Plant Biotechnology

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Plan t Biotechnology :

t h e geneti c manipulatio n o f plant s

Second edition

Adrian Slater

Nige l W . Scott

Mar k R. Fowle r

De Montfort University

DAI HOC THAI NGUYEN

TRUNGTAM HOC LIEU

OXFORD

UNIVERSITY PRESS

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© Adrian Slater, Nigel W. Scott, and Mark R. Fowler 2008

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First edition 2003

Second edition 2008

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British Library Cataloguing in Publication Data

Data available

Library of Congress Cataloging in Publication Data

Slater, Adrian.

Plant biotechnology: the genetic manipulation of plants / Adrian Slater,

Nigel W. Scott, Mark R. Fowler. — 2nd ed.

p.; cm.

Includes bibliographical references and index.

ISBN 978-0-19-928261-6 falk. paper)

1. Plant biotechnology. 2. Plant genetic engineering. I. Scott, Nigel W.

II. Fowler, Mark R. III. Title.

[DNLM: 1. Plants, Genetically Modified—genetics.

2. Crops SB , Agricultural—genetics. ISBN 978-0-19-928261-6 Printed in Grea Typeset Britai t byTP248.27.P55S5 n Graphicraf b631.3*23—dc2 123.5 y200800336 Ashfor 7 S631 td Limited 2 9 Colou p2 20020088 r, HonPres ] sg Ltd. Kon,g Gosport, Hants.

3 3 7 9 10 8 6 4 2

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Foreword

The Green Revolution, led by Norman Borlaug, Monkombu Swaminathan, and

Gurdev Khush, enabled the world's food supply to be tripled during the last three

decades of the 20th century. The extraordinary increase in agricultural productivity

was made possible by the adoption of genetically improved varieties coupled with

advances in crop management. In many countries food supply increased faster than

demand and the technological progress contributed to a decrease in the unit cost of

production so that farmers were able to share the benefits of the advances with con￾sumers, by offering food to them at lower prices. Intensive (high-input/high-yield)

agriculture has served the populations of the developed countries well but two prob￾lems have come to occupy these people. First, the full-scale exploitation of intensive

agriculture protocols does deliver high yields of high-quality produce, but the envir￾onmental impact of the processes is often high. Second, the farmers have, in fact, the

need to dispose of surplus food so that there is a downward pressure on prices in the

world market that undermines farmers' incentives.

The position of the people in the low-income countries contrasts starkly with that

in the developed countries. The world's population has increased from 2.5 billion to

6.1 billion in the last 50 years and it is unlikely to stabilize before 2100 by which time

another 3 billion people will inhabit the planet. Most of the increase will occur in the

low-income countries where poverty and hunger are already widespread. Each night

800 million people go to bed hungry and suffer from malnutrition, and one-fifth of

humankind (about 1.2 billion people) lives on earnings of less than a dollar per day.

We must satisfy the need for more food in an environmentally friendly way but we are

confronted by major challenges. Prime agricultural land is being diverted to non￾agricultural uses to meet the growing demand from housing, urbanization, and

industrialization. Countries inhabited by half the world's population are already

experiencing water crises, while the high agrochemical inputs that maximize yields

exert a high environmental impact, which is not acceptable. There is a desperate need

to produce more food from less land with less water and reduced agrochemical

inputs.

The majority of agricultural scientists, led by Borlaug, Swaminathan, and Khush,

are convinced that the required crops of high yield, high quality, low cost, and low

environmental impact can be delivered by the exploitation of the techniques for plant

biotechnology in molecular breeding strategies. The commercial adoption of trans￾genic crops by farmers has been one of the most rapid cases of technology diffusion

Số hóa bởi Trung tâm Học liệu – ĐHTN http://www.lrc-tnu.edu.vn in the history of agriculture. Between 1996 and 2002, the area planted commercially

Foreword

with transgenic crops increased from 1.7 million to 58.7 million ha. Some 6 million

farmers in 16 countries grow transgenic crops and more than a quarter of such crops

are grown in developing countries.

The Norman Borlaug Institute was established to provide an international frame￾work for co-operation in development of molecular breeding strategies. The core

research activities provide a perfect environment for training students and research

scientists who will respond to the challenges and opportunities outlined above. This

book is based upon courses taught by the authors in The Norman Borlaug Institute.

The text defines the concepts and describes the technologies that enable the genetic

manipulation of crop plants. It describes in detail the development of the two traits

(herbicide and pest resistance) that are most prevalent in commercial genetically

modified (GM) crops and examines the reasons for their success. The potential for

developments in other crop traits such as disease resistance, abiotic stress tolerance,

and improvements of yield and quality are considered and the possibility of using

plants as factories for molecular farming is also explored.

The book is strengthened by confronting the wider social aspects of GM crops, and

several of the controversies surrounding this new technology are thoroughly aired.

The eco-terrorist fringe has constrained the development and exploitation of crop

biotechnology but the measured discussion in this book will enable readers to deal

with the self-serving campaigners whose actions undermine the undernourished,

while they themselves benefit from three full meals each day. Meanwhile someone

dies of starvation every 2.1 seconds.

Norman Borlaug himself is convinced that the world has the technology to permit

a population of 10 billion people to be sustained but he is concerned that farmers may

be prevented from exploiting the new technology by small, vociferous, well-financed

groups of anti-science zealots. These affluent campaigners can afford to pay high

prices for poorly regulated 'organic' food production. On the other hand the billion

chronically poor and hungry people already in the world cannot do so and the crisis

seems likely to grow as the population increases. The new technology described in

this text will be the salvation of the undernourished, freeing them from obsolete, low￾yielding, and more costly production technology.

Professor M.C. Elliott

Director

The Norman Borlaug Institute for Plant Science Research

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Preface t o the first edition

Plant biotechnology has made tremendous progress in recent years and has enjoyed a

previously unknown level of public awareness. Unfortunately, much of this awareness

has arisen from the negative publicity that surrounds genetically modified (GM)

crops. One only has to think of the media coverage of food safety issues (so-called

frankenfoods) or environmental concerns (the Monarch butterfly affair) to gain

some appreciation of the public antipathy to this technology. As a result, the GM

debate has been fuelled largely by misinformation, and has generated much more

heat than light. It is surprising that in the course of this debate, there has not been an

accessible textbook available to which serious students of the subject could turn for

information and understanding.

The aim of this book is to provide enough information and examples to give the

reader a sound knowledge of plant biotechnology in all its various guises, but particu￾larly those related to the genetic manipulation of crop plants. It is not intended to

provide an encyclopaedic coverage of the subject; such a task would require a volume

far larger than this. As such, this is a textbook, and therefore a learning and teaching

aid, rather than an academic treatise. Hopefully, the book also encourages a critical

appraisal of plant biotechnology: not just the scientific aspects, but also the eco￾nomic, social, moral, and ethical issues that surround and, some would say, plague

the subject. Although this book is aimed at undergraduate and postgraduate stu￾dents, we do not assume a huge amount of prior knowledge, and hope that other peo￾ple will find the book to be accessible, informative, and enjoyable. We hope that this

book will make a contribution to the GM crop debate, facilitating a rational exchange

of views between informed people.

The first four chapters of the book are designed to provide a more technical intro￾duction to subjects such as gene expression, tissue culture, and plant transformation

that enables the remainder of the book to be fully appreciated. They can, of course, be

read in their own right and contain information relevant to other areas of biology.

The rest of the book looks in detail at various aspects of plant genetic manipulation

applied to crop improvement. There are four chapters which deal in depth with the

application of plant genetic manipulation to agronomic traits (herbicide, pest, and

disease resistance), which can be considered as first-generation plant biotechnology.

Three of the remaining chapters deal with more challenging and diverse advances in

the areas of stress resistance, crop yield and quality, and molecular farming. It is in

these areas that GM crops have the potential to produce real, widespread social and

Số hóa bởi Trung tâm Học liệu – ĐHTN http://www.lrc-tnu.edu.vn economic benefits, particularly for developing countries. Chapter 12 attempts to give

Preface to the first edition

an overview of plant biotechnology, past, present, and future, with reference to the

legislative framework and economic, social, moral, and ethical issues.

Throughout the book, case studies are used as extended illustrative examples of

particular points that have been made in the main text. These are meant to be read as

part of the body of each chapter. However, background information for clarification

of advanced concepts, or more advanced information itself, is clearly presented in

boxes that can be read as and when required. There is, at the end of each chapter, a

Further reading section, which is not intended to be an exhaustive reference section,

but gives enough pointers to allow and encourage further investigation. Various web￾links to informative websites are also included in the further reading section. The

World Wide Web provides an invaluable resource for investigating plant biotechno￾logy, but care should be exercised when accessing information of dubious provenance.

We would, however, encourage the use of the excellent online-journal sites to enable,

in many cases freely, an interchange of knowledge and ideas, one of the cornerstones

of science.

There is a website associated with this book, which will contain hyperlinked chap￾ter web-links, further references, downloadable figures from the book, and update

sections. We hope that this website will help to keep the reader of this book fully

informed and up to date with developments in plant biotechnology.

We would like to thank all those who have contributed to the making of this book,

not least the people whose original investigations are the basis of this book. We apo￾logize unreservedly for any mistakes, all of which are ours, omissions or failure to

acknowledge fully. Finally we would like to thank our friends and families, in par￾ticular Arlene, lane, and Hilary, for their continued support and encouragement

throughou February 200t th3 e writing of this book.

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Preface t o the second edition

When we were invited by Oxford University Press to prepare this second edition, we

must admit to approaching the task with mixed feelings.

On the one hand, we have been gratified by the success of the first edition and the

complimentary reviews it has received. This confirmed our perception that there had

been a conspicuous lack of an accessible textbook on plant biotechnology at a time

when the field had reached a level of maturity that demanded one. It has been particu￾larly pleasing to note that the book has been popular not only in the USA (the home

of commercial genetically modified (GM) crops) and in Europe (where there is so

much antipathy to them), but also in the developing economies where the potential

value of this technology is widely appreciated.

On the other hand, the fact that we ourselves have been unable to exploit this book

fully in our own teaching has been a source of great regret. Shortly after publication

of the first edition, De Montfort University was one of a number of UK universities

that was compelled to close its Departments of Biological Sciences and Chemistry,

due in part to a lack of demand from UK school leavers to study science. This means

that we no longer have the opportunity to fully 'road test' the textbook by teaching an

entire module on plant biotechnology, although it is still of value in other areas of our

teaching.

De Montfort University also closed the Norman Borlaug Institute for Plant Sci￾ence Research in which we were based, and which Professor Elliott writes about so

passionately in the Foreword. The Institute was an ambitious venture to develop

high-yield/high-quality/low-cost/low-environmental-impact crops to address the

desperate need to increase world food production. The Institute faced insuperable

odds in trying to sustain its funding stream in a post-1992 UK university during the

era of public backlash against GM crops. One of the knock-on effects of the rejection

of GM food by the UK public described in Chapter 12 was the drying up of public and

industrial funding for research directly focused on crop improvement. For good or

ill, competitive plant science funding in the UK has been focused on the genomics

of model systems such as Arabidopsis (see Chapters 1 and 13). At the same time, the

distribution of central research funds to UK universities via the Research Assessment

Exercise meant that the dice was always loaded against an institute whose primary

mission was crop improvement for the benefit of mankind, rather than research

publications as ends in themselves. As a result, our teaching and research in plant

biotechnology now overlaps with other groups and departments in the university,

Số hóa bởi Trung tâm Học liệu – ĐHTN http://www.lrc-tnu.edu.vn including forensic science, biomedical science, pharmacy, and textiles, in areas such

Preface to the second edition

as food and medicinal plant authentication and the genetic modification of non-food

crops for pharmaceutical and fibre production.

One of the unforeseen consequences for us of no longer being directly involved in

GM food crop research is that we have been able to cast a more dispassionate eye over

progress in the past few years. It is striking, for example, that the rapid rate of adop￾tion of GM crops worldwide (a 60-fold increase in area grown in the first 11 vears of

commercialization) has not been matched by an increase in the number of GM traits.

The 'big two' traits - herbicide tolerance and pest resistance - still predominate, with

the most rapid advance being the development of stacked herbicide-/pest-resistant

crops. The predominant GM crop species also remain the major US commercial crops

(soybean, maize, cotton, and oilseed rape), rather than the most important world

food crops (rice and wheat). Of particular note is the fact that several of the import￾ant agronomic traits such as disease and stress resistance that we discussed at length

in the first edition appear to have foundered at the pre-commercialization stage.

Some of the more novel 'molecular farming' traits about which there was consider￾able excitement a few years ago have also still to make a commercial impact.

So what is new in the field of plant biotechnology that justifies a second edition?

There have been some important technological advances in the rationalization of

transformation vectors around the Gateway platform, and the clarification of the

miRNA mechanisms underpinning sense and antisense transgene silencing. However,

the major developments on the horizon are those that encompass 'post-genomics' in

their deeper understanding of biological systems and the effect of genetic modifica￾tion upon them. Indeed, some of the advances such as TILLING could be described

as 'post-GMics' in the sense that they point the way forward to selecting mutants

in targeted genes without GM technology. For this reason, we have written a new

chapter that reviews the current status of plant genomics and its potential impact on

crop improvement.

We have also taken this opportunity to improve the format and presentation of the

book. The use of colour has emphasized the structural components of chapters such

as boxes and case studies. It has also allowed us to show important features in the

figures and to highlight ke\vvords in the text. The wider margins also permit kev con￾cepts to be drawn out. We trust that these improvements will help to make the text

more attractive and accessible.

We would like to dedicate this second edition to all of our colleagues over the vears

who shared the Norman Borlaug Institute vision of harnessing plant biotechnologv

for humanitarian goals.

lanuary 2008

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Contents

List of abbreviations xix

1 Plant genomes: the organization and expression of plant genes 1

Introduction 1

DNA, chromatin, and chromosome structure 1

Chromatin

4

An introduction to gene structure and gene expression 6

Gene structure and expression in a eukaryotic protein-coding gene 6

Translation 10

Regulation of gene expression 16

Chromatin conformation 16

Gene transcription '6

RNA modification, splicing, turnover, and transport 18

Translation 20

Post-translational modification 21

Localization 21

Protein turnover 21

Conclusions 22

Implications for plant transformation 22

Examples of promoter elements used to drive transgene expression 26

Protein targeting 26

Heterologous promoters 26

Genome size and organization 27

Arabidopsis and the new technologies 28

Genome-sequencing projects—technology, findings, and applications 28

Biotechnological implications of the AGI 31

Crop plant genome sequencing 31

Summary 33

Further reading 34

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Contents

2 Plant tissue culture 37

Introduction 37

Plant tissue culture 37

Plasticity and totipotency 37

The culture environment 38

Plant cell culture media 39

Plant growth regulators 41

Culture types 44

Callus 44

Cell-suspension cultures 45

Protoplasts 46

Root cultures 46

Shoot tip and meristem culture 46

Embryo culture 46

Microspore culture 47

Plant regeneration 48

Somatic embryogenesis 48

CASE STUDY 2.1 Cereal regeneration via somatic embryogenesis

from immature or mature embryos 50

Organogenesis 51

Integration of plant tissue culture into plant transformation protocols 51

Summary 52

Further reading 53

3 Techniques for plant transformation 54

Introduction 54

Agrobacren'um-mediated gene transfer 54

The biology of Agrobacterium 54

TheTiplasmid 56

Ti-plasmid features 56

The process of T-DNA transfer and integration 59

Step 1. Signal recognition by Agrobacterium 60

Step 2 Attachment to plant cells 60

Step 3 Induction of vir genes 60

Step 4. T-strand production 60

Step 5 Transfer of T-DNA out of the bacterial cell 60

Step 6. Transfer of the T-DNA and Vir proteins into the plant cell and nuclear

localization 60

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Contents {

Practical applications of Agrobacter/um-mediated plant transformation 61

CASESTUDV3.1 /Agrobocter/um-mediated transformation of tobacco 62

Transformation 64

Direct gene-transfer methods 66

Particle bombardment 67

CASE STUDY 3.2 Biolistic transformation of rice 68

Polyethylene glycol-mediated transformation 72

Electroporation 73

Silicon carbide fibres: WHISKERS'" 73

Summary 74

Further reading 74

Vectors for plant transformation 77

Introduction 77

Desirable features of any plasmid vector 77

Development of plant transformation vectors 79

Basic features of vectors for plant transformation 79

Promoters and terminators 79

Selectable markers 86

Reporter genes 87

Origins of replication 91

Co-integrative and binary vectors 91

Families of binary vectors 91

Optimization 92

Arrangement of genes in the vector 95

Transgene copy number 98

Transgene position 98

Transgene features 98

Clean-gene technology 100

Summary 100

Further reading 101

The genetic manipulation of herbicide tolerance 105

Introduction 105

The use of herbicides in modern agriculture 106

What types of compounds are herbicides? 107

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Strategies for engineering herbicide tolerance

CASE STUDY 5.1 Glyphosate tolerance m

CASESTUDY5.2 Phosphinothricin 121

Prospects for plant detoxification systems 123

Commercialization of herbicide-tolerant plants to date 124

CASE STUDY 5.3 Engineering imidazolinone tolerance by targeted

modification of endogenous plant genes 126

The environmental impact of herbicide-tolerant crops 127

The development of super-weeds 129

Summary 130

Further reading 131

6 The genetic manipulation of pest resistance 133

Introduction 133

The nature and scale of insect pest damage to crops 134

GM strategies for insect resistance: the Bacillus thuringiensis

approach 134

The use of B. thuringiensis as a biopesticide 138

Bt-based genetic modification of plants 138

CASE STUDY 6.1 Resistance of Bt maize to the European corn borer

and other pests 140

The problem of insect resistance to Bt 141

The environmental impact of Bt crops 145

The Copy Nature strategy 146

CASE STUDY 6.2 Cowpea trypsin inhibitor 149

Insect-resistant crops and food safety 153

Summary 153

Further reading 153

7 Plant disease resistance 156

Introduction 156

Plant-pathogen interactions 157

Prokaryotes 158

Fungi and watermoulds 158

Viruses 160

Existing approaches to combating disease 160

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Natural disease-resistance pathways: overlap between pests a

diseases

Anatomical defences

Pre-existing protein and chemical protection

Inducible systems

Systemic responses

Biotechnological approaches to disease resistance

Protection against pathogens

Antimicrobial proteins

Transgenic crops for food safety

Induction of HR and SAR in transgenic plants

CASE STUDY 7.1 The BASF potato

Developments for the future

Other transgenic approaches

Future prospects for breeding

CASE STUDY 7.2 Xanthomonas spp.

Summary

Further reading

8 Reducing the effects of viral disease

Introduction

Types of plant virus

RNA viruses

Entry and replication: points of inhibition

How has the agricultural community dealt with viruses?

CASE STUDY 8.1 Developments in the sugar beet industry

The transgenic approach: PDR

Interactions involving viral proteins

CASE STUDY 8.2 Arabis mosaic virus

RNA effects

Some non-PDR approaches

CASE STUDY 8.3 DNA viruses

What has been commercialized in Western agriculture?

Yellow squash and zucchini

Papaya

Potato

Risk

Summary

Further reading

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Contents

9 Strategies for engineering stress tolerance 212

Introduction 212

The nature of abiotic stress 214

The nature of water-deficit stress 214

Different abiotic stresses create a water deficit 215

CASE STUDY 9.1 Glycine betaine production 218

Targeted approaches to manipulating tolerance to specific

water-deficit stresses 222

Alternative approaches to salt stress 222

CASE STUDY 9.2 Na*/H* antiporters improve salt tolerance in

transgenic plants 223

Alternative approaches to cold stress 224

CASE STUDY 9.3 TheCORregulon 224

Tolerance to heat stress 228

Secondary effects of abiotic stress: the production of ROS 229

Strategy 1: Expression of enzymes involved in scavenging ROS 232

Strategy 2: Production of antioxidants 234

Summary 234

Further reading 234

10 The improvement of crop yield and quality 237

Introduction 237

The genetic manipulation of fruit ripening 238

CASESTUDY 10.1 Thegeneticmanipulationoffruitsoftening 240

CASE STUDY 10.2 The genetic modification of ethylene biosynthesis 243

CASESTUDY 10.3 Modification of colour 247

CASESTUDY 10.4 Golden Rice 251

Engineering plant protein composition for improved nutrition 256

The genetic manipulation of crop yield by enhancement of

photosynthesis 258

Manipulation of light harvesting and the assimilate distribution: phytochromes 258

Direct manipulation of photosynthesis: enhancement of dark reactions 261

Summary 263

Further reading 263

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