campbell et al., 1996; Wilmut et al., 2007) brought a paradigm shift in the biological science in generating transgenic livestock. In somatic cell nuclear transfer, enucleation of oocytes is followed by the fusion of the donor cell and activation of this reorganized embryo. SCNT has been efficacious in many animal species, including those reared for domestic, wildlife, or laboratory research (Galli et al., 2012). It has been the method of choice for generating larger transgenic animals, beginning with the cloning of Dolly (the first cloned animal) and later carried out in other animals, producing transgenics with greater muscle mass and therapeutic applications (Schnieke et al., 1997). It is a potent tool for the extension of superior breeding stock of farm animals for food.

Transposon-Mediated Gene Silencing Transgenesis can be induced in livestock through transposons. Transposons, or “jumping genes,” are the unique DNA elements that traffic around the genome. This inherent property of these elements can be harnessed to induce genetic modification in genome generating transgenic livestock (Ivics et al., 2009). With the discovery of sleeping beauty, PiggyBac- and Tol2-like transposons, interest have been reinvigorated in this domain (Ivics et al., 1997). Successful generation of transgenic swine has been achieved through the introduction of double stranded RNAi (ribonucleic acid mediated interference) cassette silencing the cyctic fibrosis transmembrane conductance regulator (CFTR) through the introduction of transposons (Sleeping Beauty/Tol2/ PiggyBac, etc.) flanking the nucleic acid construct containing the transcriptional unit on both sides. Transgenic rabbits (Ivics et al., 2014) and pigs carrying a fluorescent reporter (Garrels et al., 2011) have also been generated using a similar approach.

APPLICATIONS OF TRANSGENIC ANIMALS Genetically modified animals have found greater applications in medical research with many animal models being generated for different diseases. However, due to lower costs, efficiency, reliability, and reproducibility, biotechnology companies have invested in GM animals as well, taking greater interest in the applications of GM animals for food, therapeutics, organ farming, recreation, environmental sustainability, etc. Numerous commercialization applications are filed every year with the regulating agencies (Figure 2).

Biological Research Most GM animals that have been developed as of this writing fall under the category of biological research, with the most number of transgenic rodents and to some extent rabbits, guinea pigs, and pigs being developed to act as disease models for neuropsychiatric, hearing, and vision research, as well as, many types of cancers, immunodeficiency, and cardiovascular

FIGURE 2  Schematic representation of various applications of genetically modified animals.

Genetically Modified Food Animals Chapter | 3  23

and metabolic disorders (Bedell et al., 1997; Holliday, 1992). The review by Bedell et al. (1997) listed rodent models for around 110 different human disease conditions; of these, 91 generated models were a result of transgenesis. Taconic Biosciences, a research model provider, offers 223 mouse models generated through conditional and constitutive knock-outs along with constitutional and targeted transgenesis (Taconic, 2015).

Xenotransplantation Being evolutionarily close to humans, pigs are the most common animal species projected to be used in this regard. A revolutionary step has been the generation of the galα(1-2)gal epitope lacking α(1-3)galactosyltransferase enzyme through the generation of gal-knockout pigs (Lai et al., 2002). Xenotransplantation of porcine islet cells is being considered as a new option for treating type-I diabetes (Reichart et al., 2015).

Biopharming The commercial production and extraction of pharmaceuticals from body secretions of GM animals is called biopharming. The production of exogenous protein in milk was first reported in 1980 with the production of human tissue plasminogen activator in mouse milk (Gordon et al., 1987). Ever since, transgenic animals have been put to use as bioreactors for many vital pharmaceutical compounds (Houdebine, 1994; Mishra et al., 2014). Many arguments in favor of biopharming are its costeffectiveness, its easy scale up, and correct molecular folding by virtue of being produced in a mammal (Wall, 1999). A few examples are α-1 antitrypsin, antithrombin, human growth hormone, human insulin, albumin, and fibrinogen, among others.

Environmental Sustainability The generation of Enviropig™ was a major step in making biotechnology more environmentally sustainable through reducing its environmental footprint. Enviropig™ produce 70% less phosphorus in their manure due to the introduction of the phytase enzyme from Escherchia coli, which enables the pig to produce a plant phytic acid digesting enzyme in its salivary glands (Forsberg et al., 2013), reducing the cost of mineral phosphate supplements.

Food Farm animals are reared at a large scale for human consumption but the growing demand for animal meat, milk, and other products is difficult to meet. AquAdvantage™, a GM Atlantic salmon (a product of AquaBounty Technologies in Maynard, Massachusetts), is all set to become the first commercialized GM animal approved by the Food and Drug Administration (FDA) for consumption. AquAdvantage contains a promoter sequence from ocean pout and the growth hormone gene sequence from the Pacific Chinook salmon (Oncorhynchus tshawytscha). AquAdvantage and Chinook salmon belong to different genera, ruling out the possibility of natural inbreeding (Entis, 1998). It is slated to clear the 7-step FDA approval process (Fox, 2010; Ledford, 2013; Van Eenennaam et al., 2011). Other animals have also been genetically tinkered with to produce quality meat (meat rich in omega-3 fatty acids, as compared to the high omega-6 fatty acid present in normal or non-transgenic animal meat (Lai et al., 2006) (Table 1).

TABLE 1  Genetically Modified Animals for Food GM Animal

Transgene/Source

Composition

Reference

Salmon

α-form of opAFP-GHc2 rDNA construct from Pacific Chinook salmon

Higher growth rate

(FDA, 2015)

Cow

N-3 fatty acid desaturase gene from Caenorhabditis elegans

Higher omega-3 fatty acid content

(Wu et al., 2012)

Cow

Additional copies of β- and κ-casein gene (CSN2, CSN3)

Higher casein content in milk

(Brophy et al., 2003)

Pig

N-3 fatty acid desaturase (fat-1) gene from Caenorhabditis elegans

Higher omega-3 fatty acid content

(Lai et al., 2006)

Pig

Fatty acid desaturation 2 gene from spinach

Higher linoleic acid content

(Saeki et al., 2004)

Pig

Bovine α-lactalbumin gene

Higher milk yield

(Bleck et al., 1998)

24  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

CONCERNS Genetically modified food animals, though harmless, can produce serious health risks if introduced without proper risk assessment and without being proven to be safe after a thorough examination. There are concerns about the naturalness of genetically engineered animals, since the introduced genes are not the natural composite of the host genome. There are species integrity concerns as well, confounded by the fear that the transgenic species might breed with wild animals and infiltrate their habitat and survival. This brings into force the “moral veto” against the perusal of biotechnology and is the major bone of contention for animal rights activists. The possibility of replication competent viral vectors infecting nontargeted species and bringing about a host of novel lethal human infections has also prevented the introduction of GM animals for consumption. Global agencies such as the FDA, Organization for Economic Co-operation and Development, Codex Alimentarius Commission, World Trade Organization, and United Nations Environmental Program-Global Environment Facility are among the major regulators and policymaking bodies ensuring biosafety and applicability and issue guidelines for the acceptance and safeguard of GM animals.

FUTURE DIRECTIONS Transgenesis for the improvement of animal production is still in its infancy. In the case of GM animals, there is a huge gap between the regulatory controls expected and those actually exercised in reality. The current regulatory network takes a very stringent outlook, with dismal prospects of reflecting on the basic animal research in the wider context. Several GM animal lines can be recommended for human consumption but none of them have been commercialized. Transgenics in animals offer an added advantage of homologous recombination-based allele replacement not fully mastered in plants. The prospect of the transgenic fish AquAdvantage™ ending up on the dining table looks promising, being the first GM animal food product to be approved. This would open new avenues toward the likelihood of acceptance of GM meat (pork, beef, seafood, and chicken) and genetically fortified milk with enhanced protein and lipid content. Fears in the public’s opinion of negative evolutionary consequences, increased allergenicity, and threats to already endangered animal species are not entirely unfounded, as no conclusive answers are available for the unknowns and the what ifs. Effective and accountable dialog between the stakeholders (scientists, industry, and government) is pertinent to reach a public accord on the satisfactory usage of transgenesis in animals. Without this, the hopes of addressing the future needs of food and therapeutics will hang in a balance of uncertainty.

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Taconic, 2015. Genetically Engineered Models Collection. Te Riele, H., Maandag, E.R., Berns, A., 1992. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. 89, 5128–5132. Tessanne, K., Golding, M., Long, C., Peoples, M., Hannon, G., Westhusin, M., 2012. Production of transgenic calves expressing an shRNA targeting myostatin. Mol. Reprod. Dev. 79, 176–185. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. United Nations, 2014. Concise Report on the World Population. The Department of Economic and Social Affairs of the United Nations. Van Eenennaam, A.L., Muir, W.M., 2011. Transgenic salmon: a final leap to the grocery shelf? Nature Biotechnol. 29, 706–710. Wall, R., 1999. Biotechnology for the production of modified and innovative animal products: transgenic livestock bioreactors. Livest. Prod. Sci. 59, 243–255. Wesley, R.D., Tang, M., Lager, K.M., 2004. Protection of weaned pigs by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of H3N2 swine influenza virus. Vaccine 22, 3427–3434. Wilmut, I., Schnieke, A., McWhir, J., Kind, A., Campbell, K., 2007. Viable offspring derived from fetal and adult mammalian cells. Cloning Stem Cells 9, 3–7. Wolf, E., Schernthaner, W., Zakhartchenko, V., Prelle, K., Stojkovic, M., Brem, G., 2000. Transgenic technology in farm animals—progress and perspectives. Exp. Physiol. 85, 615–625. Wu, X., Ouyang, H., Duan, B., Pang, D., Zhang, L., Yuan, T., Xue, L., Ni, D., Cheng, L., Dong, S., 2012. Production of cloned transgenic cow expressing omega-3 fatty acids. Transgenic Res. 21, 537–543. Zohary, D., UCKO, P., Dimbleby, G., 1969. The progenitors of wheat and barley in relation to domestication and agricultural dispersal in the Old World. In: Domestication and Exploitation of Plants and Animals, pp. 47–66.

Chapter 4

Genetically Modified Aubergine (Also Called Brinjal or Solanum melongena) Lalitha R. Gowda Chief Scientist (Former), CSIR-Central Food Technological Research Institute, Department of Protein Chemistry and Technology, Mysore, Karnataka, India

GENERAL DESCRIPTION OF BRINJAL The edible fruit of Solanum melongena L., belonging to the genus Solanum, is described by several names. Solanum is among the largest genera, having more than 1500 described plant species (Chen, 1997). Known as “aubergine” in Europe, the name “brinjal” is common to Southeast Asia, South Asia, and Africa. The name “eggplant” in the USA and Canada is derived from the egg-like shape of the fruit of some varieties. The other known names are melongen, garden egg, and guinea squash. There are three main botanical varieties under the species melongena. The round or egg-shaped cultivars are grouped under esculentum, common eggplant. The long, slender types are included under serpentinum, snake eggplant, and the small and straggling plants are put under depressum, dwarf eggplant (Chen, 1997). Brinjal, native to India (Tsao and Lo, 2006; Doijode, 2001), is among the most common, is popular, and is a very important common man’s vegetable in India. It is the second largest vegetable crop in India. The annual production of 8–9 million tons amounts to one quarter of the global production (Choudary and Gaur, 2009). Brinjal is a versatile crop, adapted to different agro-climatic regions and grown throughout the year and throughout the country. It is primarily grown by small farmers and holds a coveted position as it is an important source of income for them. Along with tomato and onion it ranks as the second most consumed vegetable in India after potato. Brinjal features in the dishes of virtually every household in India, irrespective of food preferences, income levels, or social status. The versatile use of brinjal in Indian cuisine, for both everyday and festive occasions, has led to it being described as the “king of vegetables.” A variety of cultivars, varying in tastes, shapes, colors, and sizes, are grown in India. The cultivars range from small to large and pendulous, from oblong to round, from oval or egg-shaped to long and club-shaped; they come in colors such as green, white, or yellow, degrees of purple pigmentation to almost black, among others, or even striated shades and color gradients (Herbst, 2001). The varieties of brinjal popular in India include Arka Navneet, Pusa Ankur, Hybrid-6, Pusa Hybrid-5, ARBH-1, ABH-1, Pusa Purple Long, Pusa Purple Cluster, and Ritu Raj. West Bengal is the largest producer of brinjal followed by Maharashtra and Bihar (Kumar et al., 2011). Botanically classified as a berry, the soft white fleshy placenta contains numerous small soft seeds embedded in it. The seeds although edible, are bitter because they contain nicotinoid alkaloids.

BIOCHEMICAL AND NUTRITIONAL PROPERTIES Brinjal is primarily consumed as a cooked vegetable in various ways. It is low in calories and fats and contains mostly water, some protein, fiber, and carbohydrates. It is a good source of minerals and vitamins and is rich in total water-soluble sugars, free-reducing sugars, and amide proteins, among other nutrients. It has also been recommended as an excellent remedy for those suffering from liver complaints (Shukla and Naik, 1993). The chemical composition of the edible portion of brinjal is given in Table 1. It is reported that the long-fruited brinjal cultivars contain a higher content of free-reducing sugars, anthocyanin, phenols, glycoalkaloids (such as solasodine), dry matter, and amide proteins as compared to the oblong or ovoid cultivars (Bajaj et al., 1979). The presence of glycoalkaloids is the cause for bitterness and off flavor in brinjal and varies from 0.37 mg/100 g fresh weight to 4.83 mg (Bajaj et al., 1981). The discoloration of brinjal upon cutting it is attributed to the high polyphenol oxidase (PPO) activity. Tissue printing indicates a predominant presence of the enzyme in the exocarp and the areas surrounding the seeds in the mesocarp of brinjal fruits (Shetty et al., 2011). Immunolocalization of PPOs in the eggplant infested with shoot and fruit borer revealed localization of the PPO at the site of infection in tender shoots and fruit Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00004-X Copyright © 2016 Elsevier Inc. All rights reserved.

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and further inside the mature tissues (Shetty et al., 2011). Brinjal is known to induce IgE-mediated hypersensitive reactions in sensitized individuals with a wide spectrum of symptoms. It is a multiallergenic vegetable in that allergens occur in all the edible parts of eggplant with a predominant localization in the peel (Harish Babu and Venkatesh, 2009).

INSECT PESTS OF BRINJAL Brinjal is prone to attacks by many insect pests as well as fungal, bacterial, and viral diseases. Insect infestation is among the most limiting biotic factors for enhancing the potential yield of brinjal. The brinjal crop is prone to damage by various insects with varying degrees of infestation. The most devastating pest infestation that occurs throughout the year at all stages of plant growth is by the fruit and shoot borer (FSB) (Leucinodes orbonalis Guenee). It belongs to the insect order Lepidoptera and family Pyralidae. FSB is almost monophagous as it predominantly feeds on brinjal and on closelyrelated potato and tomato. Other solanaceous vegetable crops also serve as hosts. In the vegetative stage the terminal shoots are attacked by the larva, which bores into tender shoots and chews up the tissues. As a result the affected shoots get paralyzed, causing drooping, withering, and wilting of the affected shoots. During the reproductive stage it bores into flower buds and young fruit and feeds inside. The bored holes are plugged with excreta, which makes the fruit unmarketable and unfit for consumption. It is estimated that FSB causes losses of 60–70% in yield even after repeated insecticide sprays following the average 4.6 kg of insecticides and pesticides per hectare (Choudhary and Gaur, 2009). The plant is also attacked by several other insect pests such as Thrips, Epilachna beetle, jassids, aphids, and mites. Farmers rely exclusively on the application of pesticides to control FSB and to produce blemish-free brinjal fruit. The pesticides used belong to the class

TABLE 1  Chemical Composition of Raw Brinjal (Per 100 g Edible Portion) Constituent

Amount

Constituent

Amount

Energy

24 kcal

Calcium

18 mg

Moisture

92.7%

Iron

0.23 mg

Carbohydrates

4 g

Magnesium

15 mg

Sugars

3.53 g

Manganese

0.13 mg

Crude fiber

3 g

Phosphorus

47 mg

Fat

0.3 g

Potassium

200 mg

Protein

1.4 g

Zinc

0.22 mg

Dietary fiber

6.3 g

Oxalic acid

18 mg

 Insoluble

4.6 g

 Soluble

1.7 g

Arginine

210

Thiamine (B1)

0.04 mg

Histidine

130

Riboflavin (B2)

0.11 mg

Lysine

330

Niacin (B3)

0.90 mg

Tryptophan

60

*Pantothenic acid (B5)

0.28 mg

Phenyl Alanine

250

Vitamin B6

0.084 mg

Tyrosine

240

Folate (B9)

34 μg

Methionine

70

Vitamin C

12 mg

Cysteine

30

Vitamin E

0.3 mg

Threonine

230

β-Carotene

74 μg

Leucine

380

Vitamin K

3.5 μg

Isoleucine

270

Choline

52 mg

Valine

230

Essential amino acids (mg/g N)

Source: Nutritive Value of Indian Foods, Revised Edition, National Institute of Nutrition, Hyderabad, India.

Genetically Modified Brinjal Chapter | 4  29

of organophosphates and synthetic pyrethroids. Pesticide use is very intensive for killing the larvae before they bore inside shoots or fruits. It is difficult to control the damage through insecticide sprays as the larvae lead a concealed life within the fruit. Among the 15 recommended insecticides for brinjal more than 50% are prescribed only for FSB. Several insecticides such as cypermethrin, endosulfan, deltamethrin fenvalerate, carbaryl, dichlorvos, phorate, carbofuran, lindane, fenitrothion, dimethoate, and malathion are used to control FSB (Choudhary and Gaur, 2009). The brinjal fruit borer (Helicoverpa armigera) is polyphagous. The larvae feed first on leaves and fruiting bodies and bore into the fruits, completely eating away the internal contents. The sucking pests include Aphids (Lipaphis erysimi), Jassids (Amrasca bigutella), and White Fly (Bemisia tabaci). In India the “Root Knot Nematodes” are the most common plant parasitic nematodes (Meloidogyne spp., i.e., incognita, javanica), and infestation of these nematodes is common in brinjal. Despite the extensive use of chemical pesticides, FSB is difficult to control by the application of pesticides because the larvae, often hidden in the fruit, do not come in contact with the insecticides. The extensive use of chemical pesticides has led to health hazards and unacceptable levels of pesticide residues in edible fruit (Kabir et al., 2008). Pesticide residue in fruits and vegetables has become a food safety issue and consumers demand pesticide-free fruit. In two supervised field trials, brinjal was sprayed with the field doses of Diazinon and Carbosulfan (1.5 mL/L of water). The level of residue was above the maximum residue level. Pesticides are associated with a wide spectrum of human health hazards, ranging from short-term impacts such as headaches, dizziness, nausea, vomiting, contact dermatitis (Sharma and Kaur, 1990) and lack of coordination, tremors, mental confusion, seizures, and coma to chronic impacts like cancer, reproductive harm, and endocrine disruption (Mathur et al., 2002; Khan et al., 2013). Kaur (2008) reported recurring health problems of farmers associated with pesticide exposure.

DEVELOPMENT OF INSECT-RESISTANT Bt-BRINJAL The practices of using extensive pesticides are not only harmful to health and the environment but also unsustainable. Therefore, there was a need to develop alternative control strategies. Bt-toxins as insecticides are well known; however, it was the development and commercialization of insect-resistant transgenic Bt-crops expressing Cry toxins that revolutionized the history of agriculture. As an insect-resistance management strategy, these crops are incorporated with one or more modified Bt genes sourced originally from naturally occurring soil bacterium, Bacillus thuringiensis, a spore-forming bacteria with entomopathogenic properties. Vegetative Bt cells undergo sporulation during stress, synthesizing a protein crystal during spore formation. Proteins in these crystals are called Cry (from Crystal) proteins. The crystalline inclusions formed in the cells of B. thuringiensis subsp. kurstaki consist of different types of Cry proteins with highly specific insecticidal activity. The Cry proteins are pore-forming toxins that are secreted as water-soluble proteins; they undergo conformational changes in order to insert into or to translocate across cell membranes of their host. Cry proteins are specifically toxic to the insect orders Lepidoptera, Coleoptera, Hymenoptera, and Diptera as well as nematodes. The primary action of Cry toxins is to lyse mid-gut epithelial cells in the target insect by forming pores in the apical microvilli membrane of the cells. These proteins are highly specific to their target insect, are innocuous to humans, vertebrates, and plants, and are completely biodegradable. Therefore, Bt is a viable alternative for the control of insect pests in agriculture (Bravo et al., 2005) to provide an effective and specific built-in control for pests like the brinjal fruit and shoot borer. Bt-brinjal with the lepidopteron-specific cry1Ac gene from B. thuringiensis is tolerant to FSB, the major pest, which attacks the brinjal crop throughout its life cycle. It was created by the Maharashtra Hybrid Seed Company Ltd (Mahyco), a leading Indian seed company. Mahyco licensed and used the cry1Ac gene obtained from Monsanto. These Bt-brinjal plants have a built-in mechanism of protection against targeted pests as the protein would be endogenously produced and would not get washed away nor be destroyed by sunlight, unlike exogenously applied pesticides. The DNA construct of Bt-brinjal contains a gene sequence encoding the expression of an insecticidal protein in all parts of the brinjal plant throughout its life. The cry1Ac gene and two supporting genes, nptII and aad genes, are aligned to work in tandem to produce an insecticidal protein that is toxic to FSB. The cry1Ac gene is under the transcriptional control of the enhanced CaMV 35S promoter (P-E35S), which works as an on/off switch and regulates the spatial and temporal expression of the cry1Ac gene. This new DNA, called the “gene construct,” is illustrated in Figure 1. The gene construct is comprised of the following: (1) A cry1Ac gene isolated from B. thuringiensis sub-sp. kurstaki (B.t.k) strain HD73, introduced into the plant after suitable modification. It encodes for the insecticidal protein Cry1Ac. (2) The virus (CaMV) 35S promoter, which controls the expression of cry1Ac gene. (3) A nptII gene, which encodes the

FIGURE 1  Schematic showing the recombinant construct of Bt-brinjal (EE-1).

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selectable marker enzyme neomycin phosphotransferase II, which is used to identify transformed cells that contain the cry1Ac gene. It has no insecticidal properties and is derived from prokaryotic transposon Tn5. And finally, (4) An aad gene 3”(9)-O-aminnoglycoside adenyltransferase (AAD), which allows for the selection of bacteria containing the pMON 10518 plasmid on media containing spectinomycin or streptomycin. The aad gene is under the control of a bacterial promoter and is therefore not expressed in Bt-brinjal. The insertion of the gene into the Brinjal cell in young cotyledons was initially through an agrobacterium-mediated vector. A vector containing a cry1Ac gene, nptII gene, CaMV 35S promoter, and aad gene was used to transform young cotyledons of brinjal plants using agrobacterium followed by standard tissue culture techniques for plant regeneration. Mahyco improved the agrobacterium-mediated brinjal transformation based on a method of Fari et al. (1995). The transformed plants were regenerated and analyzed for the presence of the gene through southern blotting and progeny were also analyzed to identify elite lines segregating in a Mendelian fashion. The single copy elite event selected was named Elite Event-1 (EE-1), which could easily be identified and tracked by a polymerase chain reaction (PCR)-based event identification system (ID) in the greenhouse and field. The Tamil Nadu Agricultural University, Coimbatore, and the University of Agriculture, Dharwad (Karnataka), introgressed the EE-1 event into local brinjal varieties through plant breeding. The event EE-1 was backcrossed with the seven best-performing brinjal hybrids (MHB-4 Bt, MHB-9 Bt, MHB-10 Bt, MHB-11 Bt, MHB-39 Bt, MHB-80 Bt, and MHBJ-99 Bt) at the Mahyco Research and Life Sciences Center, Jalna, Maharashtra (Kumar et al., 1998; Mahyco, 2008). Under a public-private partnership (PPP) program supported by the Agricultural Biotechnology Support Program II (ABSP-II), Mahyco transferred the technology to public sector institutions in India, Bangladesh, and the Philippines (Report of the Expert Committee [EC-II] on Bt brinjal event EE-1) (www.moef.nic.in/sites/default/files/Report%20on%20Bt%20brinjal_2.pdf). In 2007–2008 the following Bt-brinjal hybrids/ varieties underwent confined field trials in India: (1) MHB 4 Bt, MHB 9 Bt, MHB 10 Bt, MHB 80 Bt, MHB 99 Bt, MHB 11Bt, MHB 39 Bt, and MHB 112 Bt, developed by M/s Mayhco; (2) Malapur local (S) Bt, Manjarigota Bt, Rabkavi local Bt, Kudachi local Bt, Udupigulla Bt, and GO112 Bt, developed by University of Agricultural Sciences, Dharwad; and (3) Co2-Bt, MDU1Bt, KKM1-Bt, and PLR1-Bt, developed by Tamil Nadu Agricultural University, Coimbatore. The timeline of events toward the development of Bt-brinjal by Mahyco is available in the ISAAA Brief 39 (Choudhary and Gaur, 2009).

FRUIT AND SHOOT BORER MANAGEMENT IN Bt-BRINJAL The constitutive cry1Ac gene expression in Bt-brinjal hybrids occurs in all parts of the plant and throughout its life cycle. During feeding the insect larvae ingest the endogenous inactive Cry1Ac protein along with the plant tissue. The schematic showing the mode of action of Cry toxins is illustrated in Figure 2. The alkaline pH (>9.5 in the insect gut) solubilizes protein, which is then acted on by the gut proteases, generating the toxic protein. This proteolysis cleaves an N-terminal peptide of 25–30 amino acids of Cry1Ac to yield a 60–70 kDa protease-resistant protein (Bravo et al., 2002, 2005, 2007) and approximately half of the remaining protein from the C-terminus. An additional cleavage in the N-terminal end of the toxin (helix α-1) facilitates the formation of a pre-pore oligomeric structure that is important for insertion into the membrane and for toxicity (Gómez et al., 2002; Rausell et al., 2004). The activated toxin travels across the peritrophic matrix and binds to specific receptors on the brush border membrane of the mid-gut epithelium columnar cells before inserting into the membrane. It binds to two different yet specific receptor proteins present in the insect gut membrane in a sequential manner. This results in the formation of a pre-pore oligomeric structure that is insertion competent (Bravo et al., 2004, 2007). Binding to the cadherin-like protein (CADR) receptor triggers the formation of toxin oligomers that bind to a glycosylphosphatidyl-inositol (GPI)-anchored aminopeptidase-N (APN). Toxin oligomers display high binding affinity toward N-acetylgalactosamine (GalNAc) residues on GPI-anchored NPN (Pardo-Lopez et al., 2006), resulting in a concentration of toxin oligomers on specific membrane regions called lipid rafts, where they insert into the membrane, forming an ion-pore that leads to osmotic cell death (Zhuang et al., 2002; Ning et al., 2010). Toxin insertion leads to the formation of lytic pores in microvilli of apical membranes (Aronson and Shai, 2001; Bravo et al., 2005). These events disrupt digestive processes such as loss of transmembrane potential, cell lysis, leakage of the mid-gut contents, and paralysis that, in turn, cause untimely insect death (Soberon et al., 2009). Alternatively, binding of toxin monomers to cadherin has been reported to activate intracellular signaling pathways that resulted in cell death by oncosis (Zhang et al., 2006). It is important to note that Cry protein, whether in the form of an insecticide spray or a Bt-crop, does not function on contact as most chemical insecticides do but rather acts as a mid-gut toxin. Therefore, either the Bt-plant tissue or spray formulation must be ingested by the target insect to be effective. Solubilization of the Cry protein occurs at alkaline pH, and the presence of specific receptors, particularly cadherin and APN, in the insect mid-gut epithelial cells must be present for the Cry protein to bind and oligomerize (Choudhary and Gaur, 2009). In the absence of both an alkaline milieu and specific receptors in all nonlepidopteran insects, birds, fish, animals, and human beings the protein remains inactive. The advantages of integrating the cry1Ac gene into the Bt-brinjal plant over spray formulations include an endogenous bioactive protein ubiquitously expressed

Genetically Modified Brinjal Chapter | 4  31

FIGURE 2  The mode of action of Cry1A protein in insect pests that feed on Bt-plants. Model figure reproduced with permission from Prof. Juan Luis Jurat-Fuentes. http://web.utk.edu/~jurat/.

in all tissues throughout the season for an effective and integrated FSB management, no wash off, unlike insecticide sprays during rain, degradation in sunlight, and most importantly a reduction in human exposure to the insecticide (Soberon et al., 2009).

FUNGAL-RESISTANT Dm-AMP1-AUBERGINE PLANTS Defensins are antimicrobial compounds capable of inhibiting the growth of phytopathogenic fungi by reducing hyphal elongation (Broekaert et al., 1995). Plant defensins are small peptides (45–54 amino acids) with a characteristic threedimensional folding pattern stabilized by disulfide-linked cysteines (Broekaert et al., 1995). The presence of defensins in seeds or plant tissue is a powerful built-in tool that enhances plant resistance against fungal pathogens. Expressing these in plant tissues as an alternate to chemical pesticides to control fungal disease would be safer and environmental friendly. Dm-AMP1 is an antimicrobial nonmorphogenic defensin (from Dahlia merckii), which slows down hyphal extension. DmAMP1-aubergine plants were produced by Agrobacterium tumefaciens-mediated genetic transformation on aubergine leaf explants to obtain the constitutive expression of the protein Dm-AMP1. The antibiotic resistance gene nptII was used as a selectable marker for genetic transformation to express the Dm-AMP1 defensin from D. merckii (Turrini et al., 2004). DmAMP1 protein was expressed in all tissues of the transgenic aubergines, which showed foliar resistance to Botrytis cinerea and was released into root exudates, which reduced the growth of the root pathogen Verticillium albo-atrum (Turrini et al., 2004). This expression did not interfere with recognition responses and symbiosis establishment by the arbuscular mycorrhizal fungus Glomus mosseae. These genetically modified plants have only been developed in the laboratory and no information on safety assessment is available.

DETECTION OF Bt-BRINJAL Detection methods to facilitate effective regulatory compliance for the identification of genetic traits, risk assessment, management, and postrelease monitoring also address consumer concerns, and resolution of legal disputes is essential. ­Randhawa et al. (2012) developed a multiplex PCR system that simultaneously amplifies the cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos), aminoglycoside adenyltransferase (aadA) marker gene, and a taxon-specific beta-fructosidase gene in event EE-1. The method performance was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. Ballari

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et al. (2013) developed an event-specific detection method based on the 3′ transgene insertion flanking sequence of the EE-1 brinjal event. The development of these event-specific qualitative simplex, multiplex, and real time-polymerase chain reaction methods for EE-1 brinjal is unique in that it includes two construct-specific and one event-specific sequence motif. Event-specific PCR based on the junction region between recombinant DNA insertion and adjacent host genomic DNA is the preferred method, as this event is identified as the integration locus between the exogenous DNA and host genome, which is the only unique signature of a transformation event (Xu et al., 2009).

CURRENT REGULATORY FRAMEWORK OF INDIA FOR RECOMBINANT DNA TECHNOLOGY India has very stringent rules and regulations for recombinant DNA technology. The Ministry of Environment and Forest (MoEF) and the Department of Biotechnology (DBT) coordinate the implementation of various rules and regulations, which are assigned to relevant ministries, state governments, and public sector institutions (Figure 3). The manufacture, import, use, research, and release of genetically modified organisms (GMOs) as well as products using GMOs are governed by the EPA Rules “Manufacture/Use/Import/Export and Storage of Hazardous Microorganisms, Genetically Engineered Organisms or Cells,” notified by the Ministry of Environment and Forests (MoEF now redesignated as Ministry of Environment, Forests and Climate Change [MoEF&CC]), Government of India, on December 5, 1989, under the Environment (Protection) Act 1986 (EPA). Commonly referred to as “Rules 1989,” they cover the areas of research, large-scale applications of GMOs, and products made therefrom throughout India. The regulatory agencies implement the Rules 1989 through the statutory committees (Table 2). The Ministry of Health and Family Welfare (MoHFW) in India is primarily responsible for ensuring the safety of food. The Food Safety and Standards Authority of India was established and the Food Safety and Standards Act was promulgated in 2006. Genetically modified foods have been included within the Food Safety Rules and Regulations (2011). Indian Council of Medical Research (ICMR), in its capacity as the scientific and advisory body to MoHFW, formulated “Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants” in 2008, taking into cognizance the International Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants (www.fao.org/fileadmin/user_upload/gmfp/docs/CAC.GL_45_2003.pdf). These guidelines were adopted by RCGM and Genetic Engineering Approval Committee (GEAC) in 2008 (http://icmr.nic.in/guide/­Guidelines%20for%20Genetically %20Engineered%20Plants.pdf). DBT has also prepared the following companion documents for these guidelines: (1) Acute Oral Safety Limit Study in Rats and Mice, (2) Sub-chronic Feeding Study in Rodents, (3) Protein Thermal Stability, and (4) Pepsin Digestibility Assay and Livestock Feeding Study (available at http://dbtbiosafety.nic.in). These guidelines were used by Mahyco to guide the safety assessment of Bt-brinjal for regulatory purposes.

FOOD SAFETY ASSESSMENT OF Bt-BRINJAL It was necessary for Mahyco to demonstrate that Bt brinjal is equivalent to native brinjal varieties in composition and agronomic performance and that it has no adverse effects on environment and human health following the guidelines described above prior to environmental release. M/s Mahyco carried out proximate analysis, amino acid composition, fatty acid,

FIGURE 3  Regulatory agencies involved in implementing Rules 1989 of EPA 1986 for transgenic production, release, and monitoring. The abbreviations used are the same as Table 2.

TABLE 2  The Role of the Statutory Committees Under Rules 1989 in the Context of Developing GMOs No

Name of the Committee

Function

1

Recombinant DNA advisory committee (RDAC)

l

2

Review committee on genetic manipulation (RCGM)

l

3

Genetic engineering appraisal committee (GEAC)

l

Institutional biosafety committees (IBSC)

l

5

State biotechnology coordination committees (SBCC)

l

6

District level committees (DLC)

l

Monitoring cum evaluation committee (MEC)

l

4

7

to review developments in biotechnology at national and international levels to recommend suitable and appropriate safety regulations for India in r-DNA research, use, and applications

l

to bring out manuals of guidelines specifying producers for regulatory processes on GMOs in research, use, and applications, including industry, with a view to ensuring environmental safety l to review all ongoing r-DNA projects involving high-risk category and controlled field experiments l to lay down producers for restriction or prohibition, production, sale, import, and use of GMOs both for research and applications l to permit experiments with category III risks and above with appropriate containment l to authorize imports of GMOs/transgenes for research purposes l to authorize field experiments of 20 acres in multilocations in one crop season with up to one acre at one site l to generate relevant data on transgenic materials in appropriate systems l to undertake visits of sites of experimental facilities periodically, where projects with biohazard potentials are being pursued and also at a time prior to the commencement of the activity to ensure that adequate safety measures are taken as per the guidelines to permit the use of GMOs and products thereof for commercial applications to adopt producers for restriction or prohibition, production, sale, import, and use of GMOs both for research and applications under EPA l to authorize large-scale production and release of GMOs and products thereof into the environment l to authorize agencies or persons to have powers to take punitive actions under the Environment Protection Act l

to note and to approve r-DNA work to ensure adherence of r-DNA safety guidelines of government l to prepare emergency plans according to guidelines l to recommend to RCGM about category III risk or above experiments and to seek RCGM’s approval; to inform DLC and SBCC as well as GEAC about the experiments wherever needed l to act as nodal points for interaction with statutory bodies l to ensure experimentation at designated locations, taking into account approved protocols l

to inspect, investigate, and take punitive action in case of violations of statutory provisions through the state Pollution Control Board or the Directorate of Health, etc. l to review periodically the safety and control measures in various institutions handling GMOs l to act as a nodal agency at the state level to assess the damage, if any, due to the release of GMOs and to take on site control measures l to coordinate activities related to GMOs in the state with the central ministries to monitor the safety regulations in installations to inspect, investigate, and report to the SBCC or the GEAC about compliance or noncompliance of r-DNA guidelines or violations under EPA l to act as a nodal agency at the district level to assess the damage, if any, due to the release of GMOs and to take on-site control measures l

to undertake field visits at the experimental site(s) approved by the RCGM for the purpose of collecting scientific information on the comparative agronomic advantages of transgenic plants l to prepare formats for collecting scientific information on transgenic crops in limited field trials based on the experimental design approved by RCGM l to advise, time to time, the RCGM on the risks and benefits from the use of the transgenic plants put into evaluation; to suggest new experimental design(s) to RCGM and also assist in collecting, consolidating, and analyzing the field data for evaluating the environmental risks emanating from transgenic plants l to apprise the RCGM on those transgenic crops which would be found to be environmentally safe and economically viable for recommending the same to GEAC for consideration for release into the environment l to undertake field visits at the experimental site(s) approved by GEAC for the purpose of collecting scientific information, based on specific requests made by the GEAC; to submit its report to GEAC directly with information to RCGM l to constitute, if necessary, monitoring team(s) to visit experimental sites for the purpose of collecting scientific information l to undertake occasionally specific jobs on the request of RCGM, which are not enumerated above

34  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

composition and analysis of minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, selenium, and zinc), vitamins (Vitamin C, Thiamin, Riboflavin, Niacin, Vitamin B6, Folic acid, Beta-carotene, Vitamin A, lycopene, Vitamin E, and Vitamin K) and lipids, estimation of alkaloid content, and the effect of cooking on various leaf and fruit collected from brinjal (S. melongena Linn.) hybrid MHB 80 Bt expressing Cry1Ac protein and the near isogenic line MHB–80 non-Bt counterpart hybrid to establish substantial equivalence between them. The two were comparable (http:// www.envfor.nic.in/divisions/csurv/geac/bt_brinjal.html). Bt-brinjal was subjected to toxicity, allergenicity, and feeding tests from 2003 to 2008 (Choudhary and Gaur, 2009; Kumar et al., 2011), which included acute, subchronic, and chronic testing. Mahyco used both public sector and private testing houses to evaluate Bt-brinjal. Acute oral toxicity in studies in Sprague Dawley rats, mucous membrane irritation tests in female rabbits, and primary skin irritation tests in rabbits were assessed by Intox, Pune, in 2003, whereas subchronic (90 days) feeding studies using New Zealand rabbits and goats were carried out by Advinus Therapeutic, Bangalore. CSIRIndian Institute of Chemical Technology, Hyderabad, prepared a chemical fingerprint of both the non-Bt and Bt-brinjal that included the alkaloids. Central Avian Research Institute, Izatnagar, Central Institute of Fisheries Education, and GB Pant University of Agriculture and Technology, Pantnagar, were the other public sector institutes that provided the safety assessment data. More precisely, Bt-brinjal was included in the feed and tested on fish, chickens, rabbits, rats, goats, and cows. No signs of toxicity were reported in the toxicity tests (Report of the Expert Committee [EC-II] on Bt brinjal event EE-1) (www.moef.nic.in/sites/default/files/Report%20on%20Bt%20brinjal_2.pdf). Bt brinjal was also subjected to tests for allergenicity by Rallis Bangalore. The Cry1Ac sequence was compared to known protein toxins in the PIR, EMBL, SwissProt, and GenBank protein databases. No biologically significant homologies were observed between the Cry1Ac protein sequence and the protein sequence of all known toxins in the current protein databases. Searches of protein sequence allergen databases also did not show any significant matches of the Cry1Ac protein to known allergens (Metcalfe et al., 1996). The foliage and fruit feeding studies were undertaken in goats and cows. Other studies included protein expression and quantification, substantial equivalence, and protein estimation in cooked food. Through all these studies the seed company concluded that the Bt-protein expressed by cry1Ac gene in brinjal causes no adverse effects when consumed by domestic and wild animals, nontarget organisms, and beneficial insects. From these studies Mahyo concluded that Bt protein is absolutely safe for human consumption. The detailed analysis of various studies on toxicity, allergenicity, nutritional composition, and feeding are available on the GEAC website at: http://www.envfor.nic.in/divisions/csurv/geac/bt_brinjal.html.

ENVIRONMENTAL RISK ASSESSMENT OF Bt-BRINJAL There is a concern that GMOs may pose ecological risks to the ecosystem (Icoz and Stotzky, 2008). The potential risk associated with the impact of transgenic crops on nontarget microorganisms and flora and fauna in the environment is a matter of concern. Gupta (2012) outlines the major environmental risks that are generally perceived to be associated with the commercialization of genetically modified crops. A series of open field trials are mandatory to meet regulatory requirements for agronomic performance after safety and efficacy have been established. The open field trials are divided into three categories: (1) confined field trials, (2) Biosafety Research Level-I or BRL-I trials (multilocation research trials), and (3) Biosafety Research Level-II or BRL-II trials (large-scale field trials). The greenhouse and confined field trials of Bt brinjal were undertaken during 2001–2003 at the Mahyco Research and Life Sciences Center, Jalna, Maharashtra. Environmental impact studies on pollen flow, germination, aggressiveness, and weediness, soil analysis covering the effects on soil microbiota and the presence of Cry1Ac protein in the soil, the effect on nontarget and beneficial insects, and baseline susceptibility were conducted by Mahyco and the Indian Institute of Vegetable Research, Varanasi, and different public sector institutions and accredited private laboratories from 2001 to 2008 (Choudhary and Gaur, 2009). These studies revealed that the maximum distance traveled by pollen was between 15–20 m and outcrossing varied from 1.46 to 2.7%. Though outcrossing was demonstrated in these studies, Samuels (2013) suggests that the use of appropriate pollinators—bumble bees as opposed to honey bees, which are less effective pollinators—would have favored considerably higher levels. The studies indicated that Bt-brinjal does not show any weediness characteristics and behaves in a similar fashion as any of its non-Bt counterparts. Random Amplification of Polymorphic DNA analysis has shown that Solanum incanum and Solanum viarum are closest to S. melongena. The varieties S. incanum and S. viarum occur infrequently in the wild in India but are hardly sympatric and panmictic with the cultivated varieties. When artificial hybrids were produced, the progeny were sterile, leaving no chances for gene flow among these related species. In the environment risk assessment tests only four spiny species were tested for interfertility with Bt-brinjal; only S. incanum L. (the nearest wild relative of brinjal) was found to be crossable (http://www.envfor.nic.in/divisions/csurv/geac/bt_brinjal.html). Samuels (2011) opines that the studies on the production of hybrid progeny were insufficient, although to date, six wild relative species and four cultivated

Genetically Modified Brinjal Chapter | 4  35

spiny Solanum species found in India are known to cross with brinjal to produce reproductively fit hybrids. Although no instances of natural interspecific hybridization with wild species were reported for cultivated brinjal, this does not preclude the possibility of this phenomenon. Therefore, careful consideration should be given to the study of the potential for crosstransference of genes between Bt-brinjal and its wild and weedy relatives and the possible implications. The soil impact study reported the half-life of cry1Ac protein to be 9.3–40 days, depending on the soil types. Insect larvae were used for the bioassays and enzyme-linked immunosorbent assay for the detection of Bt protein in the soil. No Bt protein was detectable in any of the samples tested. Singh et al. (2013) observed significant variation in the organic carbon between the non-Bt and Bt-brinjal, which they attributed to changes in root exudates’ quality and composition. Changes in the organic carbon, which also affected the actinomycetes population size and diversity associated with rhizospheric soils of both the crops, needed a long-term study. Field trials data indicated that the amount of insecticides used against FSB was reduced by 80%, translating into an overall insecticide reduction of 42% for the crop (Krishna and Qaim, 2008).

COMMERCIALIZATION OF Bt-BRINJAL Bt brinjal was evaluated for its efficacy and safety as per the protocols and procedures prescribed under the Rules 1989 and relevant biosafety guidelines. Following the recommendations of an expert committee that reviewed the reports submitted by Mahyco, the Appraisal GEAC of the Ministry of Environment and Forest on October 15, 2009, approved Bt-brinjal, the first GM crop for human consumption in India, for commercial use. This decision also sparked protests. Environmental and health concerns were cited that extend to other GM crops in the doubts against the technology as well as the interpretation of biosafety tests. Mr Jairam Ramesh, the then Minister of State for Environment and Forests, announced that a series of consultations with scientists, agricultural experts, farmers’ organizations, consumer groups, and nongovernmental organizations would be held in January and February, 2010, before a final decision. On February 9, 2010, the government of India decided to impose a moratorium on Bt-brinjal until “such times independent scientific studies establish, to the satisfaction of both the public and professionals, the safety of the product from the point of view of its long-term impact on human health and environment” (http://moef.nic.in/downloads/public-information/Annex_BT.pdf). On October 30, 2013, a historic decision of the government of Bangladesh made it the first country in the world to approve the commercial planting of four varieties of Bt-brinjal. Ms Matia Chowdhury, Union Minister of Agriculture, Bangladesh, supported and endorsed the commercial approval of Bt-brinjal as a step in the right direction. Bangladesh farmers have since then substantially cut down pesticide use to control FSB and significantly increased their marketable yield by mitigating economic losses. Bt gene is to be introduced in five other popular brinjal varieties, including Dohazari, Shingnath, Chaga, Islampuri, and Khatkatia to meet the growing requirement of Bt-brinjal seeds that will be planted in different brinjal growing areas (Choudhary et al., 2014). Field trials of GM Bt-brinjal, also known as Bt-talong, have officially ceased in the Philippines following a major ruling by the nation’s Court of Appeals (http://www.naturalnews.com/040878_GM_ eggplant_Bt_talong_Greenpeace. html##ixzz3UiOhWj9W). Herring (2014) opines that although India promotes agricultural biotechnology, the regulation of Bt crops has rested in a section of the state operating more on precautionary than developmental logic. Bt cotton was a success, yet Bt-brinjal encountered a moratorium on deployment despite approval by the regulatory scientific body designated to assess biosafety. It is now a little over five years since the moratorium on Bt-brinjal, and the unanswered question is whether or not Bt-brinjal will be approved for commercialization in India.

REFERENCES Aronson, A.I., Shai, Y., 2001. Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action. FEMS Microbiol. Lett. 195, 1–8. Bajaj, K.L., Kaur, G., Chadha, M.L., 1979. Glycoalkaloid content and other chemical constituents of the fruits of some egg plant (Solanum melongena L.) varieties. J. Plant Foods 3, 163–168. Bajaj, K.L., Kaur, G., Chadha, M.L., Singh, B.P., 1981. Polyphenol oxidase and other chemical constituents in fruits of eggplant (S. melongena L) varieties. Veg. Sci. 8, 37–44. Ballari, R.V., Martin, A., Gowda, L.R., 2013. Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR real-time PCR. J. Sci. Food Agric. 93, 340–347. Bravo, A., Gill, S.S., Soberón, M., 2005. Comprehensive Molecular Insect Science. Elsevier BV. Bacillus thuringiensis Mechanisms and Use; pp. 175–206. Bravo, A., Gill, S.S., Soberón, M., 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423–435.

36  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

Bravo, A., Gómez, I., Conde, J., Muñoz-Garay, C., Sánchez, J., Zhuang, M., Gill, S.S., Soberón, M., 2004. Oligomerization triggers differential binding of a pore-forming toxin to a different receptor leading to efficient interaction with membrane microdomains. Biochem. Biophys. Acta 1667, 38–46. Bravo, A., Sánchez, J., Kouskoura, T., Crickmore, N., 2002. N-terminal activation is an essential early step in the mechanism of action of the B. thuringiensis Cry1Ac insecticidal toxin. J. Biol. Chem. 277, 23985–23987. Broekaert, W., Terras, F.R.G., Cammue, B.P.A., Osborn, R.W., 1995. Plant defensins: novel antimicrobial peptides as components of the host defence system. Plant Physiol. 108, 1353–1358. Chen, N.C., 1997. Cultivation and seed production of eggplant. In: Training Workshop on Vegetable Cultivation and Seed Production Technology, Shanhua. Asian Vegetable Research and Development Centre (AVRDC), Taiwan. Choudhary, B., Gaur, K., 2009. The Development and Regulation of Bt Brinjal in India (Eggplant/Aubergine). ISAAA Brief No. 38. Ithaca, NY. Choudhary, B., Nasiruddin, K.M., Gaur, K., 2014. The Status of Commercialized Bt Brinjal in Bangladesh. ISAAA Brief No. 47. ISAAA, Ithaca, NY. Doijode, S.D., 2001. Seed Storage of Horticultural Crops. Haworth Press. Expert Committee (EC-II) report on Bt-Brinjal, 2009. www.moef.nic.in/sites/default/files/Report%20on%20Bt%20brinjal_2.pdf. Fári, M., Nagy, I., Csányi, M., Mitykó, J., 1995. Agrobacterium mediated genetic transformation and plant regeneration via organogenesis and somatic embryogenesis from cotyledon leaves in eggplant (Solanum melongena L. cv. ‘Kecskeméti lila’). Plant Cell. Rep. 82–86. Gómez, I., Sánchez, J., Miranda, R., Bravo, A., Soberón, M., 2002. Cadherin-like receptor binding facilitates proteolytic cleavage of helix α-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin. FEBS Lett. 513, 242–246. Gupta, P.K., 2012. Regulating the dual-use and dual-impact life science research: influenza virus versus biotech crops. Curr. Sci. 103, 995–1002. Harish Babu, B.N., Venkatesh, Y.P., 2009. Clinico-immunological analysis of eggplant (Solanum melongena) allergy indicates preponderance of allergens in the peel. World Allergy Organ. J. 2 (9), 192–200. Herbst, S.T., 2001. The New Food Lover’s Companion: Comprehensive Definitions of Nearly 6,000 Food, Drink, and Culinary Terms. Barron’s Cooking Guide. Barron’s Educational Series, Hauppauge, NY. Herring, R.J., 2014. On risk and regulation: Bt crops in India. GM Crops Food 5, 204–209. Icoz, I., Stotzky, G., 2008. Fate and effects of insect-resistant Bt crops in soil ecosystems. Soil Biol. Biochem. 40, 559–586. Kabir, K.H., Rahman, M.A., Ahmed, M.S., Prodhan, M.D.H., Akon, M.W., 2008. Determination of residue of diazinon and carbosulfan in brinjal and quinalphos in yard long bean under supervised field trial Bangladesh. J. Agril. Res. 33, 503–513. Kaur, R., 2008. Assessment of Genetic Damage in Workers Occupationally Exposed to Pesticides in Various Districts of Punjab. Department of Human Biology, Punjabi University, Patiala. Khan, D.A., Ahad, K., Ansari, W.M., Khan, H., 2013. Pesticide exposure and endocrine dysfunction in the cotton crop agricultural workers of southern Punjab, Pakistan. Asia Pac. J. Public Health 25, 181–191. Krishna, V.V., Qaim, M., 2008. Potential impacts of Bt eggplant on economic surplus and farmers’ health in India. Agric. Econ. 38, 167e180. Kumar, P.A., Mandaokar, A.D., Sharma, R.P., 1998. Genetic engineering of eggplant (Solanum melongena L). AgBiotech News Inf. 10, 329–331. Kumar, S., Misra, A., Verma, A.K., Roy, R., Tripathi, A., Ansari, K.M., Das, M., Dwivedi, P.D., 2011. Bt brinjal in India A long way to go. GM Crops 2, 92–98. Mahyco, 2008. Development of Fruit and Shoot Borer Tolerant Brinjal. Maharashtra Hybrid. Mathur, V., Bhatnagar, P., Sharma, R.G., Acharya, V., Sexana, R., 2002. Breast cancer incidence and exposure to pesticides among women originating from Jaipur. Environ. Int. 28, 331–336. Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., Fuchs, R.L., 1996. Assessment of the allergenic potentials of foods derived from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36 (S), S165–S186. Ning, C., Wu, K., Liu, C., Gao, Y., Jurat-Fuentes, J.L., Gao, X., 2010. Characterization of a Cry1Ac toxin-binding alkaline phosphatase in the midgut from Helicoverpa armigera (Hubner) larvae. J. Insect Physiol. 56, 666–672. Pardo-Lopez, L., Gomez, I., Rausell, C., Sanchez, J., Soberon, M., 2006. Structural changes of the Cry1Ac oligomeric pre-pore from Bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion. Biochemistry 45, 10329–10336. Randhawa, G.J., Sharma, R., Singh, M., 2012. Qualitative and event-specific real-time PCR detection methods for Bt brinjal event EE-1. J. AOAC Int. 95, 1733–1739. Rausell, C., Pardo-López, L., Sánchez, J., Muñoz-Garay, C., Morera, C., Soberón, M., Bravo, A., 2004. Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane inserted pore channel. J. Biol. Chem. 279, 55168–55175. Samuels, J., 2011. Bt brinjal, wild relatives and biodiversity. Curr. Sci. 100, 603–604. Samuels, J., 2013. Bt-brinjal: a risk assessment worth taking? Curr. Sci. 104, 571–572. Sharma, V.K., Kaur, S., 1990. Contact sensitization by pesticides in farmers. Contact Dermatitis 23, 77–80. Shetty, S.M., Chandrashekar, A., Venkatesh, Y.P., 2011. Eggplant polyphenol oxidase multigene family: cloning, phylogeny, expression analyses and immunolocalization in response to wounding. Phytochemistry 72, 2275–2287. Shukla, V., Naik, L.B., 1993. Agro-techniques of solanaceous vegetables. In: Chadha, K.L., Kalloo, G. (Eds.), Advances in Horticulture. Vegetable Crops, Part 1, vol. 5. Malhotra Pub. House, New Delhi, p. 365. Singh, A.K., Singh, M., Dubey, S.K., 2013. Changes in Actinomycetes community structure under the influence of Bt transgenic brinjal crop in a tropical agroecosystem. BMC Microbiol. 13, 122. Soberón, M., Gill, S.S., Bravo, A., 2009. Signaling versus punching hole: how do Bacillus thuringiensis toxins kill insect midgut cells? Cell. Mol. Life Sci. 66, 1337–1349. Tsao, Lo, 2006. In: Hui, Y. (Ed.), Handbook of Food Science, Technology, and Engineering. Taylor & Francis, Boca Raton.

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Turrini, A., Sbrana, C., Pitto, L., Ruffini Castiglione, M., Giorgetti, L., Briganti, R., Bracci, T., Evangelista, M., Nuti1, M.P., Giovannetti, M., 2004. The antifungal Dm-AMP1 protein from Dahlia merckii expressed in Solanum melongena is released in root exudates and differentially affects pathogenic fungi and mycorrhizal symbiosis. New. Phytol. 163, 393–403. Xu, W., Yuan, Y., Luo, Y., Bai, W., Zhang, C., Huang, K., 2009. Event-specific detection of stacked genetically modified maize Bt11×GA21 by UP-M-PCR and real-time PCR. J. Agric. Food Chem. 57, 395–402. Zhang, X., Candas, M., Griko, N.B., Taussig, R., Bulla Jr., L.A., 2006. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proc Natl. Acad. Sci. U.S.A. 103, 9897–9902. Zhuang, M., Oltean, D.I., Gomez, I., Pullikuth, A.K., Soberon, M., 2002. Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation. J. Biol. Chem. 277, 13863–13872.

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Chapter 5

Nutritional Assessment of Genetically Modified Crops Using Animal Models R.D. Ekmay, S. Papineni, R.A. Herman Dow AgroSciences LLC., Indianapolis, IN, USA

CROP COMPOSITION, NUTRITIONAL CONTEXT, AND THE SUITABILITY OF ANIMAL STUDIES The majority of genetically modified (transgenic) crops contain genetic modifications intended to improve yield through crop protection (Fernandez-Cornejo et al., 2014). This is accomplished via the introduction of genes that confer herbicide tolerance and/or insect protection. The mechanism of crop improvement is usually dependent on the expression of a critical protein that produces a desired effect, for example, the expression of the insecticidal Bacillus thuringiensis (Bt) protein by the plant. Therefore, the phenotype of the transgenic crop is pest resistance or herbicide tolerance accomplished via the expression of the protein of interest. As biotechnologies advance, nutritional assessments will not only need to consider the expression of novel proteins but will also need to consider the use of RNAi as a mode of action to confer insect protection or nutritional improvement. It must be shown whether the introduced gene significantly alters the nutritive value of the crop under its intended use. Crops with nutritional improvements, such as a healthier fatty acid profile or improved vitamin content, are in development (Fernandez-Cornejo et al., 2014). If a nutritional change is intended, then it must be shown that the genetically modified (or GM) crop provides the intended nutritional benefit. Compositional analysis is at the heart of nutritional assessments to investigate substantial equivalence (Flachowsky and Wenk, 2010). A comparative approach is used to establish substantial equivalence for safety and nutritional assessments. A non-transgenic, near-isogenic line (isoline), that is, the same genetic background but lacking the transgenic insert, is the preferred comparator. This is done under the assumption that the non-transgenic comparator has a history of safe consumption. Testing is not performed to ensure complete safety since crops may inherently contain toxins or antinutritional factors. Instead, the comparative approach only ensures that the new genetically modified variety is “as safe and nutritious as” non-transgenic varieties. Crop varieties arising from conventional breeding techniques, that is, non-transgenic, have a wide range of composition (Table 1). Yet, the lack of regulation would appear to indicate that these compositional differences do not cause concern. Geographical differences, and thus differences in soil type, rain amount, pest infestation, etc., in which the cultivar is grown, will also result in differences in crop composition. The wide range in composition for food crops and their history of safe use calls into question the general assumption that alterations to composition are inherently disadvantageous (Schnell et al., 2014). As differences in crop composition are assessed, it is sometimes expected that compositional differences exist due to geographic or biotic stress differences, and these should be considered normal biological variation (Herman and Price, 2013). Indeed, a statistical difference should not necessarily be interpreted as biologically meaningful or adverse. For example, the maximum reported iron content of non-transgenic corn is 45 ppm (or 0.0045%), with the lowest being ∼9 ppm (or 0.0009%) (Table 2). Assuming a worst-case scenario where corn represents 100% of the diet, an average pregnant woman would need to consume between 600 and 3000 kg of corn to meet her daily iron requirements (27 g/day): a physiologically impossible amount. Thus, substantial variation in the iron content of corn is inconsequential in a real-life scenario. However, if compositional differences are determined to exist, that is, if substantial equivalency cannot be demonstrated, or if there is indication that there may be adverse unintended effects, then examination of whole food or feed, for example, animal studies, may be warranted if the differences might reasonably alter the nutrition or safety of the resulting food in a biologically meaningful manner. Animal models have long been used for nutrition research and are the basis for the majority of our knowledge in “nutrient–nutrient interactions, bioavailability of nutrients and nutrient precursors, and tolerance levels for excessive intakes of nutrients” (Baker, 2008). Work in pigs and chickens led to the identification of an essential nutrient present in Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00005-1 Copyright © 2016 Elsevier Inc. All rights reserved.

39

40  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

TABLE 1  Range in the Composition of Conventionally Bred Maize and Soybean Maize

Soybean

Min-Max

Min-Max

Moisture

6.1–40.5

4.7–34.4

Crude protein

5.67–15.5

27.1–42.6

Crude fat

1.47–5.34

7.18–21.3

Carbohydrates

49.6–83.0

22.0–46.1

Crop Composition, Conventionally Bred Proximates (%)

Crude fiber

3.51–11.00

Amino acids (mg/g FW) Alanine

4.03–12.8

12.1–17.2

Arginine

1.09–5.87

19.1–31.3

Aspartic acid

3.07–11.10

30.2–46.6

Cystine/cysteine

1.12–4.61

3.43–7.29

Glutamic acid

8.85–32.5

47.8–74.0

Glycine

1.69–4.95

11.9–17.4

Histidine

1.26–3.98

7.05–10.8

Isoleucine

1.64–6.36

12.1–19.0

Leucine

5.95–22.9

21.5–31.5

Lysine

1.58–5.88

17.3–26.6

Methionine

1.07–4.2

3.76–6.14

Phenylalanine

2.24–8.55

13.6–20.5

Proline

4.24–15.0

13.1–21.2

Serine

2.17–7.07

9.81–22.8

Threonine

2.07–6.02

10.1–15.1

Tryptophan

0.242–1.86

2.86–4.7

Tyrosine

0.94–5.90

8.7–14.3

Valine

2.44–7.86

12.4–20.5

Caprylic acid

0.133–0.34

0.148–0.148

Lauric acid

0.687–0.687

0.082–0.132

Myristic acid

0.14–0.284

0.071–0.238

Myristoleic acid

ND

0.121–0.125

Pentadecanoic acid

ND

ND

Palmitic acid

7.94–20.71

9.55–15.77

Palmitoleic acid

0.095–0.447

0.086–0.194

Heptadecanoic acid

0.078–0.111

0.085–0.146

Heptadecenoic acid

ND

0.073–0.087

Fatty acids (% total FA)

Nutritional Assessment of Genetically Modified Crops Using Animal Models Chapter | 5  41

TABLE 1  Range in the Composition of Conventionally Bred Maize and Soybean—cont’d Maize

Soybean

Crop Composition, Conventionally Bred

Min-Max

Min-Max

Stearic acid

1.02–3.40

2.70–5.88

Oleic acid

17.4–40.2

14.3–32.2

Linoleic acid

36.2–66.5

42.3–58.8

Gamma linoleic acid

0.387–0.568

ND

Linolenic acid

0.57–2.25

3.00–12.52

Arachidic acid

0.279–0.965

0.163–0.482

Eicosenoic acid

0.17–1.917

0.140–0.350

Eicosadienoic acid

0.116–0.533

0.077–0.245

Eicosatrienoic acid

0.275–0.275

ND

Arachidonic acid

0.465–0.465

ND

Behenic acid

0.110–0.349

0.277–0.595

Erucic acid

0.14–0.17

ND

Lignoceric acid

0.14–0.23

ND

Beta-carotene

0.017–3.103

ND

Total tocopherols

0.585–12.236

ND

Thiamin

0.110–3.556

0.090–0.230

Vitamins (mg/100 g FW)

Riboflavin

0.045–0.216

0.170–0.290

Vitamin B3

0.936–4.290

ND

Pyridoxine

0.334–1.02

ND

Folic acid

0.0127–0.1322

0.2040–0.4290

Vitamin E

0.0013–0.0605

0.0014–0.0559

Calcium

10.9–169.0

1000.0–2770.0

Chloride

320–800

ND

Copper

0.64–14.78

ND

Iron

9.21–44.7

49.1–97.8

Magnesium

534.6–1726.6

1900–2850

Manganese

1.43–13.0

ND

Phosphorus

1323.0–4743.7

4510–8520

Potassium

1629.0–5366.7

16700–21100

Selenium

0.05–0.69

ND

Sulfur

454.0–1170.0

ND

Zinc

5.6–34.3

ND

Minerals (ppm FW)

ILSI (2010). International Life Institute Crop Composition Database (2010), Version 4.2, www.cropcomposition.org (accessed 09.07.13.).

Fatty acids Vitamin E (as α-tocopherol) Vitamin K1

Amino acids

Fatty acids

Calciume

Phosphoruse

Phytic acid

Minerals

Vitamins

Amino acids

Fatty acids

Phytic acid

Raffinose Lectins Isoflavones

Furfural

Ferulic acid

p-coumaric acid

Fatty acids

ADFe

Lectins

Trypsin inhibitor

Phytic acid

Raffinose

Stachyose

Phosphorus

Calcium

Fatty acids

Dihydrosterculic acid

Sterculic acid

Malvalic acid

Gossypol (total and free)

Vitamin E (as α-tocopherol)

NDFe

Amino acids

Proximatesb

Food

cRefers

bProximates

Malvalic acid

Dihydrosterculic acid

Sterculic acid

Gossypol (total and free)

Phosphorus

Calcium

Fatty acids

Amino acids

Proximatesb

Feed

Cotton

Proximatesb,e

analytes refer to whole seed or grain unless otherwise indicated. include dry matter, ash, crude protein, crude fat, crude fiber, and carbohydrates. to whole seed. For soybean meal, remove fatty acids and lectins. dRefers to oil, as rapeseed grain or meal is not consumed by the human population in appreciable quantities. eOnly these analytes should be considered for forage and silage.

aAll

Stachyose

Raffinose

Phytic acid

Amino acids

Proximatesb

Proximatesb,e

Food

Feed

Proximatesb

Feedc

Soybean

Food

Maize

Total sterols

Vitamin E (as α-tocopherol)

Phytic acid

Sinapine

Tannins

Glucosinolates

Minerals

Fatty acids (including erucic acid)

Amino acids

Proximatesb

Feed

Rapeseed

Fatty acids (including erucic acid)

Foodd

TABLE 2  Suggested Compositional Analytes by the OECD (2001a,b, 2002, 2004) for the Determination of Substantial Equivalency in Food and Feed of Select Cropsa

42  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

Nutritional Assessment of Genetically Modified Crops Using Animal Models Chapter | 5  43

animal proteins and fermentation products but not in plant proteins, later identified as vitamin B-12. Beriberi in chicks fed a polished rice diet led to the discovery of thiamin. Many other vitamin deficiency diseases such as scurvy, pellagra, rickets, night blindness, hemorrhagic disease, and anemia were also characterized and understood with the help of animal models, as were many mineral, vitamin, and amino acid interrelationships. One can view whole foods as vehicles for delivery of their component parts. The requirement of an animal for energy, amino acids, vitamins, and minerals for optimal growth and health is often well-defined. No crop’s composition fully matches the nutrient requirement of an animal. Some crops may provide some nutrients but are often limiting in others, and the pairing of ingredients may provide the desired dietary balance. Crops, therefore, provide a piece of the puzzle for matching a diet with the nutrient requirements of the animal. For this reason, changes to composition should be viewed in this context. A true nutritional assessment determines how well a dietary ingredient fulfils nutrient requirements, which nutrients are limiting, and how readily any limitations are correctable. In many instances, the presence of a few constituents within a food drives its nutritional value. For example, soy is primarily valued for its protein and amino acid content in animal nutrition, while maize is a highly digestible energy source. More relevant to risk assessments is an animal model’s ability to detect negative effects. A prime example of this is the discovery of the antinutritive properties of trypsin inhibitors in raw soybeans. In 1917, Osborne and Mendel observed that unless soybeans were cooked for several hours, they would not support normal growth in rats. The identification of a heat-labile protein in soybeans that inhibits the proteolytic activity of trypsin, and experiments showing that the nutritive quality of soybeans improved with heating, led to the conclusion that trypsin inhibitors were the main cause of depressed growth in experimental animals (Bowman, 1944; Ham and Sandstedt, 1944; Westfall and Hague, 1948). Poulsen et al. (2007) investigated whether a 90-day rodent feeding study was capable of detecting kidney bean PHA-E lectin toxicity from transgenic rice expressing PHA-lectin. PHA-E is the toxic component in uncooked kidney beans. The authors showed significant differences in small intestine, pancreas, stomach weight, and plasma biochemistry in rats fed genetically modified rice, confirming the sensitivity of the 90-day rodent study to detect food proteins with known adverse effects.

Processed Products Crops intended for human and animal consumption typically undergo some type of processing before reaching the food chain. This is true regardless of any genetic modification. Apples may become apple juice and apple sauce, wheat becomes flour, etc. This is especially true of soybeans and is done for a variety of reasons, including safety. Soybeans contain the antinutritive compounds trypsin inhibitor, lectin, phytic acid, raffinose, and stachyose. These are naturally occurring compounds that are unrelated to the transgenesis process but nevertheless interfere with the nutritional value of soybeans. Soybeans must, therefore, be heavily processed (heat, mechanical) to inactivate or reduce these antinutritive compounds to levels suitable for consumption. The result is a product that is suitable for human and animal consumption. The composition of processed products, therefore, does not necessarily resemble the nutrient composition of the original crop, as they often only represent a specific fraction of the original grain. For example, soybean meal is the most commonly used soybean processed product in animal feed. Soybean meal is the by-product of oil extraction and toasting, rendering a product that contains only 5% of the original oil content, increased protein and amino acids, and lower levels of antinutritive compounds. Furthermore, the final consumer products may also be enriched or fortified. Nevertheless, a minimally processed raw agricultural commodity still has a place in meeting nutrient requirements and will reach the marketplace. One example is dried, cracked whole corn, which retains much of its original nutritional profile. Whole soybeans can also be incorporated into the diet of monogastric animals (humans included), provided they have been properly heated to ensure the destruction of the antinutritive compounds. Ruminants have a greater tolerance for the antinutritional compounds present in soybeans, thus, whole raw soybeans can be fed to mature ruminants, provided that strict feeding guidelines or regimens are followed.

HISTORY OF ANIMAL STUDIES FOR NUTRITIONAL ASSESSMENT OF GENETICALLY MODIFIED CROPS Animal studies investigating the effects of genetically modified crops on nutrition and animal health have long been conducted by both industry and academic scientists and the results extensively scrutinized by academic and government organizations (Aumaitre et al., 2002; Flachowsky et al., 2005, 2007; Flachowsky and Wenk, 2010; EFSA, 2008a; Van Eenennaam and Young, 2014). A tremendous amount of work in this area has been produced by Flachowsky and Associates through the Institute of Animal Nutrition at the Federal Agricultural Research Center in Germany. The history of animal studies and the

44  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

key nutritional and safety considerations for future studies with genetically modified plants were reviewed in some detail by Aumaitre et al. (2002), Flachowsky et al. (2005), Flachowsky and Wenk (2010), and ultimately in Flachowsky (2013). Assessments by Flachowsky et al. (2007) concluded that after 16 internal animal studies investigating the nutritional value of genetically modified crops for broiler chickens, laying hens, growing and laying quails, growing and finishing pigs, growing bulls, and dairy cows, results were in agreement with more than 100 animal studies showing their safety and nutritional equivalency. The assessments of Flachowsky et al. (2007) also included a 10-generation experiment with quail and a 4-generation experiment with laying hens, with no effects being observed on reproduction. Comprehensive summaries, reviews, and meta-analysis by CAST (2006), Clark and Ipharraguerre (2001), EFSA (2008a), and Van Eenennaam and Young (2014) have concluded that there is no negative impact of commercialized genetically modified crops on animal nutrition, safety, or health. In 2008, the European Food Safety Administration (EFSA) concluded that based on studies in poultry, swine, ruminants, fish, and rabbits, if transgenic lines and the near isogenic line are compositionally comparable, then “nutritional equivalence…can be assumed. Further animal feeding studies will add little to their nutritional assessment, and that this is equally applicable to plants that have been genetically modified through the insertion of one or more genes.” Since the EFSA’s 2008 publication, further individual work has been published supporting the same conclusion (Steinke et al., 2010; Guertler et al., 2010, 2012; Buzoianu et al., 2012a,b,c,d, 2013a,b; Walsh et al., 2011, 2012a,b, 2013). More convincingly, in a meta-analysis of studies spanning over 20 years and 100 billion animals, Van Eenennaam and Young (2014) concluded that genetically modified feed “did not reveal unfavorable or perturbed trends in livestock health and productivity.” Indeed, based on the author’s calculations, only 0.33% and 0.1% of broilers and hogs in 2011, respectively, were reared organically. Thus, it can be credibly assumed that the remaining population has been exposed to genetically modified feed. Van Eenennaam and Young (2014) estimated that from 2000 to 2011, over 100 million animals consumed some level of genetically modified feed. They hypothesized that should genetically modified feed be detrimental, animal performance and health would be negatively impacted compared with performance prior to the introduction of genetically modified crops. Furthermore, this type of assessment encompasses a wide array of exposure times, from short-term (broiler) to long-term (dairy cow).

REGULATORY ASSESSMENTS Prior to commercialization, genetically modified crops must undergo safety and nutritional assessments as part of required precommercialization evaluations. The information required for registration varies by country but almost always includes plant history (history of safe use), phenotype, chemical composition, details of the transformation process (source of the inserted gene(s), DNA construct, and consequences of DNA insertion), details of the newly expressed proteins (identity, mode of action, toxicity, and allergenicity), and agronomic, phenotypic, and nutritional characteristics of the genetically modified plant (EFSA, EPA, etc.). Herman and Price (2013) indicate that over the past 20 years, all of the 148 transgenic events that were evaluated by the United States Department of Agriculture (USDA) and all 189 submissions that were evaluated by Japanese regulators were found to be substantially equivalent to non-transgenic counterparts. Canadian regulators outlined the molecular basis supporting this expectation (Schnell et al., 2014). Most regulatory agencies reference international guidelines for nutritional assessments. Some regulatory agencies have stated that the use of animal studies to evaluate the safety of GM crops should not generally be required and that they offer little scientific value (Australia/New Zealand, United States, and the EFSA). Nevertheless, a nutritional assessment involving an animal model is required by some regulatory authorities (e.g., European Commission). Unless specific country requirements dictate otherwise, internationally recognized standards and procedures such as Codex Alimentarius (2008, 2011), the Organisation for Economic Co-Operation and Development (OECD), or the International Life Science Institute (ILSI) typically drive the conduct and design of nutritional assessments (Table 2). The European Union (EU) (EFSA, 2011a,b), Australia/New Zealand (FSANZ, 2007), and to an extent Brazil (CTNBio) and Japan (Regarding Establishment of Assessment Standards of Feed additives, 1992) have recommendations or clarifications beyond what is found in OECD or ILSI guidelines. Flachowsky and Wenk (2010) detailed many of the experimental procedures for nutritional assessments of genetically modified crops that are described by ILSI (2003) and EFSA (2008a, 2011a), further elaborated below. In all cases, it was recommended to perform animal studies only when existing information (crop composition, agronomic, phenotypic, or molecular results) provides sufficient scientific justification and a testable hypothesis. If compositional equivalence cannot be established, the nutritional and biological impact of the observed changes must be assessed. Nutritional hazards can come in several forms: (1) the introduction of the transgene leads to biologically significant compositional changes that could cause an adverse effect, (2) the transgene alters metabolism and nutrient utilization/digestibility, or (3) the transgene introduces a toxicant or antinutrient. In most cases, the effects of crop composition changes on animal health and performance can be predicted. For animal production, nutrient requirements and the impact

Nutritional Assessment of Genetically Modified Crops Using Animal Models Chapter | 5  45

of deviations away from the requirements are frequently well-characterized. As previously discussed, animal nutritionists frequently see variation in the composition of sourced ingredients (whether transgenic or non-transgenic) and are able to identify these differences and correct them. The possibility of unintended effects, whether in the form of altered metabolism, digestibility, or toxicity, is the main driver for the conduct of animal studies to support a safety assessment. From these studies, one can ascertain nutritional quality, that is, whether nutrients function as expected, and can provide a reliable indication of the presence of any unintended effects. Some have questioned the sensitivity of these studies in detecting hazards in genetically modified crops. The reasons that have been cited include longevity of the study, low exposure rates, and relevance of test species. However, each of these concerns can be alleviated by the selection of the proper animal model. Indeed, key differences in nutritional physiology among species would dictate that the selection of the appropriate species is critical in a nutritional assessment. For example, adipose tissue is the site of fatty acid synthesis in pigs and ruminants, whereas synthesis occurs in the liver in chicks; β-carotene conversion to vitamin A is more efficient in rats than in pigs; chicks, rats, and mice respond more rapidly to vitamin and mineral deficiencies than pigs or humans; and so on. This highlights the importance of hypotheses-driven investigations. Another key question that often drives the need for animal models is the impact of the transgenic protein on digestion (Hammond and Jez, 2011). It should be kept in mind that the transgenic protein is often denatured and inactivated prior to consumption, and therefore, digested as any other protein. In addition, the results of simulated gastric and intestinal fluid digestions, that is, could the protein reach potential targets, should also be considered. Finally, consideration should be made on whether the intended purpose of the crop is for food (for human consumption) versus feed (for animal consumption). Nevertheless, nutritional assessments using animal models lack the power to be hypothesis generating; specific hypotheses should drive the conduct and design of animal studies instead (Herman and Ekmay, 2014).

42-Day Broiler Study Doubling of starting body weight is generally considered a minimum requirement for animal growth bioassays. The modern broiler chick, such as the modern Cobb-Vantress or Aviagen strains, experiences tremendous growth during the first few weeks of life. During the first six weeks of its life, a broiler chicken undergoes a 65-fold increase in body weight. In comparison, a human neonate will undergo a 0.5-fold increase during the same time frame. In other terms, it takes a broiler chicken 3 days to double in body weight, but it would take 5 years for a six-year-old child to double in body weight. This rapid weight increase results in a high demand for nutrients in the broiler chicken and is thus sensitive to deficiencies in nutrient availability or the presence of antinutrients. The initial 6 weeks of a broiler’s life also represent the most sensitive period in a bird’s life. For example, the tolerable upper limit for aflatoxin is set to 20 ppb for immature animals but up to 100 ppb for mature birds (USDA, 2011). The 42-day broiler study serves as the primary animal model to assess nutritional value in feed (animal consumption) but also serves as a secondary assessment for food (human consumption). There is consensus that the period to be evaluated should represent the point under which the greatest growth occurs and is likely to reveal nutritional inadequacies. The first 6 weeks of a broiler chicken’s life represent the period of greatest growth; this is also the commercially relevant life-stage. The test crop, for example, soy, corn, etc., is incorporated into a balanced diet at the highest rate that does not cause a nutritional imbalance. This is a critical point since many feedstuffs inherently contain antinutritive properties, and it is important to differentiate normal, expected dietary effects from those that may pose new concerns. These antinutritive properties are naturally occurring compounds that are unrelated to the transgenesis process. As previously stated, soybeans must be heavily processed (heat, mechanical) to inactivate or reduce these antinutritive compounds to levels suitable for consumption. A by-product of the heating process is also the denaturation of the transgenic protein into an inactive form; thus, destroying any functional capacity (Hammond and Jez, 2011). Even so, processing does not remove all antinutritive properties and there is an upper limit in which soy can be incorporated into a diet. The upper limit is not dictated by antinutritive compounds alone but must also reflect the correct proportion of nutrients needed by the animal. Corn can theoretically be 100% of the diet without fear of toxicity concerns, but inclusion at this proportion would result in deficiencies of several critical nutrients. The OECD (2009) has suggested incorporation rates for chemical residue studies; however, these recommendations are not always reflective of current practices (FAO, 2006). The experimental design for a genetically modified crop that confers agronomic benefit (believed to be nutritionally neutral) is a completely randomized design or a randomized complete block design where the variable (fixed effect) is the test crop, that is, soybean, corn, etc. that either contains the genetic modification or is a non-transgenic, near-isogenic comparator (isoline). The isoline should be genetically similar to the crop containing the event. Sex should also factor into the statistical model. Additional treatment groups may be added that contain commercial reference varieties to aid in the comparison and interpretation of results. In each case, the incorporation rate of the test crop should be held constant

46  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

across treatments. The experimental unit should be a pen containing n birds of one sex. According to some guidelines, the remaining feed ingredients should also be held constant (ILSI, 2003). Under this approach, the only ingredient variable is the source of the test crop. Other guidelines state that the goal in diet formulations should be in creating nutritionally and compositionally comparable treatments (FSANZ, 2007; EFSA, 2008a). Under the former approach, if there is a compositional difference among the test crops, direct replacement will result in compositional differences between the dietary treatments. There will then be expected performance differences and no additional information that could not otherwise be determined through compositional analysis. As such, this approach convolutes the interpretation between expected and unintended effects. It would appear that these two objectives can, in certain cases, be in direct conflict with each other. The alternative would be to equalize the nutrient profile, and not necessarily the ingredient list, of the treatments with only the test crop being held constant (FSANZ, 2007; EFSA, 2008a). The EFSA (2008a) suggests that, “When the diet is balanced, potential ‘noise’ arising from the difference in composition of one or several nutrients should be removed, which is a prerequisite for the detection of unintended effects.” The disadvantage under this scenario is that the differing ingredient list necessary to achieve equivalent nutrient profiles introduces a new source of variation. Guidance suggests that diets be formulated to meet minimum nutrient requirements such as those dictated by the NRC (1994). Modern broiler strains tend to have higher nutrient requirements than what is suggested by the National Research Council (NRC), and caution should be taken that deficiencies are not unintentionally created (Cobb-Vantress, 2013; Aviagen, 2013). On the other side of the argument, Food Standards Australia New Zealand (FSANZ) has argued that diets formulated beyond the animal’s requirements may mask nutritional deficiencies of the test crop and, thus, a de minimis diet should be used (FSANZ, 2007). The experimental design for a genetically modified crop that confers a nutritional benefit is similar to that of agronomic traits. The key difference is the inclusion of two treatments for each of the non-transgenic varieties: one that is supplemented to match the nutritional profile of the genetically modified crop and one that is not supplemented. The design of the experiment is meant to demonstrate the claimed benefit. This methodology is also used to determine nutrient digestibility or bioavailability. There has not been advocacy for direct measurement of digestibility through precision-fed animal studies or the use of inert markers and the collection of ileal or fecal digesta to support the safety assessment of GM crops (Leeson and Summers, 2001). Borzelleca (1996) indicated that the best way to evaluate overall animal health is through monitoring body weight, food consumption, and food efficiency. Endpoints should include feed intake, feed conversion efficiency (the efficiency in which feed is converted to body mass), and body weight (at a minimum) and should ideally also include organ (liver, pancreas) and carcass (breast, wing, thigh, leg, fat pad) component weights and observation. Feed intake and feed conversion ratios both serve as good indicators of nutritional quality, as they are sensitive to the presence of toxicants or antinutrients. The presence of elevated antinutritional factors such as phytic acid, trypsin inhibitor, gossypol, or fiber content is known to reduce feed intake in broilers (Selle and Ravindran, 2007; Loeffler et al., 2012; EFSA, 2008b; Mateos et al., 2012; Perryman et al., 2014). Changes to feed conversion may also be indicative of alterations to metabolism. For example, the suppression of digestive enzymes by trypsin inhibitor can lead to compensatory overstimulation of pancreatic enzymes, leading to increased endogenous losses of amino acids beyond that from decreased digestion (Barth et al., 1993) and an increase in the amount of nutrients required to maintain body weight gain (Yen et al., 1974, Pacheco et al., 2014). As the primary site for nutrient metabolism in the chicken, the liver is sensitive to capturing differences of a wide array of nutritional impairments. For example, a pale liver with the accumulation of fat is a classic sign of Fatty Liver Kidney Syndrome and biotin deficiency (Leeson and Summers, 2001). Fatty livers may also be indicative of vitamin A, thiamin, pyridoxine, vitamin E, choline deficiency, or the presence of mycotoxins. The inclusion of high oil sunflower meal into the broiler diet was found to consistently lower liver weight when compared with soybean meal (Senkoylu and Dale, 2006). Fava bean inclusion in the broiler chick diet has historically resulted in deleterious effects, including reduced liver weight (Marquardt et al., 1974; Moschini et al., 2005), even in low tannin varieties (Usayran et al., 2014). The incremental additions of an expeller-extracted canola meal to the broiler diet led to a linear increase in liver weight (Woyengo et al., 2011). It is clear that liver data can be quite meaningful in any nutritional/health assessment. Although poor growth can be indicative of a host of nutritional maladies, diminished skeletal muscle accretion is usually indicative of amino acid imbalance or deficiency. The modern broiler has been selected for increased breast muscle accretion, and thus has a larger requirement for amino acids: the building blocks of lean protein mass. As such, the presence of imbalances or limiting amino acids can result in reduced muscle accretion. Muscular dystrophy can also be a sign of vitamin E deficiency (Leeson and Summers, 2001). The collective data gathered by compositional analysis, in vitro digestions, and the results of the broiler study can be used to accurately assess the nutritive value of the genetically modified crop.

Nutritional Assessment of Genetically Modified Crops Using Animal Models Chapter | 5  47

90-Day Rodent Study Rodents have a long history in animal research. The 90-day rodent whole-food study, also known as the repeated-dose 90-day oral toxicity study, is the primary nutritional and safety study in food and is often the study discussed in the popular press. It is considered a toxicological study rather than a nutritional study due to the historical use of such studies and the nature of the analytical endpoints measured. However, the experimental design of a 90-day rodent study does not significantly differ from that of a pure nutritional study, for example, broiler chicken, and similar nutritional information can be gleaned (EFSA, 2008a, 2011b; OECD, 1998). Similar questions to those regarding a 42-day broiler study have been posed regarding the ability of a 90-day rodent study to detect unintended effects. The EFSA has concluded, based on the historical lowest-observed-effect level of known toxicants, that a 90-day rat study has a large capacity to detect unintended changes (EFSA, 2008a). Investigation into whether a 3-month study would yield similar information as a 24-month study across 40 different substances determined that for 70% of the studies, all of the findings found at 24 months were observed at 3 months (Betton et al., 1994). Poulsen et al. (2007) used the SAFOTEST protocol to determine whether a 90-day rodent feeding study is capable of detecting PHA-E lectin toxicity from genetically modified rice expressing PHA-lectin. The authors showed significant differences in small intestine, pancreas, stomach weight, and plasma biochemistry in rats fed genetically modified rice, confirming the sensitivity of the 90-day rat study. Some have pointed out that the most conservative no-observed-effect levels often occur with a 90-day study (Munro et al., 1996). Much like the 42-day broiler study, countries aside from the EU, India, and Australia/New Zealand do not provide significant guidance for the conduct of a 90-day rodent study. The EFSA requirements draw heavily from the OECD 408 guideline (OECD TG 408, 1998) for a repeated dose 90-day oral toxicity study in rodents but have a distinct set of requirements (EFSA, 2011b). A 90-day oral toxicity study in rodents on whole genetically modified (GM) food/feed is required for all GM plants containing single transformation events or stacked transformation events not obtained by conventional crossing, following the adoption of the Implementing Regulation (IR) (EU 503/2013). This EU legal requirement changes the study from a hypothesis-driven case-bycase exercise, as indicated previously by the EFSA GMO Panel “Guidance for risk assessment of food and feed from genetically modified plants” (EFSA, 2011a) and technically detailed in the Scientific Committee “Guidance on conducting repeated-dose 90-day oral toxicity study in rodents on whole food/feed” (EFSA, 2011b), into a mandatory requirement. However, to clarify the objectives of the 90-day toxicity studies the EFSA illustrated two possible scenarios, based on the identification (scenario one) or not (scenario two) of hazards in previous analyses and provided information on the study design, conduct, and interpretation (EFSA, 2014). Under either scenario, the objective of a 90-day study is the detection of toxicologically relevant differences between animals fed diets containing the whole GM food/feed in comparison to those fed a diet containing an appropriate control (EFSA, 2011a; IR (EU) 503/2013). The EU, India, and Australia/NZ direct applicants toward the OECD guidelines outlining considerations regarding the conduct of 90-day rodent studies (OECD TG 408, 1998). These 90-day oral toxicity studies are typically conducted with rats; however, the mouse is a viable alternative. Strain selection is dependent on the researcher, but consideration needs to be given to whether an inbred (isogenic) strain or outbred strain is appropriate. Typically, an outbred strain is selected, with popular strains for 90-day studies being Sprague–Dawley or Wistar. Oftentimes, strains are chosen based on the historical data available for that particular strain to allow for a better understanding of the natural variability within that strain. Weaned rats should be selected but no older than 9 weeks of age at the beginning of the study. The OECD 408 guideline indicates that individually housing animals is acceptable; however, the EFSA has indicated that rats should be housed in pairs to avoid animal stress. The OECD 408 recommends 20 animals (10 of each sex) per treatment, whereas the EFSA requires sufficient animals to achieve a minimum power of 80% (which is problematic for a study without a specific hypothesis). The test diet should be formulated in a similar fashion to that of the broiler study, with the test crop included in the diet at the highest level that does not create nutritional imbalances. A secondary formulation may be created with the test crop included at a level 25–50% of the high incorporation rate but no less than the anticipated human intake. Nutritionists typically rely on suggestions by the NRC (1995) for dietary formulation. As the 90-day rodent study is intended to be a toxicity study, the endpoints are more exhaustive, including body weight and body weight gains, feed consumption, detailed clinical observations, ophthalmology and neurobehavioral evaluations, including a functional observational battery and motor activity assessment, hematology, clotting potential, urinalysis, clinical chemistry, organ weights, and gross and histopathological evaluations that are beyond the scope of this chapter.

Other Animal Models Other livestock animals, including fish, can be used in the assessment of the nutritional quality of genetically modified food and feed. The growing and/or finishing periods of pigs and cattle represent other options, as do studies representing a portion of the lactation cycle in dairy cows or the egg-laying period in laying hens. A similar approach to that taken with a

48  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

42-day broiler study and the methodology described by ILSI (2003) and Flachowsky and Wenk (2010) should be employed. The appropriate endpoints should be selected given the animal model chosen and the results of previous compositional, agronomic, phenotypic, and molecular analyses. For example, body weight gain would be of little scientific value in studies conducted with laying hens; the types of data collected should focus instead on egg production and quality. When considering a nutritional assessment using ruminant animals, one can also take into account degradable protein, soluble protein, nonprotein nitrogen, NDIN, ADIN, starch, sugars, and possibly short-chain acids such as lactic, acetic, butyric, and isobutyric acid, along with the analytes under consideration in Table 1 for forage and silage. A typical dairy cow ratio may include 50% corn silage, 20% corn grain, and 10% dehulled soybean meal (Van Eenennaam and Young, 2014). Unlike in monogastric animals, a significant portion of feed is digested by the microflora present in the rumen of ruminant animals. The microflora metabolizes feedstuffs into individual components and by-products and the extent and quality of the resulting nutrient profile is dependent on the microflora present in the rumen of each animal. Thus, any nutritional assessment involving ruminants is assessing the nutritive value to the rumen microflora, which in turn influences the nourishment of the animal itself. The rumen also often acts as a detoxifier of many antinutritional compounds. Thus, the mature ruminant is not an apt model for anticipating negative effects in humans and monogastric livestock. The value of using a ruminant model to assess human safety and nutrition would be to elucidate the impact of genetically modified crops on commercially important foods such as milk and beef. A portion of feedstuff is able to bypass the rumen and enter the remaining gastrointestinal (GI) tract, where nutrients are metabolized as would be observed in a monogastric animal. Understanding the amount of the total feedstuff that can bypass the rumen is a critical piece of information needed for properly formulating diets for ruminants. Understanding how to accurately predict the nutritional contribution from the microflora remains a challenge, but large advances have been made in this area (Tedeschi et al., 2005). The GI tract of an immature ruminant more closely mimics that of a monogastric animal. As such, the antinutritional limitations that often escape ruminants are applicable to their young.

CONCLUSION The use of animal models to assess nutritional quality can play an important role in the regulatory safety assessment of genetically modified crops when other sources of information leave meaningful uncertainty relating to potential health or safety concerns. The use of animal models should be justified by existing data from other analyses, including compositional analysis, and is a poor choice as a general screen for unanticipated adverse effects. Animal feeding studies should be hypothesis driven. The value of animal models in assessing the intended improvement in the nutrition of food and feed is well-established in the literature, and application to nutritionally enhanced GM varieties requires no modification since the end product is what is evaluated, rather than the process for producing the new crop variety.

REFERENCES Aumaitre, A., Aulrich, K., Chesson, A., Flachowsky, G., Piva, G., 2002. New feeds from genetically modified plants: substantial equivalence, nutritional equivalence, digestibility, and safety for animals and the food chain. Livest. Prod. Sci. 74, 223–238. Aviagen, 2013. Ross 308 Nutrition Specifications. Aviagen, Huntsville, AL. Baker, D.H., 2008. Animal models in nutrition research. J. Nutr. 138, 391–396. Barth, C.A., Lunding, B., Schmitz, M., Hagemeister, H., 1993. Soybean trypsin inhibitor(s) reduce absorption of exogenous and increase loss of endogenous protein in miniature pigs. J. Nutr. 123, 2195–2200. Betton, G., Cockburn, A., Harper, E., Hopkins, J., Illing, P., Lumley, C., Connors, T., 1994. A critical review of the optimum duration of chronic rodent testing for the determination of non-tumourigenic toxic potential: a report by the BTS working party on duration of toxicity testing. Hum. Exp. Toxicol. 13, 221–232. Borzelleca, J.F., 1996. A proposed model for safety assessment of macronutrient substitutes. Regul. Toxicol. Pharmacol. 23, 15–18. Bowman, D.E., 1944. Fractions derived from soy beans and navy beans which retard tryptic digestion of casein. Proc. Soc. Exp. Biol. Med. 57, 139–140. Buzoianu, S.G., Walsh, M.C., Rea, M.C., O´Donovan, O., Gelencsér, E., Ujhelyi, G., Szabó, E., Nagy, A., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2012b. Effects of feeding Bt maize to sows during gestation and lactation on maternal and offspring immunity and fate of transgenic material. PLoS One 7, E47851. Buzoianu, S.G., Walsh, M.C., Rea, M.C., O’Sullivan, O., Cotter, P.D., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2012d. High throughput sequence-based analysis of the intestinal microbiota of weanling pigs fed genetically modified MON810 maize expressing Bacillus thuringiensis Cry1Ab (Bt maize) for 31 days. Appl. Environ. Microbiol. 78, 4217–4224. Buzoianu, S.G., Walsh, M.C., Rea, M.C., O’Sullivan, O., Crispie, F., Cotter, P.D., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2012c. The effect of feeding Bt MON810 maize to pigs for 110 days on intestinal microbiota. PLoS One 7, E33668. Buzoianu, S.G., Walsh, M.C., Rea, M.C., Cassidy, J.P., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2012a. Effect of feeding genetically modified Bt MON810 maize to approximately 40-day-old pigs for 110 days on growth and health indicators. Animal 6, 1609–1619.

Nutritional Assessment of Genetically Modified Crops Using Animal Models Chapter | 5  49

Buzoianu, S.G., Walsh, M.C., Rea, M.C., Cassidy, J.P., Ryan, T.P., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2013a. Transgenerational effects of feeding genetically modified maize to nulliparous sows and offspring on offspring growth and health. J. Anim. Sci. 91, 318–330. Buzoianu, S.G., Walsh, M.C., Rea, M.C., Quigley, L., O’ Sullivan, O., Cotter, P.D., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2013b. Sequence-based analysis of the intestinal microbiota of sows and their offspring fed genetically modified maize expressing a truncated form of Bacillus thuringiensis Cry1Ab protein (Bt Maize). Appl. Environ. Microbiol. 79, 7735–7744. Clark, J.H., Ipharraguerre, I.R., 2001. Livestock performance: feeding biotech crops. J. Dairy Sci. 84, 237–249. Cobb-Vantress, 2013. Cobb 500 Broiler Performance & Nutrition Supplement. Cobb-Vantress, Siloam Springs, AR. Codex Alimentarius Commission, 2008. Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants. CAC/ GL 45–2003. Codex Alimentarius Commission. Codex Alimentarius Commission, 2011. Principles for the Risk Analysis of Food Derived from Modern Biotechnology. CAC/GL 44–2003. Codex Alimentarius Commission. Council for Agricultural Science and Technology (CAST), 2006. Safety of Meat, Milk, and Eggs from Animals Fed Crops Derived from Modern Biotechnology. Issue paper no. 34. CAST, Ames, IA. European Food Safety Authority (EFSA), 2008a. Safety and nutritional assessment of GM plants and derived food and feed: the role of animal feeding trials. Food Chem. Toxicol. 46, S2–S70. European Food Safety Authority (EFSA), 2008b. Gossypol as an undesirable substance in animal feed. EFSA J. 908, 1–55. European Food Safety Authority (EFSA), 2011a. Scientific opinion on guidance for risk assessment of food and feed from GM plants. EFSA J. 9 (5), 2150–2187. European Food Safety Authority (EFSA), 2011b. Guidance on conducting repeated-dose 90-day oral toxicity study in rodents on whole food/feed. EFSA J. 9 (12), 2438–2459. European Food Safety Authority (EFSA), 2014. Explanatory statement for the applicability of the Guidance of the EFSA Scientific Committee on conducting repeated-dose 90-day oral toxicity study in rodents on whole food/feed for GMO risk assessment. EFSA J. 12 (10), 3871. FAO, 2006. Livestock in Geographic Transition. Livestock’s Long Shadow—Environmental Issues and Options. FAO, Rome, Italy. pp. 23–77. Fernandez-Cornejo, J., Wechsler, J.J., Livingston, M., Mitchell, L., 2014. Genetically Engineered Crops in the United States. Economic Research Report ERR-162. United States Department of Agriculture, Washington, DC. Flachowsky, G., 2013. In: Flachowsky, G. (Ed.), Animal Nutrition with Transgenic Plants. CABI Biotechnology Series. CABI, Oxfordshire, UK. Flachowsky, G., Wenk, C., 2010. The role of animal feeding trials for the nutritional and safety assessment of feeds from genetically modified plants— present stage and future challenges. J. Anim. Feed Sci. 19, 149–170. Flachowsky, G., Aulrich, K., Böhme, H., Halle, I., 2007. Studies on feeds from genetically modified plants (GMP) contributions to nutritional and safety assessment. Anim. Feed Sci. Tech. 133, 2–30. Flachowsky, G., Chesson, A., Aulrich, K., 2005. Animal nutritional with feeds from genetically modified plants. Arch. Anim. Nutr. 59, 1–40. Food Standards Australia New Zealand (FSANZ), 2007. The role of animal feeding studies in the safety assessment of genetically modified foods. In: Rep. Workshop of Food Standards Australia New Zealand, 15th June 2007, Canberra 19 p. Guertler, P., Brandl, C., Meyer, H.D., Tichopad, A., 2012. Feeding genetically modified maize (MON810) to dairy cows: comparison of gene expression pattern of markers for apoptosis, inflammation and cell cycle. J. Verbr. Leb. 7, 195–202. Guertler, P., Paul, V., Steinke, K., Wiedemann, S., Preißinger, W., Albrecht, C., Spiekers, H., Schwarz, F.J., Meyer, H.H.D., 2010. Long-term feeding of genetically modified corn (MON810)—fate of cry1Ab DNA and recombinant protein during the metabolism of the dairy cow. Livest. Sci. 131, 250–259. Ham, W.E., Sandstedt, R.M., 1944. A proteolytic inhibiting substance in the extract from unheated soybean meal and some characteristics of the trypsin inhibitor in soybeans. J. Biol. Chem. 154, 505. Hammond, B.G., Jez, J.M., 2011. Impact of food processing on the dietary risk assessment for proteins introduced into biotechnology-derived soybean and corn crops. Food Chem. Toxicol. 49, 711–721. Herman, R.A., Price, W.D., 2013. Unintended compositional changes in genetically modified (GM) crops: 20 years of research. J. Agric. Food Chem. 61, 11695–11701. Herman, R.A., Ekmay, R., 2014. Do whole-food animal feeding studies have any value in the safety assessment of GM crops? Regul. Toxicol. Pharmacol. 68, 171–174. ILSI, 2003. Best Practices for the Conduct of Animal Studies to Evaluate Crops Genetically Modified for Input Traits. International Life Sciences Institute. International Life Institute Crop Composition Database, 2010. Version 4.2, www.cropcomposition.org (accessed 09.07.13.). Leeson, S., Summers, J.D., 2001. Scott’s Nutrition of the Chicken, fourth ed. University Books, Guelph, Ontario, Canada. PO Box 1326. Loeffler, T., Baird, S.R., Batal, A.B., Beckstead, R., 2012. Effects of trypsin inhibitor levels in soybean meal on broiler performance. Poult. Sci. 91 (Suppl. 1), 42. Marquardt, R.R., Campbell, L.D., Stothers, S.C., McKirdy, J.A., 1974. Growth response of chicks and rats fed diets containing four cultivars of raw or autoclaved faba beans. Can. J. Anim. Sci. 54, 177–182. Mateos, G.G., Jiménez-Moreno, E., Serrano, M.P., Lazaro, R.P., 2012. Poultry response to high levels of dietary fiber sources varying in physical and chemical characteristics. J. Appl. Poult. Res. 21, 156–174. Moschini, M., Masoero, F., Prandini, A., Fusconi, G., Morlacchini, M., Piva, G., 2005. Raw Pea (Pisum sativum), raw Faba bean (Vicia faba var. minor) and raw Lupin (Lupinus albus var. multitalia) as alternative protein sources in broiler diets. Ital. J. Anim. Sci. 4, 59–70. Munro, I.C., Ford, R.A., Kennepohl, E., Sprenger, J.G., 1996. Correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern. Food Chem. Toxicol. 34, 829–867.

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National Research Council, 1994. Nutrient Requirements of Poultry, ninth revised ed. Natl. Acad. Press, Washington, DC. National Research Council, 1995. Nutrient Requirements of Laboratory Animals, fourth revised ed. Natl. Acad. Press, Washington, DC. OECD, 1998. Guideline for the Testing of Chemicals—Repeated Dose 90-day Oral Toxicity Study in Rodents. 408. OECD, 2001a. Series on the Safety of Novel Food and Feed No. 1. Consensus Document on Key Nutrients and Key Toxicants in Low Erucic Acid Rapeseed (Canola). Organisation for Economic Cooperation and Development (OECD), Paris. OECD, 2001b. Series on the Safety of Novel Food and Feed No. 2. Consensus Document on Compositional Considerations for New Varieties of Soybean: Key Food and Feed Nutrients and Anti-nutrients. Organisation for Economic Cooperation and Development (OECD), Paris. No. 2 ENV/JM/MONO, p. 15. OECD, 2002. Series on the Safety of Novel Food and Feed No. 6. Consensus Document on Compositional Considerations for New Varieties of Maize (Zea mays): Key Food and Feed Nutrients and Antinutrients and Secondary Metabolities. Organization for Economic Cooperation and Development (OECD), Paris. OECD, 2004. Series on the Safety of Novel Food and Feed No. 11. Consensus Document on Compositional Considerations for New Varieties of Cotton (Gossypium hirsutum and Gossypium barbadense): Key Food and Feed Nutrients and Anti-nutrients. Organization for Economic Co-operation and Development (OECD), Paris. OECD, 2009. Guidance Document on Overview of Residue Chemistry Studies. Organization for Economic Co-operation and Development (OECD), Paris. Pacheco, W.J., Stark, C.R., Ferket, P.R., Brake, J., 2014. Effects of trypsin inhibitor and particle size of expeller-extracted soybean meal on broiler live performance and weight of gizzard and pancreas. Poult. Sci. 93, 2245–2252. Perryman, K.R., Berry, W.D., Olanrewaju, H.A., Dozier III, W.A., 2014. Growth performance and meat yield responses of broilers fed diets containing low oligosaccharide soybean meals from one to forty-two days of age. Poult. Sci. 91 (Suppl. 1), 42. Poulsen, M., Schrøder, M., Wilcks, A., Kroghsbo, S., Lindecrona, R.H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Taylor, M., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007. Safety testing of GM-rice expressing PHA-E lectin using a new animal test design. Food Chem. Toxicol. 45, 364–377. Schnell, J., Steele, M., Bean, J., Neuspiel, M., Girard, C., Dormann, N., Pearson, C., Savoie, A., Bourbonnière, L., Macdonald, P., 2014. A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments. Transgenic Res. 1–17. Sell, P.H., Ravindran, V., 2007. Microbial phytase in poultry nutrition. J. Anim. Feed Sci. 135, 1–41. Senkoylu, N., Dale, N., 2006. Nutritional evaluation of a high-oil Sunflower meal in broiler starter diets. J. App. Poult. Res. 15, 40–47. Steinke, K., Guertler, P., Paul, V., Wiedemann, S., Ettle, T., Albrecht, C., Meyer, H.H., Spiekers, H., Schwarz, F.J., 2010. Effects of long-term feeding of genetically modified corn (event MON810) on the performance of lactating dairy cows. J. Anim. Physiol. Anim. Nutr. (Berl.) 94, E185–E193. Tedeschi, L.O., Fox, D.G., Sainz, R.D., Barioni, L.G., de Medeiros, S.R., Boin, C., 2005. Mathematical models in ruminant nutrition. Sci. Agric. 62, 76–91. Usayran, N.N., Sha’ar, H., Barbour, G.W., Yau, S.K., Maalouf, F., Farran, M.T., 2014. Nutritional value, performance, carcass quality, visceral organ size, and blood clinical chemistry of broiler chicks fed 30% tannon-free fava bean diets. Poult. Sci. 93, 2018–2027. USDA-GIPSA (U.S. Department of Agriculture Grain Inspection, Packers, and Stockyards Administration), 2011. Aflatoxin Handbook. Van Eenennaam, A.L., Young, A.E., 2014. Prevalence and impacts of genetically engineered feedstuffs on livestock populations. J. Anim. Sci. 92, 4255–4278. Walsh, M.C., Buzoianu, S.G., Gardiner, G.E., Rea, M.C., Gelencser, E., Janosi, A., Epstein, M.M., Ross, R.P., Lawlor, P.G., 2011. Fate of transgenic DNA from orally administered Bt MON810 maize and effects on immune response and growth in pigs. PLoS One 6, E27177. Walsh, M.C., Buzoianu, S.G., Gardiner, G.E., Rea, M.C., O’Donovan, O., Ross, R.P., Lawlor, P.G., 2013. Effects of feeding Bt MON810 maize to sows during first gestation and lactation on maternal and offspring health indicators. Br. J. Nutr. 109, 873–881. Walsh, M.C., Buzoianu, S.G., Gardiner, G.E., Rea, M.C., Ross, R.P., Cassidy, J.P., Lawlor, P.G., 2012a. Effects of short-term feeding of Bt MON810 maize on growth performance, organ morphology and function in pigs. Br. J. Nutr. 107, 364–371. Walsh, M.C., Buzoianu, S.G., Rea, M.C., O’Donovan, O., Gelencser, E., Ujhelyi, G., Ross, R.P., Gardiner, G.E., Lawlor, P.G., 2012b. Effects of feeding Bt MON810 maize to pigs for 110 days on peripheral immune response and digestive fate of the cry1Ab gene and truncated Bt toxin. PLoS One 7, E36141. Westfall, E.J., Hague, S.M., 1948. The nutritive quality and trypsin inhibitor content of soybean flour heated at various temperatures. J. Nutr. 35, 374–380. Woyengo, T.A., Kiarie, E., Nyachoti, C.M., 2011. Growth performance, organ weights, and blood parameters of broilers fed diets containing expellerextracted canola meal. Poult. Sci. 90, 2520–2527. Yen, J.T., Hymowitz, T., Jensen, A.H., 1974. Effects of soybeans of different trypsin-inhibitor activities on performance of growing swine. J. Anim. Sci. 38, 304–309.

Chapter 6

Noncoding RNA-Based Genetically Modified Crops: Concepts and Challenges S.V. Ramesh1, Shelly Praveen2 1Indian Council of Agricultural Research-ICAR, Directorate of Soybean Research, Indore, India; 2Indian Agricultural Research Institute, Advanced Centre for Plant Virology, Division of Plant Pathology, New Delhi, India

INTRODUCTION Genetically modified (GM) crops originated in 1983 with the concept of introducing a trait by expressing protein in the plant, which does not occur naturally in species. From 1996 onward the success story of genetically modified plants by expressing foreign protein revolutionized the concept of genetic engineering, for example, Bt crops, which are genetically engineered to produce a toxin with the goal of protecting the crop from insects dominate. Many more traits were addressed by expressing a foreign protein. The first genetically engineered crop product approved for sale (in 1994) was the FlavrSavr tomato. It was developed based on the concept of noncoding RNA and the need for the introduction of noncoding RNAs (ncRNAs) for viral protection and to fine-tune host gene expression, was felt to incorporate the desired trait more effectively. Noncoding RNAs are indispensable effector molecules of many biological processes like gene expression, defense against invading nucleic acids (viruses, transposons), chromatin maintenance, etc. in eukaryotic organisms (Lee et al., 1993; Baulcombe et al., 1996; Reinhart et al., 2002). The significance of ncRNAs in gene regulatory mechanisms came from the inadvertent discovery of the dsRNA-induced gene silencing phenomenon called RNA interference (RNAi) in Caenorhabditis elegans (Fire et al., 1998) and in plants (Waterhouse et al., 1998). Prior to dsRNA-mediated gene silencing effects in plants, occurrences of antisense RNA- and sense RNA (co-suppression)-induced gene repression were known in plants as posttranscriptional gene silencing (PTGS) (Napoli et al., 1990) and in fungi as quelling (Romano and Macino, 1992). The discovery of the RNAi phenomenon coupled with research in the fields of transgenic plants and plant molecular virology has led to a greater understanding of small ncRNAs (sncRNA)-based gene silencing (Baulcombe et al., 1996; Hamilton and Baulcombe, 1999). Studies have proven that RNAi-derived ncRNAs are evolutionarily conserved in plants and play diverse gene regulatory roles (Vaucheret, 2006). This led to the development of ncRNA-based genetically engineered crops. ncRNAs are deployed as potential effectors of gene regulation whenever expression of certain proteins or associated traits is undesired. Major achievements of ncRNA-based crop genetic engineering are in the field of pathogen resistance wherein down-regulation of viral, fungal, or viroid gene expression is imperative in developing resistant crop phenotype. Besides the microbial pathogens, the phenomenon of ncRNA genetically modified crops has played a greater role in expressing dsRNA as plant incorporated protectants (PIPs) against insects, nematodes, and insect vectors that spread deadly viral diseases. In addition, expression of small ncRNAs has resulted in the elimination of toxic allergenic compounds in plants, adding to the nutritive value of many crops, and in secondary metabolite engineering. The role of ncRNA-based genetic modification in developing male sterile genetic lines is another salient application of ncRNAs in accelerating the process of crop breeding (Table 1).

VARIOUS ncRNA-BASED SILENCING PLATFORMS The development of various ncRNA-based gene silencing platforms owes much to the scientific approaches associated with the development of virus-resistant transgenics (Figure 1). For developing virus resistance, as early as in 1986 the concept of coat protein-mediated resistance appeared (Powell-Abel et al., 1986). It involves expression of complete coat protein gene derived from viral genome to confer resistance against a particular virus. Genetically engineered papaya plants were developed using this concept for conferring Papaya ringspot virus resistance. Besides coat protein, other viral-derived nonstructural enzymatic proteins, like RNA-dependent RNA polymerase (RdRP), showed a similar response (Golemboski et al., 1990; Fitchen and Beachy, 1993). During the same period the role of RNA in conferring resistance has gained Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00006-3 Copyright © 2016 Elsevier Inc. All rights reserved.

51

52  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

TABLE 1  Various Instances of ncRNA-Based Crop Genetic Modifications S. No.

Traits

Target Gene

Crop and Year

1

Virus resistance

Potato virus Y

Potato (1998, 2004)

Mungbean yellow mosaic India virus

Black gram (2003)

African cassava mosaic virus, Pepper mild mottle virus, Alfalfa mosaic virus, Tobacco etch virus, Plum pox virus, Cucumber green mottle mosaic virus

Tobacco (2003, 2007)

Tomato yellow leaf curl virus, Tomato leaf curl New Delhi virus, Cucumber mosaic virus

Tomato (2006, 2010, 2014)

Papaya ringspot virus type W

Muskmelon (2007)

Rice tungro bacilliform virus

Rice (2008)

African cassava mosaic virus

Cassava (2009)

Citrus tristeza virus

Mexican lime (2010)

Alfalfa mosaic virus, Bean pod mottle virus, Soybean mosaic virus

Soybean (2011)

Bean golden mosaic virus

Common bean (2014)

Cowpea severe mosaic virus, Cowpea aphid-borne mosaic virus

Cowpea (2014)

2

Viroid resistance

Potato spindle tuber viroid

Tomato (2004, 2009)

3

Fungal disease resistance

Blumeria graminis f. Sp. Tritici, Blumeria graminis Avra10, P. triticina, P. graminis, P. striiformis, PtMAPK, PtCYC1, PtCNB

Wheat (2007, 2010, 2013)

Phytophthora parasitica var. nicotianae,

Tobacco (2009)

Blumeria graminis Avra10

Barley (2010)

Fusarium graminearum CYP51A, CYP51B and CYP51C

Arabidopsis and Barley (2013)

Agrobacterium tumefaciens

Arabidopsis (2006)

Agrobacterium tumefaciens

Walnut (2013)

Cotton bollworm(Helicoverpa armigera)

Tobacco (2007, 2012, 2013)

Corn rootworm (Diabrotica virgifera virgifera LeConte)

Maize (2007)

Helicoverpa armigera

Cotton (2011, 2013)

Myzus persicae

N. benthamiana A. thaliana (2011)

Nilaparvata lugens

Rice (2011)

Sitobion avenae

Wheat (2014)

Root knot nematode

Tobacco (2007, 2013)

Soybean cyst nematode, H. glycines, root knot nematode

Soybean (2006, 2010, 2012, 2013)

H. schacbii

Arabidopsis (2009)

Striga asiatica L.

Maize (2009)

Orobanche aegyptiaca M6PR

Tomato (2009)

Cuscuta pentagona STM

Tobacco (2012)

Gly m Bd 30 K

Soybean (2003)

Mal d 1

Apple (2005)

4

5

6

7

8

Bacterial diseases resistance

Insect resistance

Nematode resistance

Parasitic weeds

Allergen removal

ncRNA Based GM Crops Chapter | 6  53

TABLE 1  Various Instances of ncRNA-Based Crop Genetic Modifications—cont’d S. No.

9

10

11

Traits

Male sterility

Secondary metabolite engineering

Nutritional improvement

Target Gene

Crop and Year

Lyc e 1, Lyc e 3

Tomato (2006)

Ara h 2

Peanut (2008)

Reduced linamarin, cytochrome P450

Cassava (2003)

LFS tearless onion

Onion (2008)

TA29

Tobacco (2007)

OsGEN-like

Rice (2005)

Codeinone reductase genes

Papaver somniferum (2004)

Berberine bridge enzyme (BBE)

Eschscholzia californica (2007)

Squalene synthase (SQS)

Artemisia annua (2009)

Patatins

Potato (2008)

Desaturases in FA biosynthesis

Cotton (2002)

22-kDa zein storage proteins

Maize (2003)

β-carotene hydroxylase gene

Potato (2007)

GmMIPS1

Soybean (2006)

Enhanced synthesis of amylose type starch

Wheat (2006)

UDP-Glc:sinapate glucosyltransferase gene

Brassica Napus (2005)

DE-ETIOLATED1 (DET1)

Brassica napus (2009)

12

Fruit quality

ACC oxidase

Tomato (2005)

13

Toxins removal

CaMXMT1

Coffea arabica Coffea canephora (2003)

Cadinene synthase

Cotton (2006)

OsPCS1

Oryza sativa L. ssp. Japonica (2007)

Glutelin mutant line LGC-1

Rice (2003)

γ-gliadins in wheat

Wheat (2008)

BjMYB28

Brassica juncea (2013)

JC sugar-dependent 1 ( JcSDP1)

Jatropha curcas (2014)

CCoAOMT

Maize (2013)

14

Industrial application

momentum, when the noncoding viral genome sequence, such as the satellite RNAs, has been shown to confer resistance against virus infection (Harrison et al., 1987). Nontranslatable coat protein was also found to be superior in conferring antiviral resistance when compared to translatable coat protein (Lindbo and Dougherty, 1992). Similarly, using antisense as well as truncated viral-derived proteins, like replicas and movement protein, yielded effective resistance (Cooper et al., 1995; Beachy, 1997; Palukaitis and Zaitlin, 1997; Praveen et al., 2005). Besides developing virus resistance, down-regulation of host gene expression was achieved by incorporating DNA in antisense orientation and expressing RNA to repress the cognate RNA. The FlavrSavr tomato was derived by ectopic expression of tomato’s polygalactouranase gene in antisense orientation, leading to down-regulation of cognate sense gene expression (Redenbaugh et al., 1992). Thus the phenomenon of RNA-based gene silencing was exploited to engineer crop traits during the initial stages of GM crop development. However, many such experiments of expressing either sense or antisense RNA in plants could initiate the process of PTGS only in low frequencies.

54  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

FIGURE 1  Journey of various ncRNA-based silencing platforms from the concept of pathogen-derived resistance to deployment of syn-tasiRNAs in crop improvement.

Simultaneous expression of sense and antisense RNA in plants was achieved by engineering transgene to express duplex RNAs, thereby increasing the frequency of gene silencing observed (Waterhouse et al., 1998). Such high-frequency gene silencing was conferred by inverted repeats (IR) transgenes that were designed to express transcript, which folds back to form dsRNA. Thus, dsRNA molecules are the initiator of the cascade of molecular events resulting in RNA-based gene silencing. Subsequently, the role of protein machinery, comprising RNAses like Dicers and RNA polymerases like RNAdependent RNA polymerases (RDR), in dicing the dsRNA into effector small interfering RNAs (siRNAs) and amplifying the silencing signals was deciphered (Zamore et al., 2000; Bernstein et al., 2001). The resultant siRNAs bind to homologous RNAs to down-regulate the expression with the help of slicers, like Argonautes, forming a complex called an RNA-induced silencing complex (RISC). In addition, the siRNAs are primed for further amplifying the silencing signals and they are also mobile (Palaqui et al., 1997). The method was refined by deploying intron spliced hairpin RNA (ihpRNA) gene construct that yielded more consistent gene silencing (Smith et al., 2000) (Figure 2). In addition to siRNA-based crop genetic modification, endogenous microRNAs (miRNAs) have also been utilized in crop genetic engineering (Alvarez et al., 2006; Niu et al., 2006; Schwab et al., 2006). miRNAs are a class of endogenous sncRNAs transcribed from miRNA genes that are involved in gene regulation. The Micro RNAs precursor RNA is characterized and the endogenous gene regulatory network is altered to express artificial miRNAs, targeting any gene of interest (Ossowski et al., 2008). Since the silencing signals are immobile, miRNA-based gene silencing is suitable for tissuespecific down-regulation of gene expression (Figure 2). Trans-acting siRNAs (tasiRNAs) are another class of endogenous siRNAs, with their generation involving miRNA-mediated cleavage of trans-acting siRNA (TAS) transcript. The resultant tasiRNAs act in trans (Peragine et al., 2004; Vazquez et al., 2004). This trans-acting potential of tasiRNAs is effectively exploited in developing a single AtTAS1c-based genetic construct expressing multiple synthetic tasiRNAs (syn-tasiRNAs), each targeting a different transcript (Carbonell et al., 2014). Hence, syn-tasiRNAs are the preferred mode of ncRNA-based genetic modification where multiple, unrelated targets are required to be modulated, as in a complex metabolic engineering. Furthermore, partial silencing effects are also possible by manipulating the promoters expressing artificial tasiRNAs (de la Luz Gutieorrez-Nava et al., 2008).

APPREHENSIONS OF NONCODING RNA-BASED GENETICALLY MODIFIED CROPS The deployment of small RNAs in crop genetic modification poses various considerations. It differs significantly from the first-generation transgenic crops, which are based on the expression of protein. Genetic modification based on ncRNAs

ncRNA Based GM Crops Chapter | 6  55

FIGURE 2  sncRNA-based gene silencing tools. siRNA and amiRNA are the most widely adopted tools in crop genetic modifications. The figure depicts the biogenesis of amiRNAs and intron spliced hpRNA-derived siRNAs, their mode of action, and the systemic spread of silencing signals along with the protein machinery involved.

involves targeted down-regulation of a gene expression. The down-regulation of one gene expression might result in complex changes in a metabolic network; hence, careful selection of the gene to be repressed is very important. During the repression of a gene, silencing signals (siRNAs) are produced, which are also mobile. The amplification of these signals by host RdRP and the generation of secondary siRNAs enhances the effect of silencing manifold. Small ncRNAs have also been implicated in triggering methylation of genome in a sequence specific manner, leading to transcriptional gene silencing. The mobile silencing signals generated in the form of siRNAs also promote de novo methylation of the corresponding genome (Mette et al., 2000). RNA silencing generally functions as a mechanism to defend plants from viral infection. However, viruses have evolved proteins, called viral suppressors of RNA silencing (VSRs), as a potent weapon to disrupt RNA silencing at various stages (Brigneti et al., 1998; Baulcombe, 2002). VSRs deliberately sabotage the RNA silencing pathway by binding to dsRNA

56  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

(Merai et al., 2006) and siRNA, sequestering sRNAs (Silhavy et al., 2002), preventing the spread of silencing signals (Voinnet et al., 2000), etc. Virus infection in plants is prevalent in the field conditions and VSRs function nonselectively, hence the efficacy of ncRNA-based transgenics is limited under the influence of virus infections. Effective performances of GM crops based on small RNAs greatly rely on the ability to repress the expression of cognate mRNAs with a specific nucleic acid sequence. Hence, the selection of the target gene is the main key. For silencing the gene(s) of viruses, the main criteria should be the selection of viral gene sequences indispensable for their existence and conserved among the various viral strains. Since viruses are evolving very quickly, careful selection of the gene for silencing will help in developing effective silencing strategies. While targeting the host gene(s), the probable impact in its metabolic path should be weighed before selection. After the selection of the target gene, extensive in silico analysis of the key parameters, like prediction of potent siRNAs formation and possible off-targets in the host genome as well as in nontarget organisms, is very crucial to avoid any inadvertent effects (Lin et al., 2005; Birmingham et al., 2006). Although RNAi gene silencing functions on the principle of sequence homology, sometimes sncRNAs also silence genes from nontarget organisms (Jackson et al., 2003; Jackson and Linsley, 2004). siRNA-based gene silencing of the v-ATPase gene of western corn root worm resulted in nontarget gene silencing in related insects, like southern corn root worm and Colorado beetle (Baum et al., 2007). The studies on inadvertent gene silencing revealed little nontarget effects on the soil actinomycetes in the vicinity of Papaya ringspot virus-resistant transgenics (Hsieh et al., 2006). Similarly, insects in the environment of the Plum pox virus-resistant plants are found to be free of nontarget effects (Capote et al., 2008). Besides nontarget gene silencing, RNAi also induces other unwanted perturbations in the host through immune stimulation and saturation of RNAi machinery (Jackson and Linsley, 2010). Since sequence identity of six or seven consecutive matches between the siRNA guide strand and any cognate host transcripts could potentially down-regulate, the possibility of off-targets affecting other vital gene functions cannot be ruled out (Lin et al., 2005; Birmingham et al., 2006). Phenotypic aberrations were observed in tomato when short hairpin RNA targeting AC4 ORF of Tomato leaf curl New Delhi virus (ToLCNDV) was expressed constitutively (Praveen et al., 2010). Similarly, Glycine max MIPS one silencing, though it resulted in the intended effect of reduced phytate, also led to impaired seed development (Nunes et al., 2006). Hence, off-targets effects are to be precluded at an early stage. Screening for homology between nematode and host tobacco genes revealed little sequence homology, which paved the way for developing RNAi-based nematode-resistant plants (Fairbairn et al., 2007). Another important factor that determines the trait stability of ncRNA-based crops is the effect of mutations. Various potential scenarios have been identified: (1) the breakdown of gene silencing due to mutations in the transgene, (2) offtarget effects of mutated small sRNAs, and (3) target gene mutations that not only leave the silencing system ineffectual but might also lead to resurgent pests and diseases. Mutations in amiRNA-based crops are more common, though single or double mutation has also been recognized in siRNA-based gene silencing (Elbashir et al., 2001; Amarzguioui et al., 2003). The Turnip mosaic virus (TuMV) population, under the influence of amiRNA mediated resistance, has circumvented the RNA silencing by the accumulation of mutations in the target genomic regions (Lin et al., 2009). Furthermore, the rate of accumulation of mutations is accelerated when the viral population is exposed to suboptimal concentrations of amiRNAs (Lafforgue et al., 2011). Not withstanding these apprehensions, as of this writing 12 different crops based on ncRNAs with various traits are being grown in different parts of the globe (Table 2).

PERSISTENCE OF ncRNAs On the issue of persistence, nucleic acids have been exempted from the studied tolerance levels as no ill effects due to nucleic acid consumption have been demonstrated. The ingestion of transgene encoded dsRNA and dsRNA in feeding assays has demonstrated it as a potential crop pest control measure (Baum et al., 2007; Mao et al., 2007; Gordon et al., 2007). In addition, the process of the amplification of toxicity was also observed wherein dsRNA was primed to produce secondary RNAs with random targets within the insect body (Baum et al., 2007; Gordon et al., 2007). The secondary structural features associated with hairpin RNA and RNA sequence mismatches require examination with respect to their environmental stability. In addition, the process of dsRNA transfer from plants to insects via feed, ultimately resulting in animal gene silencing, adds to the problem of the persistent nature of dsRNA (Whyard et al., 2011).

PREDICTIVE ENVIRONMENTAL RISK ASSESSMENT The above mentioned characteristic features of ncRNA-based crop genetic modifications pose a serious concern in defining predictive environmental risk assessment (ERA).

ncRNA Based GM Crops Chapter | 6  57

TABLE 2  Current Status of ncRNA-Based GM Crops Approved Worldwide for Cultivation, Food, and Feed Uses S. No.

Crop and Traits Modified

Event or Trade Name

Developer

Country

1

Medicago sativa KK179

Monsanto Company and Forage Genetics International

Australia 2014, Canada 2014, New Zealand 2014, USA 2013

EMBRAPA 5.1

EMBRAPA (Brazil)

Brazil 2011

66

Florigene Pty Ltd.

Australia 1995 Norway 1998

Resistance to Papaya ringspot virus (PRSV)

Rainbow, SunUp

Cornell University and University of Hawaii

Canada 2003, Japan 2011, USA 1996

Resistance to PRSV

Event 63-1

Cornell University and University of Hawaii

USA 1996

Resistance to PRSV

Huanong no. 1

South China Agricultural University

China 2006

Resistance to PRSV

X17-2

University of Florida

USA 2008

Petunia-CHS

Beijing University

China 1998

C-5

United States Department of Agriculture

USA 2007

Modified starch/carbohydrate, reduced acrylamide potential, black spot bruise tolerance

Innate™ Russet Burbank Potato (event E12, E24)

J.R. Simplot Co.

USA 2014

-do-

Innate™ Ranger Russet Potato (event F10, F37)

J.R. Simplot Co.

USA 2014

-do

Innate™ H Potato (event: H37, H50)

J.R. Simplot Co. Event

USA 2014

-do-

Innate™ Atlantic Potato (event: J3, J55)

J.R. Simplot Co. Event

USA 2014

Reduced acrylamide potential, black spot bruise tolerance

Innate™ G Potato (event G11),

J.R. Simplot Co.

USA 2014

-do-

Innate™ Atlantic Potato (event J78)

J.R. Simplot Co.

USA 2014

Reduced levels of amylose and increase in the levels of amylopectin in starch granules

Amflora™ (event EH92-527-1)

BASF

European Union 2010

Monsanto Company

USA 1998

Reduced lignin content

2

Phaseolus vulgaris Bean golden mosaic virus (BGMV) resistance

3

Dianthus caryophyllus Delayed senescence

4

5

Carica papaya

Petunia hybrida Co-suppression of chalcone synthase (CHS) gene

6

Prunus domestica Resistance to Plum pox virus (PPV)

7

Solanum tuberosum L.

Resistance to Potato virus Y (PVY)

Hi-Lite NewLeaf™ Y potato (event HLMT15-15, HLMT15-3 HLMT15-46)

Continued

58  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

TABLE 2  Current Status of ncRNA-Based GM Crops Approved Worldwide for Cultivation, Food, and Feed Uses—cont’d S. No.

8

Crop and Traits Modified

Event or Trade Name

Developer

Country

-do-

New Leaf™ Y Russet Burbank potato (event RBMT15-101)

Monsanto Company

Australia 2001, Canada 1999, Japan 2003, Mexico 2001, New Zealand 2001, Philippines 2003, S. Korea 2004, USA 1997

Resistance to Potato leaf roll virus (PLRV)

Trade Name: New Leaf™ plus Russet Burbank potato (event RBMT21-129, RBMT21-350 RBMT22-082)

Monsanto Company

Australia 2001, Canada 1999, Japan 2001, Mexico 2001, New Zealand 2001, Philippines 2004, S. Korea 2004, USA 1997

-do-

New Leaf™ plus Russet Burbank potato (event RBMT21-152 RBMT22186 RBMT22-238 RBMT22-262)

Monsanto Company

USA 1998

Resistance to Potato virus Y (PVY)

ShepodyNewLeaf™ Y potato (event SEMT1502 SEMT15-15)

Monsanto Company

Australia 2001, Canada 1999, Japan 2003, Mexico 2001, New Zealand 2001, Philippines 2003, S. Korea 2004, USA 1997

-do-

ShepodyNewLeaf™ Y potato (event SEMT15-07)

Monsanto Company

USA 1998

Gm-fad2 gene silencing blocks the conversion of oleic acid to linoleic acid and allows accumulation of monounsaturated oleic acid in the seed

Event: 260-05 (G94-1, G94-19, G168)

DuPont (Pioneer Hi-Bred International Inc.)

Australia 2000, Canada 2000, Japan 2001, New Zealand 2000, USA 1997

-do-

Trade Name: Treus™, Plenish™ (event: DP305423)

DuPont (Pioneer Hi-Bred International Inc.)

Australia 2010, Canada 2009, China 2011, Japan 2010, Mexico 2008, New Zealand 2010, Philippines 2013, Singapore 2014, S. Africa 2011, S. Korea 2010, Taiwan 2010, USA 2009

-do-

Event DP305423 x GTS 40-3-2

DuPont (Pioneer Hi-Bred International Inc.)

Canada 2009, Japan 2010, Mexico 2011, S. Africa 2011, S. Korea 2011, Taiwan 2012,

-do-

Vistive Gold™ MON87705

Monsanto Company

Australia 2011, Canada 2011, Colombia 2012, Japan 2012, Mexico 2011, New Zealand 2011, S. Korea 2013, Taiwan 2013, USA 2011

Reduces desaturation of 18:1 oleic acid to 18:2 linoleic acid; increases the levels of monounsaturated oleic acid and decreases the levels of saturated linoleic acid in the seed

Event: MON87705 x MON89788

Monsanto Company

Mexico 2012, S. Korea 2013, Taiwan 2014

Glycine max L.

ncRNA Based GM Crops Chapter | 6  59

TABLE 2  Current Status of ncRNA-Based GM Crops Approved Worldwide for Cultivation, Food, and Feed Uses—cont’d S. No.

Crop and Traits Modified

9

Cucurbita pepo

10

Event or Trade Name

Developer

Country

Multiple virus (Cucumber mosaic cucumovirus (CMV), Zucchini yellow mosaic potyvirus (ZYMV), Watermelon mosaic potyvirus 2 (WMV2) resistance

Event: CZW3

Seminis Vegetable seeds (Canada) and Monsanto Company (Asgrow)

Canada 1998, USA 1994

Resistance to Zucchini yellow mosaic potyvirus (ZYMV), Watermelon mosaic potyvirus 2 (WMV2)

Event: ZW20

Seminis Vegetable seeds (Canada) and Monsanto Company (Asgrow)

USA 1994

PK-SP01

Beijing University

China 1998

Event: Vector 21-41

Vector Tobacco Inc. (USA)

USA 2002

Delayed fruit ripening

Event: 1345-4

DNA Plant Technology Corporation (USA)

Canada 1995, Mexico 1998, USA 1995

Delayed fruit softening

Event B, da

Zeneca Plant Science and Petoseed Company

Mexico 1996, USA 1994

-do-

Event Name F

Zeneca Plant Science and Petoseed Company

Canada 1996, Mexico 1996, USA 1994

-do-

FLAVR SAVR™

Monsanto Company

Canada 1995, Mexico 1995, USA 1994

Delayed fruit ripening

Event: Huafan No 1

Huazhong Agricultural University

China 1997

Resistance to Cucumber mosaic cucumovirus

Event: PK-TM8805R (8805R)

Beijing University

China 1999

Capsicum annuum Resistance to Cucumber mosaic cucumovirus (CMV)

11

Nicotiana tabacum L. Reduced nicotine content

12

Lycopersicon esculentum

Compiled from GM Approval Database, International Service for the Acquisition of Agri-biotech Applications (ISAAA), http://www.isaaa.org/ gmapprovaldatabase/default.asp.

Invasiveness and volunteerism are threats associated with incorporated traits like pest or disease resistance as they offer crops selective advantages in the receiving environment. In virus-resistant transgenic plants, transgene is derived from the virus genome, hence its effect in the environment requires study. Even though recombination between transgene-derived RNA and viral genomic RNA has been demonstrated in experimental setups, no reports of such recombination in field conditions have been found (Borja et al., 1999; Greene et al., 1994). A review of ERA features of first-generation virusresistant transgenic plants revealed no adverse impacts on the receiving environment (Fuchs and Gonsalves, 2007; Ramesh, 2013), hence it is sensible to conclude that expression of viral-derived nucleic acids should not create any novel risks in the environment. A viable approach to engineering transgene containment in vegetatively propagated plants involves silencing of meiotic-critical genes (Wang et al., 2012).

IMPACT ON PLANT PROTECTION MEASURES The induced RNA silencing mechanism in ncRNA-based transgenics constitutively utilizes molecular components of the host defense machinery (Fusaro et al., 2006), hence an unintended interruption of the plant’s defense capability, owing to saturation

60  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

of innate RNA silencing potentially impacts plant protection measures. Silencing of Glycine max gene P34, an allergenic protein with insecticidal properties, resulted in an unintentional susceptibility to Pseudomonas and could potentially cause susceptibility to other insects (Herman, 2005). Besides using ncRNAs in genetically modified crops, spraying of crude extracts of dsRNA expressed from bacteria conferring resistance to insects (Mao et al., 2007; Wang et al., 2011) and viruses (Tenllado et al., 2004; Yin et al., 2009) is a potential addition to the ways the RNAi methodology is being utilized in crop improvement programs.

FOOD AND FEED SAFETY RNA silencing-based GM crops do not produce any heterologous proteins, hence toxicity and allergenicity tests can be avoided. However, the effector molecules in ncRNA-based crops are siRNAs, amiRNAs or its precursor dsRNA, and/or transgene DNA, which are evaluated for food and feed safety concerns. The consumption of DNA through the human diet has been proven to be safe and devoid of any undesirable health effects. Transgene-derived DNA differs from diet-derived DNA in no physical or intrinsic properties, and furthermore, DNA has not been detected to be present in vertebrate tissues in food and feed safety studies (Bertheau et al., 2009). Similarly, RNA is considered to be safe for human consumption as it also forms an integral component of the human diet. There are no reports of the absorption of intact RNA in vertebrates; however, invertebrates like insect larvae and nematodes have been demonstrated to intake dsRNA. It poses a problem with small RNA-based GM crops like soybean resistant to soybean cyst nematode (Huang et al., 2006), wherein the plausibility of gene transfer to other soil microflora cannot be ruled out. Plant-derived sRNAs exhibit perfect or near perfect sequence complementarity to many of the essential mammalian genes and are consumed abundantly in daily dietary intakes, revealing a history of safe consumption (Ivashuta et al., 2009). However, transgene-derived ncRNAs are under constitutive expression and are relatively abundant. A controversial revelation of rice-derived miRNA in the mammalian bloodstream and its role in the modulation of mammalian LDLRAP1 mRNA elucidates the importance of stability of ncRNA (Zhang et al., 2012).

NUTRITIONAL COMPOSITION AND EQUIVALENCY Comparative metabolomics of RNAi-based crops and non-GM crops would provide greater insight into the non deliberate modifications in the crops and the detection of novel toxicants due to ncRNA expression (Heinemann et al., 2011). Current GM safety assessment procedures are primarily based on a targeted analysis of nutritional composition. However, nontargeted metabolite profiling involves a comparison in light of natural variability in metabolites prevalent in the conventional crop along with the nonmodified parental line. In nontargeted metabolite profiling, conventionally grown olive and GM canola expressing MUFA, which was developed as an alternative for olive, were compared to study the nutritional composition.

CONCLUSIONS AND FUTURE DIRECTIONS Although biosafety issues related to GM crops based on noncoding RNAs are relatively fewer than the foreign protein expressing GM crops, the potential concerns posed by noncoding RNA-based silencing include possible off-target gene silencing in nontarget organisms, silencing of the target gene in unintended organisms, and saturating the RNAi machinery of the host. To develop an understanding of these issues, the kinetics of the turnaround of expressing ncRNA populations at a given point of time should be properly worked out. Studies on evaluating the quantum of ncRNA accumulation at a particular point of time and its persistence in the environment will help in deciding the potential threats to unintended organisms, which appear to be at an unrealistic scale. Both the biosafety concerns as well as the economics of producing GM crops must be considered for moving forward, although there are no doubts that this tool has enhanced the power of regulating genomes for a broad spectrum of applications.

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Redenbaugh, K., Bill, H., Belinda, M., Matthew, K., Ray, S., Rick, S., Cathy, H., Don, E., 1992. Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the FlavrSavr Tomato. CRC Press. p. 288. Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., Bartel, D.P., 2002. MicroRNAs in plants. Genes. Dev. 16, 1616–1626. Romano, N., Macino, G., 1992. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol. Microbiol. 6, 3343–3353. Schwab, R., Ossowski, S., Riester, M., Warthmann, N., Weigel, D., 2006. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18, 1121–1133. Silhavy, D., Molnar, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M., Burgyan, J., 2002. A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25- nucleotide double-stranded RNAs. Eur. Mol. Biol. Organ. J. 1, 3070–3080. Smith, N.A., Singh, S.P., Wang, M.B., Stoutjesdijk, P.A., Green, A.G., Waterhouse, P.M., 2000. Total silencing by intron spliced hairpin RNA. Nature 407, 319–320. Tenllado, F., Llave, C., Diaz-Ruiz, J.R., 2004. RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Res. 102, 85–96. Vaucheret, H., 2006. Posttranscriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev. 20, 759–771. Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A.C., Hilbert, J.L., Bartel, D.P., Crete, P., 2004. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis RNAs. Mol. Cell. 16, 69–79. Voinnet, O., Lederer, C., Baulcombe, D.C., 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157–167. Wang, X., Wang, P., Sun, S., Darwiche, S., Idnurm, A., Heitman, J., 2012. Transgene induced co-suppression during vegetative growth in Cryptococcus neoformans. PLoS Genet. 8 (8), e1002885. Wang, Y., Zhang, H., Li, H., Miao, X., 2011. Second-generation sequencing supply an effective way to screen RNAi targets in large scale for potential application in pest insect control. PLoS One 6 (4), e18644. Waterhouse, P.M., Graham, M.W., Wang, M.B., 1998. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. U.S.A. 95, 13959–13964. Whyard, S.H., Cameron, F.H., Moghaddam, M., Lockett, T.J., 2011. European patent EP 2 333 061 A1 2011. Yin, G., Sun, Z., Liu, N., Zhang, L., Song, Y., Zhu, C., Wen, F., 2009. Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system. Appl. Microbiol. Biotechnol. 84, 323–333. Zamore, P., Tuschl, T., Sharp, P., Bartel, D., 2000. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33. Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., Li, J., Bian, Z., Liang, X., Cai, X., Yin, Y., Wang, C., Zhang, T., Zhu, D., Zhang, D., Xu, J., Chen, Q., Ba, Y., Liu, J., Wang, Q., Chen, J., Wang, J., Wang, M., Zhang, Q., Zhang, J., Zen, K., Zhang, C.Y., 2012. Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA. Cell Res. 22, 107–126.

Chapter 7

Agrobacterium-Mediated Alien Gene Transfer Biofabricates Designer Plants Shweta Mehrotra, Vinod Goyal University of Delhi, Delhi, India

THE BIOLOGY OF AGROBACTERIUM Agrobacterium is soil borne Gram-negative phytopathogen known to cause crown gall disease (Smith and Townsend, 1907) in dicotyledonous plants like grape vines, stone fruit, apple, peach, cherry, almond, raspberry, and nut trees. Agrobacterium has been referred to as nature’s genetic engineer because of its natural ability to transfer its genetic material to eukaryotic cells and it is the only known example of trans-kingdom DNA transfer because of which it has been used extensively in genetic engineering of plants for the introduction of foreign genes into plant cells. Agrobacterium-mediated plant transformation has revolutionized agricultural biotechnology and has led to improved yield and quality of crops thus aiding in world food security (Mohammed and Abalaka, 2011). Agrobacterium tumefaciens induces tumorous growth of plant tissues by transferring a part of its large tumor inducing (Ti)-plasmid, T-DNA, into the nucleus of infected plant cells. The T-DNA contains oncogenes encoding enzymes involved in synthesis of auxins and cytokinins, and the genes encoding for the synthesis of opines (octapine, nopaline, agropine). Agrobacterium rhizogenes, closely related to A. tumefaciens, produces hairy root disease in dicotyledonous plants in a similar way. The virulence plasmid of A. rhizogenes, the Ri-plasmid, shares extensive functional homology with the Tiplasmid. Agrobacterium has also been reported to transfer DNA to nonplant organisms like yeasts, fungi, microalgae, and mammalian cells (Soltani et al., 2008; Cheng et al., 2012). Agrobacterium-mediated plant transformation is a complex and highly regulated process involving genetic determinants of both the bacterium and the plant cell. Agrobacterium, apart from its bacterial genome, contains the tumor inducing Ti-plasmid which consists of different regions: the origin of replication (ori); the virulence region (vir), needed for transfer of T-DNA to plant genome; the opine catabolism region, necessary for the breakdown of opines; the conjugative transfer region for gene transfer; the T-DNA region, coding for oncogenes for tumor induction, genes for hormone production (auxin and cytokinin), and genes for opine synthesis. The vir region is organized into eight operons (vir A–H), having around 25 genes that regulate the processing and transfer of T-DNA. The T-DNA region is flanked by 25-base pair (bp) right and left border sequences. The T-DNA region can be cleaved and the gene of interest can be inserted between the left and right border repeats. The T-DNA region, harboring the foreign gene, can be transferred and stably integrated into the plant genome (Figure 1). The bacterial chromosomal virulence genes, chvA, chvB, and pscA, are also essential for the transfer process (Douglas et al., 1985; Thomashow et al., 1987). Wounded plant cells secrete low molecular weight compounds which are recognized specifically by the Agrobacterium as signal molecules that induce vir gene expression and subsequently activate T-DNA transfer to the plant cell.

AGROBACTERIUM-MEDIATED T-DNA TRANSFER PROCESS Gene transfer from Agrobacterium to plant cells involves six essential steps: (1) signal recognition, (2) bacterial colonization, (3) induction of bacterial virulence system, (4) generation of T-DNA transfer complex, (5) transfer of T-DNA, and (6) integration of T-DNA into the plant genome (Figure 2). Studies by Stachel et al. (1985, 1986) and Stachel and Zambryski (1986) demonstrated that wounded plant cells can induce the expression of vir genes, which are considered to be essential for the process of plant transformation.

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63

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Signal Recognition Agrobacterium perceives signal molecules like acetosyringone, phenolics, and sugars that are released by wounded plant cells. The responses of Agrobacterium to major plant-derived signals that impact Agrobacterium–plant interactions have been extensively discussed by Subramoni et al. (2014).

Bacterial Colonization Bacterial colonization is the initial step, and takes place when Agrobacterium attaches to the surface of the plant cell (Matthysse, 1986). Lipopolysaccharides and capsular polysaccharides of Agrobacterium play an important role in the colonizing process and interaction with the host plant (Whatley and Spress, 1977; Bradley et al., 1997). The chromosomal att locus (20 kb) contains chromosomal virulence genes (chv A and chv B) required for the attachment of Agrobacterium to plant cells at the wound site. Genes on the left side of att are involved in molecular signaling events and those on the right side are involved in the synthesis of fundamental components (Bradley et al., 1997).

Induction of Bacterial Virulence System T-DNA transfer is mediated by products encoded by the 30–40 kb vir region of the Ti plasmid which is composed of six essential operons (virA, virB, virC, virD, virE, virG) and two nonessential operons (virF, virH) (Nixon et al., 1986; Iuchi, 1993). The virA gene encodes a polypeptide of 829 amino acids and virG encodes a polypeptide of 267 amino acids. VirA and VirG proteins function as members of a two-component sensory-signal transduction genetic regulatory system. VirA is a transmembrane dimeric sensor protein that detects the presence of plant wound factors like acetosyringone and plant phenolic compounds, and transmits this information to the inside of the bacterium. VirG functions as a transcription factor regulating the expression of transcriptional factor regulating the expression of vir genes when it is phosphorylated by VirA (Jin et al., 1990a,b). The virA gene is constitutively expressed while regulation of virG is complex (Stachel and Zambryski, 1986; Stachel et al., 1986). The virB operon encodes the type IV secretion system that delivers the T-DNA and a number

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of virulence proteins into the plant cells. Reports also suggest that Agrobacterium uses type VI DNase as an antibacterial weapon for interbacterial competition in planta (Ma et al., 2014). The activation of the vir system is also dependent on temperature and pH.

Generation of the T-DNA Transfer Complex The activation of vir gene expression leads to a complex series of events for the transfer of T-DNA element from the bacterium to the host plant cell. Single stranded (ss) molecules representing the copy of the T-DNA strand is generated (Stachel and Zambryski, 1986; Stachel et al., 1986). VirD1/D2 recognize the 25 bp border sequence and produce ss endonucleotic nicks in the bottom strand of each border which act as initiation and termination sites for T-DNA strand production. After nicking, VirD2 remains tightly associated with the 5′ end of T-DNA strand, giving T-complex a polar nature. VirD2 serves as a pilot protein to guide the T-DNA strand from bacterium to plant cell. The T-DNA strand synthesis is initiated at the right border and proceeds in the 5′–3′ direction.

Transfer of T-DNA In the T-DNA transfer process, a specific DNA segment in ssT-DNA–VirD2 protein complex is recognized and transported through the bacterial inner and outer cell membranes and cell wall, and then through the plant cell and nuclear membranes and finally integrates into the plant nuclear genome. VirE2 is an inducible ssDNA binding protein encoded by virE, which binds tightly to the T strand thereby preventing the degradation by the nucleases. Finally, the binding of VirE2 unfolds and extends ssDNA to a narrow diameter (2 nm) facilitating its transfer through membrane channels. VirD4 protein is responsible for adenosine triphosphate-dependent linkage of the protein complex necessary for T-DNA translocation.

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The majority of the Vir B proteins form a membrane-spanning channel. VirB4 and VirB11 are necessary for active DNA transfer (Zupan and Zambryski, 1995). Studies suggest that a T-pilus is also involved in the transformation process that assists the Vir B5 and VirB2 proteins (Wu et al., 2014a). Vir F is directly translocated from the bacterium to the plant cell along with the T-DNA through the VirB/VirD4 transport system and induces targeted degradation of specific proteins inside the plant cell (Jurado Jácome, 2011).

Integration of T-DNA into the Plant Genome Once the T-DNA enters the cytoplasm of the plant cell, it receives the nuclear localization signal and passes through the nuclear pore complex. VirE2 interacting protein (VIP1) helps to dissociate VirE2 from the T-DNA and is essential for T-DNA nuclear import. However, studies suggest that VIP1 is not essential for the process (Shi et al., 2014). Inside the nucleus of the plant cell, the associated Vir proteins dissociate and the naked T-DNA produces a double stranded DNA associated with histones, which then integrates randomly into the plant genome by illegitimate recombination (Gustavo et al., 1998). VirD2 has an active role in precise integration on T-strand in the plant chromosome (Tinland et al., 1995).

COMPARISON OF T-DNA TRANSFER TO CONJUGATIVE DNA TRANSFER Conjugation is a process where DNA is transferred from a donor to a recipient bacterium by specialized cell-to-cell contact mediated by conjugative plasmids. The best-studied system is of the fertility factor (F) plasmid. Agrobacterium–plant DNA transfer and bacterial conjugative DNA transfer are considered to be evolutionarily related mechanisms. Stachel and Zambryski (1986) and Stachel et al. (1986) proposed this hypothesis based on similarities in transfer of conjugal DNA with Agrobacterium-mediated transfer. DNA transfer is initiated by ss nicks at T-DNA right border with the help of VirD1, D2 complex in case of Agrobacterium transfer, and at nick region of oriT with the help of TraI and TraJ in case of bacterial conjugative DNA transfer, respectively. There occurs a site-specific cleavage by VirD2 and TraI, both of which remain attached to the 5′ end of the DNA to be transferred. Both T-DNA transfer and conjugal transfer occur in 5′–3′ direction. In conjugal system, DNA transfer and conjugative DNA synthesis are initiated in response to a signal generated by the formation of a stable mating pair. In Agrobacterium transfer, nicking at T-DNA borders is dependent on plant induced vir gene expression. Products of virB and tra2 are involved in the export of DNA. The VirB proteins have a structural role in the transformation process and the T-DNA transfer proceeds when a close contact is formed between bacterial and plant cell with the help of specific VirB polypeptides. This may be similar to the F pili formed during conjugal transfer. The products of virB probably form a membrane-associated complex, which may presumably be functionally similar to a pilus (Zupan and Zambryski, 1995). However, the analogy between Agrobacterium–plant cell transformation and bacterial conjugation still needs to be further studied.

FACTORS INFLUENCING AGROBACTERIUM-MEDIATED TRANSFORMATION Various factors act synergistically to influence the transformation efficiency by controlling T-DNA transfer and its integration into the plant genome (Sood et al., 2011; Mehrotra and Goyal, 2012).

Osmotic Treatment of Explants Supplementation of coculture medium with sucrose, glucose, or mannitol enhances the transformation efficiency by increasing the competency of plant cells to T-DNA delivery and the subsequent recovery of plant cells.

Desiccation of Explants Desiccation treatment by air-drying of the precultured immature embryos, suspension culture cells, and embryonic and embryogenic calluses greatly enhances T-DNA delivery and plant tissue recovery, leading to a more stable transformation frequency.

Culture Medium Composition of the culture medium, concentration of salts and sugars, growth regulators, acetosyringone, and polyvinylpolypyrrolidone (PVP) greatly affect the transformation efficiency. Ethylene is known to inhibit Agrobacterium efficiency.

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Antinecrotic Treatments Antinecrotic treatments for preinduction reduce the oxidative burst. Treatment of explants with ascorbic acid, cysteine, and silver nitrate improves the transformation efficiency and explant viability. Thiol compounds like l-cysteine, dithiothreitol, and sodium thiosulphate result in stable transformations.

Temperature Temperature of cocultivation is a critical factor to achieve high frequency of transformation. Optimal cocultivation temperatures for dicot species vary between 19 and 22 °C, whereas for monocots it ranges from 24 to 25 °C.

Surfactants Surfactants like Silwet L77, Tween 20, and pluronic acid F68 in the inoculation medium assist in attachment of A. tumefaciens to the host cells.

Antibiotics Various antibiotics cefotaxime, carbenecillin, hygromycin, and kanamycin have been used in Agrobacterium-mediated transformation to suppress or eliminate Agrobacterium and to improve transformation efficiency.

Promoters Promoters facilitate expression of the gene in the tissues of the host. Constitutive promoters that are commonly used for plants include Cauliflower mosaic virus (CaMV) 35S, cassava vein mosaic virus promoter, opine promoters, plant ubiquitin (Ubi), rice actin 1 (Act-1), and maize alcohol dehydrogenase 1. CaMV 35S is the most commonly used constitutive promoter for high levels of gene expression in dicot plants. Maize Ubi and rice Act-1 are being currently used as constitutive promoters for monocots. Strong super-promoters that are hybrids of triple repeat of octopine synthase activator sequence plus mannose synthase activator elements (mas) fused to a mas promoter have also been designed for enhancement of gene expression.

Selectable Markers Selectable marker genes are conditionally dominant genes that confer an ability to grow in the presence of applied selective agents that are normally toxic to plant cells or inhibitory to plant growth, such as antibiotics and herbicides. Genes that are frequently used to select transformed plant tissues include nptII, hpt, bar, and gox, that confer resistance to kanamycin, hygromycin, phosphinothricin, and glyphosate, respectively. Commonly used selectable marker systems include neomycin phosphotransferase, hygromycin phosphotransferase, phosphoinothricin acetyltransferase, and glyphosate oxidoreductase. Use of nptII in concert with the antibiotic kanamycin has become the most widely used selectable marker gene system in dicotyledonous plants. Several positive selection systems, such as isopentenyl transferase, benzyladenine N-3-glucuronide, phosphomannoseisomerase, D-serine dehydratase, and D-amino acid oxidase, have been set up to avoid the use of antibiotic or herbicide resistance genes and to achieve high cotransformation efficiency using Agrobacterium.

Reporter Genes Reporter genes are frequently used as indicators of transformation and also to assess gene expression. They should have no endogenous activity in the plant to be transformed. Commonly used reporter genes are β-glucuronidase (gus), luciferase (luc), and green fluorescent protein (gfp).

ADVANCES IN AGROBACTERIUM-MEDIATED TRANSFORMATION Agrobacterium-mediated genetic transformation is the most extensively used method for transformation of various agronomically and horticulturally important plant species including both dicotyledons and monocotyledons. It exploits tissue culture and tissue culture independent in planta techniques which have been discussed in the subsequent sections.

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Agrobacterium-Mediated Transformation of Dicotyledonous Plants Agrobacterium has been enormously exploited for transformation of dicotyledonous plants. The earliest dicot species to be transformed was Nicotiana tabacum (Herrera-Estrella, 1983), later followed by Arabidopsis thaliana. To transform Arabidopsis, young flowering plants are dipped into Agrobacterium solution under a vacuum, which causes the bacteria to infiltrate into the flowers, and transfers the T-DNA into the DNA of the developing seeds. The seeds are then germinated on a selection medium to obtain transgenic plants. Agrobacterium-mediated transformation of other dicots is a three to four month procedure which involves the cocultivation of cut leaf discs with Agrobacterium strain, which is applied on the surface of leaf discs. The cutting of leaf discs produces a wound-related compounds, such as acetosyringe, that induce virulence genes. The leaf discs are then transferred to selection media containing antibiotic or herbicide. Transformation occurs along the cut edges of leaf discs resulting in callus formation. Tissue of callus carries foreign DNA integrated randomly into the plant genome. The callus tissue is then transferred to regeneration medium containing an antibiotic that allows only the transgenic plants to develop. Various methods, like sonication-assisted agrobacterium-mediated transformation (SAAT) and fast agro-mediated seedling transformation (FAST), have been developed for efficient Agrobacterium-mediated transformation. SAAT involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium (Trick and Finer, 1997). FAST involves cocultivation and transient transformation of young seedlings (Li et al., 2009; Weaver et al., 2014). Agrobacterium-mediated transformation has been successfully used for improvement of various economically important plants including food crops, legumes, fruits, vegetables, and orchids using leaves, roots, axillary buds, cotyledons, and hypocotyls as explants (Tian et al., 2013; Yu et al., 2010; Parmesha et al., 2012; Mano et al., 2014; Thiruvengadam et al., 2013; Nyaboga et al., 2014; Gnasekaran et al., 2014).

Agrobacterium-Mediated Transformation of Monocotyledonous Plants Monocotyledons initially had been recalcitrant to this system because they are not naturally susceptible to Agrobacterium. Various factors such as genotype, explant, age, and physiological state of explant, Agrobacterium strain, binary vector, selectable marker gene and promoter, inoculation and coculture conditions (temperature and pH), coculture medium, irradiance, osmotic treatment, desiccation, necrosis, Agrobacterium density, antioxidants (dithiothreitol, PVP) and surfactants, tissue culture, and regeneration medium may influence the Agrobacterium-mediated transformation especially in monocotyledonous species. Oxidative burst, phenolization, and subsequent cell death are the frequent phenomena during the interaction of Agrobacterium with monocot plant cells. Improvements in technological development in Agrobacterium-mediated genetic transformation have led to successful transformations of commercially important monocots has been attained in rice, maize, barley, sorghum, wheat, triticale, millet, and sugarcane. Various artificial intelligence approaches like artificial neural networks, genetic algorithms and neurofuzzy logic have been suggested which can be used for modeling, and optimizing Agrobacterium-mediated transformation procedure and understanding the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation (Pérez-Piñeiro et al., 2012).

Agrobacterium-Mediated In Planta Transformation In planta transformation refers to the tissue culture-independent transformation of a plant in vivo with Agrobacterium, in which the inoculation and cocultivation process with Agrobacterium takes place as the plant develops normally. It does not involve regeneration procedures and therefore the tissue culture-induced somatic mutations or somaclonal variations are circumvented. In planta transformation protocol was introduced by Feldmann and Marks (1987) and Clough and Bent (1998) in Arabidopsis thaliana. Arabidopsis seeds were inoculated with Agrobacterium, and plants were raised in the absence of selection medium. The progeny was germinated on antibiotic containing media and transformants were obtained. Later, Chang et al. (1994) and Katavic et al. (1994) used “clip-and-squirt” techniques wherein reproductive inflorences were clipped off and Agrobacterium was applied to the center of plant rosettes. The new inflorences formed were removed and Agrobacterium was reapplied and the plants were allowed to set seeds. Vacuum infiltration has also been used for highly efficient transformation (Bechtold et al., 1993; Bechtold and Pelletier, 1998). Arabidopsis plants were uprooted at the early stages of flowering and were placed in a jar containing Agrobacterium. A vacuum was applied and then released to cause air trapped inside the plant to bubble off and be replaced by Agrobacterium solution. Plants were sown back in the soil and were allowed to set seeds. Stably transformed lines were selected in the next generation using antibiotics or herbicides. Another significant achievement was the floral dip method developed by Clough and Bent (1998)

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in which Agrobacterium is applied to flowering plants that subsequently set seed, and transgenic plants are then selected among the progeny seedlings. The female tissues such as developing ovules within the gynoecium of young flowers have been reported to be the primary targets of Agrobacterium-mediated floral-dip transformation of Arabidopsis (Desfeux et al., 2000), whereas another is the primary target of the floral-dip transformation in rice (Rod-in et al., 2014). Pollen has also been an important target of transformation. Cocultivation of pollen grains with Agrobacterium or application of Agrobacterium prior to or after pollination has been successfully reported to give rise to transgenic plants, predominantly in cotton and also in various other plant species like maize, papaya, wheat, and tobacco by the process of pollen tube-mediated gene transfer (Eapen, 2011). Pistil drip transformation has also been a successful method for cotton transformation, where the application of Agrobacterium onto the pistil after pollination gives rise to stable transformants (Zhang and Chen, 2012). Meristem cells can be targeted successfully for delivery of DNA and are competent for stable transformation. Agrobacterium can be directed toward the apical meristem or the meristems of axillary buds. In planta inoculation of embryo axes of germinating seeds and allowing them to grow into seedlings ex vitro has also been demonstrated. Agrobacterium transfers the gene into the genome of diverse cells that are already destined to develop into specific organs and the meristematic cells still to be differentiated (Keshamma et al., 2008; Rohini and Rao, 2008; Keshamma et al., 2012; Naseri et al., 2012). In planta transformation technology has gained immense importance and widespread acceptance in the recent years because of the minimal effort, expense, and expertise. Moreover, it is widely reproducible and facilitates positional cloning and insertional mutagenesis and reduced unintended mutagenesis.

Agrobacterium-Mediated Transformation of Nonplant Organisms Agrobacterium has been reported to transform prokaryotic and eukaryotic nonplant organisms through its type IV secretion system. Different strains of Agrobacterium like LBA4404, EHA105, LBA1100, and super-virulent A281 have been used for this purpose. Successful transformations have been reported in prokaryotes like Gram-positive bacteria, Streptomyces lividans, and eukaryotes like Chlamydomonas reinhardtii (algae), Phytophthora infestans, Aspergillus niger, Agaricus bisporus (fungi), Saccharomyces cerevisae (yeast), HeLaR19, and embryonic kidney 290 cells (mammalian cells) (Soltani et al., 2008). Agrobacterium-mediated genetic modification of yeast has been the major subject of interest as it allows targeted integration of foreign DNA compared with the random integration in eukaryotes. Along with the T-DNA, various proteins like Vir E2, E3, and F are also transferred from Agrobacterium to the nonplant hosts like yeast. Proteins like Vir A, B, D, and G are also involved in the transformation process. Therefore, it is suggested that Agrobacterium has great potential for use in protein therapies (Soltani et al., 2008). Agrobacterium is a powerful tool for insertional mutagenesis, gene tagging, and gene targeting. Moreover, the ability of Agrobacterium to transfer T-DNA to eukaryotes and prokaryotes may have evolutionary consequences as it may contribute to horizontal gene transfer.

Challenges in Agrobacterium-Mediated Transformation Recent years have witnessed a remarkable progress in Agrobacterium-mediated genetic engineering. Great efforts have been made to improve the techniques of Agrobacterium-mediated gene transfer although complete understanding of the complex mechanisms that underlie the interkingdom DNA process is still far from being reached. Some challenges remain to be addressed (Mehrotra and Goyal, 2012). Various economically important plant species are highly recalcitrant to Agrobacterium and genotype-independent transformation of these crop species and forest species is difficult to achieve. Sitespecific/targeted integration of T-DNA at specific chromosomal locations to avoid random T-DNA integration has not yet been established. Moreover, stable integration of the transgene and its consistent inheritance in advanced generations without loss or alteration of expression is another milestone to be achieved. The Agrobacterium-mediated genetic transformation of plastids is another challenging task. Moreover, genetic transformation of fungi, animal, and human cells is still in its infancy and needs further research. It will be interesting to elucidate the complex signal transduction mechanisms and regulatory circuits which synergistically control the interkingdom DNA transfer to improve the efficiency of the transformation process and to address the challenges in Agrobacterium-mediated transformation.

APPLICATIONS Agrobacterium-mediated transformation has played a pivotal role in revolutionizing the agriculture sector of the world by improving crop productivity and quality, and solving environment related issues. The various applications of the process have been briefly discussed in this section.

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1. Production of economically useful products like biopharmaceuticals like anticoagulants, human epidermal growth factor, edible vaccines and interferons, bioethanol, biodegradable plastics, primary (phytohormones, ubiquinones) and secondary metabolites (terpenoids), antimicrobial agents, and high-quality paper (Banta and Montenegro, 2008). 2. Production of high-quality foreign proteins in plant cell cultures including recombinant proteins and metabolites. 3. Detection and removal of toxic contaminants from water and soil thus serving in bioremediation. Poplars with enhanced glutamylcysteine synthetase show high potential for uptake and detoxification of heavy metals (Peuke and Rennenberg, 2005). Also poplars engineered for over expression of cytochrome P450 2E1 help in removal of environmental pollutants like volatile hydrocarbons, vinyl chloride, chloroform, and benzene. They help to remediate sites contaminated with variety of pollutants at low costs (Doty et al., 2007). 4. Remarkable increases in crop productivity by enhancing photosynthetic capacity, levels of hormones, brassinosteroids, antiripening polyamines and salicylic acid, stress tolerance against abiotic and biotic factors like drought, salinity, heavy metals, parasites, and pathogens, and by increasing shelf- and vine-life characteristics. 5. Reduction in the use of harmful agrochemicals by enhancing resistance of plants to herbicides, insects, pests, and diseases. Herbicide-resistant soybean and glyphosate-resistant crops include maize, canola, oilseed rape, sugarbeet, tobacco, and cotton and Bt crops, including maize and cotton, have been developed 6. Enhancement of nutritional content in crop plants, including increased levels of β-carotene in Golden Rice, canola, soybean and corn; vitamin E in Arabidopsis, corn, and soybean; starch content in potatoes; amino acids such as lysine and antioxidants such as lycopene in tomatoes. 7. Improvement of commercially important traits like novel flower color in horticultural plants, and quality of wood in conifers and other trees (Malabadi and Nataraja, 2007).

BIOSAFETY ASPECTS Recent advances in the development and commercialization of transgenics have raised various biosafety concerns for humans, animals, and environment (Mehrotra and Goyal, 2013). Regulatory agencies that govern the field trials and release of transgenics demand uniformity and stability of T-DNA integration and expression. The ideal transgenic plant for most research and breeding purposes and for commercialization should contain a single intact copy of the desired transgene without associated vector back-bone, inserted into a single non-functional region of the plant genome. Agrobacterium-mediated transformation is the most promising technology for generating “biosafe” transgenics. It a highly efficient process of gene transfer and it predominantly results in the integration of transgenes at a single locus. The lower rearrangement frequency and singly or low copy number helps to reduce the possibility of gene silencing and increase the stability of transgene in advanced generations. The resulting transgene insertion sites tend to have simpler structures. Because of simple integration patterns, Agrobacterium has also been used to develop marker free transgenics through “clean gene” technology which eliminates the risk of horizontal gene transfer and could mitigate vertical gene transfer. Moreover, introducing T-DNA through Agrobacterium minimizes the chances of integrated T-DNA vector backbone sequences in transformed plants. In planta Agrobacterium-mediated transformation methods decrease the chances of genome-wide mutations introduced into transgenic plants. However, Agrobacterium-mediated transformation may also result in variable number, nature, and genomic location of the T-DNA insertion events but the frequency is comparatively low (Mehrotra and Goyal, 2012).

SUMMARY AND FUTURE PROSPECTS Agrobacterium has become the most popular and indispensible part of genetic engineering because of its natural tendency of interkingdom gene transfer. The unique characteristic of Agrobacterium is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals which act stringently and synergistically to activate the virulence system of the bacterium responsible for transfer and integration of T-DNA into the plant genome. Agrobacteriummediated transformation is the most preferred method of transferring desirable characteristics to crop plants to improve agricultural production. It is the most facile and versatile method of transformation for commercial biotech product development. Agricultural biotechnology has greatly benefitted from Agrobacterium-mediated transformation. The enduring success of Agrobacterium-mediated transformation is attributable to the improvements in techniques and understanding of the mechanisms underlying the process. Regulation of plant-derived signals has been demonstrated to increase the transformation efficiency (Nonaka and Ezura, 2014). Efforts have been made to efficiently integrate foreign genes and develop stable transgenics of various plant species with desirable characteristics. Various nonplant species have also been transformed with this approach. Introduction of in planta transformation approaches that evade tissue culture

Agrobacterium-Mediated Alien Gene Transfer in Plants Chapter | 7  71

have facilitated the development of efficient and stable transformation systems for successful transformation of recalcitrant crops. Combined with other powerful techniques such as bacterial artificial chromosome, very large DNA fragments can now be transformed into the plant genome by Agrobacterium (Shibata and Liu, 2000; Ali and Bakkeren, 2015). Efforts are being made to develop various artificial intelligence technologies to study the interaction of various factors involved in transformation and to understand the regulatory processes controlling the molecular, biochemical, and developmental processes involved in Agrobacterium-mediated transformation to improve the efficiency and stability of transformation. Wu et al. (2014b) reported a fast, simple, sensitive, and reliable method “Agrobacterium-mediated enhanced seedling transformation (AGROBEST),” for achieving high transformation and expression efficiency which will be useful in dissecting the intricacies of the Agrobacterium-mediated gene transfer process. Significant progress has been made to understand various aspects of Agrobacterium like plant–microbe signaling, interkingdom DNA transfer, T-DNA integration, tumorigenesis, opine production, proteins involved in bacterial cell–host cell communication, intracellular molecular transport, and DNA repair and recombination, which occur during the process of Agrobacterium-mediated transformation, although the details of the key molecular events and plant-encoded factors involved in T-DNA transfer are still elusive. The complex interplay of signaling pathways and regulatory events governing interkingdom DNA transfer needs further research. It will be worthwhile to develop new high-throughput technologies for Agrobacterium-mediated transformation to promote further research and development in this area. Recent advances and the ongoing research into plant genetic transformation will help to reduce crop losses from pests and diseases, improve nutrient efficiency of food and feed, avoid postharvest losses by improving shelf life of fruit and vegetables, and increase stress tolerance of crop plants and hence substantially improve crop production for sustainable agricultural development to address commercial, environmental, and humanitarian issues.

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Jin, S., Prusti, R.K., Roitsch, T., Ankenbauer, R.G., Nester, E.W., 1990a. The VirG protein of Agrobacterium tumefaciens is phosphorylated by the autophosphorylated VirA protein and this is essential for its biological activity. J. Bacteriol. 172, 4945–4950. Jin, S., Roitisch, T., Christie, P.J., Nester, E.W., 1990b. The regulatory VirG protein specifically binds to a cis acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes. J. Bacteriol. 172, 531–562. Jurado Jácome, E., 2011. Agrobacterium infection: translocation of virulence proteins and role of VirF in host cells. Doctoral Thesis, Leiden University. Katavic, V., Haughman, G.W., Reed, D., Martin, M., Kunst, L., 1994. In planta transformation of Arabidopsis thaliana. Mol. Gen. Genet. 245, 363–370. Keshamma, E., Sreevathsa, R., Kumar, A.M., Kumar, A.R.V., Udayakumar, M., 2012. 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Genetic transformation of conifers: applications in and impacts on commercially forestry. Transgenic Plant J. 1, 289–313. Mano, H., Fujii, T., Sumikawa, N., Hiwatashi, Y., Hasebe, M., 2014. Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica. PLoS One 9 (2), e88611. Matthysse, A.G., 1986. Initial interactions of Agrobacterium tumefaciens with plant host cells. Crit. Rev. Microbiol. 13, 281–307. Mehrotra, S., Goyal, V., 2012. Agrobacterium-mediated gene transfer in plants and biosafety considerations. Appl. Biochem. Biotechnol. 168 (7), 1953–1975. Mehrotra, S., Goyal, V., 2013. Evaluation of designer crops for biosafety-a scientist’s perspective. Gene 515 (2), 241–248. Mohammed, A., Abalaka, M.E., 2011. Agrobacterium transformation: a boost to agricultural biotechnology. J. Med. Genet. Genomics 3 (8), 126–130. Naseri, G., Sohani, M.M., Pourmassalehgou, A., Allahi, S., 2012. In planta transformation of rice (Oryza sativa) using thaumatin-like protein gene for enhancing resistance to sheath blight. Afr. J. Biotechnol. 11 (31), 7885–7893. Nixon, B.T., Ronson, C.W., Ausubel, F.M., 1986. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad. Sci. U.S.A. 83, 7850–7854. Nonaka, S., Ezura, H., 2014. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer. Front. Plant Sci. 5, 681. Nyaboga, E., Tripathi, J.N., Tripathi, L., 2014. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement. Front. Plant Sci. 5, 463. Parmesha, M., Habeebulla, M., Khan, M., 2012. A preliminary attempt for efficient genetic transformation and regeneration of legume Mucuna pruriens L. mediated by Agrobacterium tumefaciens. Turk. J. Biol. 36, 285–292. Pérez-Piñeiro, P., Gago, J., Landín, M., Gallego, P.P., 2012. Agrobacterium-mediated transformation of wheat: general overview and new approaches to model and identify the key factors involved. In: Çiftçi, Y.O. (Ed.), Agrobacterium-Mediated Transformation of, Transgenic Plants – Advances and Limitations. InTech, ISBN: 978-953-51-0181-9. http://dx.doi.org/10.5772/35232. Peuke, A.D., Rennenberg, H., 2005. Phytoremediation with transgenic trees. Z. Naturforsch. C. 60 (3–4), 199–207. Rod-in, W., Sujipuli, K., Ratanasut, K., 2014. The floral-dip method for rice (Oryza sativa) transformation. J. Agric. Technol. 10 (2), 467–474. Rohini, V.K., Rao, K.S., 2008. A novel in planta approach to gene transfer for legumes. In: Kirti, P.B. (Ed.), Handbook of New Technologies for Genetic Improvement of Legumes. CRC, NewYork, pp. 273–286. (Chapter 18). Shi, Y., Lee, L.-Y., Gelvin, S.B., 2014. Is VIP1 important for Agrobacterium-mediated transformation? Plant J. 79, 848–860. Shibata, D., Liu, Y.-G., 2000. Agrobacterium-mediated plant transformation with large DNA fragments. Trends Plant Sci. 5, 354–357. Smith, E.F., Townsend, C.O., 1907. A plant tumor of bacterial origin. Science 25, 671–673. Soltani, J., van Heusden, G.P., Hooykaas, P.J., 2008. Agrobacterium-mediated transformation of non-plant organisms. In: Tzfira, T., Citovsky, V. (Eds.), Agrobacterium: From Biology to Biotechnology. Springer, New York, pp. 649–675. (Chapter 18). Sood, P., Bhattacharya, A., Sood, A., 2011. Problems and possibilities of monocot transformation. Biol. Plant. 55, 1–15. Stachel, S.E., Zambryski, P.C., 1986. Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell 47 (2), 155–157. Stachel, S.E., Nester, E.W., Zambryski, P.C., 1986. A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc. Natl. Acad. Sci. U.S.A. 83, 379–383. Stachel, S.E., Messens, E., Montagu, M.V., Zambryski, P., 1985. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318, 624–629. Subramoni, S., Nathoo, N., Klimov, E., Yuan, Z.C., 2014. Agrobacterium tumefaciens responses to plant-derived signaling molecules. Front. Plant Sci. 5, 322. Thiruvengadam, M., Jeyakumar, J., Kamaraj, M., Ill-Min, C., Kim, J., 2013. Optimization of Agrobacterium-mediated genetic transformation in gherkin (‘Cucumis anguria’ L.). Plant Omics 6 (3), 231–239. Thomashow, M.F., Karlinsey, J.E., Marks, J.R., Hurlbert, R.E., 1987. Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment. J. Bacteriol. 169, 3209–3216.

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Tian, N., Liu, S., Huang, J., van der Krol, A.R., Bouwmeester, H.J., Liu, Z., 2013. An improved Agrobacterium tumefaciens mediated transformation of Artemisia annua L. by using stem internodes as explants. Czech J. Genet. Plant Breed. 49, 123–129. Tinland, E., Schoumacher, F., Gloeckler, V., Bravo-Angel, A.M., Hohn, B., 1995. The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome. EMBO J. 14, 3585–3595. Trick, H.N., Finer, J.J., 1997. SAAT: sonication assisted Agrobacterium-mediated transformation. Transgenic Res. 6, 329–336. Weaver, J., Goklany, S., Rizvi, N., Cram, E.J., Lee-Parsons, C.W., 2014. Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings. Plant Cell Rep. 33 (1), 89–97. Whatley, M.H., Spress, L.D., 1977. Role of bacterial lipopolysaccharide in attachment of Agrobacterium to moss. Plant Physiol. 60, 765–766. Wu, H.Y., Chen, C.Y., Lai, E.M., 2014a. Expression and functional characterization of the Agrobacterium VirB2 amino acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency. PLoS One 9 (6), e101142. http://dx.doi.org/10.1371/journal.pone.0101142. Wu, H.Y., Liu, K.H., Wang, Y.C., Wu, C.F., Chiu, W.L., et al., 2014b. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 10, 19. Yu, Y., Liu, L.S., Zhao, Y.Q., Yang, P., Zhao, B., Guo, Y.D., 2010. A highly efficient in vitro plant regeneration and Agrobacterium-mediated transformation of i var. botrytis. N. Z. J. Crop Hortic. Sci. 38, 1–11. Zhang, T., Chen, T., 2012. Cotton pistil drip transformation method. Methods Mol. Biol. 847, 237–243. Zupan, J.R., Zambryski, P., 1995. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol. 107 (4), 1041–1047.

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Chapter 8

Understanding the Factors Influencing Attitudes toward Genetically Modified Rice Latifah Amin, Hasrizul Hashim Pusat Citra Universiti, Universiti Kebangsaan Malaysia, Selangor, Malaysia

INTRODUCTION Rice is a major staple crop for most parts of the world and has become part of their cultural identity. As a staple food, a single serving of 100 g of rice consists of 80 g of carbohydrates and provides 365 kcal, or about one-fifth of human calorie needs per day. Rice also contains numerous trace elements that could be crucial to human health such as thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), and vitamin B6 (USDA, 2014). There are four major varieties of rice including indica, japonica, aromatic, and glutinous. The most common species, Oryza sativa, has both the indica and japonica varieties and they are mainly cultivated and exported by Asian countries such as China, India, Thailand, Indonesia, and Japan. In Malaysia, it is estimated that the country can only generate about 73% of the total rice for local consumption (Matthews et al., 1995) therefore the remaining 27% is imported. Between 2012 and 2013, the global rice production was reported to stand at around 469 million tons (milled basis) which had to meet the worldwide consumption demand of 468 million tons (IGC, 2013), contributing to 21% of global human per capita energy (Maclean et al., 2002). It is projected that the worldwide consumption of rice will surpass its production by 2016–2017. This is attributed to an expected decline in rice production in China (IGC, 2013). Meanwhile, Rejesus et al. (2012) forecasted that the global rice consumption is set to increase to about 490 million tons in 2020, and to about 650 million tons by 2050. While the projections show substantial increases of rice demand for future populations, producers and scientists have taken steps to solve this matter. One of the alternatives still under consideration is to produce a rice variety capable of a high yield while being disease- and pest-resistant. Oryza rufipogon is a wild type rice species indigenous to Malaysia. However, its aggressiveness competes with and reduces the yield of cultivated rice (Federal Noxious Weed Disseminules, 2014), therefore it has been listed as an “invasive weed” in the United States (Freese et al., 2004). Both O. sativa and O. rufipogon are analogous and vegetatively similar which is why they share the same genus of Oryza, hence both could be hybridized easily (Fasahat et al., 2012). Since early 2000, local researchers have been developing a high yielding superior Red rice. The process involves conventional crossbreeding of O. sativa ssp. indica cv. MR219, an elite local variety which is known for its high grain yield, short maturation period, major pest resistance, and good eating quality, with O. rufipogon (Fasahat et al., 2012). It was also reported that the new high-yielding superior Red rice was ready to be commercialized (UKM, 2012). Scientists have presumed that the Os11Gsk gene in O. rufipogon is the one which is responsible for the improvements of yield in rice, as research shows a significant link between the genes present and high yield traits (Thalapati et al., 2012). Through this finding, it is now possible for any commercial variant of rice around the world other than O. sativa ssp. indica cv. MR219 to undergo transformation by inserting this gene directly into the rice variant of interest. This technique of gene modification is different from the conventional breeding as it is more targeted (involving only the desired trait) and can allow the transfer of genes between organisms of any species (EuropaBio, 2014). Although gene modification can provide benefits to economy and society, public perceptions of the potential benefits and risks of products resulting from this technique are still mixed and vary across geographic regions throughout the world (Kikulwe et al., 2011). Past studies have shown that public attitudes toward\GM rice such as Golden Rice (containing Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00008-7 Copyright © 2016 Elsevier Inc. All rights reserved.

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provitamin A) and vitamin C-enhanced rice are driven by certain predictors such as perceived benefit and religious acceptance (Amin et al., 2014). Thus the purpose of this study is to assess the factors which potentially influence the attitudes held by the Malaysian public to GM rice containing a “yield gene” from the wild type, O. rufipogon.

THEORETICAL FRAMEWORK AND HYPOTHESES DEVELOPMENT The theoretical framework of public attitudes toward GM rice, containing a “yield gene” from the wild type, O. rufipogon, has been developed based on the model of attitudes toward biotechnology application and product proposed by Pardo et al. (2002) and Brehdahl (1999), based on Fishbein’s multi-attribute attitude model (1963). The variables in the model are arranged according to their assumed effects upon the subsequent variables, beginning with factors that are known to trigger attitudes. The magnitude of the influence from one variable to another variable will be determined by the regression weights of structural equation modeling (SEM). The attitude toward GM rice is determined by the specific perceptions of risks and benefits (Chen and Li, 2007; Grunert et al., 2000), religious acceptance (Kelley, 1995; Nicholas, 2000), and possible moral concerns from the application (Amin et al., 2011; Gott and Monamy, 2004; Sjoberg, 2004). General attitudinal factors, such as engagement, confidence toward key players, attitudes to technology, attitudes to nature and religiosity are also included, since past research has shown their causal relationship with risk and benefit perceptions (Amin et al., 2005, 2011; Chen and Li, 2007; Gaskell et al., 2003; Siegrist, 2000). Figure 1 represents the conceptual research framework of public attitudes toward GM rice labeled with the corresponding hypotheses.

Engagement The variable engagement in this study consists of three subfactors which are past and intended behavior, awareness, and knowledge, as suggested by previous literature (Amin et al., 2011; Gaskell et al., 2003). At first, Gaskell et al. (2003) found that engagement with biotechnology issues was an important factor associated with attitudes toward biotechnology among the Europeans. Previous studies also showed that engagement in biotechnology was positively associated with encouragement of biotechnology applications and other positive predictors such as perceived benefits (Amin et al., 2011; Christoph et al., 2008; Pardo et al., 2002). Due to the importance of this factor in the studies of public perception, the following hypotheses are proposed for the association between engagement and other factors:

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Attitudes toward Genetically Modified Rice Chapter | 8  77

H1: When the public has a higher engagement in biotechnology, then they will be more accepting of GM rice from their religious perspective. H2: When the public has a higher engagement in biotechnology, then they will have higher positive attitudes to GM rice. H3: When the public has a higher engagement in biotechnology, then they will perceive higher benefits associated with GM rice. H4: When the public has a higher engagement in biotechnology, then they will perceive lower risks associated with GM rice. H5: When the public has a higher engagement in biotechnology, then they will perceive lower moral concerns associated with GM rice.

Confidence in Key Players Past studies found that consumer confidence in science and institutions, involved in gene technology regulation, is associated with risks and benefits perceptions related to gene technologies (Chen and Li, 2007; Frewer et al., 2000). Gaskell et al. (2003) had reported that confidence in industry, regulation and other civil society groups has led to the encouragement of biotechnology applications. Meanwhile, Amin et al. (2011) demonstrated that those who have confidence in the key players tended to perceive the application as having a higher risk acceptance, benefit, and encouragement. Chen and Li (2007) also demonstrated that social confidence in institutions is a variable predictor for perceived benefits and perceived risks. Due to its importance, the following hypotheses were developed for the association of confidence in key players with other attitude variables. H6: When the public has more confidence in the key players involved in modern biotechnology, then they will be more accepting of GM rice from their religious perspective. H7: When the public has more confidence in the key players involved in modern biotechnology, then they will perceive higher benefits associated with GM rice. H8: When the public has more confidence in the key players involved in modern biotechnology, then they will have a higher positive attitude to GM rice. H9: When the public has more confidence in the key players involved in modern biotechnology, then they will perceive lower risks associated with GM rice. H10: When the public has more confidence in the key players involved in modern biotechnology, then they will perceive lower moral concerns associated with GM rice.

Attitudes to Technology The impact of technologies is considered an important variable because it has been said to provide a picture for the formation of public views about biotechnology (Gaskell et al., 2003). A previous study of Malaysian attitudes toward biotechnology applications showed several general attitudes as the predictors for the attitudes including a predisposition toward science and technology (Amin et al., 2005). Respondents who have a negative predisposition toward science and technology were found to have more general concerns and viewed the GM product as risky and had higher moral concerns but low benefits, risk acceptance, and encouragement. In this study, attitudes to technology consist of four items that reflect the negative impact brought by technology (Amin et al., 2005, 2011). A higher score in this construct indicates a higher negative predisposition toward science and technology from the respondent. H11: When the public has a higher negative predisposition toward science and technology, then they will be less accepting of GM rice from their religious perspective. H12: When the public has a higher negative predisposition toward science and technology, then they will perceive lower benefits associated with GM rice. H13: When the public has a higher negative predisposition toward science and technology, then they will have a higher negative attitude to GM rice. H14: When the public has a higher negative predisposition toward science and technology, then they will perceive higher risks associated with GM rice. H15: When the public has a higher negative predisposition toward science and technology, then they will perceive higher moral concerns associated with GM rice.

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Attitudes to Nature Attitudes to nature consist of items which reflect the respondents’ preferences toward nature and material values and are sometimes referred to as societal values or nature values in previous studies (Gaskell et al., 2000; Rohrmann, 1994). A higher score for this construct means that the respondent is more inclined toward the material (as opposed to more inclined toward nature for a lower score). Societal value has been shown as one of the predictors of encouragement of six b­ iotechnology applications (Gaskell et al., 2000) and has exerted considerable influence on both the perceived risk magnitude and risk acceptance of technological risks (Rohrmann, 1994). The public, who placed materialistic value above nature value, also tended to perceive more benefits and fewer risks from the GM product such as GM food (Amin et al., 2006). In this study, the following hypotheses are proposed. H16: When the public are more inclined toward materials, then they will be more accepting of GM rice from their religious perspective. H17: When the public are more inclined toward materials, then they will perceive higher benefits associated with GM rice. H18: When the public are more inclined toward materials, then they will perceive lower risks associated with GM rice. H19: When the public are more inclined toward materials, then they will have higher positive attitudes to GM rice. H20: When the public are more inclined toward materials, then they will perceive lower moral concerns associated with GM rice.

Religiosity Stakeholders in Malaysia were once cited to have claimed themselves highly attached to their religion (Amin et al., 2011). A previous study also reported that the public who were more attached to their religion tended to be more critical regarding biotechnology issues as they saw more general promise of biotechnology and more risk of GM products such as GM food (Amin et al., 2005, 2011). Due to the important role of religious beliefs in the public opinion study regarding modern biotechnology, the following hypotheses are proposed: H21: When the public claim they are more religious, then they will be more accepting of GM rice from their religious perspective. H22: When the public claim they are more religious, then they will perceive higher benefits associated with GM rice. H23: When the public claim they are more religious, then they will perceive higher risks associated with GM rice. H24: When the public claim they are more religious, then they will have a higher positive attitude to GM rice. H25: When the public claim they are more religious, then they will perceive higher moral concerns associated with GM rice.

Perceived Moral Concerns According to Furedi (1997), individual and societal risk perceptions correspond to a system of moral values. However, Gaskell et al. (2000) noticed that moral acceptability appeared to act as a veto for support for different biotechnology applications. The results of the United States public survey suggests there is a possibility for the Americans to use moral reasoning in forming opinions toward six applications of biotechnology (Priest, 2000), while a Canadian study shows that moral acceptance was the strongest predictor for encouragement of animal cloning (Einsiedel, 1997). The moral concern variable is added to the model since past studies have confirmed its role as a major predictor for risk perception and encouragement of biotechnology products in Malaysia (Amin et al., 2006, 2011). Therefore, the following hypotheses are proposed: H26: When the public perceive higher moral concerns associated with GM rice, then they will perceive higher risks associated with GM rice. H27: When the public perceive higher moral concerns associated with GM rice, then they will perceive lower benefits associated with GM rice. H28: When the public perceive higher moral concerns associated with GM rice, then they will be less accepting of GM rice from their religious perspective. H29: When the public perceive higher moral concerns associated with GM rice, then they will have higher negative attitudes to GM rice.

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Perceived Risks and Perceived Benefits Perceived benefits and perceived risks have been identified as important predictors in attitude assessment (Bredahl, 1999; Einsiedel, 1997; Gaskell et al., 2003; Rowe, 2004). Both predictors are difficult to be conceptualized separately due to the complexity of risks and benefits perception, in fact, few researchers believe both variables are dependent (Gaskell et al., 2003; Rowe, 2004). The perception of benefits revolves around producers, consumers, health, and societal issues while risks are extended to long-term unknown effects, health, environment, and societal and moral issues (Rowe, 2004). Due to the importance of risk and benefit perception, the following hypotheses are proposed: H30: When the public perceives higher risks associated with GM rice, then they will perceive lower benefits associated with GM rice. H31: When the public perceives higher risks associated with GM rice, then they will be less accepting of GM rice from their religious perspective. H32: When the public perceives higher risks associated with GM rice, then they will have higher negative attitudes to GM rice. H33: When the public perceives higher benefits associated with GM rice, then they will be more accepting of GM rice from their religious perspective. H34: When the public perceives higher benefits associated with GM rice, then they will have a higher positive attitude to GM rice.

Religious Acceptance Religious denomination and religious belief have shaped many opinions on a wide range of topics including genetic engineering (Kelly, 1995). Other than confidence in scientists, corporations, and government, people’s religious and social views also have a significant influence on public attitudes toward biotechnology (Hossain et al., 2002). Certain religions do not allow uncontrolled interference with life such as genetic engineering (Epstein, 1998). In Islam, scientific research is encouraged in order to understand natural phenomena and the universe. However, not everything is considered permissible; it is important to consider the purpose of the research and any harmful effects toward humans, the environment, and society and must be in line with the rules of Shari’ah (Hajj Mustafa, 2001). On the other hand, issues of “halal” sources of genes are also important for the Muslims. Due to its significant role in shaping the positive attitudes toward genetic engineering, the following hypothesis has been proposed: H35: When the public are more accepting of GM rice from their religious perspective, then they will have a higher positive attitude to GM rice.

RESEARCH METHODOLOGY Survey Data Collection Data were collected by means of a face-to-face survey of 509 adults of age 18 years and above, residing in the Klang Valley region, Malaysia, from March to December 2012. The Klang Valley was selected because it is the center of Malaysia’s economic and social development and people who reside in the area met the requirement of diverse backgrounds required in this study. The respondents were stratified according to stakeholders’ groups which consisted of producers, scientists, policy makers, nongovernmental organizations, media, religious scholars, university students, and consumers (general public).

Instrument The multidimensional instrument measuring attitudes to GM rice was developed and adopted based on earlier studies. The instrument incorporated 10 variables, five of which are general dimensions which include engagement, confidence in key players (Gaskell et al., 2003), attitudes to technology (Gaskell et al., 2003; Rohrman, 1994), attitudes to nature (Rohrmann, 1994), and religiosity (Amin et al., 2011; Kelley, 1995). Five specific variables for the application include perceived risk (Rohrmann, 1994), perceived benefit (Macer, 2000), perceived moral concern (BABAS, 1999; Comstock, 2000), religious acceptance (Kelley, 1995; Nicholas, 2000), and attitudes to GM rice (Amin et al., 2011; Gaskell et al., 2003). All items were measured on 7-point Likert scales, except for engagement, which was measured based on a total score of 10.

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Statistical Analysis Initial reliability tests and confirmatory factor analysis were carried out using Statistical Package for the Social Sciences software to assess the consistency and unidimensionality of the construct. Correlation analyses were then carried out at a bivariate level followed by SEM analysis to explore the initial relationships among the variables. A single step SEM analysis, as recommended by Hair et al. (2010), was carried out to estimate the measurement and structural model using AMOS version 20 with maximum likelihood function.

RESULTS Measurement Model (Confirmatory Factor Analysis) Confirmatory factor analysis was carried out to test the adequacy of the measurement model (Anderson and Gerbing, 1988) and a total of 10 constructs has been established. The hypotheses presented in Figure 1 constitute the “model 1” of attitudes to GM rice in SEM.

Structural Equation Modeling SEM is a powerful multivariate analysis technique where causal relationships among variables are sought (Levy and Varela, 2003; Luque, 2000). One of the advantages of SEM compared with other general linear modelings is that SEM is capable of determining the relationships among a group of latent constructs that are represented by their measurements (Lei and Wu, 2007). In this study, a single-step SEM analysis was carried out to estimate the measurement and structural model using AMOS version 20 with maximum likelihood function. The model generation strategy as recommended by Joreskog and Sorbom (1996) was used to specify the model but the modifications of the nested models were only carried out when they were substantively meaningful. A series of four nested models were tested to identify the best model for attitudes to GM rice. The first model was specified according to the research framework described earlier (Figure 1). It contains 35 proposed hypotheses which were analyzed to examine the relationship among the variables. Throughout SEM, the suggestion has been to remove non-significant parameters of the original model and to add additional paths suggested by the modification index to improve the model fit as long as they were corroborated by the theory (Byrne, 2001). During model 1, 5 out of 35 hypotheses were eliminated due to being statistically insignificant at the probability level of 0.05. The changes were then saved as model 2. In model 2, another 6 hypotheses were eliminated due to being statistically insignificant at the probability level of 0.05, whereas one item under attitudes to GM rice was deleted. Another 10 hypotheses were eliminated due to the same reason, as in model 3. Besides, eight items were found to have a number of high residual covariances with other items, therefore they were all deleted. Correlated errors among the items in the same dimension were allowed, thus two correlated errors have been added. The model was then named as model 4, and this model constitutes the final version of the attitudes to GM rice model. According to Hair et al. (2006), a well-fitting model should have GFI, AGFI, and CFI greater than 0.90, while an RMSEA value less than 0.05 supported with narrow confidence interval. Costa-Font and Gil (2009) also used several commonly used fit indices to assess the overall model fit which includes Chi-square (χ2), CMIN/DF (χ2/df), NFI and NNFI, while Carmines and McIver (1981) suggested a good model should have the value of χ²/df less than 3. The measurement model for attitudes to GM rice was found to have a good fit as summarized in Table 1.

Construct Reliability and Validity Three types of reliabilities, measured in this paper, are the internal consistency (Cronbach alpha), item reliability and construct reliability. The Cronbach’s alpha coefficients for all constructs are above 0.60 and are considered good (Table 1). The corrected item-total correlations for all items in each dimension were considered acceptable. The construct reliability is represented by the composite reliabilities and the average variance extracted (AVE). From Table 1, it is clear that the composite reliabilities for all constructs are above 0.7 while the variance extracted (AVE) are above 0.4 (except for perceived benefit which has an AVE of 0.343) indicating good construct reliability (Hair et al., 2010).

Relationships among the Variables Figure 2 shows the final structural model of public attitudes toward GM rice. Perceived benefit emerges as the most important direct predictor of attitudes to GM rice (β = 0.70, p