Plant Biotechnology

Plant Biotechnology

PROLOGUE

Plant biotechnology emerged as a new scientific discipline in the late 1980s after the achievement of gene transfer in plants and outcomes deriving from recombinant DNA technologies combined with basic sciences such as molecular biology, biochemistry, development and plant physiology. The book “Plant Biotechnology” introduces the reader to and explains the principles of plant biotechnology, and defines, describes and analyzes a wide range of applications. Through basic principles, it aims to introduce the research strategy and help to understand the results of this dynamic technology. It also discusses how basic as well as applied results can essentially have a direct impact on our own life and way of thinking.

After the impulsiveness of its youth, plant biotechnology as a discipline is still maturing in paths of thoughts and applications. This is due to new data arising from basic research and from various early applications. This experience has given this scientific discipline a mature experience based on given principles, so that any subsequent changes and applications will be wiser and sensitive to humanity and the environment.

While in the early days of plant biotechnology stable transformation was the core approach in generating transgenic plants, transient transformation was later found to be equally important and effective in producing valuable bioactive molecules that can be exploited for human health. The ease of transient transformation and gene expression may possibly have led to potential outcomes and applications being underestimated. A new revolutionary application based on targeted modification at the level of a single base of DNA was very recently found to give satisfactory results, with highly significant applications in plant biotechnology. The use of programmable endonucleases, together with the CRISPR/Cas9 system, could alleviate fears regarding genetic interventions.

Although the principles of using programmable endonucleases are mentioned, a thorough presentation of the results of this new approach appears in several different chapters of “Plant Biotechnology”, describing the challenges and the response of the scientific community to most issues, such as those that directly affect the environment.

The effectiveness of this technology is emphasized in response to rapid climate changes. Although on the face of it, it sounds like an oxymoron, the use of plant biotechnology to remediate environmental hazards is becoming a reality.

It may be early days still, but the combination of bioinformatics, systems biology and other basic plant sciences with biotechnology may lead us to synthetic plant biology that can take us to other horizons. It should be mentioned that this latter branch, biotechnology, and its applications, also encompasses an economic dimension that not only can generate growth but can also be artificially used to exert pressure. It can also play an essential role in the increasingly significant field of bio-politics. This uncontrolled mixture of economic dimension and bio-politics should be properly and more wisely applied. All are responsible in this endeavor. Each scientist comes from a defined cultural, philosophical and educational background, determining their morality. Science does not have a homeland, it educates people, and advances culture and philosophy.

Tedious and detailed protocols are avoided in this book. However, data and strategies are used to clarify key approaches and concepts, and to relate current achievements and results with future implications. At the same time, various biotechnological achievements are explained through a two-way relationship of the transfer of knowledge, and communication between different disciplines of biological sciences on the activity of various biomolecules and the function of different genes/proteins and that of plant biotechnology. Some examples are selected from the vast number that are mentioned in the literature to illustrate the essential points of plant biotechnology and to provide a solid foundation of knowledge in current research and future applications. The book serves the needs of a textbook for the corresponding course and can be used in different courses such as Plant Molecular Genetics, Modern Trends in Gene Manipulation, Molecular Ecophysiology, and Human Nutrition and Health. The topics developed and elaborated in the pages of this book can be understood by students who have already grasped basic concepts of molecular biology, genetics, biochemistry and plant physiology.

The book “Plant Biotechnology” refers to previous knowledge and elaborates on new dimensions and new approaches. It tries to convey basic knowledge and principles that govern plant biotechnology and through them to identify necessary and future applications. In addition to various application topics, special emphasis is placed on applications that have a direct impact on human health and nutrition, as well as on the environment.

This book is divided into eighteen Chapters. Chapter 1 serves as an introduction to molecular approaches and strategies for the isolation and analysis of genes that can then be used to transfer them into plants. Chapters 2 and 3 provide information about the Agrobacterium system and the vectors used for stable transformation. Various transformation approaches are also analyzed. Chapter 4 is a reference to the totipotency of plant cells and the regeneration of plants, as well as the use of plant cells as factories for the production of biomolecules, mainly secondary metabolites of high added value. Chapters 5-7 discuss the modes of abiotic transformation (transient or stable). Chapter 8 provides information about the use of molecular markers as a means for plant breeding, identification of cultivars and varieties, and authenticity of products, as well as the use of such molecular tools to accelerate genetic crosses. Chapter 9 discusses the importance of gene targeting and the transfer of genes to specific chromosomal loci without generating mutations. Chapter 10 provides features about the advancement of science that shifted methods towards holistic molecular approaches. It is now widely accepted that holistic approaches are the cornerstones in understanding life. Perpetuation of genetic information without additional new genetic material and targeted gene replacement with programmable endonucleases are also discussed in this Chapter. Chapter 11 provides more in-depth coverage about new molecular approaches and strategies to understand the multifaceted roles of proteins and genes and to appropriately design wiser plant biotechnology applications.

Chapters 12-14 extensively describe the achievements of plant biotechnology in generating plant resistance to herbicides, insects, bacteria, fungi and viruses by conventional transgenic strategies and via novel approaches to recover next-generation non-transgenic plants using programmable endonucleases. Accumulated knowledge about the complexity of plant disease resistance is also used to generate resistant plants to bacteria, fungi and viruses.

Chapter 15 establishes a solid bridge connecting plant biotechnology to the environment. There are examples of plant resistance to (a)biotic stresses based on accumulated data and knowledge while the use of transgenic plants in environmental protection (phytoremediation) is beginning to emerge. Chapter 16 focuses on human and animal nutrition and the use of transgenic plants in nutritional biofortification. It is clear that the growth of the population on Earth and nutritional trends drives the need for increased crop production and increased levels of nutritional quality. Chapter 17 thoroughly discusses the use of plants to produce bioactive molecules of high added value and biopharmaceuticals to fight human (and animal) diseases.

Chapter 18 covers bioethics, patents and regulations governing this technology and its extensions. Specific reference about targeted changes of bases of DNA is elaborated, so that the reader can contemplate these, without blinkers and clichés. This technology has a direct and dynamic impact on the social and cultural fabric of ethics while the economic as well as bio-political dimensions begin to affect all societies on our planet. Selected bibliography at the end of each Chapter is provided for those who would like to delve further.

I would like to express through these few lines my appreciation to all the people who worked and helped the Molecular Biology Laboratory. Over the years, young enthusiastic scientists have flourished and kept the momentum going for new endeavors. All of them, with the help of students, have had a catalytic impact on the writing of “Plant Biotechnology”. I will deliberately not mention specific individuals in case I forget somebody. I would like to believe that the time of all those who have passed through the laboratory will be engraved in their minds. The author would like to thank the editorial, production, marketing, and sales teams at EMBRYO Publications for their guidance in the preparation of this book and especially Stelios Vasiliadis for the excellent technical support and the appearance of the book. They have been helpful in all aspects of this project. Georgia Papaioannou deserves special mention for the production and the appearance of the book. Susan Coward for valuable advice and making specific editing changes to the language.

Finally I would like to express my gratitude and apologies to my wife Rania, to our two children Pari and Dimitris, and to our two grand-children Sonia and Janko for the years of imposition and neglect. Without their help, tolerance, patience and love, this book could not have been completed.

CONTENTS

PROLOGUE 7

CHAPTER 1 PRINCIPLES OF GENE MANIPULATION 21

1.1 Introduction 22

1.2 Basic Methods 23

1.3 Type II Restriction Enzymes 26

1.4 Other Enzymes Used in DNA Manipulation 27

1.5 Joining DNA Fragments 30

1.6 Plasmids 32

1.7 Genomic and cDNA Libraries 35

1.8 Bacteriophages 43

1.9 Other Vectors 48 

1.9.1 Cosmids 48

1.9.2. Yeast Artificial Chromosomes (YACs) 50

1.9.3 Bacterial Artificial Chromosomes (BACs) 53

1.10 Nucleic Acid Detection and Gene Identification 54

1.11 Nucleic Acid Hybridization and Detection 59

1.12 Screening Methods to Isolate Genes from a Library 63

1.12.1. Screening Depending on Quantity 64

1.12.2 Heterologous Hybridization 65

1.12.3 Antibodies Can Be Used to Isolate a Gene 67

1.12.4 Chromosome Walking 68

1.12.5 Screening of Libraries to Isolate Regulatory Genes 70

1.12.6 Isolation of a Gene Using Insertional Mutagenesis 70

1.12.7. Differential Screening - Subtractive Libraries - Differential Display 72

1.13 Gene Analysis 76

1.13.1 Dynamics of Gene Expression 77

1.13.2 DNA Sequencing 78

1.13.3 Structural Analysis of a Promoter 81

1.13.4 Functional Analysis and Promoter Dynamics 83

1.14 Analysis, Detection and Identification of Proteins 85

Selected Bibliography 89

CHAPTER 2 AGROBACTERIUM AND GENETIC COLONIZATION 91

2.1 Introduction 92

2.2 Infectious Plasmids 93

2.3 Phytopathogenesis 94

2.4 Properties of Crown Gall Cells 95 

2.5 Opines, Permeases and Types of pTi Plasmid 98

2.6 T-DNA Structure 102

2.7 Analysis and Gene Expression of the T-DNA Region 106

2.8 Function of the Genes from the T-DNA Region 110

2.8.1 Functional Analysis of the Genes in the T-DNA Region 110

2.8.2 Genes of Bacterial Origin that Promote Plant Growth 113

2.9 Genetic Colonization 114

2.10 Molecular Mechanism of T-DNA Transfer 115

2.10.1 Virulence Genes 115

2.10.2 Agrobacterium-Plant Cell Recognition 116

2.10.3 Mutual Recognition Between Agrobacterium and Monocotyledons 119

2.11 Regulation of Virulence Gene Expression 120

2.11.1 VirA and VirG 121

2.11.2 Interactions Between VirA, VirG and ChvE to Induce vir Genes 122

2.12 The T-DNA Transfer Trajectory 126

2.13 Bacterial-to-Plant Cell Transfer 130

2.14 Vir Proteins that Define the Host Range Are Not Absolutely Essential for Toxicity and T-DNA Transfer 134

2.15 T-DNA Entry into the Nucleus 135

2.16 Distribution of T-DNA Inserts in the Plant Genome 137

2.17 Molecular Analysis of T-DNA Insertion 138

2.18 Mechanistic Models of T-DNA Insertion 140

2.18.1 Vir Proteins Implicated in the Insertion of T-DNA 141

2.18.2 Single-stranded or Double-stranded Insertion Models 142

2.19 Identification of Transgenic Plants 146

Selected Bibliography 151

CHAPTER 3 VECTORS AND TRANSGENIC PLANTS 153

3.1 Introduction 154

3.2 The vir and T-DNA Regions Are Linked on the pTi Plasmid 155

3.3 Oncogenic Properties Can Be Uncoupled from T-DNA Transfer 155

3.4 The Cis or Co-integrating Vector System 157

3.5 The Binary Vector System 162

3.6 The pGREEN Binary Vector System 171

3.7 Selectable Marker Gene-free Vectors Are Commonly Used to Generate Transgenic Plants 173

3.8 Vectors Used in Abiotic Transformation 177

3.9 Selectable Marker Genes 180

3.10 Plant Selection and Resistance Mechanisms 183

3.11 Marker or Reporter Genes 183

3.11.1 β-Glucuronidase 185

3.11.2 Chloramphenicol Acetyltransferase 186

3.11.3 Luciferase 187

3.11.4 Green Fluorescent Protein 187

3.11.5 Other Reporter Genes 189

3.12 Empirical Knowledge Is Needed for the Transformation of Cultivated Plants 190

3.13 Plant Transformation with Oncogenic Wild-type Agrobacterium Strains 192

3.13.1 Choosing the Transformation Method by Using Wild-type Agrobacterium strains 193 

3.13.2 Selection of Explant Size. 194

3.14 Transformation and Transgenic Plants 195

3.14.1 Transformation of Tobacco Using Leaf Discs 197

3.14.2 Selection of Transgenic Plants Through Callus 197

3.14.3 Transformation of Small Homogeneous Plant Explants 199

3.14.4 Protoplast Transformation 199

3.14.5 Transformation of Root Explants 201

3.14.6 Floral Dip Transformation 201

3.14.7 Seed Transformation 202

3.15 Regulation of ‘Foreign’ Gene Expression in Transgenic Plants 204

3.16 Inducible, Tissue-specific and Developmentally-regulated Promoters 205

3.17 mRNA Leader Sequence 209

3.18 3’ Untranslated Region Sequences 210

3.19 Codon Usage and Intron Effect 210

3.20 Subcellular Targeting Sequences 212

3.21 Chloroplast Transformation 212

3.22 Artificial Plant Chromosomes 215

Selected Bibliography 217

CHAPTER 4 PLANT CELL AND TISSUE CULTURE 219

4.1 Introduction 220

4.2 Basics of Tissue Culture 220 4.3 Methods of Tissue Culture 222

4.4 Callogenesis 225

4.5 Somatic Embryogenesis 228

4.6 Micropropagation and Somatic Organogenesis 230

4.7 Protoplasts 234

4.7.1 Protoplast Isolation 234

4.7.2 Protoplast Culture 235

4.7.3 Protoplast Uses 236

4.8 Anther Culture 239

4.9 Synthetic Seeds 241

4.10 Plant Regeneration 243

4.11 Somaclonal Variation 243

4.12 In Vitro Production of Secondary Metabolites 245

4.13 Tissue Culture and Production of Healthy Virus-free Elite Germplasm 249

4.14 Mutations and Selection 251

4.15 Tissue Culture and Generation of Transgenic Plants 254

4.16 Tissue Culture and Transient Transformation 257

Selected Bibliography 258

CHAPTER 5 PLANT ELECTROPORATION 261

5.1 Introduction 262

5.2 Electric Pulses 263

5.3 Creation of Membrane Pores 264

5.4 Membrane Permeability to DNA 266

5.5 Bacterial Transformation Using Electric Pulses 269 

5.6 Electroporation of Plant Protoplasts 271

5.7 Protoplast Electroporation Parameters 274

5.7.1 Electric Settings 275

5.7.2 Protoplast Density and Other Parameters 278

5.8 DNA Electroporation and Homologous Recombination 280

5.9 Electroporation of Pollen 283

5.10 Transformation Through Tissue Electroporation 287

Selected Bibliography 289

CHAPTER 6 PARTICLE BOMBARDMENT 291

6.1 Introduction 292

6.2 DNA Transfer Through Bombardment 292

6.3 Gene Transfer in Plants Using Particle Bombardment 293

6.4 Principles of Particle Bombardment in Plants 295

6.5 Biological Principles in Transient Transformation 301

6.6 Biological Principles of Gene Transfer in Plants 304

6.7 Selection Following Bombardment 307

6.8 Pattern of DNA Insertion Using Bombardment 309

6.9 Bioballistic Co-transformation 310

6.10 Recovery of Transformed Plants 313

6.11 Bombardment of Dicotyledonous Plants 314

6.12 Bombardment of Monocotyledonous Species 315

6.13 Transformation Efficiency in Tree Species by Using the Gene Gun Delivery System 316

6.14 Bioballistic Approaches to Inducing RNA Silencing and Targeted Genome Editing 317

6.15 Gene Transfer in Mammals 318

6.15.1 Particle Bombardment and Gene Transfer in Mammalian Cells 318

6.15.2 Biotic Gene Transfer in Animal Systems 319

6.15.3 Transfection by Retrovirus, Adenovirus and Herpes-Simplex Virus (H-SV) 320

6.16 Particle Bombardment and Gene Therapy 321

6.17 Applications and Strategies in Gene Therapy 322

6.18 Genetic Immunization 324

Selected Bibliography 325

CHAPTER 7 CHEMICAL AND DIRECT PHYSICAL GENE TRANSFER 327

7.1 Introduction 328

7.2 PEG-Mediated DNA Uptake 328

7.3 Elimination of Additional DNA Copies 331

7.4 Efficiency of PEG-mediated DNA Transfer 334

7.5 PEG-mediated Transient Expression 335

7.6 Vectors Used in Transformation with PEG 339

7.7 Protoplast Fusion Using PEG 340

7.8 Microinjection 341

7.9 Microinjection of Protoplasts 343

7.10 Cell-penetrating Peptides and Cell Nanotransporters 346

Selected Bibliography 349 

CHAPTER 8 MOLECULAR BREEDING 351

8.1 Introduction 352

8.2 A Brief Overview of Molecular Markers 353

8.3 Features of Molecular Markers 354

8.4 Restriction Fragment Length Polymorphism (RFLP) 355

8.5 Rapid Amplified Polymorphic DNA (RAPD) 358

8.6 Amplified Fragment Length Polymorphism (AFLP) 360

8.7 Molecular Markers Based on Simple Sequence Repeats (SSR) and Short Tandem Repeats (STR) 364

8.8 Cloned Sequences Can Be Used as Markers 364

8.9 Molecular Markers Based on a SNP and on Insertions/Deletions (in/del) 366

8.10 Molecular markers and Identification of Pathogens 367

8.11 Advantages and Disadvantages of Using Molecular Markers 368

8.12 Applications of Molecular Markers 369

8.13 Identification and Discrimination of Different Varieties 370

8.14 Contribution of Marker-assisted Selection (MAS) 371

8.15 Strategies in Developing MAS 375

8.16 Molecular markers and Applications 376

8.16.1 Maize (Zea mays L.) 376

8.16.2 Barley (Hordeum vulgare) 377

8.16.3 Rice (Oryza sativa) 377

8.16.4 Soft Wheat (Triticum aestivum) 379

8.16.5 Tomato (Lycopersicon esculentum) 380

8.16.6 Soybean (Glycine max) 381

8.16.7 Bean (Phaseolus vulgaris) 382

8.16.8 Apple (Malus domestica) 382

8.16.9 Peach (Prunus persica) 383

8.16.10 Grapevine (Vitis) 384

8.16.11 Other Species 384

8.17 Selecting the Mapping Population 385

Selected Bibliography 387

CHAPTER 9 GENE TARGETING 391

9.1 Introduction 392

9.2 Recombination Events During Gene Targeting 393

9.3 Gene Replacement and Targeted Insertion 393

9.4 Models of DNA Recombination 395

9.5 Homologous Recombination in Plants 400

9.5.1 Extrachromosomal Homologous Recombination 401

9.5.2 Intrachromosomal Homologous Recombination 402

9.6 Methods of Gene Targeting 404

9.6.1 Gene Targeting Using Direct DNA Transfer 404

9.6.2 Gene Targeting Using Agrobacterium-mediated Transformation 404

9.7 Challenges in Gene-targeting Experiments 405

9.8 Precision of Gene Targeting 406

9.9 Gene Targeting Using Various Selection Schemes 407

9.9.1 Positive Selection Using PCR 407

9.9.2 Positive/Negative Selection 407 

9.10 Factors Affecting Gene Targeting in Plants 409

9.10.1 DNA-Transfer Method 409

9.10.2 Target Site and Genome Size 411

9.10.3 DNA Size and Sequence Similarity 411

9.10.4 Induction of Homologous Recombination Events 412

9.11 Future Prospects of Gene Targeting 413

Selected Bibliography 415

CHAPTER 10 HOLISTIC AND TARGETED MOLECULAR APPROACHES 417

10.1 Introduction 418

10.2 Next-generation Sequencing 419

10.3 Genomics 423

10.4 Transcriptomics 425

10.5 RNA Sequencing (RNA-seq) 428

10.6 Proteomics 431

10.7 Metabolomics 442

10.8 Epigenomics 443

10.9 RNA interference (RNAi) 448

10.9.1 Biogenesis of miRNAs 449

10.9.2 Biogenesis of siRNAs 451

10.9.3 Biogenesis of Secondary siRNAs 451

10.9.4 Silencing Mechanisms 451

10.10 Virus-induced Gene Silencing (VIGS) 454

10.11 Gene and Genome Editing and Site-specific Modifications 455

10.11.1 Zinc-finger Nucleases (ZFNs) 457

10.11.2 Transcription Activator-like Effector Nucleases (TALENs) 458

10.11.3 CRISPR/Cas9 460

10.12 Types of Gene and Genome Editing Through DNA Double-strand Breaks 466

10.13 Viral Vectors and Gene Editing 468

Selected Bibliography 471

CHAPTER 11 FUNCTIONAL ANALYSIS OF GENES AND PROTEINS 473

11.1 Introduction 474

11.2 Functional Complementation in Yeast 475

11.3 Functional Complementation in Homologous Systems 478

11.4 Protein-Protein Interactions Using the Yeast Two-Hybrid System 479

11.5 Multifunctional Protein Activities 484

11.6 cDNA Library Screening Using Protein-Protein Interactions 485

11.7 Functional biochemical pathways are transferred in yeast 487

11.8 Co-immunoprecipitation and Protein Interactions 489

11.9 Protein Interaction Network (Interactome) 491

11.10 Reporter Genes and Functional Analysis 492

11.11 Protein Localization in Different Cellular Compartments 494

11.12 Protein Co-localization 500

11.13 Split GFP 503

11.14 Electrophoretic Mobility Shift Assays (EMSA) 505

11.15 Chromatin Immunoprecipitation (ChIP) 506 

11.16 Heterologous Expression 508

11.17 Map-based Cloning 508

11.18 Targeting Induced Local Lesions in Genomes (TILLING) 514

11.19 Polymerase Chain Reaction (PCR) 516

Selected Bibliography 520

CHAPTER 12 HERBICIDE-RESISTANT TRANSGENIC PLANTS 523

12.1 Introduction 524

12.2 Weeds and Herbicides 525

12.3 Development of Herbicide-resistant Crop Plants 526

12.4 Biodegradation and Detoxification of Herbicides 530

12.5 Herbicide-detoxifying Enzymes of Bacterial Origin 530

12.6 Herbicide-detoxifying Enzymes of Plant Origin 535

12.7 Modification of the Herbicide Target 537

12.7.1 Glyphosate 538

12.7.2 Sulphonylureas 541

12.7.3 L-Phosphinothricin 542

12.7.4 Atrazine 543

12.7.5 Clethodim 545

12.7.6 Trifluralin 545

12.7.7 Synthetic Auxins 546

12.7.8 Fluridone 546

12.7.9 Lactofen 546

12.8 Mechanisms Conferring Non-Target-site Herbicide Resistance 547

12.9 Aspects Concerning Herbicide-resistant Transgenic Plants 549

12.10 Applications Using ZFN, TALEN and CRISPR/Cas9 551

12.11 Applications Using Modified Cas9 555

12.12 Advantages and Disadvantages in Generating Herbicide-resistant Plants Using Genome Editing 561

12.13 Effects of Genome Editing in Agriculture 562

Selected Bibliography 564

CHAPTER 13 PLANT RESISTANCE TO INSECTS 567

13.1 Introduction 568

13.2 Plant–Insect Interactions 569

13.3 Factors that Contribute to the Rapid Increase of Insect Populations 570

13.4 Plant Defense Mechanisms Against Insects 573

13.5 Bacterial Toxins 577

13.5.1 The Bt Toxin 578

13.5.2 Activity of Bt Toxin 580

13.5.3 Conditions Required for the Action of Bt Toxin 582

13.6 Cry Toxin Genes of Bacillus thuringiensis 584

13.6.1 Modifying Bt Gene Expression in Bacillus 585

13.6.2 Gene Stacking and Chimeric Toxins Made from Various Bt Gene Domains 585

13.6.3 Expression of Bt Toxin Genes in Other Microorganisms 587

13.7 Vegetative Insecticidal Proteins (VIPs) 588

13.8 Insect-resistant Transgenic Plants 589

13.9 Transgenic Plants Expressing Bt Toxins 591 

13.9.1 The Transfer of cry Genes in Plants 592

13.9.2 Modified cry Genes Result in Insect-resistant Transgenic Plants 595

13.9.3 Environmental Issues and Approaches 596

13.10 Transgenic Plants Expressing Different Insecticidal Proteins 601

13.10.1 Protease Inhibitors 601

13.10.2 Inhibition of α-Amylase 605

13.10.3 Oxidation of Cholesterol 606

13.10.4 Other Miscellaneous Inhibitors 606

13.11 Plant Resistance to Insects via RNAi 609

13.12 ZFN, TALEN, CRISPR/Cas9 Applications to Control Insect Pests 611

Selected Bibliography 616

CHAPTER 14 PLANT RESISTANCE TO VIRUSES, BACTERIA AND FUNGI 619

14.1 Introduction 620

14.2 Plant Resistance to Viruses 620

14.3 Viral Infection 622

14.4 Cross-protection of Plants Against Viral Infection 627

14.5 Transgenic Plants Resistant to Viruses 628

14.6 The Integration of Diverse Viral Genes in Plant Genomes Provides Plant Resistance to Virus 633

14.7 Antisense DNA Technology, RNAi and Resistance to Virus 634

14.8 Crosstalk Between Biotic and Abiotic Stress 638

14.9 Molecular Interactions Underlie Plant Resistance to Pathogens 638

14.10 Complex Plant Resistance to Pathogenic Diseases 642

14.11 Plant Resistance to Bacteria 643

14.12 Plant Resistance to Fungi 646

14.13 Plant Resistance Using ZFN, TALEN and CRISPR/Cas9 Programable Nucleases 652

14.14 Plant Resistance to Virus Using Gene-editing Approaches 658

14.15 Editing Susceptibility Host Genes and Resistance to Bacteria 660

14.16 Plant Resistance to Fungi Using Genome Editing 663

14.17 Limitations in the Development of Plant Resistance Using Programmable Nucleases and Future Perspectives 666

14.18 Bioethical Issues in the Development of Plant Resistance Using Programmable Nucleases 667

Selected Bibliography 669

CHAPTER 15 TRANSGENIC PLANTS AND ENVIRONMENT 671

15.1 Introduction 672

15.2 Enhancing the Absorption of Nutrients 673

15.3 Plant Biotechnology and Atmospheric Pollution 675

15.4 Pluralism in Productivity 676

15.5 Tree Improvement Using Antisense RNA 678

15.6 Phytoremediation 680

15.6.1 Mercury 682

15.6.2 Organic Compounds 684

15.6.3 Cadmium 684

 15.6.4 Ozone 685

15.7 Plants as Biomarkers for Environmental Contamination 685

15.8 Starch, Cell Wall and Biofuels 688

15.8.1 Biodiesel 689

15.8.2 Bioethanol 689

15.9 Photosynthesis 694

15.10 Plant Resistance to Salinity and to Drought 695

15.11 Multiple Response Pathways to Environmental Stress 703

15.11.1 Induced Gene Activation 704

15.11.2 Osmoprotectants 705

15.11.3 The Modulation of Signaling Pathways Can Confer High Plant Resistance to Stressors 706

15.12 Resistance of Transgenic Plant to Abiotic Stress.....................................................708

15.13 Use of ZFN, TALEN or CRISPR/Cas9 Technology in Generating Plant Tolerance to Abiotic Stress 710

15.14 Synthetic Approaches in Generating Plant Resistance to Abiotic Stresses 714

Selected Bibliography 715

CHAPTER 16 FOOD AND TRANSGENIC PLANTS 717

16.1 Introductio 718

16.2 Modification of Plant Metabolism 719

16.3 Storage Proteins 720

16.4 Amino Acids 723

16.5 Sugars 727

16.6 Starch Modification 729

16.7 Fatty Acids 730

16.7.1 Modification of Fatty Acid Composition 731

16.7.2 Increasing the Content of Fatty Acids 734

16.7.3 Polyunsaturated Fatty Acids 736

16.8 Transgenic Plants and Flavor 739

16.9 Transgenic Plants and Diet 741

16.10 Nutritional Biofortification 742

16.11 Antisense RNA and Co-suppression Approaches to Modifying Plant Physiology 744

16.11.1 Modification of Flower Color 744

16.11.2 Modification of Fragrance 747

16.12 Flower Meristem 748

16.13 Modification of Fruit Ripening Using Antisense RNA 749

16.14 Change in Fruit Appearance Using Antisense and RNAi technology 752

16.15 Reshaping Synthesis of Toxic Metabolites 753

16.16 Protection Scheme or Terminator Technology 755

16.17 Nutritional Improvements 756

16.18 Vitamins and Antioxidants 760

16.19 Yield Improvement 762

16.20 Gene Editing Using Programable Nucleases and Food Production 764

Selected Bibliography 768

CHAPTER 17 CONTRIBUTION OF TRANSGENIC PLANTS TO HEALTH 771

17.1 Introduction 772

17.2 Transferring Multiple Genes to Reconstitute a Complex Biochemical Pathway 773

17.3 Edible and Other Vaccines 774

17.4 Plants Used as Bioreactors and Molecular Agriculture. 780

17.5 Production of Secondary Metabolites With High Added Value 786

17.5.1 Saponins 787

17.5.2 Beneficial Lignans: Etoposide 789

17.5.3 Flavones: Wogonin and Baicalein 791

17.5.4 Artemisinin 791

17.5.5 Catharanthus 793

17.5.6 Oleosides and Olive 796

17.6 Transient Expression in Heterologous Plant Systems to Produce Biopharmaceuticals 797

17.7 Modifications of Plant Metabolism to Produce Secondary Metabolites 800

17.8 Production of Biomolecules and Antibodies Through Transient Expression 801

17.9 Programable Nucleases and Genome Editing in Producing Healthcare Biomolecules 809

Selected Bibliography 812

CHAPTER 18 BIOETHICS, PATENTS AND REGULATIONS, AND DIALECTICS 815

18.1 Introduction 816

18.2 New Technologies and Society 816

18.3 Biotechnology and the Evolution of Society 818

18.4 Biotechnology and Environment 819

18.5 Impact of Plant Biotechnology on Humans and the Environment 819

18.6 Plants, Animals and Humans 823

18.7 The Labyrinth of Patents Governing Biological Functions 825

18.8 Biosecurity and Biosafety 828

18.9 Bioethical Issues in Generating New Plant Varieties Using Programmable Nucleases .829

Selected Bibliography 832

INDEX I 833

Abbreviation of DNA nucleotide and degenerate bases or ambiguity during sequencing 833

INDEX II 834

Genetic code 834

INDEX III 835

Amino acids 835

INDEX IV 836

Type II Restriction Enzymes 836

INDEX V 843

Thermostable Polymerases for PCR 843

GLOSSARY 845

INDEX WORDS 859 

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