Plant Biotechnology and Genetics

C. Neal Stewart, PhD, is Racheff Chair of Excellence in Plant Molecular Genetics and Professor, Department of Plant Sciences, University of Tennessee. In addition to the prior edition of Plant Biotechnology, he has written Weedy and Invasive Plant Genomics, Plant Transformation Technologies, and Research Ethics for Scientists: A Companion for Students, all published by Wiley.

Foreword xvi

Contributors xviii

Preface xx

1. The Impact of Biotechnology on Plant Agriculture 1
Graham Brookes

1.0 Chapter Summary and Objectives 1

1.0.1 Summary 1

1.0.2 Discussion Questions 1

1.1 Introduction 1

1.2 Cultivation of Biotechnology (GM) Crops 2

1.3 Why Farmers Use Biotech Crops 4

1.4 GM’s Effects on Crop Production and Farming 7

1.5 How the Adoption of Plant Biotechnology has Impacted the Environment 8

1.5.1 Environmental Impacts from Changes in Insecticide and Herbicide Use 8

1.5.2 Impact on GHG Emissions 11

1.6 Conclusions 13

Life Box 1.1 Norman E. Borlaug 14

Life Box 1.2 Mary-Dell Chilton 15

Life Box 1.3 Robert T. Fraley 17

References 19

2. Mendelian Genetics and Plant Reproduction 20
Matthew D. Halfhill and Suzanne I. Warwick

2.0 Chapter Summary and Objectives 20

2.0.1 Summary 20

2.0.2 Discussion Questions 20

2.1 Overview of Genetics 20

2.2 Mendelian Genetics 23

2.2.1 Law of Segregation 26

2.2.2 Law of Independent Assortment 26

2.3 Mitosis and Meiosis 27

2.3.1 Mitosis 29

2.3.2 Meiosis 29

2.3.3 Recombination 30

2.3.4 Cytogenetic Analysis 31

2.3.5 Mendelian Genetics and Biotechnology Summary 32

2.4 Plant Reproductive Biology 32

2.4.1 History of Research in Plant Reproduction 32

2.4.2 Mating Systems 32

2.4.3 Hybridization and Polyploidy 36

2.4.4 Mating Systems and Biotechnology Summary 38

2.5 Conclusion 38

Life Box 2.1 Richard A. Dixon 39

Life Box 2.2 Michael L. Arnold 40

References 42

3. Plant Breeding 43
Nicholas A. Tinker and Elroy R. Cober

3.0 Chapter Summary and Objectives 43

3.0.1 Summary 43

3.0.2 Discussion Questions 43

3.1 Introduction 44

3.2 Central Concepts in Plant Breeding 45

3.2.1 Simple vs. Complex Inheritance 45

3.2.2 Phenotype vs. Genotype 46

3.2.3 Mating Systems, Varieties, Landraces, and Pure Lines 47

3.2.4 Other Topics in Population and Quantitative Genetics 49

3.2.5 The Value of a Plant Variety Depends on Many Traits 51

3.2.6 A Plant Variety Must Be Environmentally Adapted 51

3.2.7 Plant Breeding is a Numbers Game 52

3.2.8 Plant Breeding is an Iterative and Collaborative Process 52

3.2.9 Diversity, Adaptation, and Ideotypes 53

3.2.10 Other Considerations 56

3.3 Objectives in Plant Breeding 56

3.4 Methods of Plant Breeding 57

3.4.1 Methods of Hybridization 58

3.4.2 Self‐Pollinated Species 58

3.4.3 Outcrossing Species 63

3.4.4 Clonally Propagated Species 67

3.5 Breeding Enhancements 68

3.5.1 Doubled Haploidy 68

3.5.2 Marker‐Assisted Selection 68

3.5.3 Mutation Breeding 70

3.5.4 Apomixis 71

3.6 Conclusions 71

Life Box 3.1 Gurdev Singh Khush 72

Life Box 3.2 P. Stephen Baenziger 74

Life Box 3.3 Steven D. Tanksley 75

References 77

4. Plant Development and Physiology 78
Glenda E. Gillaspy

4.0 Chapter Summary and Objectives 78

4.0.1 Summary 78

4.0.2 Discussion Questions 78

4.1 Plant Anatomy and Morphology 79

4.2 Embryogenesis and Seed Germination 80

4.2.1 Gametogenesis 80

4.2.2 Fertilization 82

4.2.3 Fruit Development 83

4.2.4 Embryogenesis 83

4.2.5 Seed Germination 85

4.2.6 Photomorphogenesis 85

4.3 Meristems 86

4.3.1 Shoot Apical Meristem 86

4.3.2 Root Apical Meristem and Root Development 88

4.4 Leaf Development 89

4.4.1 Leaf Structure 89

4.4.2 Leaf Development Patterns 91

4.5 Flower Development 92

4.5.1 Floral Evocation 92

4.5.2 Floral Organ Identity and the ABC Model 93

4.6 Hormone Physiology and Signal Transduction 94

4.6.1 Seven Plant Hormones and Their Actions 94

4.6.2 Plant Hormone Signal Transduction 96

4.7 Conclusions 100

Life Box 4.1 Deborah Delmer 100

Life Box 4.2 Natasha Raikhel 102

Life Box 4.3 Brenda S.J. Winkel 103

References 105

5. Tissue Culture: The Manipulation of Plant Development 107
Vinitha Cardoza

5.0 Chapter Summary and Objectives 107

5.0.1 Summary 107

5.0.2 Discussion Questions 107

5.1 Introduction 107

5.2 History of Tissue Culture 108

5.3 Media and Culture Conditions 109

5.3.1 Basal Media 109

5.3.2 Growth Regulators 110

5.4 Sterile Technique 111

5.4.1 Clean Equipment 111

5.4.2 Surface Sterilization of Explants 112

5.5 Culture Conditions and Vessels 113

5.6 Culture Types and Their Uses 113

5.6.1 Callus and Somatic Embryo Culture 113

5.6.2 Cell Suspension Cultures 117

5.6.3 Anther/Microspore Culture 119

5.6.4 Protoplast Culture 119

5.6.5 Somatic Hybridization 120

5.6.6 Embryo Culture 120

5.6.7 Meristem Culture 121

5.7 Regeneration Methods of Plants in Culture 121

5.7.1 Organogenesis 121

5.7.2 Somatic Embryogenesis 123

5.7.3 Synthetic Seeds 123

5.8 Rooting of Shoots 123

5.9 Acclimation 124

5.10 Problems that can Occur in Tissue Culture 124

5.10.1 Culture Contamination 124

5.10.2 Hyperhydricity 124

5.10.3 Browning of Explants 124

5.11 Conclusions 125

Acknowledgments 125

Life Box 5.1 Glenn Burton Collins 125

Life Box 5.2 Martha S. Wright 127

Life Box 5.3 Vinitha Cardoza 128

References 129

6. Molecular Genetics of Gene Expression 133
Maria Gallo and Alison K. Flynn

6.0 Chapter Summary and Objectives 133

6.0.1 Summary 133

6.0.2 Discussion Questions 133

6.1 The Gene 133

6.1.1 DNA Coding for a Protein via the Gene 133

6.1.2 DNA as a Polynucleotide 134

6.2 DNA Packaging into Eukaryotic Chromosomes 134

6.3 Transcription 135

6.3.1 Transcription of DNA to Produce Messenger Ribonucleic Acid 135

6.3.2 Transcription Factors 140

6.3.3 Coordinated Regulation of Gene Expression 140

6.3.4 Chromatin as an Important Regulator of Transcription 141

6.3.5 Regulation of Gene Expression by DNA Methylation 142

6.3.6 RNA‐Directed Gene Silencing by Small RNAs 143

6.3.7 Processing to Produce Mature mRNA 143

6.4 Translation 144

6.4.1 Initiation of Translation 147

6.4.2 Elongation Phase of Translation 147

6.4.3 Translation Termination 147

6.5 Protein Postranslational Modification 147

Life Box 6.1 Maarten Chrispeels 150

Life Box 6.2 David W. Ow 152

References 154

7. Plant Systems Biology 155
Wusheng Liu and C. Neal Stewart, Jr.

7.0 Chapter Summary and Objectives 155

7.0.1 Summary 155

7.0.2 Discussion Questions 155

7.1 Introduction 155

7.2 Defining Plant Systems Biology 157

7.3 Properties of Plant Systems 158

7.4 A Framework of Plant Systems Biology 159

7.4.1 Comprehensive Quantitative Data Sets 160

7.4.2 Network Analysis 161

7.4.3 Dynamic Modeling 161

7.4.4 Exploring Systems and Models Toward Refinement 161

7.5 Disciplines and Enabling Tools of Plant Systems Biology 162

7.5.1 Plant Genomics 162

7.5.2 Plant Transcriptomics 166

7.5.3 Plant Proteomics 168

7.5.4 Plant Metabolomics 170

7.5.5 Bioinformatics 172

7.6 Conclusions 176

Life Box 7.1 C. Robin Buell 177

Life Box 7.2 Zhenbiao Yang 178

References 179

8. Recombinant DNA, Vector Design, and Construction 181
Mark D. Curtis and David G.J. Mann

8.0 Chapter Summary and Objectives 181

8.0.1 Summary 181

8.0.2 Discussion Questions 181

8.1 DNA Modification 181

8.2 DNA Vectors 186

8.2.1 DNA Vectors for Plant Transformation 188

8.2.2 Components for Efficient Gene Expression in Plants 190

8.3 Greater Demands Lead to Innovation 192

8.3.1 “Modern” Cloning Strategies 192

8.4 Vector Design 197

8.4.1 Vectors for High‐Throughput Functional Analysis 197

8.4.2 Vectors for Gene Down‐Regulation Using RNA Interference (RNAi) 199

8.4.3 Expression Vectors 199

8.4.4 Vectors for Promoter Analysis 200

8.4.5 Vectors Derived from Plant Sequences 201

8.4.6 Vectors for Multigenic Traits 203

8.5 Targeted Transgene Insertions 204

8.6 Prospects 205

Life Box 8.1 Wayne Parrott 206

Life Box 8.2 David Mann 207

References 208

9. Genes and Traits of Interest 211
Kenneth L. Korth

9.0 Chapter Summary and Objectives 211

9.0.1 Summary 211

9.0.2 Discussion Questions 211

9.1 Introduction 212

9.2 Identifying Genes of Interest via Genomics and other Omics Technologies 212

9.3 Traits for Improved Crop Production Using Transgenics 214

9.3.1 Herbicide Resistance 215

9.3.2 Insect Resistance 218

9.3.3 Pathogen Resistance 220

9.3.4 Traits for Improved Products and Food Quality 222

9.4 Conclusion 227

Life Box 9.1 Dennis Gonsalves 227

Life Box 9.2 Ingo Potrykus 229

References 231

10. Promoters and Marker Genes 233
Wusheng Liu, Brian Miki and C. Neal Stewart, Jr.

10.0 Chapter Summary and Objectives 233

10.0.1 Summary 233

10.0.2 Discussion Questions 233

10.1 Introduction 234

10.2 Promoters 234

10.2.1 Constitutive Promoters 235

10.2.2 Tissue‐Specific Promoters 236

10.2.3 Inducible Promoters 237

10.2.4 Synthetic Promoters 239

10.3 Marker Genes 239

10.3.1 Selectable Marker Genes 242

10.3.2 Reporter Genes 246

10.4 Marker‐Free Strategies 250

10.5 Conclusions 254

Life Box 10.1 Fredy Altpeter 255

Life Box 10.2 Taniya Dhillon 257

References 259

11. Transgenic Plant Production 262
John J. Finer

11.0 Chapter Summary and Objectives 262

11.0.1 Summary 262

11.0.2 Discussion Questions 262

11.1 Overview of Plant Transformation 263

11.1.1 Introduction 263

11.1.2 Basic Components for Successful Gene Transfer to Plant Cells 263

11.2 Agrobacterium Tumefaciens 265

11.2.1 History of Agrobacterium Research 266

11.2.2 Use of the T‐DNA Transfer Process for Transformation 268

11.2.3 Optimizing Delivery and Broadening the Taxonomical Range of Targets 269

11.2.4 Strain and Cultivar Compatibility 270

11.2.5 Agroinfiltration 271

11.2.6 Arabidopsis Floral Dip (Clough and Bent 1998) 271

11.3 Particle Bombardment 272

11.3.1 History of Particle Bombardment 272

11.3.2 The Fate of the Introduced DNA into Plant Cells 274

11.3.3 The Power and Problems of Direct DNA Introduction 275

11.3.4 Improvements in the Control of Transgene Expression 276

11.4 Other Methods of Transformation 276

11.4.1 The Need for Additional Technologies 276

11.4.2 Protoplasts 277

11.4.3 Whole Tissue Electroporation 278

11.4.4 Silicon Carbide Whiskers 278

11.4.5 Viral Vectors 278

11.4.6 Laser Micropuncture 279

11.4.7 Nanofiber Arrays 279

11.5 The Rush to Publish 280

11.5.1 Controversial Reports of Plant Transformation 280

11.5.2 Criteria to Consider in Judging Novel Plant Transformation Methods 284

11.6 A Look to the Future 286

Life Box 11.1 Ted Klein 286

Life Box 11.2 John Finer 287

Life Box 11.3 Kan Wang 289

References 291

12. Analysis of Transgenic Plants 293
C. Neal Stewart, Jr.

12.0 Chapter Summary and Objectives 293

12.0.1 Summary 293

12.0.2 Discussion Questions 293

12.1 Essential Elements of Transgenic Plant Analysis 293

12.2 Assays for Transgenicity, Insert Copy Number, and Segregation 295

12.2.1 Polymerase Chain Reaction 295

12.2.2 Quantitative PCR 295

12.2.3 Southern (DNA) Blot Analysis 296

12.2.4 Segregation Analysis of Progeny 300

12.3 Transgene Expression 301

12.3.1 Transcript Abundance 301

12.3.2 Protein Abundance 302

12.4 Knockdown or Knockout Analysis Rather than Overexpression Analysis 304

12.5 The Relationship Between Molecular Analyses and Phenotype 305

Life Box 12.1 Hong S. Moon 305

Life Box 12.2 Neal Stewart 306

Life Box 12.3 Nancy A. Reichert 308

References 310

13. Regulations and Biosafety 311
Alan McHughen

13.0 Chapter Summary and Objectives 311

13.0.1 Summary 311

13.0.2 Discussion Questions 311

13.1 Introduction 311

13.2 History of Genetic Engineering and Its Regulation 313

13.3 Regulation of GM Plants 315

13.3.1 New Technologies 316

13.3.2 US Regulatory Agencies and Regulations 317

13.3.3 European Union 319

13.3.4 Canada 321

13.3.5 International Perspectives 321

13.4 Regulatory Flaws and Invalid Assumptions 323

13.4.1 Conventional Plant Breeding has Higher Safety than Biotechnology‐Derived GM 324

13.4.2 GMOs Should Be Regulated Because They’re GMOs and Un‐natural 324

13.4.3 Even though Product Risk is Important, It is Reasonable that Process (GMO) Should Trigger Regulation 324

13.4.4 Since GM Technology is New, It Might Be Hazardous and Should Be Regulated 325

13.4.5 If We Have a Valid Scientific Test, Then It Should Be Used in Regulations 326

13.4.6 Better Safe than Sorry: Overregulation is Better than Underregulation 326

13.5 Conclusion 327

Life Box 13.1 Alan McHughen 328

Life Box 13.2 Raymond D. Shillito 329

References 331

14. Field Testing of Transgenic Plants 333
Detlef Bartsch, Achim Gathmann, Christiane Saeglitz and Arti Sinha

14.0 Chapter Summary and Objectives 333

14.0.1 Summary 333

14.0.2 Discussion Questions 333

14.1 Introduction 334

14.2 Environmental Risk Assessment Process 334

14.2.1 Initial Evaluation (Era Step 1) 334

14.2.2 Problem Formulation (ERA Step 2) 335

14.2.3 Controlled Experiments and Gathering of Information (ERA Step 3) 335

14.2.4 Risk Evaluation (ERA Step 4) 335

14.2.5 Progression through a Tiered Risk Assessment 335

14.3 An Example Risk Assessment: The Case of Bt Maize 336

14.3.1 Effect of Bt Maize Pollen on Nontarget Caterpillars 337

14.3.2 Statistical Analysis and Relevance for Predicting Potential Adverse Effects on Butterflies 339

14.4 Proof of Safety Versus Proof of Hazard 340

14.5 Modeling the Risk Effects on a Greater Scale 340

14.6 Proof of Benefits: Agronomic Performance 341

14.7 Conclusions 342

Life Box 14.1 Tony Shelton 343

Life Box 14.2 Detlef Bartsch 344

References 346

15. Intellectual Property in Agricultural Biotechnology: Strategies for Open Access 347
Monica Alandete‐Saez, Cecilia Chi‐Ham, Gregory Graff, Sara Boettiger and Alan B. Bennett

15.0 Chapter Summary and Objectives 347

15.0.1 Summary 347

15.0.2 Discussion Questions 347

15.1 Intellectual Property and Agricultural Biotechnology 348

15.1.1 What is Intellectual Property? 349

15.1.2 What is a Patent? 349

15.2 The Relationship Between Intellectual Property and Agricultural Research 351

15.3 Patenting Plant Biotechnology: Has an Anti‐Commons Developed? 352

15.3.1 Transformation Methods 352

15.3.2 Selectable Markers 353

15.3.3 Promoters 354

15.3.4 Subcellular Localization 354

15.3.5 The Importance of Combining IP‐Protected Components in Transgenic Crops 355

15.4 What is Freedom to Operate (FTO)? 355

15.4.1 The Importance of FTO 355

15.4.2 FTO Case Study: the Tomato E8 Promoter 356

15.5 Strategies for Open Access 358

15.6 Conclusions 359

Life Box 15.1 Alan Bennett 360

Life Box 15.2 Maud Hinchee 361

References 363

16. Why Transgenic Plants Are So Controversial 366
Jennifer Trumbo and Douglas Powell

16.0 Chapter Summary and Objectives 366

16.0.1 Summary 366

16.0.2 Discussion Questions 366

16.1 Introduction 367

16.1.1 The Frankenstein Backdrop 367

16.1.2 Agricultural Innovations and Questions 367

16.2 Perceptions of Risk 368

16.3 Responses of Fear 370

16.4 Feeding Fear: Case Studies 372

16.4.1 Pusztai’s Potatoes 372

16.4.2 Monarch Butterfly Flap 373

16.5 How Many Benefits are Enough? 373

16.6 Continuing Debates 375

16.6.1 Process vs. Product 375

16.6.2 Health Concerns 375

16.6.3 Environmental Concerns 376

16.6.4 Consumer Choice 376

16.7 Business and Control 376

16.8 Conclusions 377

Life Box 16.1 Tony Conner 378

Life Box 16.2 Channapatna S. Prakash 379

References 381

17. The Future: Advanced Plant Biotechnology, Genome Editing, and Synthetic Biology 383
Wusheng Liu and C. Neal Stewart, Jr.

17.0 Chapter Summary and Objectives 383

17.0.1 Summary 383

17.0.2 Discussion Questions 383

17.1 Introduction: The Birth of Synthetic Biology 384

17.2 Defining Synthetic Biology for Plants 385

17.2.1 Design Cycles of Synthetic Biology 385

17.2.2 Foundations of Synthetic Biology 387

17.2.3 Components of Plant Synthetic Biology 388

17.3 Enabling Tools for Plant Synthetic Biology 389

17.3.1 Computer‐Aided Design 389

17.3.2 Synthetic Promoters 389

17.3.3 Precise Genome Editing 389

17.4 Synthetic Biology Applications in Plants 393

17.4.1 Synthetic Inducible Promoters 394

17.4.2 A Device for Monitoring Auxin‐Induced Plant IAA Degradation in Yeast 395

17.4.3 Circuits for Phytosensing of Explosives or Bacterial Pathogens in Transgenic Plants 395

17.5 Conclusions 397

Life Box 17.1 Joshua Yuan 397

Life Box 17.2 Wusheng Liu 398

References 399

Index 402