The Effect of Compositional Traits on the Survivability

March 2004

The Effect of Compositional Traits on the Survivability and

Persistence of GM Crops

Final Report

GM Compositional Traits Final report

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Preface This study which reviews the development of genetically modified crops with altered compositional traits, and the effects the alterations have on the persistence and/or survival of the crops in the environment, has been produced as part of the Department for Environment, Food and Rural Affairs (Defra) Genetically Modified Organisms (GMO) Research Programme.

The study was conducted by Atkins Environment in association with staff from the Functional Genomics Group at the University of Bristol, the School of Plant Sciences at the University of Reading, and CNAP at the University of York.


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CONTENTS

EXECUTIVE SUMMARY VII

1. INTRODUCTION 1-1

Aims and Objectives of the Study 1-4

2. REVIEW OF CURRENT AND FUTURE APPLICATIONS OF GM CROPS WITH MODIFIED COMPOSITIONAL OR STRESS TOLERANCE TRAITS 2-5

Modified Protein Content 2-7 Introduction 2-7 Changing the amino acid composition of a single protein, or altering the effect of one or more existing plant proteins 2-8

Modification of wheat proteins 2-8 Modification of vegetative storage proteins 2-11 Allergens 2-11

Modified Carbohydrate Content 2-13 Modified starch 2-13

Introduction and overview of starch biosynthesis 2-14 Changes to starch quantity - modification of the enzyme ADP-glucose pyrophosphorylase 2-18 Changes to starch quantity - modification of sucrose degradation 2-21 Changes to starch quantity - modification of pyrophosphatase expression 2-22 Changes to starch quantity � modification of adenylate supply to the plastid 2-23 Changes to starch quality - modification of amylose:amylopectin ratio 2-24 Changes to starch quality - modification of starch lipid content 2-27 Indirect alteration of starch quality - modification of grain hardness 2-27 Side effect to starch modification (changes to quality and quantity) 2-29

Modified sugar 2-30 Modification of glucose and fructose content 2-30 Modification of hexose accumulation � alteration of post-harvest changes and inhibition of cold-induced starch degradation 2-32 Modified sucrose content 2-34 Modification of palatinose content 2-35

Modified fructan content 2-37 Modified cellulose 2-39 Modification of other cell wall polysaccharides 2-40 Modification of raffinose family oligosaccharides 2-41

Modified Lignin Content 2-43 Introduction 2-43 Shikimate pathway 2-48 Phenylpropanoid pathway 2-49

Modification of 4CL 2-50 Modification of C4H 2-51


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Modification of C3H 2-52 Modification of OMT and CCoAOMT 2-52 Modification of F5H 2-56

Lignin-specific pathway 2-58 Modification of CCR 2-58 Modification of CAD 2-59

Multiple gene manipulation and targeted gene manipulation 2-61 Modification of OMT and CCR 2-62 Modification of CCR and CAD 2-62 Modification of OMT, CCR and CAD 2-63

Enzymes involved in the regulation, final production and deposition of lignin 2-64 Peroxidases 2-65 Gibberellins 2-65 Laccase 2-66 Lignin regulatory factors 2-67

Conclusions and final thoughts on genetic manipulation of lignin biosynthesis and deposition 2-67

Modified Oil and Fat Content 2-68 Biosynthesis of fatty acids in plants 2-69

Note on fatty acid nomenclature 2-70 Genetic modification of fatty acid composition 2-74

Limitations of single gene modifications 2-76 Protein engineering 2-77 Increasing fatty acid yield 2-78

Modified oil content for food and cooking applications 2-78 Modified oil content for industrial applications 2-81

Wax production 2-84 Modified Micronutrient Content 2-86

Modified amino acid content 2-86 Modification of amino acid biosynthetic pathways 2-88 Modification of endogenous seed storage proteins 2-90 Modification using naturally occurring proteins 2-90 Modification using synthetic proteins 2-93

Modified fatty acid content 2-94 Modified flavonoid content 2-95 Modified vitamin content 2-97

Vitamin E 2-97 Vitamin A 2-98 Vitamin C 2-101

Modified mineral content 2-102 Iron 2-102

Production of Speciality Compounds for Medical Applications 2-104 Transgene expression strategies 2-106

Vaccines 2-110 Enteric disease vaccines 2-112 Hepatitis B virus 2-116 Measles vaccine 2-117 Malaria 2-118 Protection against autoimmune diseases and responses 2-119 HIV Vaccine 2-121 Lymphoma Vaccine 2-122 Caveat on production of vaccines in plants 2-124

Antibodies 2-124 Cancer Diagnosis and Therapy 2-125 Diagnostic antibody for crossmatching donors/receivers 2-127 Diagnosis and treatment of alloimmunisation or haemolytic disease of the newborn 2-128


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Antibodies for the Treatment of HIV 2-129 Biopharmaceutical Proteins 2-129

Modification of protein glycosylation 2-130 Anti-hypertensive protein 2-132 Human plasma proteins 2-132 Other medical proteins 2-133

Production of Speciality Compounds for Non-medical Applications 2-138 Use of Plants for the Production of Compounds with Agronomic Applications 2-139

Production of glucanase 2-139 Production of phytase 2-140 Modification of pest resistance 2-142

Use of Plants for the Production of Polyhydroxyalkonates (PHAs) 2-144 Biosynthesis of PHB in plant cytoplasm 2-145 Biosynthesis of PHB in the chloroplast 2-146 Biosynthesis of PHB in the leucoplast 2-147 Biosynthesis of PHBV in plants 2-147

Use of Plants for the Production of Spider Silk Protein 2-149 Use of Plants for the Production of Industrial Enzymes 2-150

Cellulase 2-150 Xylanase 2-151 Production of beer and spirits 2-151

Use of Plants for the Production of Collagen 2-152 Use of Plants for the Production of Squalene 2-153 Production of Trehalose as a Stabilising Agent 2-154 Use of Plants for the Production of Biofuels 2-154

Modified Plant Growth 2-156 Modified tolerance to drought stress 2-158

Improved drought tolerance using osmoprotectants 2-159 Osmoprotectants - modification of glycine betaine production 2-161 Osmoprotectants - modification of trehalose production 2-163 Osmoprotectants � modification of fructan production 2-165 Osmoprotectants � modification of D-ononitol production 2-165

Modified tolerance to salt stress 2-166 Improving salt tolerance through detoxification 2-167 Improving salt tolerance through homeostasis 2-169 Improving salt tolerance through growth regulation 2-170

Modified tolerance to cold stress 2-171 CBF pathway 2-174 Modification of cold-induced genes 2-176 LEA (late-embryogenesis abundant) proteins 2-176 Plant antifreeze proteins 2-177

3. ASSESSMENT OF THE EFFECTS OF COMPOSITIONAL CHANGES ON PERSISTENCE AND SURVIVAL IN THE ENVIRONMENT 3-179

Modified Protein Content 3-183 1,3-1,4-β-glucanase 3-183 Vegetative storage proteins (VSPs) 3-183

Modified Carbohydrate Content 3-184 Modified starch or sugar content 3-184

General effects of starch or sugar modification 3-184 Sugars as signalling compounds 3-185 Changes to AGPase 3-185 Changes to triose-phosphate phosphate translocator (TPT) 3-186 Increasing starch accumulation by reducing starch degradation 3-187


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Modification of starch branching enzyme 3-188 Modification of grain hardness 3-188 Modification of invertase enzymes 3-188 Modification of sucrose synthase activity 3-189 Modification of fructan content 3-189

Modified cellulose 3-189 Modification of pectic polysaccharides 3-189 Modification of raffinose family oligosaccharides 3-190

Modified Lignin Content 3-190 Modified Oil and Fat Content 3-192

Fatty acid modification and altered sensitivity to cold 3-194 Fatty acid modification and altered seed germination 3-195

High stearate oilseed rape � reported altered effects on seed performance 3-196 High laurate oilseed rape � reported altered effects on seed performance 3-197

Modified Micronutrient Content 3-198 Modified α-linoleic acid content 3-198 Modified flavonoid content 3-199 Modified iron content 3-199

Modification for Speciality Medical Applications 3-199 Persistence of the products of the genetic modification 3-200

Modification for Speciality Industrial (non-medical) Applications 3-201 Modification of pest resistance 3-202 Production of biodegradable plastics 3-203 Production of trehalose as a stabilising agent 3-203

Modified Plant Growth 3-204 Improved drought tolerance through accumulation of osmoprotectants 3-205 Improved salt tolerance 3-207

Detoxification 3-207 Homeostasis 3-207

Improved cold tolerance 3-208 Conclusions 3-209

4. COMMERCIALISATION OF GM CROPS WITH ALTERED COMPOSITIONAL TRAITS 4-218

Current situation in Europe 4-218 Within the next five years 4-219 In five to ten years 4-219 Beyond ten years 4-221

5. RECOMMENDATIONS FOR FUTURE WORK 5-222

6. REFERENCES 6-224

List of Tables Table ‎2.1 � Current understanding on the fatty acids, plants, enzymes

and gene regulation available for modification to alter the fatty acid composition in plants (adapted from Thelen and Ohlrogge, 2002 [147]) 2-73


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Table ‎2.2 - Fatty acid composition of oil from soybean seeds of different transgenic lines (adapted from Kinney, 1996 [149]) 2-79

Table ‎2.3 - Medical applications of transgenic plants 2-107 Table ‎2.4 � Examples of genetic modification to express osmoprotectants

in higher plants (adapted from [312]) 2-160 Table ‎3.1 � Summary of effects of modified compositional traits on the

persistence or survival of crop plants in the environment 3-210

List of Figures Figure ‎2.1 � Schematic representation of pathways leading to the accumulation

of starch in mesophyll cells (adapted from [34, 44, 46]). 2-17 Figure ‎2.2 � Schematic representation of pathways leading to the accumulation

of starch in storage cells (such as potato tubers) (adapted from Müller-Röber and Kobmann, 1994 [34]). 2-18

Figure 2.3 - Schematic representation of the lignin biosynthetic pathway 2-47 Figure ‎2.4 � Structure of triacylglycerol molecule [149]. 2-70 Figure ‎2.5 � Simplified scheme of plastid fatty acid synthesis (adapted from Thelen

and Ohlrogge, 2002 [147]) (see Table 2.1 for key to number references) 2-71 Figure ‎2.6 � Simplified scheme of reactions for modification of fatty acids in

oilseeds and their assembly into triacylglycerols (adapted from Thelen and Ohlrogge, 2002 [147]) (see Table 2.1 for key to number references) 2-72

Figure ‎2.7 - Biosynthetic pathway of the aspartate family of amino acids (adapted from Tabe and Higgins (1998) [170]). 2-89

Figure ‎2.8 � Schematic overview of the flavonoid biosynthesis pathway (adapted from Sevenier, 2002 [7]). 2-96

Figure ‎2.9 - Biosynthetic pathway for polyhydroxybutyrate-co-hydroxyvalerate (PHBV) in transgenic plants (adapted from Poirier, 2002 [298]). 2-148

Figure ‎2.10 - Synthesis of glycine betaine and its precursors in plants (adapted from Rontein et al., 2002) [312]. This pathway applies to almost all biological systems, although Arthrobacter sp. produce GlyBet through the N-methylation of glycine. 2-162

Figure ‎2.11 - Synthesis and metabolism of trehalose (adapted from Rontein et al., 2002) [312] 2-163

Figure ‎2.12 - CBF cold-acclimation pathway (adapted from Thomashow 2001) [343] 2-175

Figure ‎4.1 � Number of permits issued by USDA (and distribution of crops) for experimental field trials in the USA involving transgenic crops modified for the production of medical-related products (1991 to June 2002). Data taken from Lheureux et al. (2003) [4]. 4-220


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EXECUTIVE SUMMARY

Objectives of the Study

The purpose of this study is to review the current and potential future applications of genetic modification technology to change the compositional or key stress tolerance traits of crop plants, and to identify those plants and traits that may have a future as commercially grown varieties of crop plants. The second component of the report is an assessment of the effect(s) that such changes might have on the survival and/or persistence of the modified crops in the environment.

What are Modified Compositional Traits?

Compositional traits are those that define the content and/or quality of particular compounds produced by the plant. Most conventional (non-GM) plants grown today are cultivated for a characteristic compositional trait. Potatoes for example are grown for the starch produced in their tubers, with specific varieties cultivated to provide starch with particular processing or cooking properties; and different varieties of oilseed rape for example are grown for the different fatty acids produced in their seeds. Because compositional traits are characterised by the production of a particular substance or compound they are also described as �output� traits.

The modification of compositional traits in crop plants has been ongoing since man first started cultivating wild varieties of plants. The purpose of this report is to review the developments in compositional trait modification that have been, are, or may be brought about through the use of genetic modification. These developments involve either the alteration of existing compositional traits, such as the modification of oilseed rape to produce different fatty acids; or alteration of plants to produce new compounds such as pharmaceuticals or industrial enzymes.

To date (2003), the large majority of genetically modified (GM) crop plants reported to be under cultivation worldwide are modified for changes in agronomic traits such as pest resistance or tolerance to specific herbicides. These traits, also described as �input traits� as they affect inputs to the crops (chemicals, manpower and time), are the key characteristics of the so called �first generation� of GM crops to be developed.

GM crops with altered compositional traits represent the �second generation� of transgenic crop plants, and offer potentially many more applications to agriculture, the environment, human health and industry than the �first generation� crops. As recognised by the recent


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Cabinet Office report1, GM crops with modified compositional traits will expand significantly the range of transgenic crops relevant to UK agriculture over the next 10-15 years.

Which Stress Tolerance Traits?

Stress tolerance traits are those that improve a plant�s ability to withstand adverse environmental conditions such as drought, high salt content and freezing. Whilst stress tolerance traits are defined as �input� traits they have been included in this report because of the significant effect they should, by design, have on the persistence or survival of the plant in the environment. The focus of this report on modifications to drought, salinity and freezing reflects the relevance of these stresses to UK and European agriculture.

Scope of the Report

The report is divided into three parts: a review of current and future applications of GM crops with modified compositional and key stress tolerance traits; an assessment of the effects of those modifications on the persistence and/or the survival of the plant in the environment; and a review of possible future commercialisation of GM crops with altered compositional or key stress tolerance traits.

Information to compile the review of current and future GM compositional and stress tolerance traits has been taken from peer-reviewed journals and reports, as well as various patent databases. The review presents plants and traits at various stages of the development process required for the production of GM crops with the required agronomic characteristics. These range from those where only the gene of interest has been identified, to those that have undergone field trials or are grown commercially.

Modifications with the most immediate commercial future are those that have been expressed in plants such as oilseed rape, potatoes, maize or rice, whose non-GM varieties are already cultivated widely. Such traits include altered starch or fatty acid content in potatoes, oilseed rape and soybeans; and the production of phytase in oilseed rape2. However, in many cases the modifications and the plants described are unlikely to have a commercial future and represent just one of the many stages of development required for the production of GM crops with the required agronomic characteristics. This is particularly true for studies involving the plant Arabidopsis thaliana which is not grown as a crop plant, but has been well-studied at a genetic level and is therefore often used in the early stages of trait identification development. The use of plants such as tobacco may also indicate that the development programme is at an early stage. Tobacco is relatively easy to modify genetically and is often used in the development programme prior to the modification of the target crop plant. Where the modification reported is unlikely to have a future in an agricultural context (because the plants used are not appropriate, or the modification does not have the desired effect, or the modification causes unwanted adverse effects) this point is made clear in the text.

1 Cabinet Office Strategy Unit (2003). Weighing up the costs and benefits of GM crops. Available at www.strategy.gov.uk 2 Already commercially available under the trade name Phytaseed®.


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In addition to an evaluation of the potential future of the modification in terms of whether it may be used agriculturally, as assessment is also made as to whether the modification will alter the persistence or survival of the transgenic crop in the environment. Because the modification of compositional traits can involve the alteration of key characteristics of the plant, such as lignin content, levels of storage compounds such as starch, changes to sugars (which play key roles as plant signalling compounds), and the composition and formation of seeds (with subsequent effects the number of progeny produced and the seed germination and development), then the alteration of survival or persistence is the key area of environmental risk assessment for such GM plants.

Changes to stress tolerance traits are by design likely to improve the ability of the plant to persist or survive in the environment, although behaviour of the plant in a non-stressed environment, for example one with good water availability, is an important consideration in assessing the overall improvement in persistence or survival. (Issues such as pollen dispersal or hybridisation with wild relatives are the same as those for other GM plants and have therefore not been addressed in this report3).

Modifications Reviewed

The review of modified compositional and key stress tolerance traits is presented in eight sections, divided according to the purpose of the modification. This division was felt to be more useful to users of the report than if it were divided according to crop or specific gene. The groups of modifications addressed are:

• modified protein content � to include changes to total protein and changes in specific amino acids. Examples include the modification of glutenin proteins in wheat for altered bread-making characteristics, and the reduction in levels of specific allergens such as AraH1 in peanuts;

• modified carbohydrate content � to include changes in starch, sugars, fructan, cellulose and other compounds. The modification of starch involves altering both starch quantity and starch quality (amylose:amylopectin ratio), and the low-calorific value of fructans means these compounds have potentially a very significant commercial market as fat replacers in spreads and ice-creams;

• modified lignin content � this has applications to both silviculture (forestry) and fodder crops such as maize as changes to their lignin content affects their digestability and therefore nutritional benefits to livestock. Most of the modifications related to lignin alteration involve attempts to reduce the lignin content. Removal of lignin is one of the most energy intensive components of paper manufacture, and therefore reduced-lignin wood may offer a more energy efficient feedstock for the paper industry. Increased lignin content though has also been reported, with hyperlignified wood offering a more efficient renewable fuel source;

3 Such characteristics would of course be addressed as part of the assessment process conducted by the regulatory authorities should any crop with modified compositional traits be submitted for experimental release or commercial planting.


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• modified oil and fat content � has applications to the food industry in the production of oils with altered cooking properties; to the �nutraceutical� industry in the production of less saturated fats and low cholesterol oils; and the non-food industry for the production of agents for use in paints, varnishes and inks;

• modified micronutrient content � addresses the alteration of plants to increase existing levels (or produce new forms) of compounds reported to have particular benefits to both human health and animal husbandry. Modifications include changes to amino acid content, fatty acid levels and production of flavonoids, as well as the production of vitamins (E, A and C) and increased levels of minerals such as iron. This section also includes a review of the �golden rice� designed to improve levels of vitamin A, and described by the plant biotechnology industry as a key example of a beneficial application of GM plants;

• production of speciality compounds for medical applications � such as vaccines (hepatitis B, malaria and measles for example), antibodies (for cancer diagnosis and therapy) and biopharmaceutical proteins (human plasma proteins, anti-hypertensive proteins). The use of plants in this area is currently very dynamic field of investigation with an increasing number of products and applications reported. As a consequence of the significant number of reports published in this area, this report has focused on those produced in the last few years rather than attempting to address all work to date. The use of plants as the �production unit� is often replacing existing microbially-based fermentation production systems as plants offer a much cheaper approach. Initial commercialisation of such plants is likely to be in glasshouses rather than in the field, although field-based cultivation has been proposed for some of the plants and products reported. The toxic nature of many of the compounds that may potentially be produced does however have implications to the growth of such plants in a non-contained field environment;

• production of speciality compounds for non-medical applications � includes the modification of plants for altered pest resistance and the production of compounds used as animal feed supplements such as glucanase and phytase; as well as the use of plants to produce biodegradable plastics, spider silk protein, collagen and squalene. The use of GM plants to produce squalene for example has potential environmental benefits as this compound (used in the cosmetics industry) is sourced currently from shark�s liver. Plants such as oilseed rape and soybean modified to produce phytase are already grown commercially (although not in the UK), and are described as the most advanced example (in terms of commercial application) of a system designed to improve animal feed diets; and

• modified plant growth � reviews those modifications designed to have a direct effect on the plant�s tolerance to the key environmental (abiotic) stresses of water availability, salt levels and low temperature (including freezing). Many of the modifications reviewed are at an early stage of development, but do in most cases offer the potential to improve agronomic performance of the plants in environments


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where drought, high salt content and low temperature occur. As traditional breeding approaches are reported to have had limited recent impact on improving stress tolerance, the application of genetic modification techniques may provide a key pathway to improve plant characteristics in this area.

Potential Effects on Persistence and Survival

Information on the potential effects of the modified compositional trait on persistence and/or survival has been taken, where available, from studies reported in the scientific literature. However, for many of the modifications reported, investigations into the potential changes to persistence and/or survival have not been conducted, with the studies focusing on agronomic performance or, as in the case of the modifications at an early stage of development, the stability and success of the genetic modification.

Therefore, information to support the assessment has also been taken from studies involving non-GM plants. Whilst such studies are not applicable for evaluation of the effects that the expression of non-plant products (such as vaccines) may have on persistence or survival, information derived from conventional (i.e. non-GM) plant studies on the effect of expression of high levels of stearic acid (fatty acid 18:0) on seed performance are for example applicable to transgenic crops modified for the same purpose.

Key plant traits that, if modified, are likely to have an effect on the survival or persistence of the plant are:

• growth rate;

• seed production;

• germination time;

• seed dormancy;

• pest resistance;

• decay resistance;

• herbivory;

• flowering time;

• development of storage structures;

• overwintering survival;

• drought tolerance;

• high salt tolerance;

• low temperature tolerance.

Modification of any of these traits, either directly or indirectly is expected to alter the persistence or survival of the GM plant in the environment. A summary of the effects identified is presented in Table 1 at the end of this executive summary. A minority of the modifications to compositional traits identified are assessed as having a direct effect on the persistence or survival of the plant (i.e. altered persistence or survival is a consequence of the intended purpose of the modification) , with the majority of the changes to persistence or survival identified as being a consequence of indirect effects of the modification. Some modifications to both compositional and stress tolerance traits are reported to have both direct and indirect effects on the persistence or survival of the transgenic plant.

Examples of direct effects include the alteration of pest resistance (positive effect on survival), reduction in cellulose content (negative effect on survival and persistence) and the


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reduced expression of AGPase which causes reduced plant growth and flower development (negative effect on survival). Such direct effects are probably easier to assess as they are more likely to be identified being an intended consequence of the modification.

Indirect effects are often less immediately obvious. In some cases they may only become apparent under particular environmental conditions and this makes them harder to identify and assess. The greater induced seed dormancy that occurs in oilseed rape modified to express high levels of stearic acid for example only occurs under conditions of high temperature, high nutrients and full light; or low nutrients and darkness.

Modifications to stress tolerance traits are by design likely to have a positive effect or persistence/survival, although the occurrence of adverse phenotypic effects, or poor behaviour of the plant when not under the particular stress may negate the positive effect.

What is more difficult to assess is the significance of the identified changes to persistence or survival. Modifications that result in reduced starch content in potato for example are proposed as having a negative effect on persistence and survival as the changes will reduce the energy reserves required by the plant to survive overwinter and to initiate growth at the start of the following growing season. However, what is not known is the level of reduction in starch levels that can be sustained by the potato before if its overwintering survival is impaired. Understanding the significance of such changes will though require field trial studies.

Commercialisation of GM Crops with Modified Compositional Traits?

GM plants with modified compositional traits are described as a key component of the �second generation� of GM crops. Although many of these crops are 10-15 years away from commercial cultivation, a limited number being grown commercially now in countries such as the USA and Canada4. Within the EU, the Amylogene potato (C/SE/96/3501)5 modified for altered starch content (high amylopectin levels) has received favourable approval from the EC�s Scientific Committee on Plants pending authorisation under the old Directive EC 90/220/EEC. This plant may therefore be the first GM plant with a modified compositional trait (other than the pest resistant plants) available for commercial cultivation within the EU.

Within the next five years more potatoes modified for altered starch content, oilseed rape modified for altered oil content and tomatoes with altered fruit ripening characteristics are predicted to be available commercially in the EU (although possibly as imported products rather than cultivated crops). The other modifications reviewed in this report (including the modified stress tolerance traits) are however predicted to take in excess of ten years to reach commercialisation, should they prove to have no adverse agronomic or environmental effects, and complete the required regulatory approvals process.

4 Such as the oilseed rape modified to produce phytase. 5 Identification number designated by the EC for this potato.


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Table 1 � Summary of effects of modified compositional traits on the persistence or survival of crop plants in the environment General purpose of

the modification Gene or enzyme modified Crop6 Effect observed Assessed effect on persistence or survival in the field

Protein content Increased production of 1,3-1,4-β-glucanase

Potato Reduced tuber yield and adverse cell wall morphology.

Negative (i.e. the modification will reduce the ability of the plant to persist or survive in the environment). In this case the reduction is due to diminished energy stores.

Protein content Reduced formation of vegetative storage proteins (VSPs)

Soybean No effects looked for, and therefore none reported.

Proposed to cause a negative effect on plant persistence or survival.

Starch or sugar content

Sucrose-phosphate synthase Tomato Increased yield. Positive (competitive advantage) or negative (greater exposure to herbivory).

Sugar content Increased sucrose isomerase (resulting in reduced sucrose levels). Crops modified using constitutive and phloem-specific promoter.

Tobacco and potato

Sever retardation in growth (tobacco) and severe impairment in tuber sprouting (potato).

Negative (reduced growth and development).

Sugar content Increased sucrose isomerase. Tuber specific promoter.

potato No adverse effects observed Negative due to reduction in starch levels. This is assessed to reduce survival of the plant due to the importance of starch as an energy storage material.

Sugar content Increased fructan accumulation through expression of sacB gene from B. subtilis

Sugar beet Improved drought tolerance Positive effect in areas with low water availability. No significant effect under non-drought conditions.

Starch content Expression of Sh2r6hs (increased AGPase activity)

Wheat and rice Improved CO2 fixation Positive due to improved photosynthesis and therefore improved plant growth.

Starch content Increased AGPase activity Wheat and rice Reduced seed abortion Positive due to greater numbers of seeds produced per plant.

6 Refers to the crop in which the modification has been reported. Depending on the trait(s) altered the modification may be applicable to other crops. Where a crop is not listed (for example as in the case of lignin modification), then the modification and its associated reported effect is applicable to a wide range of plants.


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General purpose of the modification Gene or enzyme modified Crop Effect observed Assessed effect on persistence or

survival in the field Starch content Decreased AGPase activity Potato Smaller but greater number of

tubers and earlier flower formation

Negative or positive. The effects on survival depend whether early flowering will provide a competitive advantage, and whether a reduction in tuber size will reduce overwintering survival of the tubers (and therefore next season�s growth).

Sucrose modification Decreased activity of triose-phosphate translocator

Potato Increased growth rate during the dark period

Positive or negative. Increased growth rate may confer a competitive advantage but may also make the plant more prone to herbivory.

Starch content Expression of yeast invertase, bacterial glucokinase or bacterial sucrose phosphorylase

Induction of glycolysis and greater partitioning of carbon into respiration. No increase in starch accumulation

Negative due to reduction in starch accumulation and therefore reduced energy storage capacity in the plant.

Starch content Expression of inorganic pyrophosphatase enzyme

Potato Accelerated sprouting Positive or negative. If sprouting occurs too early in the season then conditions may be too cold or wet for the potatoes to survive. Otherwise, the modification should improve survival as it will provide a competitive advantage.

Starch quality Inhibition of starch branching enzyme

Potato Reduced tuber yield and decreased total starch content


Starch quality Increased expression of pinA and pinB

Maize and rice Increased grain hardness (direct effect), and improved resistance to specific fungal pathogens (indirect effect)

Positive (reduced herbivory and greater fungal resistance).

Sucrose content Reduced expression of the invertase enzyme β-fructosidase

Tomato Reduced fruit size and increased ethylene production (causing faster fruit ripening)

Negative (smaller fruits and shorter time for the fruits to remain on the plant. Therefore seed dispersal by herbivores will be reduced).

Sucrose content Inhibition of sucrose synthase Tomato Reduced fruit setting Negative � modification assessed to reduce seed development.


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survival in the field Cellulose content Not reported Increased or decreased

cellulose content Positive if cellulose increased (due to improved resistance to decay). Negative if cellulose decreased (due to reduced plant strength and greater herbivory).

Pectic polysaccharide content

Expression of eRGL (confers overproduction of rhamnogalacturonan lyase

Potato Reduced pollen fertility and formation of smaller, more wrinkled tubers

Negative due to reduced ability of the plant to produce fertile pollen and a reduction in energy stores (smaller tubers).

Raffinose family oligosaccharide content

Reduced production of raffinose family oligosaccharides (RFOs)

alfalfa Reduced ability to deal with cold and drought

Negative due to reduced resistance to environmental stresses (cold and drought).

Lignin modification Any change to lignin biosynthetic pathway

Reduction in lignin content (direct effect)

Negative (reduced resistance to adverse weather, attack by pests and herbivory).


Reduction in lignin content (indirect effect)

Negative due to reduction in overwintering capability because of possible link between the genes controlling lignin synthesis and those involved in winter survival traits [1]

Lignin modification Inhibition of phenylalanine ammonia lyase (PAL)

Reduction in levels of phenolic compounds

Negative (due to the role of phenolics in pest resistance and therefore greater susceptibility of the plant to pests).

Lignin modification Inhibition of CCoAOMT and O-methyltransferase (OMT)

Reduction in plant growth and flowering activity

Negative due to reduced plant growth.

Lignin modification Alteration of levels of CAD, CCR or OMT

Increased decay. Greatest effect observed with OMT

Negative (plant will persist for less time in the environment once it has died).

Lignin modification Inhibition of hydroxycinnamoyl CoA ligase (4CL)

Arabidopsis thaliana and aspen

Collapsed cell walls and stunted growth in Arabidopsis but elevated growth rate in aspen. Only a limited number of plants were used in the aspen study [2] and the results should be viewed with caution.

Negative (in the Arabidopsis) but positive in the aspen.


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survival in the field Lignin modification Downregulation of caffeoyl CoA

O-methyltransferase (CCoAOMT)

Tobacco and poplar

Reduced cross-linking in the lignin

Negative due to reduced strength of the lignin. This would make the plant more susceptible to adverse weather events, and possibly also to decay, pest attack and herbivory.

Lignin modification Downregulation of hydroxycinnamoyl CoA reductase (CCR)

Indirect effects likely to occur. Positive or negative depending on the nature of the indirect effect. Due to the involvement of CCR in a range of plant processes a number of effects could occur.

Oil or fat modification Any alteration of membrane fatty acids

Reduced level of seed germination. Altered membrane fatty acids will also affect the plant�s response to temperature change (and other membrane transport processes, thereby causing a range of adverse effects).

Negative due to reduction in seed germination and therefore development of the plant.

Oil or fat modification Production of a fatty acid not normally produced by the plant

Poor sequestration of the novel fatty acid into membrane lipids

Negative due to reduced ability to deal with changes in environmental conditions (temperature and water availability).

Oil or fat modification Increased production of lauric acid (12:0)

Oilseed rape Delayed and a reduced level of germination. Once germinated the plants exhibit increased growth rate 2-4 weeks post emergence. Effects only observed under conditions of low temperature (10°C).

Negative. This assumes that the increased growth rate is not sufficient to compensate for the reduced germination levels.

Oil or fat modification Reduced expression of fad2 leading to increased production of oleic acid (18:1)

Arabidopsis sp., soybean and oilseed rape. Similar effects were not seen in sunflower

No survival at temperature <6°C. The effect though is plant and gene specific, and depends on the level of inhibition that occurs.

Negative if the plant is to survive overwinter. The effect is only of relevance to winter sown or perennial crop plants.


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survival in the field Oil or fat modification Increased production of stearic

acid (18:0) Oilseed rape Greater induced dormancy and

reduced germination of the seeds. Effects only observed under conditions of high temperature, high nutrients and full light; or low nutrients and darkness.

Negative due to reduction in germination, but positive if the conditions are sufficient to cause an increase in induced dormancy.

Modified micronutrient (linoleic acid) content

Reduction in level of fatty acid α-linoleic acid (18:2∆9,12)

Reduced resistance of the plant to plant pathogens and reduced pollen development.

Negative.

Modified micronutrient (flavonoid) content

Reduced flavonoid content Reduced resistance to plant pathogens and decreased UV protection.

Negative.

Modified micronutrient (iron) content

Expression of ferritin gene Lettuce Enhanced growth during early development.

Positive unless the larger plants are more attractive to herbivores.

Production of compounds for medical applications

Production of the antigen merozoite surface protein (MSP1)

Tobacco The plant flowered but did not set seed.

Negative due to reduced seed formation.


Expression of ribosome inactivating proteins (RIPs)

Potato Inhibition of protein synthesis and leaf rolling at the point of infection.

Negative due to impaired leaf development.

Modified pest resistance

Expression of resistance to beet necrotic yellow vein virus (BNYVV)

Sugar beet Reduced rate of bolting Negative. This assumes that reduced bolting will lead to reduced seed dispersion from the plant.


Expression of Bt toxin Sunflower Increased seed production (up to 50 percent). Effect only observed in the field environment in the presence of lepidopteran pests.

Positive. The observed effect is an indirect knock-on effect of the reduction in pest attack. It was not observed in a glasshouse environment (in the absence of the pest) and is therefore not a genetic consequence of the modification.


Expression of any pest resistant characteristic

Improved pest resistance (direct effect).

Positive, but only of relevance in environments containing the pest(s).


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survival in the field Modified pest resistance

Expression of Bt toxin Maize Increased lignin content Positive. This is also an indirect effect of the production of the Bt toxin. Unlike the previous example it is probably due to expression of the cry gene affecting other plant processes and is therefore viewed as a genetic consequence of the modification.

Production of biodegradable plastic

Expression of polyhydroxyalkonates (PHAs)

Reduced growth and development. However, the effects have only been observed during early stages of the modification process. The most recently developed plant exhibited no adverse phenotypic characteristics.

Negative where impaired growth occurred.

Improved drought tolerance through accumulation of any osmoprotectant

Any gene or enzyme leading to increased accumulation of an osmoprotectant

Improved tolerance to drought and high salt levels. May confer further tolerance to other environmental stresses such as low temperature.

Positive if the environmental stress is realised. Zero or possibly negative effect in environments where the stress is not realised. If the trait is expressed constitutively then the likelihood of a negative effect is higher in non-stressed environments.

Improved drought tolerance through accumulation of glycine betaine

Expression of the Arthrobacter enzyme COX

Production of hydrogen peroxide Negative if the H2O2 has a deleterious effect on the plant. Such effects were however not reported by the study.


Expression of betA from E. coli or codA from Arthrobacter

Oilseed rape and tobacco

Accumulation of glycine betaine, and improved tolerance to salinity, freezing and drought.

Positive effects in environments where drought, high salt or freezing temperatures are encountered.

Improved drought tolerance through accumulation of trehalose

Constitutive expression of the gene encoding TPS enzyme

tobacco Some accumulation of trehalose, but also stunted growth (poor leaf and root development), altered sugar metabolism and altered fertility.

Increased tolerance to drought, but the adverse morphological effects mean that persistence/survival is likely to be reduced.


xx


survival in the field Improved drought tolerance through accumulation of trehalose


Tobacco Better accumulation of trehalose than with expression of TPS, but adverse morphological effects still realised.

Negative, although the stunted plants did exhibit greater drought tolerance.


Constitutive expression of the otsB gene from E. coli (encoding TPP enzyme)

Tobacco Low level accumulation of trehalose, but heightened drought tolerance. Plants exhibited substantial changes in morphology and accumulated higher levels of non-structural carbohydrates.

Negative due to changes in plant morphology.

Improved drought tolerance through accumulation of fructan

Constitutive expression of sacB gene from Bacillus subtilis.

Tobacco and sugar beet

Increased fructan accumulation and improved drought tolerance. Plants exhibited more rapid growth rate.

Positive under drought conditions due to great drought tolerance. Increased growth rate is also likely to confer a selective advantage over other plants. Not known whether any changes are expressed when no drought stresses are imposed.

Improved drought and salt tolerance through accumulation of D-ononitol

Expression of cDNA encoding D-myo-inositol methyltransferase.

Tobacco Increased accumulation of D-ononitol. Photosynthetic fixation of CO2 was inhibited to a lesser extent under conditions of drought or salt stress.

Positive under conditions of drought or salt stress.

Improved salt tolerance

Expression of mutant version of pst1 gene

Arabidopsis

Improved tolerance to salt.

Positive under conditions of high salt levels. Plants likely to have improved tolerance to other abiotic stresses (including drought).


Overexpression of AtSOS1 Arabidopsis


Positive under conditions of high salt levels.


Overexpression of AtNHX1 Arabidopsis, tomato and oilseed rape

Improved tolerance to salt. fruit loss when exposed to salt stress.

Positive under conditions of high salt levels. No adverse effects on plant growth or phenotype reported.


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survival in the field Improved salt tolerance

Overexpression of HLA gene from yeast

Watermelon and tomato

Improved salt tolerance. Modified plants expressed less fruit loss when exposed to salt stress.

Positive under conditions of high salt levels. Greater fruit production is assessed to confer a selective advantage to the long-term survival (over several generations) of the plant. Greater fruit production may of course result in increased herbivory and therefore result in reduced survival of individual plants in the environment.

Improved salt tolerance through accumulation of mannitol

Expression of mt1D gene from E. coli in the chloroplast

Tobacco Increased accumulation of mannitol and improved tolerance to salt. Under non-stressed conditions the plants were 20-25 percent smaller than wild type.

Positive under conditions of high salinity, but probably negative under non-stressed conditions due to the smaller size of the plants.

Improved stress tolerance through accumulation of proline

Modification of natural proline inhibition systems

Mothbean and Arabidopsis

Increased accumulation of proline. Plants exhibited greater tolerance to salt and also cold. No adverse phenotypic effects reported.

Positive under conditions of high salt or low temperature.

Improved drought tolerance through accumulation of proline

Expression of P5CS gene from mothbean

Tobacco Overproduction of proline, and also enhanced root biomass and flower development.

Positive under drought conditions. The enhanced root growth and flower development are also likely to confer a selective advantage to the modified plants.

Improved salt tolerance through accumulation of sorbitol

Expression of a cDNA for sorbitol-6-phosphate dehydrogenase (S6PDH) from apple

Tobacco Increased accumulation of sorbitol (0.2-130 µmol g-1 fw). Plants expressing >3 µmol g-1 fw developed necrotic lesions on their leaves, infertility, and/or the inability to regenerate roots.

Positive under conditions of salt stress if the level of expression is restricted to <3 µmol g-1 fw. Higher levels of expression are likely to have a negative effect on persistence/survival.

Cold tolerance in the absence of low temperature stimulus

Constitutive overexpression of CBF/DREB

Arabidopsis Severe stunting of phenotype, decrease in seed yield and a delay in flowering.

Negative effect on persistence or survival.


1-1

1. INTRODUCTION

1.1 The large majority of the genetically modified (GM) crops reported to be under cultivation worldwide are modified for changes in agronomic traits such as improved tolerance to herbicides or greater resistance to insect pests [3, 4]. These so called �first generation� transgenic crops [5] consist predominantly of GM maize, soybean and cotton modified for altered herbicide tolerance or pest resistance [6, 7] and account for the majority of those GM crops assessed to date for release in the UK and EU for research trials or commercial cultivation. Whilst these �first generation� GM crops are reported to provide improved efficiencies of production and therefore potentially reduced costs [3], the transgenic traits employed are designed predominantly for the improvement of agronomic properties [4].

1.2 The �second generation� of GM crops7 [8, 7] now in development include a much wider range of modified traits and involve plants other than the bulk commodity crops such as maize, cotton and soybean [6]. These �second generation� crops include those modified for changes in compositional or quality traits such as flavour, starch content, protein or vitamin levels and oil production. Because such traits are designed to provide a particular novel substance or product they have been described as �output traits�, whereas the herbicide tolerance or pest resistance traits have been labelled �input traits� as they are concerned primarily with affecting inputs to the crop (chemicals, manpower and time) [9, 4]. The modification of plants to alter their response(s) to environmental stress(es) or to increase yields is referred to as the alteration of an �input� trait [4]. However, because the modification of tolerance to environmental stress(es) is likely to have such a significant effect on the persistence and/or survival of the modified crops in the environment, then a review of these modifications has been included in this report for completeness.

1.3 The range of compositional traits that could potentially be altered or changed is limited only by the level of understanding and identification of the genes and plant processes involved. Evaluation of the types of GM plants and their modifications being submitted for regulatory approval worldwide demonstrates the increased level of interest and research in plants with altered quality traits. For example, in Canada in 1994, 72 percent of all field trials of GM crops were for plants with modified herbicide tolerance. In 1998, herbicide tolerant GM crops accounted for just 24

7 Defined as those without selective antibiotic resistance markers (Dunwell et al., 2001).


1-2

percent of the GM crops undergoing field trials [3]. The large biotechnology companies have stated that they expect the development of GM crops to focus more on those with �output� traits [10, 4].

1.4 The purpose of this study is to review the range of modifications that are or may be used to alter the compositional and key stress tolerance traits of crop plants grown in the UK and EU, with particular attention being made to any modifications that may alter the persistence or survival of the GM crop in the environment.

1.5 The report is presented in two sections. The first section is intended to review current and future applications of GM crops with modified compositional or stress tolerance traits and provide an indication of the commercialisation potential of that particular trait. The information for the review has been compiled from peer-reviewed scientific publications, from patent databases and from discussions with researchers.

1.6 Some of the compositional modifications reported have to date only been investigated in plants such as Arabidopsis thaliana. Although such studies are important in identifying the genes and biosynthetic or regulatory pathways involved, the modification is only likely to have a commercial future (i.e. be used by farmers or growers on an agronomic scale) if it can be applied to widely used crop plants such as oilseed rape, potatoes, maize, soybean or rice.

1.7 For each of the modifications or groups of modifications addressed in this report a statement has been made (in italics at the end of the relevant section) as to the possible commercial future of the modification(s) described. The purpose of this statement is to provide the reader with an indication of whether crops modified with that particular trait might be seen in field trials or in commercial cultivation in the near future, or are a longer term development currently at the early stages of research.

1.8 The second section of the report assesses the information available on the known or potential effects that changes to compositional traits may have on persistence and/or survival of the GM crop in the environment. As with the review in the first section, information for the risk assessment has been obtained from the scientific literature and where possible includes details on both direct and indirect effects of the modified compositional traits on the potential persistence and survival of the GM plant in the environment.

1.9 As a footnote to this study, it is recognised that the use of GM crops in the UK and some parts of the EU has been the subject of intense public and political interest. Much of this interest has resulted from both a lack of information and a misunderstanding of the information available on the uses of GM crops and the potential implications to the environment and human health. Whilst this study will focus entirely on the scientific issues involved, and is not intended as a forum for the


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review of the advantages and disadvantages of genetic modification and GM crops, it is recognised that the public can only benefit from access to current, objective and comprehensive reviews of the application of GM crops and the likelihood of adverse effects to the environment being realised.


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AIMS AND OBJECTIVES OF THE STUDY

1.10 The aims of the study are to review the current and potential future applications of genetic modification to change the compositional or stress tolerance traits of crop plants, and to assess the effect(s) that such changes might have on the survival and/or persistence of those crops in the environment.

1.11 The aims of the study have been addressed through completion of the following objectives:

• identification and review of existing and potential future applications of genetic modification to alter compositional or stress tolerance traits of crop plants either as the target alteration or as a side effect. The review has covered the different crop species and the genes involved, as well as the relevant donor organisms and promoter and terminator sequences; and

• review of the information available on the known or potential effects that changes in compositional or stress tolerance traits may have on the persistence and/or survival of the GM crop in the environment, and an assessment of the risks posed to the environment by GM crops with altered compositional or stress tolerance traits with respect to changes in their persistence and/or survival.


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2. REVIEW OF CURRENT AND FUTURE APPLICATIONS OF GM CROPS WITH MODIFIED COMPOSITIONAL OR STRESS TOLERANCE TRAITS

2.1 The purpose of this section of the report is to review the current and future applications of genetically modified (GM) crops with modified compositional or stress tolerance traits. For the purposes of the review, the current and future applications of such GM crops have been presented as eight sub-sections divided according to the intended purpose of the modification, rather than by crop species or specific gene. Although such an approach is not clearcut (some modifications may for example be viewed as relevant to several sections), it is intended as a means to address what is a very diverse and rapidly developing area of research and development.

2.2 The eight principal groups of modifications that are covered in the review are:

• modified protein content � to include alterations of total protein and changes in specific amino acids. Examples include the modification of cereals for improved bread-making by increasing levels of glutenin present [11], and the reduction of levels of specific proteins with known allergenic properties, such as AraH1 in peanuts [12];

• modified carbohydrate content � to include alterations in starch and sugar content for both food and industrial applications;

• modified lignin content � to review modifications of lignin content in plants. Although this is of relevance predominantly to silvicultural applications, changes in lignin composition of other crops such as maize have applications for altered processing properties of the plant;

• modified oil and fat content � to include alterations for both food and industrial applications, such as the production of increased levels of plant sterols and stanols for low cholesterol margarines, and the formation of fatty


2-6

acids with conjugated double bonds which have applications as drying agents in paints, varnishes and inks;

• modified micronutrient content � such modifications are usually designed to provide improved health or nutritional characteristics of the plant and include for example rice modified to express higher concentrations of vitamin A or tomatoes altered to express increased concentrations of particular flavonoids [13];

• production of speciality compounds for medical applications � such as recombinant blood factors and vaccines;

• production of speciality compounds for non-medical applications � such as cellulase, biodegradable plastics, spider silk and chymosin; and

• modified plant growth � to review those modifications designed to have a direct effect on the plant�s tolerance to the key environmental (abiotic) stresses of water availability, salt levels and low temperature (including freezing).


2-7

MODIFIED PROTEIN CONTENT

Introduction Changing the amino acid composition of a single protein, or altering the effect of one or more existing plant proteins

Modification of wheat proteins Modification of storage proteins Allergens

Introduction

2.3 Almost all of the GM crop modifications addressed in this review involve alterations to proteins or amino acids in one form or another. The purpose of this section of the report (Modified Protein Content) is to review those modifications that specifically affect proteins, but do not affect any of the other groups of modifications covered by the report.

2.4 Applications that involve the modification of proteins, but that are designed specifically to alter �non-protein� properties of the plant, are reviewed in subsequent sections of this report. Examples of such modifications include:

• the manipulation of the gene encoding for the enzyme cinnamyl alcohol dehydrogenase which alters the lignin characteristics of woody plants (reviewed in the �Modified Lignin� section);

• modifications of amino acid content to improve methionine content in potatoes (reviewed in the �Modified Micronutrient Content� section); and

• the use of transgenic plants for the production of foreign proteins (reviewed in the �Production of Speciality Compounds for Medical Applications� and the �Production of Speciality Compounds for non-Medical Applications� sections).

2.5 Modification of protein can be approached either from the perspective of changing the amino acid composition, altering the effect of one or more plant proteins that already exist within the plant, or from the concept of adding an entirely new protein to a plant that occurs naturally in a plant belonging to a different genus, or in an organism belonging to an entirely different phylum or kingdom.

2.6 The review of the modifications for altered protein content in this section of the report has been divided into two parts:

• genetic modification of existing plant proteins; and


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• genetic modification to produce xenogenic (non-plant) proteins in plants.

2.7 With respect to the modification of plants to produce xenogenic proteins, plants are able to offer a number of advantages over existing protein production systems (namely microbial and animal cell cultures). Plants offer the potential to produce xenogenic (and existing plant) proteins in large quantities and at relatively low costs8 [14] The technology and expertise to grow plants in large numbers of course already exists and therefore, assuming the modified plants are no different in terms of agronomic traits to their non-transgenic relatives, the cultivation of the GM crops at a commercial scale should be straightforward.

2.8 Modifications that are addressed in this section of the report include modifications for the production of industrial enzymes for use in agriculture; transformation of wheat so that it expresses high molecular weight glutenins, the modifications of plant proteins to reduce allergenic traits and the modification of storage proteins.

Changing the amino acid composition of a single protein, or altering the effect of one or more existing plant proteins

Modification of wheat proteins

2.9 In wheat, the gluten proteins form the major storage proteins in the grain. These proteins determine the viscoelastic properties of dough (the balance between extensibility and elasticity) that allows it to be used for bread-making and in other baking applications [11, 15]. It has now been shown that the manipulation of the high molecular weight (HMW) subunits of glutenin is possible in transgenic plants [16] with resultant benefits in baking quality [12].

2.10 It is thought that the difference in the number of HMW subunits and in the properties of subunits expressed, accounts for between 45 and 70 percent of the variation in bread-making performance within European varieties of wheat [15]. The addition of further HMW subunits for example has been found to improve the dough-making characteristics of wheat [12].

2.11 An example of this comes from Blechl and Anderson (1996) [17] who expressed a HMW subunit gene (comprising of a fusion between the Dy10 and Dx5 genes) under the control of the native HMW subunit promoter and other flanking sequences in a wheat cultivar. The transgenic plants were shown to synthesise substantial amounts of hybrid seed storage protein, demonstrating the feasibility of manipulating wheat

8 The costs of growing GM crops are low both in terms of start-up and running costs. Although the cost of microbial fermentation are also low, such technology usually requires significant start-up costs.


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storage proteins. The effect of these changes in the amount of HMW protein on dough strength was however not assessed.

2.12 The effect of changes in HMW subunits on dough strength was investigated by Barro et al. (1997) [11]. They found that the genetic transformation of wheat with two additional HMW subunit genes led to increased dough strength, with a quantitative difference in dough strength being dependent on the number of transgene products expressed. In both cases the expression of the HMW genes (1Dx5 and 1Ax1) was driven by their own endosperm-specific promoters [11].

2.13 The studies described above investigated the expression of HMW subunit genes in non-commercial cultivars of wheat. The next stage of development was to express HMW subunit transgenes in cultivars in which conventional (non-GM) selection for bread-making quality has already resulted in high dough strength and the expression of four to five HMW subunit genes. It was anticipated that the expression of one or more additional genes encoding for HMW subunit proteins should raise the total amount of HMW subunit protein above the current ceiling of ~10 percent of total flour proteins, resulting in further increases in bread-making quality [11].

2.14 The effects of the expression of a HMW subunit gene (1Ax1) in a commercial cultivar of wheat (Bobwhite) was investigated by Vasil et al. (2001) [18]. As with previous studies, the increased levels of HMW subunits seen in the transgenic wheat resulted in higher quality dough for bread-making [18]. The transgenic Bobwhite was also shown to express 1Ax1 in addition to the existing five HMW subunit genes that it expresses naturally. This is the first reported example of the stable expression of more than five HMW subunit genes within a cultivar of wheat. However, Vasil et al. (2001) [18] did not look at whether this expression increased the HMW protein above the current highest level of 10 percent of total flour proteins. The third generation transgenic lines and a non-transgenic line were planted in a field trial, with analysis of the fourth generation seeds showing that 1Ax1 was stable under field conditions [18]. A strong negative correlation was noted between total protein level and grain yield. This is a well-known agronomic relationship [18] and is not specifically related to the introduction of this transgenic construct.

2.15 Vasil and Vasil have a patent [19] covering the Bobwhite wheat cultivar transformed to express 1Ax1 HMW subunit gene described. There are at least two other existing patents relating to the alteration of the properties of wheat dough by the genetic modification of the HMW glutenins [20, 21].

2.16 Shewry et al. (2002) [15] conducted a comparative study on the effect of the transformation of wheat with different HMW subunit genes and concluded that different HMW subunit genes may have fundamentally different effects on gluten structure and properties and that these were unpredictable in terms of their


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expression levels, structures and interactions with other HMW subunit proteins. However, a recent patent has been issued that covers certain modified HMW glutenins and their genetic code. The authors of the patent application claimed that the modified HMW glutenins had a predetermined and predictable effect on the viscoelastic properties of dough when expressed in GM wheat [20]

2.17 None of these experiments have looked explicitly at the effect that the changes in HMW glutenins may have on the persistence/survival of the plant; apart from the negative relationship between total protein level and grain yield noted by Vasil et al. (2001) [18]. It is unclear whether this genetic change would have any effect on persistence or survival of the transgenic plant, compared with the wild-type.

2.18 A study by Barro et al. (2002) [22] was designed specifically to address the agronomic performance of transgenic wheat plants (modified to express HMW genes) under field conditions, compared to the unmodified parent wheat lines. The study was conducted over two years and whilst it found differences for important agronomic traits between the GM and non-GM lines, these differences were either not statistically significant in the trial as a whole, or were significant for one year and not the other, meaning that the variation was most likely due to changes in the environment and weather conditions [22]. As a consequence of the small scale nature of the trials, any changes to individual plants caused by the environment or weather were therefore likely to have a greater effect on the results than if the same changes occurred in a larger trial.

2.19 The findings from the study also raised the issue of the effect of somaclonal variation9 as a cause of some of the differences observed. Changes occurring as a consequence of somaclonal variation are well documented for tissue culture [22], but have also been reported to occur following particle bombardment (Bregitzer et al. 1998) and cell electroporation (Arencibia et al. 1999), albeit at a lower level (consensus of) [23-26, 22]. Changes caused by somaclonal variation may be eliminated from the crop by backcrossing the transgenic lines by their parents and selecting for the desired genotype [22].

2.20 It is likely that such GM plants will be subject to further field trials to determine the effects on yield and persistence/survival. The findings from Barro et al. (2002) [22] reported comparable (and possibly higher) agronomic traits and yields between the transgenic and non-transgenic lines. If predictable changes can be made to GM

9 Somoclonal variation is the change(s) that occur at the chromosomal level during the regeneration of plant tissue. In theory, the regeneration of tissue explants into a mature plant should result in the production of clones of the parent plant. However, genetic mutations and chromosomal breakage(s) or rearrangements may occur during regeneration resulting in a mature plant that is not a complete clone of the plant from which the tissue was obtained.


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dough viscosity using certain HMW subunit genes, such GM plants could progress through to commercialisation.

Modification of vegetative storage proteins

2.21 It is recognised that the term �storage protein� usually refers to seed storage proteins, such as the vicilins and legumins [27]. The modification of such proteins is addressed elsewhere in this report, including for example the modification of the peanut allergenic protein discussed in the following section. This section addresses the modification of vegetative storage proteins (VSPs) in soybeans, which have been proposed to be involved in the sequestration of nitrogen and other nutrients during early plant development [28].

2.22 The two VSPs, VSPα and VSPβ in soybean have been investigated through the production of a transgenic soybean expressing a VspA antisense gene construct. The modification resulted in no differences in productivity between the transgenic plant and the wild-type, under typical growth conditions. This could be due to compensation through the increase of other proteins or non-protein N [28]. Since there was no change in productivity, it was suggested that the VSPs could be altered in either their amino acid composition to improve their nutritional content, or in other ways, without deleterious effects on persistence and survival for the plant, at least under typical growth conditions. However, VSPs may play an important role when the plant is under stress and such manipulation may affect the growth of the plant when under stress [28].

2.23 Such modifications may be best applied to the modification of nutritional content (see section on modified micronutrient content) rather than manipulating the quantities of the storage protein per se.

Allergens

2.24 It is estimated that eight percent of children under three years of age and two percent of adults are affected by food allergies. These occur through the hypersensitive response of the person�s immunoglobulin E (IgE) to particular allergens that express IgE binding sites [29]. The most severe food-induced allergic reactions are caused by peanuts and tree nuts [30], and has resulted in work to identify and characterise the allergens involved [29]. Some of the proteins that cause this allergenicity have been identified and it has been proposed that genetic modification may be used to reduce allergenicity in nuts; for example, through the manipulation of the genes encoding the allergenic proteins [30]. The identification of a high sequence similarity between the seed storage glycinin proteins of several species of legume [29] means that studies with one plant such as soybean may be applicable to peanuts for example.


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2.25 The feasibility of using transgenic techniques to reduce the levels of allergens in foodstuffs has been demonstrated by the successful reduction of the allergenic 14-16 kDa protein in rice (Nakamura and Matsuda, 1996; cited by [12]). A similar approach is now being adopted to reduce the levels of one or more of the allergenic proteins (e.g. AraH1) in peanut. The probability of success of this strategy has increased recently following the detailed analysis of the specific allergenic domains within the protein [31] together with the development of reliable transformation techniques for this crop [12].

2.26 The identification of other peanut allergenic proteins (namely AraHII) and their potential use for the detection, quantification and genetic manipulation of peanut allergens in foods has also been the subject of patents (e.g. [32]).

2.27 Similar work has been carried out for the walnut, with the identification of the gene encoding the major allergen in the English Walnut (Jug r 1) and the subsequent identification of the specific allergenic domains within this protein and its core amino acid residues [30]. Work has also been reported in the identification and preproduction of a recombinant allergen from the Olive family (rOle e 1) [33]. However, there have yet to be studies that manipulate these allergenic proteins using GM technologies in order to reduce the allergenic effects of these foods.

2.28 Although no studies have been reported to date in which plants have been modified not to express particular allergenic proteins; the ongoing identification of the proteins involved, and the increasing occurrence of nut allergies in western countries means that further work in this field may be expected.


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MODIFIED CARBOHYDRATE CONTENT

Modified starch Introduction and overview of starch biosynthesis Changes to starch quantity - modification of the enzyme ADP-glucose pyrophosphorylase Changes to starch quantity - modification of sucrose degradation Changes to starch quantity - modification of pyrophosphatase expression Changes to starch quantity - modification of adenylate supply to the plastid Changes to starch quality - modification of amylose:amylopectin ratio Changes to starch quality - modification of starch lipid content Indirect alteration of starch quality - modification of grain hardness Side effect to starch modification (changes to quality and quantity)

Modified sugar Modification of glucose and fructose content Modification of hexose accumulation � alteration of post-harvest changes and inhibition of cold-induced starch degradation Modified sucrose content Modification of palatinose content

Modified fructan content Modified cellulose Modification of other cell wall polysaccharides Modification of raffinose family oligosaccharides

2.29 The purpose of this section of the report is to review the applications reported for the

modification of carbohydrate content in plants.

Modified starch

2.30 The purpose of this section of the report is to review applications for the genetic modification of plants for altered starch content. Interest in the modification of plants to alter their starch content is a consequence of the widespread use and importance of starch and starch derived products both in foodstuffs and non-food10 [34, 35]. Information on the biosynthetic pathways for starch is available and has the potential to allow the starch content of a range of plants to be altered through genetic modification.

2.31 Interest in starch modification is concerned with altering both the quantity and quality of the starch produced by plants:

• Starch biosynthesis in plants can be modified to increase and reduce the quantity of starch produced and accumulated in the plant. As illustrated in Figures 2.1 and 2.2 (transfer of sucrose to starch in plastid and amyloplast), sucrose produced during photosynthesis is converted and accumulated as starch in the chloroplast and amyloplast. Therefore, by modifying the plant�s

10 Non-food applications include adhesives in the paper and textile industries.


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conversion of sucrose to starch, the level of starch accumulated by the plant may be altered.

Direct transgenic approaches to the modification of sucrose to starch transition reported to date include the alteration of sucrose metabolism and the manipulation of the starch synthesis pathway.

An indirect approach to increasing starch quantity, through manipulation of the adenylate supply to the plastid, has also been found to have a significant effect on starch levels [35]. The increases of 60 percent above wild type reported for this approach represent the highest changes achieved through genetic modification to date (2003) [36]

• The purpose of modifying starch quality is to alter the physical and chemical properties of the starch for industrial processing. Such changes are described as modifying the functionality of the starch and include the modification of the ratio of amylose to amylopectin, the structure of the amylopectin (degree of branching for example), the presence of endogenous lipids and the shape and size distribution of the starch granule [37]. Changing the functionality of the starch is desirable to the food and industrial processing industries as it results in starches with novel properties, such as greater freeze-thaw stability [38] and altered processing temperatures [39]. Modifications are also available that result in the starch from one crop such as potatoes to be altered to provide similar properties to starch from other (potentially more expensive) crops such as very-high-amylose maize starch (also referred to as LAPS or low-amylopectin starch) [39].

2.32 With respect to cereal plants, starch functionality can also be altered by changing the hardness of the grain. This controls the manner in which the endosperm and starch granule is fractured during the milling process which has an important affect on processing performance [37].

2.33 As with several other sections of this report, it is viewed as useful to describe the biosynthesis of starch in non-modified plants as this will help in the identification of the components of the pathway that have been targeted by genetic modification.

Introduction and overview of starch biosynthesis

2.34 In plants starch is the most abundant storage carbohydrate and is used both as an osmotically inert storage compound (termed �transitory starch�) in the chloroplasts, and as a longer-term storage compound (termed �storage starch�) in the amyloplasts (dedicated plant structures such as potato tubers and cereal seeds) [34, 40, 41]. The


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formation of starch (and sucrose) is described as a crucial stage in photosynthesis, as these compounds are the nonphosphorylated �end products� of photosynthetic metabolism and act as the interface between plant growth and development [42].

2.35 Starch is composed of two types of glucose polymer:

• amylose, which is essentially an unbranched polymer of α-1,4-glucose; and

• amylopectin, which consists of α-1,4-glucose linked chains with α-1,6-glucose branches [41].

2.36 In unmodified plants amylose normally accounts for 20-30 percent of the starch, with amylopectin accounting for the remaining 70-80 percent [34]. Amylose and amylopectin differ both in the degree of branching and in molecular size, with amylopectin being approximately 1000-fold larger than amylose. The structural differences between the two polymers results in different physical and chemical properties, and have led to research into the modification of starch quality in plants with the aim of achieving a homogenous starch substrate consisting of either amylose or amylopectin.

2.37 Starch biosynthesis in higher plants is regulated in a number of ways involving both short-term (allosteric regulation of enzyme activities) and longer-term effects (developmental changes in enzyme activities) [34]. All plants produce �transitory� starch through the photosynthetic activity taking place in the chloroplasts of green tissues, and some plants are able to store starch in non-green storage tissues (amyloplast) such as potato tubers and cereal seeds.

2.38 The biosynthesis of starch in the chloroplast is described as taking place in three stages [34]:

• the formation of glucose-1-phosphate (G1P) � in the mature leaves, CO2 is fixed through the reductive pentose phosphate (RPP) cycle to form 3-phosphoglycerate (3-PGA) which is subsequently converted to triose-phosphates (TPs). The TPs are either transported into the plant cytosol via the triose-phosphate phosphate translocator (TPT), or further metabolised within the chloroplast to fructose-6-phosphate (F6P). The F6P is used to regenerate the ribulose-1,5-biphosphate (Rib-1,5-P2) or is converted via glucose-6-phosphate to G1P (Figure 2.1);

• conversion of G1P to ADP-glucose � mediated by ADP-glucose phosphorylase (Figure 2.1); and


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• conversion of ADP-glucose to starch (amylose, amylopectin) � this is a highly complex process mediated by the concerted action of branching enzymes and several isoforms of starch synthase [43]. The starch synthase enzymes catalyse a polymerisation reaction by transferring the glucosyl residue from the ADP-glucose to α-1,4-glucans, whereas the branching enzymes introduce α-1,6-glycosidic branchpoints into the linear α-1,4-glucans. The starch synthases are divided into two classes, granule bound (designated GBSS I and GBSS II) and soluble (SSS I and SSS II) [34]. The branching enzymes, of which there are a diversity of isoforms, are generally classified into two families on the basis of structural relatedness (Burton et al., 1995; cited by [43]).

2.39 Other enzymes such as the debranching enzyme (RE)(a hydroylase enzyme), disproportionating enzyme (DE) (a glucanotransferase) and starch phosphorylase (STP)) are also involved in the determination of starch structure [44]. Although their exact role has yet to be determined, possible roles of the enzymes were proposed by Kobmann et al. (1997) [44] and are presented in Figure 2.1.

2.40 With respect to the accumulation of starch granules in plant storage organs such as potato tubers and cereal seeds, the level of accumulation is dependent on the supply of starch from the leaves through the phloem. Starch accumulation in the amyloplast therefore depends on the rate of photosynthesis, the ability of the plant to synthesise starch in the leaves and the ability of the plant to load sucrose into the phloem system [34].

2.41 On reaching the storage cells, the sucrose is unloaded from the phloem by symplastic or apoplastic (S/A)11 pathways operating across the storage cell�s membrane (Figure 2.2). The activity of these pathways results in the conversion of the sucrose to hexose phosphates which are subsequently moved via specific transport proteins into the amyloplast [34]. Although the same enzymes are thought to be involved in starch accumulation in both the chloroplast (Figure 2.1) and amyloplast (Figure 2.2), the application of genetic modification to one enzyme step in the chloroplast for example to the supposedly equivalent step in the amyloplast should not be assumed and may require a different approach [45].

11 Defined as pathways in which the sucrose is or is not cleaved by the apoplastic invertase enzyme


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Figure 2.1 � Schematic representation of pathways leading to the accumulation of starch in mesophyll cells (adapted from [34, 44, 46])12.

12 The pathway illustrated for the conversion of glucose-6-phosphate to starch is proposed to occur in the both the chloroplast and amyloplast.

sent to storage tissues

3-PGA

TP

Rib-1,5-P2

F6P

CO2 chloroplast

mesophyll cell

glucose-6-phosphate

phosphoglucomutase

STARCH GRANULE

linear glucans (amylose)

glucose-1-phosphate

ADP-glucose

branched glucans(amylopectin)

AGPase

RE

GBSS SSS/BE/DE

STP STP/BE/DE

CALVIN CYCLE

TPTTP

F6P

sucrose

PiPi


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Figure 2.2 � Schematic representation of pathways leading to the accumulation of starch in storage cells (such as potato tubers) (adapted from Müller-Röber and

Kobmann, 1994 [34]).

Changes to starch quantity - modification of the enzyme ADP-glucose pyrophosphorylase

2.42 Much of the work reported to date has focused on the modification of the enzyme ADP-glucose pyrophosphorylase (AGPase) which mediates the conversion of glucose-1-phosphate to ADP-glucose. This enzyme, which consists of two large and two small subunits [47] is present in both photosynthetic (leaf) and non-photosynthetic (potato tuber and seed) tissues13 [34]. The role of AGPase in starch accumulation is demonstrated by the low levels of starch in plants mutated to have reduced activity of the AGPase [48]. The genetic modification of plants for increased or decreased AGPase activity resulted in those plants having increased or decreased (respectively) levels of starch [34]. However, although the modification of AGPase has been reported to affect starch levels in a range of plants, with particularly good results reported for potato, it is not known whether the enzyme has the same role or degree of control in all plants. Therefore the modification of AGPase activity may not be a suitable target to alter starch accumulation in all crop plants [45].

13 ADP-ase is however not present in stomatal guard cells.

S/A

G1P/G6P

sucrose/hexoses

G1P

ADP-glucose

STARCH GRANULE

amyloplast

storage cell

G1P/G6P

SUCROSE (from leaves)


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2.43 For those plants in which AGPase does have a key role in starch accumulation, the manipulation of AGPase activity through genetic modification is reported to confer an increase in starch accumulation through the following pathways:

• increased activity of AGPase results in increased conversion of glucose-1-phosphate to ADP-glucose and ultimately greater starch accumulation;

• reduced sensitivity to inhibition by orthophosphate (Pi). Pi is the negative allosteric effector for AGPase. Reducing the sensitivity of AGPase to Pi is reported to increase activity of that enzyme [47];

• improved plant productivity; and

• improved stability of the AGPase enzyme. In maize for example, AGPase is particularly heat-labile. A single amino-acid substitution in the maize large subunit is reported to achieve greater stability of the enzyme and consequently greater AGPase activity (Greene and Hannah, 1998; cited by [47]).

2.44 Increased levels of AGPase have been achieved by genetically modifying tomato, potato and tobacco calli to express a chimaeric gene from Escherichia coli (containing a modified N-terminal targeting signal from Arabidopsis to ensure correct expression of the bacterial gene in the plant) under the control of a CaMV 35S promoter. The modification resulted in a significant increase in starch production in the transgenic calli [49, 34]. However, although starch content was increased in the calli, attempts to regenerate complete plants from the modified calli were unsuccessful; suggesting that excess starch formation in the leaves might decrease the amount of sucrose available for export and consequently lead to poor carbon supply to actively growing tissues in the plant [34].

2.45 Expression of the same chimaeric gene, but under the control of a tuber specific patatin promoter, in potato calli, resulted in no restriction on the regeneration of complete plants containing tubers with an average of 35 percent more starch. Some of the transgenic tubers contained 60 percent more starch than the wild-type plants [34].

2.46 A reported indirect effect of the modification of AGPase is the effect on seed development and overall plant productivity. In wheat for example, only a minority of the flowers present on each plant develop into seeds, with many of the embryonic seeds being aborted early in their development. This is reported to be due to poor starch accumulation within the plant as a whole. Because AGPase catalyses the rate-limiting step in starch biosynthesis, then improvements in AGPase activity within


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the developing seeds should improve seed development and overall plant productivity [47].

2.47 The potential for AGPase to improve plant productivity in wheat was assessed by the modification of wheat with a modified maize Sh2 gene (designated Sh2r6hs). The Sh2r6hs gene encoded an altered large subunit of AGPase and was designed to confer greater stability on the whole enzyme, as well as reduced sensitivity to inorganic phosphate (Pi) [47, 50]. Analysis of the transgenic plants found that expression of Sh2r6hs increased plant yield by improving the number of seeds per plant [47]. Similar findings have been reported for rice modified to express the Sh2r6hs gene [50]. The increased starch content in the transgenic rice was reported to be a consequence of decreased sensitivity to inhibition by Pi, and as with the modified wheat allowed the rice plant to carry more seed to maturity (i.e. set more seed) [50].

2.48 Increased activity of AGPase means that more of the sugars produced during photosynthesis are converted to starch. A consequence of this is the reduction in feedback inhibition of leaf sugars on photosynthesis (Sun et al. 1999; cited by [47]). The enhancement in photosynthesis in the transgenic wheat may therefore underlie the observed improvements in plant yield and productivity, rather than the more immediate increase in AGPase activity [47].

2.49 Changes in plant yield and productivity conferred by increased activity of AGPase may confer altered persistence and survival of the transgenic wheat in the environment. The reduced level of seed abortion may in itself confer a greater number of progeny from a single plant and possibly greater dissemination of the modified seed.

2.50 As may be expected, a reduction in AGPase activity results in a decrease in starch accumulation by the plant. Reductions in starch quantity through AGPase modification have been achieved using antisense RNA14 technology. Complementary DNAs (cDNAs) encoding the small subunit (AGPaseB) and one of the larger subunits (AGPaseS) of the enzyme were inserted into potato in reverse orientation under the control of the constitutive CaMV 35S promoter [51].

2.51 Modification of the potatoes with the AGPaseS subunit resulted in a weak reduction of AGPase activity in the leaves and only a slightly greater inhibition in the tubers, compared with the wild-type. However, modification of the plants with the AGPaseB subunit reduced AGPase activity in the leaves by 70-95 percent (relative to wild-type) and by almost 98 percent in the tubers [51].

14 ribonucleic acid (RNA).


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2.52 In addition to changes in the starch content in the leaves and tubers, the modification also resulted in phenotypic changes to the transgenic plants which may have implications to persistence and survival of such plants in the environment. Phenotypic changes were observed in both the shoots and tubers of the modified potatoes. The plants exhibiting the highest inhibition of AGPase showed a greater tendency to form auxiliary shoots and accumulated slightly more anthocyanins in the upper stem regions. Flower formation occurred 2-4 weeks earlier in the transgenic plants, although the overall structure of the flowers appeared to be unchanged [51].

2.53 With respect to changes to the plant tubers, the modified plants exhibited smaller but an increased number of tubers (up to 90) per plant. Whilst the wild-type potatoes formed a single tuber at the end of each stolon15, the modified potatoes had as many as ten tubers on each stolon [51].

2.54 Changes to tuber development in the transgenic plants only occurred when the antisense AGPaseB was under the control of a constitutive promoter. When the studies were repeated using a leaf-specific promoter, tuber characteristics in the transgenic plants were the same as in the wild-type plants [51], suggesting that the inability of the modified plants to synthesise normal amounts of �transitory� starch had neither an inhibitory or stimulatory effect on tuber development [34].

2.55 Reduction in AGPase activity in the modified potato plants was also found to have a concomitant increase in the level of soluble sugars present [34]. The greatest increases were observed for sucrose (up 30 percent) and glucose (up ~8 percent) compared with the wild-type. Increased sugar content resulted in tubers of the transgenic plants having a higher than average water content and meant that although tuber numbers were higher, the total dry weight of tubers per plant was significantly lower relative to the wild-type. The effects of such changes on the persistence and survival of such transgenic potatoes in the environment have not been determined. However, because of the important role of potato tubers as energy storage organs for the potato plant, then any reduction in starch content may be expected to have implications to the long-term survival of such plants in the environment. Replacing starch with soluble sugars in the tubers effectively reduces the amount of long-term food storage substrate available to the plant.

Changes to starch quantity - modification of sucrose degradation

2.56 The alteration of sucrose degradation has been reported as another approach to increasing starch accumulation in plants. Strategies reported to date have involved modifying potatoes with a yeast invertase, a bacterial glucokinase or a bacterial sucrose phosphorylase. Although each approach has resulted in the transgenic


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potato tubers exhibiting higher levels of hexose phosphates and 3PGA compared with the wild-type, starch levels remained unchanged [46, 52]. Rather than increase starch accumulation, each of the modifications was found to result in the induction of glycolysis and a massive partitioning of the increased carbon into respiration. Although such shifts in carbon flux may be expected to alter the potential of the modified potatoes to survive in the environment, the effect of such changes within the plant has not been determined.

Changes to starch quantity - modification of pyrophosphatase expression

2.57 As a consequence of the number of enzyme reactions involved in the synthesis and accumulation of starch in plants, other more indirect modifications have been reported to have an effect on starch production. The modification of potatoes and tobacco for example for increased expression of the enzyme pyrophosphatase (PPase) in the cytosol results in a reduction in cytosolic concentration of inorganic pyrophosphate (PPi) and a dramatic change to carbohydrate levels in the plant including changes to starch synthesis and accumulation [53].

2.58 Geigenberger et al. (1998) [53] hypothesised that the overexpression of PPase in the modified tobacco and potato plants (designated ppa1 transformants) would result in a reduction in starch levels compared with the wild-type plants. This reduction was proposed to be due to the role of PPi in the operation of UDP-glucose pyrophosphorylase (UGPase) in the hexose-forming direction. By reducing the level of PPi (through increased PPase activity), the operation of the UGPase would also be reduced, leading to an accumulation of UDP-glucose and a depletion of UDP, as well as an inhibition of SuSy. The restriction of UGPase activity would mean that levels of hexose-phosphates and therefore also 3-PGA would also be low. Because AGPase is activated by 3-PGA then levels of this enzyme in the transformed ppa1 plants would be reduced resulting in an inhibition of starch synthesis.

2.59 Down-regulation of the sucrose synthase (SuSy) enzyme in potatoes through antisense repression is known to reduce starch accumulation (as well as causing reduced tuber dry weight and a decreased concentration in storage proteins) [54]. Inhibition of SuSy activity (to <5 percent of wild-type levels) in potato tubers and maize endosperm can result in decreases in starch content of 70 and 40 percent respectively [55].

2.60 However, studies by Geigenberger et al. (1998) [53] with potatoes found that instead of a reduction in starch synthesis, a three-fold overexpression of the PPase resulted in the modified potatoes containing 42-55 percent more starch than the wild-type tubers. Overexpression of the PPase therefore does not result in decreased

15 Stolons are underground shoots which develop into tubers through greater starch accumulation in


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concentrations of SuSy, UGPase and AGPase in the developing tubers, with levels of AGPase actually being increased [53]. Changes in the expression of an inorganic pyrophosphatase gene in potato have however also been found to affect the sprouting behaviour of the tubers [56]. The potatoes, modified to express an inorganic pyrophosphatase gene from E. coli cytosolically under the control of a tuber-specific patatin promoter displayed significantly accelerated sprouting relative to the wild type [56]. Such behaviour may be expected to have an effect on the growth of the modified potatoes in the environment, although whether the effect will have a positive or negative effect on persistence/survival will depend on the environmental conditions when sprouting occurs.

2.61 The reasons for the observed effects that a reduction in levels of PPi has on starch levels in the transgenic potatoes have not been fully elucidated. However, it is likely that changes to PPi have much wider implications to plant metabolism and growth than has been previously suspected. The ppa1 potatoes showed changes in the activities of other enzymes such as phosphofructokinase (PFK) and pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) which may allow the conversion of adenine triphosphate (ATP) to PPi (and consequently increase PPi levels) [53]. Further studies are being conducted with respect to the effect of PPi modification, although no additional results have been identified.

Changes to starch quantity � modification of adenylate supply to the plastid

2.62 The modification of starch quantity through the alteration of adenylate supply to the plastid represents an indirect but potentially very fruitful approach to increasing starch yields.

2.63 The increased starch yields of 60 percent (fresh weight) above wild type potatoes reported by Regierer et al. (2002) [35] were achieved through the downregulation (by antisense repression) of the activity of the plastidial isoform of adenylate kinase. The modified potatoes also exhibited an 84 percent increase in tuber yield (but no increase in tuber number), and increases in certain amino acids (arginine, methionine, histidine, tryptophan, lysine, isoleucine, phenylalanine and tyrosine) of between two- and fourfold compared to wild type [35].

2.64 A possible limitation with this approach is that it is non-specific for the alteration of starch levels and may therefore have an effect on other plant processes. The observed changes in amino acid levels are an illustration of this. Some evaluation of field performance was conducted by Regierer et al. (2002) and found no major changes in either the aerial portion of the plants or in the earliness of the crop. Differences in tuber size and tuber density were observed [35].

the apical part of the stolon leading to a cessation of shoot extension and an increase in radial growth.


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2.65 On the basis of the information identified to date, some GM-based strategies offer successful approaches for improving starch yields in crops plants. Field trials (30), involving potato, tomato, maize, rice and wheat modified for increased starch content are underway in the USA. Three field trials have been conducted with crops modified for reduced starch levels [57]. Because of the importance of starch as both a foodstuff and for industrial processes, the continued development of such crops may be expected.

Changes to starch quality - modification of amylose:amylopectin ratio

2.66 Much of the work to modify starch quality has been conducted in potatoes16 and has focused on the manipulation of the starch synthase enzymes and branching enzymes to alter the amylose:amylopectin ratio in the starch produced. Antisense expression of the starch synthase GBSS I enzyme in the model alga Chlamydomonas reinhardii resulted in the production of starch that was both amylose-free and contained a modified form of amylopectin (Delrue et al. (1992); cited by [34]).

2.67 A natural mutation in the gene encoding GBSS has been identified in maize, potato, rice, wheat and barley and is reported to result in changes to the amylose:amylopectin ratio in these plants [41]. In studies with wheat the degree of change was reported to be due to the isoform(s) of GBSS I expressed by the plant. Three isoforms of the GBSS I enzyme have been identified in naturally occurring varieties of wheat. Comparison of the starch quality in varieties expressing the GBSS I-A, GBSS I-B or GBSS 1-D17 null allele has found that the greatest change in starch quality (compared with a variety containing neither null alleles) is conferred by a variety possessing all three null alleles in which the starch is almost completely amylose free. Loss of the GBSS 1-A isoform results in the smallest change in the amylose:amylopectin ratio [45].

2.68 Antisense expression of GBSS I in potatoes by genetic modification also resulted in the production of starch with no amylose [39]. Interestingly, the modification had no effect on the total starch content of the GM potatoes [58]. This latter point is an important consideration if the modified plants are to be used in industrial processes and grown on a commercial scale.

2.69 Although the studies with antisense expression of GBSS I in Chlamydomonas and potato demonstrated that initial work in model systems (such as Chlamydomonas) can be applied to higher plants, the differences in starch synthase enzymes amongst

16 Field trials involving crops other than potato have been conducted. Twenty-two trials have taken place to date in the USA for example involving crops with modified starch metabolism (www.isb.vt.edu). 17 The GBSS 1-D null allele is extremely rare in wheat and has only been detected in a Chinese cultivar, Bai Huo (Båga et al., 1998).


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higher plants mean that such model studies cannot always be applied universally [34]. For example, in pea the GBSS I and SSS II are identical proteins, whereas in maize and potato they are different polypeptides with the protein corresponding to GBSS II in potato having a significantly higher molecular weight to that of the pea GBSS II [59].

2.70 Other reported approaches for the modification of starch synthase genes have included the overexpression of bacterial glucan synthases in the plastid [60]. The expression of the glgA gene (encoding glycogen synthase) from E. coli in potato achieved a decrease in amylose content of the starch. However the modification also resulted in an increase of low molecular weight glucans in the amylopectin fraction, and an unwanted decrease in total starch content in the potato.

2.71 Because the starch branching enzymes (SBEs) are responsible for the introduction of a branched structure into the starch molecule, then the modification of the activity of these enzymes has been proposed as offering another approach to changing the amylose:amylopectin ratio in the starch [43]. The naturally occurring �wrinkled� pea mutant has no SBE I activity and has an amylopectin content in its starch of only 30 percent (compared with 70 percent in the wild-type) [43].

2.72 Multiple forms of SBEs have been identified, with two isoforms (SBE I and SBE II) identified in pea and three in maize (I, II1 and IIb) for example. Each of the isoforms are assigned to one of two families (termed �A� and �B�) on the basis of structural relatedness, rather than on plant species. Members of SBE family A include maize SBE IIa/b, pea SBE I and rice SBE III, and SBE family B includes maize SBE I, pea SBE II, rice SBE I and potato SBE I [43]

2.73 Safford et al. (1998) [43] reported that only one SBE isoform has been identified in potato (SBE I), and that the modification of amylose:amylopectin ratio in potato through the antisense inhibition of SBE I required reducing the activity of SBE I to a level below five percent that of the wild-type. Such a significant reduction is required before any alteration in the physical properties of the starch is identifiable, and indicates a large excess capacity of SBE in potato [43].

2.74 Subsequent studies have reported the occurrence of a second isoform of SBE in potato (SBE family A) (Larsson et al. 1996; cited by [61]). Although this isoform only occurs at very low levels (~2 percent relative to the family B isoform), it is reported to have a major role in the control of starch structure in potatoes [61]. Schwall et al. (2000) [39] proposed that the SBE A isoform is required to synthesise amylopectin with a normal branching structure.

2.75 The simultaneous antisense inhibition of both potato SBE isoforms has a significant affect on starch composition and results in the GM potatoes having a very high


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amylose content and negligible levels of highly branched amylopectin [39]. Phosphorus content of the modified potatoes was also altered with levels of ≤3000 µg g-1 compared with just 500 µg g-1 in the wild-type [39].

2.76 The GM potatoes described by Schwall et al. (2000) [39] were constructed by the retransformation of antisense SBE B potato lines with an antisense SBE A construct. This approach resulted in a higher amylose content than that attained from previous SBE A/SBE B inhibited potatoes where the inhibition was achieved through the insertion of a single antisense construct [43].

2.77 Inhibition of SBE activity through a two step process [39] reduced tuber SBE activity to <0.9 percent compared with wild-type and leaf activity to <0.7 percent. Although the modified plants appeared phenotypically normal the modification did have a significant effect by halving tuber yield and starch content. Tuber morphology was also altered, with the transgenic tubers exhibiting a more elongated shape and a tendency to bud off additional tubers [39]. Changes in tuber yield and morphology are likely to have implications to the persistence and or survival of the GM potato in the environment.

2.78 The modification of the amylose:amylopectin ratio in starch has many implications for the food and processing industries. A key characteristic required by the food industry is good freeze-thaw stability of the starch used in products that are frozen or stored at chilled temperatures [38]. The limitation with conventional starch is that at low temperatures the glucan chains re-associate (the starch is said to retrograde) and this results in the separation of the starch into a solid and liquid phase (termed syneresis). This of course has an adverse affect on the quality of the frozen or chilled product.

2.79 Studies have shown that the long unbranched amylose chains have a much greater tendency to retrograde than the shorter amylopectin chains, and that therefore low amylose starches have a relatively better freeze-thaw stability [38]. Syneresis of starches currently used in the food industry is controlled chemically through monosubstitution which by controlling cooperative hydrogen bonding between the helices prevents reassociation of the glucan chains.

2.80 An alternative approach is to use a starch which contains no long chain amylose, and only short branch chain amylopectin [38].

2.81 Jobling et al. (2002) [38] reported the development of a freeze-thaw stable potato starch through the simultaneous antisense downregulation of three starch synthase genes (GBSS I, SS II and SS III). Expression of each of the synthase genes was reduced to a barely detectable level with the resulting starch having zero amylose content and amylopectin with no long-chain branched chains. The presence of the


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large number of very short chain amylopectins and their role in preventing the reassociation of the glucan chains was proposed as the key reason for the good freeze-thaw stability of the modified starch [38]. The modified plants also showed no significant variation in phenotype or reduction in yield compared with the wild-type [38].

2.82 A further side benefit of this modified starch was that because it cooks at a much lower temperature than normal potato starches, then the manufacture of the food product should require less energy [38] leading to potential environmental benefits and financial savings.

Changes to starch quality - modification of starch lipid content

2.83 Lipids are present in starch complexed to the amylose component. The lipids are thought to exert important effects on the interaction of the starch granule with water and therefore may have an influence on processing properties of the starch [37].

2.84 Although the lipid content of cereal starches is low (~1 percent) the lipids, which are predominantly lysophospholipids, do have an effect on the characteristics of the starch, particularly viscosity. Current understanding of the biochemical or molecular control of lipid content in cereal starch is limited. However, the identification of two genes in wheat (designated fpl-1 and fpl-2) that have been shown to be involved in the control of starch lipid content suggests that it may soon be possible to modify wheat for altered starch lipid content [37].

Indirect alteration of starch quality - modification of grain hardness

2.85 As discussed, grain hardness has an important influence on starch quality, especially in wheat, as it determines the amount of damage to the starch that is likely to be incurred during milling [37]. Therefore, although the modification of grain hardness does not have a direct in planta effect on starch composition, it does affect the quality of the starch that is ultimately derived from the plant after processing. The modification of grain hardness has therefore been included in this report for completeness.

2.86 In so called �soft� grains the level of adhesion between the starch granules and the surrounding protein matrix is weaker than in �hard� grains. Therefore during milling the soft grains fracture along the planes between the starch granule and the protein, whereas with hard grains the starch granule itself is fractured, leading to greater water absorption of the flour. The differences in processing, water absorption and baking characteristics mean that soft grains are milled to make flour for biscuits and pastry, whereas hard grain flour is used for bread and noodles [37, 62].


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2.87 Soft grained cereals also offer advantages as animal feed. Soft grains form smaller particles than harder grains when milled [62], and studies with both cows and pigs have reported improved digestibility and greater weight gain with finer milled feed (Knowlton et al. 1996, and Wondra et al. 1995; cited by [62]).

2.88 Studies have shown that the adhesion between the starch granules and the protein matrix is determined by the protein friabilin, which prevents adhesion from occurring (Greenwell and Schofield, 1986; cited by [62]). Friabilin consists of a 1:1 molar ratio of two proteins PINA and PINB, produced by expression of the pinA and pinB genes [62]. Both genes are found naturally in wheat, oats and barley, but not in maize and rice (which are predominantly hard textured). Naturally occurring mutations in either gene in wheat result in hard-textured grains [62].

2.89 The modification of rice to express pinA and pinB (under the control of a ubiquitous promoter) resulted in a reduction in grain hardness relative to the wild-type, and demonstrated the potential for genetic modification to alter grain hardness [62].

2.90 The implications of changing grain hardness with respect to the persistence and survival of the modified cereal in the environment have not been reported. Any preferential differences in terms of herbivory may affect the survival of the GM plants in the environment.

2.91 The modification of levels of PINA and PINB in crops may however have a significant indirect effect on the persistence and survival of the transgenic crops in the environment. This is a consequence of the reported in vitro antimicrobial properties of the puroindolines (including PINA and PINB) [63].

2.92 Puroindolines have been found to be effective in vivo against several fungal plant pathogens (Dubreil et al. 1998; cited by [63]), and rice modified with pinA and pinB showed significantly increased tolerance to the fungal pathogens Magnaporthe grisea (29-54 percent increase in tolerance) and Rhizoctonia solani (11-22 percent increase) [63].

2.93 The implications of these findings are significant because rice does not naturally contain puroindolines, and both fungi are difficult to control conventionally and are the most prevalent fungal pathogens of rice. M. grisea causes rice blast and is difficult to control because of inherent instability and variable pathogenicity of the fungus. R. solani, which causes sheath blight is also difficult to control through traditional breeding of resistant cultivars due to the low levels of available host resistance [63].


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2.94 Therefore as well as altering grain hardness and milling properties, the modification of cereals to express pinA and pinB is also likely to confer some resistance to two key fungal pathogens. This resistance would offer significant advantages to the persistence and survival of the modified crop in the environment. The resistance trait would be especially advantageous to rice which naturally does not contain puroindolines.

2.95 Because of the importance of grain hardness to the food and feed industries, then it is likely that cereals genetically modified for altered grain hardness do have a future agronomically. This is likely to be particularly relevant to crops such as rice and maize which lack many naturally occurring softer grain varieties. The antimicrobial properties of the puroindolines are also likely to be viewed by farmers and industry as an additional advantage of the system and may add greater impetus to the commercialisation of this genetic modification. Field trials of wheat genetically modified to express puroindolines have been conducted in the USA [57].

Side effect to starch modification (changes to quality and quantity)

2.96 Many of the studies investigating modifications to starch quantity and quality in crop plants have also reported changes to the phosphate content of the modified starch (relative to the wild-type) [39]. Although changes in phosphate content were not addressed in these reports, Lorberth et al. (1998) [64] reported that a reduced phosphate content in potato starch had a secondary effect on its degradability and altered phenotype in both the leaves and tubers of the transgenic crop.

2.97 The study reported by Lorberth et al. (1998) focused on a 4851 bp cDNA (designated R1). The protein encoded by R1 had no significant sequence homology to known starch metabolising enzymes, but was similar to other (as yet unidentified) sequences present in Arabidopsis and rice [64]. Potatoes modified (by antisense technology) for reduced expression of R1 showed pronounced changes in their starch metabolism.

2.98 The transgenic potatoes exhibited a starch excess phenotype in their leaves, and reduced cold-induced sweetening18 in their tubers [64]. The increased starch content in the leaves was manifested in the form of a net increase (50-90 percent) in plant dry matter. No changes in the photosynthetic capacity, or in levels of soluble sugars were observed in the modified plants. Although increased dry matter levels have limited agronomic value in potatoes (a non-fodder crop), the observation of similar changes in tobacco mean that such changes may be possible in fodder crops. This is of potential benefit agronomically in providing extra grazing material. Changes in dry matter levels may also have an effect on the persistence and survival of the GM


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plant in the environment if it alters levels of herbivory of the transgenic plant relative to the wild-type.

2.99 The studies reported by Lorberth et al. (1998) are viewed as having limited agronomic application for potatoes. The modification of R1 levels in fodder crops (should such a gene sequence exist) may though offer benefits in providing increased levels of dry matter for animal feed.

Modified sugar

2.100 The modification of the sugar content in plants provides applications for both food and industrial products. As a consequence of sucrose�s high calorific value and cariogenic19 properties there has been increasing demand reported for other sugars [65]. Whilst many of the sugars that are used as sucrose replacements, such as fructose, glucose and palatinose are already produced at a commercial scale, production is often achieved through microbially mediated processes in large biofermenters. The limitations with such systems are a restricted scale of production and relatively high cost (particularly start-up costs). The use of transgenic plants as a cheaper alternative to microbial systems has been proposed for the production of sucrose substitutes. To date, modifications have been proposed for the production of fructose/glucose and palatinose in transgenic plants.

2.101 The role of sugars as signalling compounds in plants means that changes to plant sugars may have implications to all stages of a plant�s lifecycle, from seed germination and vegetative growth to reproductive development and seed formation [66]. Although no specific changes in plant development have been reported as a consequence of the genetic modification of plant sugar levels [66], such changes should be considered as part of any field release of a modified plant. Sugar signalling has been defined as the interaction between a sugar molecule and a sensor protein that generates a signal. The signal initiates signal transduction cascades that ultimately result in altered gene expression or changes in enzyme activities [66]. Although any neutral sugar or glycolytic intermediate is reported as having the potential to have a signalling function, to date such a role has only been described for hexose and sucrose [66]. Much of the work to date has been conducted in yeast but is reported to be applicable to plants [66]

Modification of glucose and fructose content

2.102 A number of approaches have been proposed for modification of glucose and fructose content in crop plants. Industrial and food uses for glucose and fructose

18 See section on Modification of starch degradation � alteration of post-harvest changes for further details on cold-induced sweetening. 19 Causes dental caries (cavities).


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have increased through greater demand for crystalline dextrose, dextrose syrups and high-fructose syrups [67]. Fructose for example is becoming an increasingly popular sweetener in a range of food products, in which it has usually replaced sucrose.

♦ Modification of starch degradation � increase in fructose/glucose concentration

2.103 Starch may be converted to glucose and then fructose by a two stage reaction catalysed by an α-amylase (starch to glucose) and a glucose isomerase (glucose to fructose) [67]. As starch is stable at ambient temperature, the hydrolysis to glucose and fructose has to be conducted at a high temperature (100 ºC), requiring the isolation of enzymes from thermophilic bacteria.

2.104 The existing system of using microbially-based processing is limited by the relatively high cost of obtaining glucose isomerase and the high temperature requirements of the process. The glucose isomerase for example must be stable at 100 ºC to ensure that the fructose produced is not converted back to glucose [67].

2.105 The alternative, plant-based system proposed by Beaujean et al. (2000) [67], involves the use of a single bifunctional enzyme generated by gene fusion and expressed in a transgenic potato. Exposure of the harvested potato to temperatures >65 ºC activates the enzyme and results in the hydrolysis of the starch to fructose. The proposed advantage of the system is that although the transgene is expressed in the potato, because the gene confers the expression of a thermotolerant enzyme then activity of the new enzyme is negligible in the plant under field conditions and therefore should not have an effect on growth or behaviour of the modified potato in the environment.

2.106 The transgenic potato described by Beaujean et al. (2000) [67] was modified by a gene fusion system encoding a glucose isomerase (from Thermus thermophilus) ligated to the 3� end of an α-amylase (from Bacillus stearothermophilus). Expression of the transgene was reported to have no effect on the development and metabolism of the modified potatoes, including starch accumulation. However, after harvesting and exposure to a temperature of 65 ºC the concentration of glucose and fructose in the processed potato was increased by a factor of 3.9 and 14.7 respectively, compared with the wild-type. Storage of the modified potatoes in an unprocessed form for two months after harvesting was found to have a further effect on fructose and glucose levels. After processing the levels of the two sugars in the stored potatoes were 5.85 and 22.05 times greater than the wild-type [67].


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♦ Modification of invertase enzymes � decrease in glucose/fructose content

2.107 Invertases (such as β-fructosidase) are a group of related enzymes that hydrolyse sucrose to glucose and fructose [68]. The genes encoding acid invertases (which have a pH optimum for activity between pH 3 and pH 5) have been cloned from a number of plants including tomato (Lycopersicon esculentum), and offer the potential to modify the sucrose content of crops [68].

2.108 Studies with the TIV1 gene, which is known to be a component of the sucr locus (the trait of sucrose accumulation) in tomato have found that the constitutive expression (CaMV 35S promoter) of the gene in an antisense orientation results in a significant change to the soluble sugar composition of the fruit and an increased accumulation of sucrose. The total sugar content of the fruit however remained unchanged [68].

2.109 Antisense expression of the TIV1 gene also resulted in a 30 percent reduction in the size of the fruit produced by the transgenic plants, and an increase in ethylene production [68]. Ethylene is produced naturally by plants and encourages fruit ripening. The increase in the concentration of this chemical may therefore have implications to the persistence of the modified tomatoes on the plants as they are likely to ripen faster than those present on non-modified plants.

2.110 The modification of potatoes for increased levels of invertase has been conducted as an approach to reduce sucrose content in the plants [46]. The expression of a yeast invertase in potatoes however resulted in increased levels of hexose phosphate and 3PGA but no change in sucrose or starch concentration.

2.111 Plants modified for altered glucose and fructose content are viewed as having future commercial applications where they can offer a cheaper alternative to microbial-based systems. Of the modifications addressed in this section, the development of plants with the high-temperature induced systems are viewed as having the greatest future potential. The modification of invertase enzymes is assessed to have less potential, based on the developments reported to date.

Modification of hexose accumulation � alteration of post-harvest changes and inhibition of cold-induced starch degradation

2.112 The modification of starch degradation for post-harvest applications is of most relevance to potatoes. As discussed, potato tubers act as the food storage organ for the plant during the winter, and as an energy supply for the generation of new progeny. When potato tubers sprout, the starch in the region of the tuber surrounding the bud is degraded to sugars to support the new growth. To prevent this, potatoes are either treated with dormancy-prolonging chemicals or stored at low temperatures [69]. However, prolonged storage at low temperatures causes a


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process known as cold-induced sweetening in which starch throughout the tuber is converted to soluble sugars [64, 69].

2.113 Cold-induced sweetening is of course not a desirable phenomenon for the potato processing industry as the hexoses produced react with free amino acids and negatively affect the processing quality of the potatoes [69]. Accumulation of the hexoses is thought to occur due to an imbalance between the rate of starch degradation and the rate of glycolysis, leading to an accumulation of sucrose which is subsequently split by an invertase to glucose and fructose (Isherwood, 1973; cited by [69]).

2.114 The modification of potatoes to either inhibit starch degradation [49, 64] or reduce the activity of the invertase [70, 69], has been proposed as the two general approaches to reduce the occurrence of cold-induced sweetening [69].

2.115 As well as exhibiting increased starch synthesis, the potatoes modified for increased expression of AGPase (by overexpression of a mutated ADP-glucose pyrophosphorylase gene (glgC16) from E. coli)20 [49] also underwent limited cold-induced sweetening with negligible accumulation of hexose (Stark et al. 1996; cited by [69]). A reduction in cold-induced sweetening was also achieved through the repression of levels of a starch granule-bound protein (designated R1)21 [64]. In these transgenic potatoes, the modification caused a reduction in phosphate content of the starch resulting in less digestible starch [64]. Although resulting in reduced cold-induced sweetening, both approaches are limited in that they involved the alteration of starch quantity or quality.

2.116 The invertase-based approach has been reported as a successful method for reducing cold-induced sweetening and also offers particular processing improvements. Zrenner et al. (1996) [70] modified potato with a cold-inducible vacuolar invertase (VI) expressed in an antisense orientation. Reduction in cold-induced sweetening was achieved when residual VI activity was inhibited to <10 percent compared with wild-type. Similar results were achieved by inserting a VI inhibitor (Nt-inhh) from tobacco into the potato under the control of a CaMV 35S promoter [69].

2.117 The modified potatoes expressing Nt-inhh exhibited no changes in starch content, growth morphology or yield. The potatoes also did not brown during frying (a problem often encountered with end of season potatoes). This characteristic is reported to be advantageous to the potato food processing industry [69].

20 see section Changes to starch quantity - modification of the enzyme ADP-glucose pyrophosphorylase. 21 see section Side effect to starch modification.


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2.118 The genetic modification strategies to overcome cold-induced sweetening have been developed because this characteristic is undesirable for the potato processing industry. Therefore successful strategies that offer a means to overcome cold-induced sweetening without other changes to starch quality/quantity and plant yield, are likely to undergo further development. The modification with the VI inhibitor is viewed as offering the only strategy with commercial potential, and is a good example of the application of genetic modification technology offering a realistic alternative to existing chemical based approaches (i.e. the use of sprouting inhibitors) and long standing post harvest problems (cold-induced sweetening).

Modified sucrose content

♦ Modification of sucrose synthase for altered sucrose content

2.119 Sucrose synthase (SuSy) catalyses the cleavage of sucrose. In fruit such as tomatoes, SuSy is important in controlling the import carbohydrate capacity of the fruit, with changes in SuSy activity reported to result in both the growth rate of the tomato fruit and the numbers of fruit produced per plant [71]. Changes in SuSy activity therefore have implications both to the production of tomatoes and to the persistence and survival of the modified tomato plants in the environment.

2.120 Reduced activity of SuSy in tomatoes was achieved by D�Aoust et al. (1999) [71] by expressing an antisense RNA fragment of the TOMSSF gene under the control of a CaMV 35S promoter. Although the transgene was under the control of a constitutive promoter, inhibition of SuSy only occurred in the fruit and flower, with little or no inhibition in the stem, leaf and seed tissues. Although other studies investigating SuSy inhibition in maize and potatoes reported a significant reduction in starch accumulation [55], the transgenic tomatoes showed no decrease in fruit dry weight and negligible change in starch accumulation [71].

2.121 Changes in SuSy activity in the modified tomatoes were found to affect the import and metabolism of sucrose in the developing tomato fruits and consequently to alter the capability of the tomato fruits to set properly. Even a ten percent reduction in SuSy activity caused a significant reduction in fruit setting with a fewer number of tomatoes produced on the mature plant relative to the wild-type [71]. This may have implications to their long-term persistence and survival in the environment.

♦ Modification of activity of sucrose-phosphate synthase activity � increase in sucrose content

2.122 The enzyme sucrose-phosphate synthase (SPS) is a key enzyme in the synthesis of sucrose. Tomatoes modified to overexpress SPS in the leaves had increased fruit


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sugar content [42]. Increased fruit sugar content only occurred when the gene encoding SPS was placed under the control of the ribulose-1,5-bisphosphate carboxylase-oxygenase small subunit promoter. The use of a constitutive CaMV 35S promoter did not achieve an increase in sucrose content [42].

♦ Modification of phosphofructokinase activity � increase in sucrose content

2.123 Modification of the activity of phosphofructokinase (PFK) in a sugar-storing plant such as sugarcane is reported to offer another strategy for the alteration of the sugar content of the plant [72]. Decreasing the concentration of the β-subunit of the enzyme results in the down-regulation of the PFK enzyme and an increase in the sucrose content of the sugarcane.

2.124 Although the modification of PFK levels in sugarcane were found to have an effect on sucrose levels, the observation that similar enzyme changes in tobacco and potato do not alter sucrose levels [73] suggests that this modification may not be applicable in all plants.

2.125 The modification of plants for altered sucrose content is currently at an early stage of development. The agronomic or commercial benefits of such developments are currently limited to altering the sugar content of plants such as tomatoes, and the effects reported on fruit setting capability for example will need to be addressed before any commercial development can take place. Field trials of tomatoes with modified sucrose phosphate synthase have been conducted in the USA [57], suggesting that studies with tomatoes in this area are progressing.

Modification of palatinose content

2.126 Palatinose (also referred to as isomaltulose and 6-O-α-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose, and occurs naturally as a minor component of honey. It has similar physicochemical characteristics to sucrose but only half the sweetness, a lower calorific value and is non-cariogenic [65]. Because of these properties, palatinose is produced industrially as an artificial sweetener and as a sucrose replacement in a wide range of applications.

2.127 Industrial production of this compound involves the use of immobilised cells of the bacterium Erwinia rhapontici which are able to produce a sucrose isomerase (α-glucosyltransferase) which catalyses the conversion of sucrose to palatinose and trehalose [65]. However, as a consequence of the high cost and relatively limited scale of this bioreactor-based production system, the bacterial formation of palatinose means that the widespread use of such sucrose isomers as alternative sweeteners is limited.


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2.128 Because of their ready internal supply of sucrose, plants are viewed as ideal systems for the production of palatinose or other sucrose isomers for food or industrial applications if they can be modified appropriately. Initial studies on the modification of plants to produce palatinose used a gene encoding a sucrose isomerase (designated palI) from E. rhapontici under the control of a constitutive promoter [74]. Although the modified tobacco plants accumulated 45 times more palatinose than sucrose, the plants were characterised by severe growth retardation [75]. This was proposed to be due to the depletion of the sucrose source for starch sink development and is the opposite to that reported by Smidansky et al. (2002) [47] who improved seed development in wheat by modifying the plants for improved sink strength. Because palatinose is non-metabolisable then the conversion by the plant of utilisable sucrose into this sugar is effectively a metabolic dead-end for the plant.

2.129 In order to avoid adverse effects on plant growth, subsequent studies with potatoes involved placing the palI gene under the control of a tuber specific promoter [65]. The system used linked the palI gene to a patatin class I B33 promoter fused to the signal peptide of the proteinase inhibitor II. Modification of the potato with this gene system resulted in up to 20 µmol g-1 (fresh weight (fw)) of palatinose being produced in the apoplasm of the tubers. Levels of palatinose in wild-type tubers are effectively zero [65]. The transgenic potatoes showed no signs of changes in growth or development, although the affect of the modification on tuber yield was not determined22 [65].

2.130 Because the modification resulted in only a minor increase in starch accumulation in the tubers, then it was proposed that palatinose synthesis occurred in the transgenic potato at the expense of starch production [65]. However, although the potatoes exhibited good palatinose production the limited availability of sucrose during the later stages of tuber development means that potatoes may not be the most suitable crop for production of sucrose isomers. Higher palatinose yields may therefore be achieved with a crop such as sugar beet or sugarcane that accumulates sucrose naturally [76, 65]. Other possibilities to improve sucrose isomer levels include increasing the efficiency of sucrose to palatinose conversion through the use of alternative sucrose isomers [65]. The sucrose isomerase enzyme from the bacterium Serratia plymuthica for example has a Km of 65 mM for sucrose (compared with 200 mM for the E. rhapontici sucrose isomerase), making this enzyme much more suitable for palatinose production on a commercial scale [65].

2.131 The modification of plants to produce palatinose is at an early stage of development with studies to date focusing on potatoes. Although field trials of potatoes modified for altered palatinose content are reported to be underway (2002), potato ultimately may not be the most suitable crop. The potential of the technology to provide an

22 The transgenic potatoes are reported to be currently undergoing field trials in which changes in tuber yield will be determined.


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alternative cheaper alternative to microbial-based systems means that crops with modified palatinose content are likely to have a commercial future. Commercialisation of this technology is most likely to be seen with crops such as sugar beet or sugarcane.

Modified fructan content

2.132 Fructans are polymers of fructose produced by about fifteen percent of flowering plants as a reserve carbohydrate [77]. There are two main groups of fructans (β2-1 and β2-6) which differ in the nature of the link between the fructose moieties [7]. The β2-1 fructans (also referred to as inulin) are the ones used in industrial processing and have both food and non-food applications. The small chain fructans (consisting of up to five fructose units)23 have a taste similar to regular sugar, and because the human digestive system does not contain the enzymes necessary to degrade fructans, then these compounds have applications as low calorie sweeteners [78, 77]. The longer chain fructan molecules (DP6-60) form emulsions that have a fat-like texture and neutral taste. These compounds have applications as low-calorie fat replacers in foods such as spreads and ice-cream [77, 7].

2.133 In addition to their application as low-calorie replacements for existing sugars and fats, β2-1 fructans are also reported to offer beneficial health effects including improved blood lipid composition, mineral uptake from food and a reduced risk of colon cancer. Ingestion of 20 g of inulin a day is reported to provide the optimum health benefits from these compounds. However, the average Western diet only provides 5 g of inulin per day. Increased production of inulin may therefore allow increased levels in the diet.

2.134 Fructans for use in the food ingredient industry are currently sourced from either the roots of chicory (Cichorium intybus) or Jerusalem artichoke, or in a bioreactor system using fungal invertases and sucrose as the feedstock [77].

2.135 The limitations with the current approaches are that the bioreactor system is relatively expensive to run, and neither chicory nor Jerusalem artichoke are ideal crops in terms of good yield, ease of harvesting and good product quality [77].

2.136 The application of GM technology to the production of fructans offers the potential to:

• adapt existing fructan-accumulating crops such as chicory with genes from other plants to improve both the quality and quantity of fructans formed [77]; or

23 The number of fructose units is described as the degree of polymerisation or DP with DP3 fructans consisting of three fructan units (Sevenier et al., 2002).


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• modify non-fructan accumulating plants that have superior agronomic performance, for example sugar beet or potatoes24 [7]; or

• modify plants with bacterial genes encoding fructosyl transferase enzymes. This approach results in the production of very high molecular weight (DP>10000) fructans (usually referred to as levans) (Caimi et al. (1996); cited by [77]). The commercial applications for these levans was not reported.

2.137 Sugar beet for example offers an ideal system for fructan production. The crop has a well established agronomy and is able to yield up to 10 tonnes of sucrose per hectare, with the sucrose being accumulated in the vacuoles of the tap root cells [7]. Studies with chicory have shown that the fructan is also synthesised in the root tap cell vacuole, with fructan biosynthesis using sucrose as the primary substrate.

2.138 Two key genes have been identified for the modification of sugar beet to produce fructans. Short chain fructans requires the insertion of the 1-sst gene which encodes the enzyme sucrose:sucrose fructosyl transferase (SST), whereas the production of the longer chain molecule requires both 1-sst and 1-fft (encoding fructan:fructan fructosyl transferase (FFT)) [7].

2.139 The modification of sugar beet with the 1-sst gene from Helianthus resulted in the accumulation of short chain fructans (DP3, DP4 and DP5) in the transgenic beet [78]. Comparisons of the sugar levels in the transgenic beet compared with the unmodified form showed that sucrose levels in the transgenic beet were 7.9 mg g-1 (fw) compared with 84.2 mg g-1 (fw) in the unmodified beet. The transgenic beet also contained 37.2, 22.5 and 4.8 mg g-1 (fw) of the fructans DP3, DP4 and DP5 respectively [78]. The difference in the sugar content was reported to be due to the modified plant converting the sucrose into fructans.

2.140 The modification of other sugar beet lines with both the 1-sst gene and the 1-fft gene from Helianthus resulted in the accumulation of fructans with a high DP (data not published) [7]. It is not known whether these high-DP fructans were similar to the levans reported by Caimi et al. (1996)(cited by [77]).

2.141 Sugar beet have also been modified with a construct containing the sacB gene from Bacillus subtilis (encoding levansucrase which generates fructan from fructose) [79]. Maximum levels of fructan accumulated in the transgenic beet were lower (~5 mg g-1 dw in the leaves and ~0.3-0.9 mg g-1 dw in the roots) than those achieved with the Helianthus genes.

24 Although potatoes have been genetically modified to produce fructans (by expression of 1-sst), the levels produced were lower than for sugar beet, indicating that sugar beet offers a more suitable system for the production of fructans (Hellwege et al., 1998; cited by Smeekens,1998).


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2.142 The developments with the GM fructan producing sugar beet described above, and the need for an alternative fructan producing crop to chicory, mean that GM fructan beet may be viewed as a GM crop likely to be grown commercially. No studies have been identified investigating the effect of the 1-sst and 1-fft modifications on the persistence or survival of the transgenic beet in the environment. However, the role of fructan as an osmoprotectant means that plants containing high concentrations of fructan may should enhanced tolerance to drought conditions. The sacB+ sugar beet showed significantly better growth under drought conditions compared to the wild-type, and may therefore be expected to survive in areas with low water availability.

2.143 Alterations to sucrose levels (caused by modifying fructan production), may have implications to the growth and survival of the GM beet in the environment as sucrose is used by the plant as a growth storage compound.

Modified cellulose

2.144 Plants with modified cellulose content have been developed [80]. However, because of the important role of cellulose as the load-bearing structure in plant cells [81, 82] such modifications are likely to be either lethal or have drastic effects on plant growth and development [83].

2.145 Cellulose is a polymer of β(1,4)-linked glucan chains in which each glucan residue is orientated 180° to its neighbour to form a polymeric repeating unit (cellobiose) [81]. The formation and deposition of cellulose in plant cells occurs both during and after cell growth (termed primary and secondary cellulose respectively), and is responsible for both the extent and orientation of cellular expansion, and therefore ultimately the shape of the plant [80, 81]. Any alteration of cellulose biosynthesis is therefore likely to have an effect on multiple aspects of the development of the plant [80].

2.146 As of 2000, the understanding of the synthesis of cellulose in plants is limited, with much of the information available having been developed from studies of the systems in two bacteria, Acetobacter xylinum and A. tumefaciens. The system in plants is reported to show substantial homology to the bacterial cellulose synthases, with the plant cellulose synthesis genes described collectively as the CesA subgroup of genes [81].

2.147 The purpose of the studies on cellulose modification that have been conducted to date has been to understand further the genetic basis of cellulose biosynthesis in plants. Reducing or modifying the cellulose content of plants does in theory have potential applications for crops used as animal fodder, for timber or in industrial processing. Such a modification should improve the digestibility of the fodder (and therefore the nutritional benefit to the animal), improve the decay resistance in timber or reduce the processing requirements (thereby leading to energy savings for


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example). However, although such advantages of cellulose modification may in theory occur, the extremely adverse effects on plant growth and development mean that these modifications are unlikely to be pursued in the field.

2.148 Interestingly US Patent 6,495,740 [84] reported the modification of the cellulose biosynthetic pathway in a range of plants (including crop plants and trees), through the antisense expression of the gene encoding a cellulose synthase enzyme. The objective of the modification was for improved decay resistance in timber and altered digestibility of foodstuffs. No information was however provided on the occurrence of any adverse effects on the growth or development of the transgenic plants. The patent is viewed as a precursor to further development of such modified plants.

Modification of other cell wall polysaccharides

2.149 With respect to other cell wall polysaccharides, modification strategies have been reported for the pectic polymers that comprise between 30-50 percent of the cell walls in dicotyledonous plants [85]. Pectic polysaccharides form a matrix within the cell wall surrounding the load-bearing network of cellulose and cross-linking glycans. Pectic molecules comprise mainly of homogalacturonan (HGA) and rhamnogalacturonan I (RG I) [86].

2.150 HGA is composed of unbranched α-1,4-linked galacturonic acid (GalA) residues, whilst RG I consists of a backbone of repeating α-(1,2)-L-rhamnose(Rha)-α-1,4-D-GalA disaccharide units with side chains of arabinan and/or galactan [86].

2.151 Although the functions of individual pectic polysaccharides are still unclear, they have been implicated in the regulation of cell wall porosity, cell separation, cell expansion, texture changes associated with fruit ripening and as a source of signalling molecules [86]. Because of their role in the composition and structure of plant cells, then improvements to industrial processing are proposed as the most likely reason for any genetic modification of pectic polysaccharides.

2.152 The potato starch processing industry for example generates large volumes of waste pulp that is rich in pectins. Whilst other plant pectins are processed for use as food gelling agents for example, potato pectin is not used due to its relatively high content of neutral galactans [85]. The modification of pectin structure in potatoes to reduce the level of neutral galactans without affecting other characteristics of the tuber therefore offers the potential to make potato pulp suitable for downstream processing.

2.153 However, because of the wide range of functions that have been attributed to pectic polysaccharides, particularly with respect to their role in the development of the periderm and cortex tissues in the plant cells [86], then it is likely that any


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modification of these compounds will have a phenotypic or morphological effect on the modified plant. For example, potatoes modified to overexpress RG 1 lyase (by the addition of the eRGL gene from the fungus Aspergillus aculeatus) produced smaller tubers with much deeper �eyes� and a wrinkled appearance [86]. Higher expression of the eRGL gene, which confers the ability to cleave the RG I backbone at specific sites, resulted in a more severe change in phenotype [86]. Pollen from the GM potatoes also had reduced fertility relative to the wild-type [86].

2.154 The modification of cell wall polysaccharides presented in this section describe studies designed to determine the role of pectic polysaccharides in planta, and not to develop crops with modified compositional traits for commercial applications. Whilst changes to pectic polysaccharides may offer improvements for industrial processes, the adverse effects of the modifications on the crops may have a significant affect on the agronomic viability of growing such modified crops, and will need to be addressed before any large scale cultivation takes place.

Modification of raffinose family oligosaccharides

2.155 The raffinose family of oligosaccharides are α-galactosides of sucrose and include the compounds raffinose, stachyose and verbascose [87]. They are found in many higher plants, especially in the seeds of leguminous plants [88]. Although limited genetic modification of plants to alter levels of these oligosaccharides has been reported to date (2003), they have been included in this review because there is a potential modification with application to the animal feed industry, and these compounds also have a role as osmoprotectants in the plants [89] and are known to accumulate in vegetative tissue during cold acclimation [88]. However, whilst they are reported to provide an important role in conferring desiccation tolerance to seeds [90, 91], their importance in improving tolerance of vegetative tissues to environmental stresses may be limited [88].

2.156 The basis of the potential modification is that because α-galactosidase activity is lacking in the intestine mucosa of non-ruminant animals [92], then any raffinose family oligosaccharides (RFOs) present in the diet are not digested, but are converted by bacteria in the gut to hydrogen, carbon dioxide and methane [87]. Consequently, RFOs are considered to be the principal flatulence causing compounds present in food of plant origin [87] and mean that soybean meal for example cannot be fed to pigs or poultry.

2.157 One strategy to reduce levels of RFOs in plant seeds is to block the expression of the gene encoding galactinol synthase (GS), through an antisense modification approach [92]. This enzyme catalyses the first committed step in the biosynthesis of RFOs and is thought to play a key regulatory role in the partitioning of carbon between sucrose and RFOs in the developing seeds [89].


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2.158 Although such a modification has the potential to reduce levels of RFOs in plant seeds, the role of these oligosaccharides as osmoprotectants in the plants [89] mean that the alteration of RFO levels in planta may have implications to the response of the transgenic plants to drought and cold stresses.


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MODIFIED LIGNIN CONTENT

Introduction Shikimate pathway Phenylpropanoid pathway

Modification of 4CL Modification of C4H Modification of C3H Modification of OMT and CCoAOMT Modification of F5H

Lignin-specific pathway Modification of CCR Modification of CAD

Multiple gene manipulation and targeted gene manipulation Modification of OMT and CCR Modification of CCR and CAD Modification of OMT, CCR and CAD

Enzymes involved in the regulation, final production and deposition of lignin Peroxidases Gibberellins Laccase Lignin Regulatory Factors

Conclusions and final thoughts on genetic manipulation of lignin biosynthesis and deposition

Introduction

2.159 Lignin is an essential major structural component in the walls of certain specialised plant cells. It imparts both strength and water impermeability to the plant and is closely associated with the polysaccharides found in the secondary cell wall. Stress-induced lignin deposition provides a mechanism for the sealing off of sites of pathogen infection and/or wounding [93-97].

2.160 The composition of lignin subunits that make up lignin compounds varies between plant species, between cell types within species and between cell types in different stages of tissue development [93, 98, 99] There are three subunits of lignin in the plant kingdom: p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S). In gymnosperms (e.g. black spruce) the lignin in the tracheary secondary wall is mainly composed of G subunits whereas H units are the dominant components of the middle lamella regions (overall G is the dominant subunit). In angiosperms (e.g. birch) the secondary cell walls are made up of G and S units, while vessel lignins are mainly composed of G units (overall S is the dominant subunit). Grasses contain all three of the G, S and H subunits. [93, 97, 100] These observations illustrate the complexity of lignin biosynthesis even within a single plant and imply that lignin assembly is a highly organised biochemical process which can predictably differ according to cell type [100].


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2.161 Although the basic pathway for lignin biosynthesis was outlined some years ago, more recent work, including work in the field of genetic modification, has resulted in revisions to the previously proposed pathway and the proposal of alternative routes for the synthesis of some intermediate molecules formed in the process [101, 102].

2.162 The reason lignin biosynthesis has been a focus for research into plant metabolism is due to its importance in industrial and agricultural applications [103, 104, 102, 100]. In the pulp and paper industry, lignins have to be removed from wood through expensive and polluting chemical processes in order to recover cellulose [93, 105-107, 95, 99, 108, 97, 1, 109]. Lignins also limit the digestibility of forage crops by livestock, resulting in the incomplete utilisation of cellulose and hemicellulose in ruminant animals [93, 95, 99, 1]. Both of these areas of modification are focused towards reducing the lignin composition of woody plants. Trees with reduced lignin content could also lead to the more efficient production of bioethanol [110].

2.163 However, there is also interest in increasing the lignin composition of plants for the purposes of an improved renewable energy source. This is because lignin has a high calorific value, and therefore a hyperlignified wood which could be used as fuel could help towards moves for more renewable fuel sources [93, 105, 97].

2.164 The lignin biosynthetic pathway is discussed in detail in Baucher et al. (1998) [101]. A simplified pathway is discussed below, (highlighting the main enzymes involved), to enable clear discussion of how the biosynthetic pathway of lignin has been manipulated genetically, with the aims of the industrial and agricultural communities in mind (as discussed above). The biosynthetic pathway for lignin biosynthesis can be simplified into three main sections (simplified from [101, 98, 100]):

(i) the Shikimate Pathway: this forms the aromatic amino acids phenylalanine, tyrosine and tryptophan from carbohydrates. Enzymes involved in this pathway include 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP);

(ii) the Phenylpropanoid Pathway: this starts from phenylalanine and leads to the biosynthesis of cinnamic acid (through the action of the enzyme phenylalanine ammonia lyase (PAL)). In some grasses and maize, cinnamic acid is produced from tyrosine (catalysed by tyrosine ammonia-lyase (TAL)).

The second part of the phenylpropanoid pathway is the processing of cinnamic acid: this occurs via a series of successive hydroxylation and methylation steps into:


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− p-coumaric acid (catalysed by cinnamic acid 4 hydroxylase � C4H);

− caffeic acid (produced from p-coumaric acid, catalysed by coumaric acid 3-hydroxylase � C3H)

− ferulic acid (produced from caffeic acid, catalysed by caffeic acid O-methyltransferase � OMT)

− 5-hydroxyferulic acid (produced from ferulic acid, catalysed by ferulate- 5 � hydroxylase � F5H)

− sinapic acid (produced from 5-hydroxyferulic acid, catalysed by 5-hydroxyferulic O-methyltransferase � OMT)

Other enzymes are also involved in the phenylpropanoid pathway, whose action results in the production of further CoA esters of cinnamic acid (HCA CoA). Additional enzymes involved include:

− hydroxycinnamoyl CoA ligase (4CL);

− p-coumaroyl CoA-3-hydroxylase (pCCoA3H); and

− caffeoyl CoA O-methyltransferase (CCoAOMT).

The products of these various hydroxylation and methylation steps are the precursors of several major classes of plant product, only one of which is lignin.

(i) the Lignin-Specific Pathway: this involves the reduction of the products described in the phenylpropanoid pathway into monolignols, followed by their polymerisation via radical coupling. The different steps in monolignol biosynthesis are well-known, although the order in which the individual reactions occur in the pathway is not. In addition, the regulation and precise cellular location of the enzymes is not known. The enzymes involved are:

− hydroxycinnamoyl CoA reductase (CCR) this catalyses the reduction of HCA CoA to the corresponding aldehydes; and

− hydroxycinnamoyl alcohol dehydrogenase (CAD) this catalyses the last step in the synthesis of the lignin precursors; the reduction of cinnemaldehydes to cinnamyl alcohols.


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The cinnamyl alcohols are the precursors of the three lignin aromatic components: syringyl (S), p-hydroxyphenyl (H) and guaicyl (G).

2.165 The lignin biosynthesis pathway described above is summarised in Figure 2.3.

2.166 Several genes involved in the lignin biosynthesis pathway were cloned by the early 1990s, and all the major enzymes involved and their corresponding genes have now been studied and isolated. The last enzyme and gene to be isolated and identified was that encoding C3H. This was identified (2001) in an Arabidopsis mutant [101, 98, 103, 107, 100, 111, 112].

2.167 Early attempts at the genetic modification of lignin biosynthesis encountered problems due to the fact that the biosynthetic pathway is more complicated than was originally thought. The role of each of the enzymes in lignin biosynthesis appears to vary between different plant species and the importance of each of these enzymes in the amount and composition of lignin is unclear. Because of this it is difficult to predict the exact impact that the down-regulation of a certain gene has on the lignin content and composition of transgenic plants [101, 98, 103, 107, 96]. This is mainly because of the lack of understanding of the complexities involved in lignin assembly (e.g. how the process is initiated and how heterologous deposition of the various lignin subunits is controlled) [100]. Even less clear is the sequence of events that leads to the insertion and polymerisation of the lignin subunits within the extracellular carbohydrate matrix [96] Recent work has identified a group of glucosyltransferase genes involved in the glycosylation of sinapic acid and sinapyl alcohol, and which therefore may be involved in the production of syringyl subunits found in lignin in cell walls [113].

2.168 The following sections (firstly divided into the three parts of the biosynthetic pathway) review the application of genetic modification to lignin biosynthesis. The most recent work is discussed in the most detail, so as to provide DEFRA with the most up to date information, with the earlier work discussed as background. The last sections review studies addressing the use of GM technology in manipulating the final stages of lignin polymerisation and deposition.


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Figure 2.3 - Schematic representation of the lignin biosynthetic pathway

DAHP= 3-Deoxy-D-Arabino-Heptulosonate-7-Phosphate synthase. PAL= Phenylalanine Ammonia Lyase. C4H= Cinnamic acid 4-hydroxylase. C3H= Coumaric acid 3-hydroxylase. OMT=Caffeic acid O-methyltransferase. F5H=

Ferulate 5-Hydroxylase. 4CL= Hydroxycinnamoyl CoA ligase. pCCoA3H= p-Coumaroyl CoA-3-Hydroxylase (C3H and associated reductase). CCoAOMT= caffeoyl CoA O-Methyltransferase. CCR= Hydroxycinnamoyl CoA reductase. CAD=

Hydroxycinnamoyl alcohol dehydrogenase. S, H and G are all subunits of lignin.

Carbohydrate Shikimate Pathway

Tryptophan Phenylalanine Tyrosine

Phenylalanine

Cinnamic acid Phenylpropanoid Pathway

p-C oumaric acid

Caffeic acid

4CLFerulic acid CoA esters of Cinnamic Acid

p CCoA3H CCoAOMT

5-Hydroxyferulic acid Further CoA esters of Cinnamic Acid

Sinapic acid

to the Lignin-Specific Pathway

DAHP

PAL

C4H

C3H

OMT

F5H

OMT


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Lignin-Specific Pathway

CoA esters of Cinnamic AcidSinapyl CoA ester p- Coumaroyl CoA ester Feruloyl CoA ester

CCR

Corresponding AldehydesSinapaldehyde p- Coumaraldehyde Coniferaldehyde

CADCinnamyl alcohols

Sinapyl alcohol p- Coumaryl alcohol Coniferyl alcohol

Lignin aromatic componentssyringyl (S) p- hydroxyphenyl (H) guaiacyl (G)

from the Phenylpropanoid Pathway

Shikimate pathway

2.169 The reduction of DAHP synthase through the use of antisense technology led to a reduction in lignin biosynthesis by 65 percent showing unsurprisingly, that the Shikimate pathway is very important for lignin biosynthesis (Jones et al., 1995; cited by [101]). However, most work on the genetic manipulation of lignin biosynthesis has concentrated on the second two sections of the lignin biosynthetic pathway (Phenylpropanoid pathway and the lignin-specific pathway). This is probably because the Shikimate pathway forms the initial stage of many biosynthetic pathways and the manipulation of this pathway is likely to result in changes to traits of the GM species other than just lignin biosynthesis [101], and may therefore have knock-on effects to other plant characteristics.


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Phenylpropanoid pathway

2.170 PAL genes have been isolated and sequenced from a number of different plant species, including gymnosperms, monocotyledons and dicotyledons; a number of different isozymes of PAL have also been identified in different species [101].

2.171 The first plants to be generated with engineered lignin were tobacco plants with sense-suppressed levels of PAL. As expected, the modification resulted in a reduction in PAL activity and a reduction in stem lignin. However, these plants were also severely affected in their development; i.e. unusual phenotypes developed with localised lesions, altered leaf shape and texture, stunted growth, reduced pollen viability and altered flower morphology and pigmentation. These plants were also more susceptible to fungal pathogens (Elkind et al., 1990 and Maher et al., 1994 both in [101])(Elkind et al., 1990; cited by [93]) [98, 105].

2.172 Such examples as this show that early steps in lignin genetic modification are unsuitable for a controlled and specific modification of the lignin content of plants. PAL is involved in the biosynthesis of other phenolic compounds, and therefore the inhibition of PAL was found to alter the production of these compounds, in addition to lignin. These other phenolic compounds were found to be required for the normal functioning of the plant [93] It has been known for more than 20 years that the inhibition of PAL results in serious consequences for the vascular integrity of plants (Amrheim et al., 1983; cited by [100]).

2.173 Similarly, the genetic modification of the enzymes C4H and 4CL might be expected to produce adverse effects in GM plants. These enzymes are also involved in biosynthetic pathways other than that of lignin biosynthesis. The products of the reactions that these enzymes catalyse are precursors of a wide range of phenolic compounds [103, 100]. Indeed, their genetic modification has led to negative effects on plant growth and development in some species. C4H, however, is also considered to potentially be an important rate-limiting step in the lignin biosynthetic process (see C4H section below) [100].

2.174 Two enzymes of the Phenylpropanoid pathway that are thought to represent important targets for the controlled manipulation of lignin biosynthesis are OMT and F5H. OMT is thought to control the production of ferulic acid in gymnosperms or the formation of ferulic and sinapic acids in angiosperms. F5H is proposed to control the production of 5-hydroxyferulic acid in both plant groups [93] There may in fact be more than one isoform of F5H which have slightly different substrate specificities [100].

2.175 Ferulic and hydroxyferulic acids lead to the production of lignins composed of guaiacyl lignin monomers, whilst synaptic acids lead to the production of lignins


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composed of syringyl lignin monomers. Therefore in gymnosperms the major lignin monomer is guaiacyl while in angiosperms it is syringyl [93, 97].

2.176 The relative proportions of the different subunits that make up lignin is important in the industrial paper-making process, as the efficiency of wood pulping is directly related to the proportion of syringyl units that make up the lignin. This is because lignins made up of more syringyl units are less condensed in nature. Gymnosperm wood (with a low proportion of syringyl and high proportion of guaiacyl units) has been found to require more chemical treatments during the pulping process than angiosperm wood that contains a higher proportion of syringyl units [93, 114, 115]. The first attempts at the manipulation of lignin composition through the genetic manipulation of the enzymes F5H and OMT were therefore designed to alter the composition of the units making up the lignin within wood, with the aim of improving the efficiency of the wood pulping industry [93].

2.177 Having outlined briefly the phenylpropanoid pathway, the following sections discuss the recent discoveries and genetic modifications of the enzymes in the phenylpropanoid pathway including 4CL, C4H, OMT, F5H, C3H and CCoAOMT

Modification of 4CL

2.178 A number of different isozymes of 4CL have been identified in different species and a number of different cDNAs for this enzyme have been isolated from plants [101]. To date, 4CL down-regulation has been carried out only for isozymes that are considered to be involved in lignin biosynthesis. Other isozymes of 4CL are involved in, for example, flavonoid biosynthesis. It is unclear whether manipulations of lignin isozymes of 4CL also affect the functions of other 4CL isozymes [100]

2.179 One of the first demonstrations of the genetic modification of 4CL was in Arabidopsis, where the suppression of 4CL gene expression resulted in a change in the subunit composition of lignin where the guaiacyl content decreased and the syringyl content stayed at approximately the same level (Lee et al., 1997 in [114])(Lee et al., 1997; cited by [98]). This resulted in reduced lignin levels, but at a cost to the plant, namely collapsed cell walls and stunted growth in the most severe lignin reductions (Kajita et al., 1996 and Piquemal et al., 1998; both in [2]).

2.180 Hu et al. (1999) [2] went on to investigate whether these adverse effects would also be seen in trees with modified 4CL genes. A patent for the alteration of the lignin pathway through the manipulation of the gene encoding 4CL was filed as part of this work [116]. The work by Hu et al. (1999) produced possibly the most interesting piece of research into lignin modification, from an industry perspective, and showed that the reduced expression of the 4CL gene in transgenic aspen led to a reduction in lignin content (up to 45 percent) without a change in lignin composition. The


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modified trees also exhibited an increased growth rate and increased cellulose content. The change in cellulose content was proposed to be a plant compensation method for the reduction in lignin and may account for the preserved plant structure in the transgenic aspen trees [2].

2.181 The genes used in the study by Hu et al. (1999) [2] had been isolated from aspen. The enzyme that this gene (Pt4CL1) codes for was thought to be devoted to lignin biosynthesis in developing xylem tissue of aspen. Genetically modified trees were produced through A. tumefaciens mediated transformation using an antisense Pt4CL1 gene construct [2].

2.182 Although the experiment provided evidence that lignin biosynthesis could be manipulated, subsequent reviews urged caution in the interpretation of Hu et al.�s (1999) [2] results, due to the small sample size involved. Further studies investigating the effects of the manipulation of various isozymes of 4CL may provide further information on how useful this could be for the manipulation of lignin biosynthesis and its application in industry and agriculture [114, 100]. The targeting of the genetic modification to those isozymes of 4CL involved in the development of lignin in key parts of a plant (e.g. the xylem), along with the use of the species specific isozyme (i.e. the use of the aspen lignin 4CL isozyme gene for the modification of aspen) may hold the key to successful lignin manipulation. The increased growth rates seen in the genetically modified plants produced by Hu et al. (1999) [2] are likely to provide a selective advantage of these GM plants over the wild-type plant, therefore the release of such plants would need to be approached with caution and further study would need to be undertaken to see how the young trees produced in Hu et al.�s experiment continue to develop.

2.183 Anterola and Lewis (2002) [100] noted that for all of the experiments where 4CL has been down-regulated in plants with the aim of altering the lignin content, the significant reductions in lignin only occur after more than a 60 percent reduction in 4CL activity. This implies that there is more 4CL activity available in the wild-type plants than necessary for the production of lignins. 4CL may therefore not be a rate-determining step in lignin biosynthesis.

2.184 Therefore further investigations looking at the genetic manipulation of this enzyme to alter lignin composition in plants may not be forthcoming.

Modification of C4H

2.185 C4H is a cytochrome P450-linked monooxygenase involved in a wide range of biosynthetic pathways. At least two different forms of C4H exist and a number of cDNAs encoding C4H have been cloned from different plant species [101, 100].


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2.186 Transgenic tobacco plants have been produced that express the alfalfa C4H gene in a sense or antisense orientation. An increase in C4H activity did not have any effect on lignin content or composition. A reduction in C4H activity, however, did lead to a reduction in lignin content and a marked difference in the lignin composition, with the proportion of syringyl units being strongly reduced. In addition, no adverse effects were reported in the growth and development of these genetically modified plants (Sewalt et al., 1997; cited by [101, 98, 100]

2.187 Anterola and Lewis (2002) [100] analysed the data from this study further and suggested that the results showed a progressive and quantitative reduction in lignin quantity with downregulation of the gene encoding C4H. This led to them suggesting, provisionally, that C4H may be a major rate-limiting step in lignin biosynthesis.

2.188 However, due possibly to the fact that the reduction in lignin is also related to a reduction in the proportion of syringyl units and that these subunits are the most desirable for their beneficial effects on the structure of lignin for industrial and agricultural processing, research has not focused on this enzyme further.

Modification of C3H

2.189 C3H was the last known enzyme and gene to be isolated and identified from the lignin biosynthetic pathway. It is thought to be responsible for the 3-hydroxylation of p-coumaric acid to form caffeic acid and was thought to be a cytochrome P450 enzyme [117]. This enzyme was identified and confirmed as a cytochrome P450-dependent monooxygenase in 2002 in an Arabidopsis mutant [100, 111, 112]. This mutation resulted in Arabidopsis plants that had a lower lignin content than the wild-type and were also extremely dwarfed and susceptible to fungal attack [100, 111]. Analysis of the lignin in the mutant found that along with a large decrease in total lignin content, there was also an almost complete reduction in both G and S subunits and the lignin almost entirely consisted of H units, suggesting that C3H is a rate-limiting step in the production of G and S units [100, 111].

2.190 Further study is likely to be undertaken to look at the effect of down-regulation of the gene encoding C3H on lignin content and structure and the growth and development of transgenic plants.

Modification of OMT and CCoAOMT

2.191 The cDNAs encoding a number of OMTs have been identified and characterised from a number of different species, mostly from angiosperms, although more recently one has also been identified from gymnosperms [101, 98]


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2.192 At least two different isozymes have been identified; the first playing a role primarily in lignification and the second playing a role in defence against infection (Jaeck et al., 1992; cited by [101]). The OMT involved in lignification is thought to be involved in the methylation of compounds that eventually result in the production of the syringyl units of lignin [100].

2.193 The genetic manipulation of OMT, and its effects on lignification have been the subject of study since the mid-90s, involving a number of different plant species [100] and subject to patent applications (e.g. US Patent no. 5.959,178 published 1999). Early manipulations of the OMT gene produced conflicting results. One research group investigating tobacco, genetically transformed with alfalfa OMT cDNA, found that the modification resulted in a significant reduction in lignin, with no change in lignin monomer composition (Ni et al., 1994; cited by [93]. Van Doorsseleare et al. (1995) [118] however, found that the expression of an antisense construct of the cDNA of OMT in poplar under the control of the CaMV 35S promoter resulted in reduced OMT activity (up to 95 percent). In this study, the lignin content of the trees was not decreased, but there was a significant reduction in syringyl units and an increase in guaiacyl units, along with the appearance of a new component of lignin, namely 5-hydroxy-guaiacyl. The xylem of these transgenic plants was also altered in colour (pale pink compared with white/yellow of wild-type). The altered colour of lignin tissue is also seen in other GM plants with down-regulated OMT, as well as mutants that lack syringyl lignin subunits. The stems of the down-regulated plants are also reduced in strength (Huang et al., 1999; cited by [100]).

2.194 The different results obtained by the two groups of researchers in the two transgenic plants could be due to heterologous expression or due to the analytical methods used by the different teams of researchers to analyse the amount and subunit composition of lignin [101]. What these experiments did demonstrate was that it is possible to modify lignin composition and/or lignin content through the genetic modification of the OMT gene.

2.195 The results of the experiments described above, along with the results of other experiments on the modification of the OMT gene were re-analysed by Anterola and Lewis (2002) [100] to look for common patterns and trends. A notable trend was that, although OMT was clearly linked to syringyl production, there was no reduction in syringyl content of genetically modified plants until the OMT activity in the GM plant was less than 30 percent of the wild-type activity. This suggests that OMT is not a rate-limiting step for the biosynthesis of syringyl lignin subunits [100].

2.196 Further experiments have also found that the expression of antisense OMT genes in transgenic poplar and aspen produced dramatic differences in lignin composition compared with wild-type, with a reduction in syringyl units and an increase in guaiacyl units in the genetically modified plants (Lapierre et al., 1999 and Tsai et al.,


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1998 in [119, 114]. With the increase in guaiacyl units, it was not surprising that the OMT down-regulation resulted in a reduction in the pulping efficiency of two year old transgenic poplars, compared with the wild-type (Lapierre et al., 1999 in [119]). This led to Jouanin et al. (2000) [119], attempting the opposite approach, i.e. over-expression of OMT in order to produce lignin with increased levels of syringyl units. However, the increase in OMT activity in transgenic poplars over-expressing OMT did not result in any changes in lignin content and/or structure.

2.197 These results led to hypotheses being put forward that OMT was not the only methyltransferase involved in the production of syringyl and guaiacyl units and that the formation of guaiacyl unit precursors involved another pathway that was catalysed by the enzyme caffeoyl CoA O-methyltransferase (CCoAOMT) (Zhong et al., 1998 in [120, 119, 114]). The gene encoding for this enzyme has been cloned and is the subject of a US patent [121].

2.198 Transgenic tobacco (Zhong et al., 1998; cited by [99]) and transgenic poplar (Meyermans et al., 2000 and Zhong et al., 1998; cited by [99]) down-regulated for CCoAOMT showed a reduction in lignin content, due to a reduction in both syringyl and guaiacyl units, confirming the role of CCoAOMT in the biosynthesis of these lignin units. Meyermans et al. (2000) [122] also found a slightly increased syringyl:guaiacyl ratio in the transgenic poplars, suggesting that CCoAOMT is most important for the catalysation of the production of guaiacyl units. This reduction in lignin did not appear to affect the morphology and the growth of the transgenic poplars [99]. Zhong et al. (2000); cited by [97]) also showed that the lignin extracted from the wood of these transgenic plants was less cross-linked than in the wild-type. These characteristics made the wood of CCoAOMT down-regulated plants easier to process during pulping than the wild-type (Petit-Conil et al., 1999; cited by [97]).

2.199 Subsequent experiments looking to increase the digestibility of alfalfa forage have provided further confirmation of the role of CCoAOMT in the production of guaiacyl units. Guo et al. (2001) [123] produced two sets of transgenic alfalfa plants. In the first, they generated transgenic alfalfa plants with strong down-regulation of the gene encoding OMT. This resulted in a reduction in lignin content, along with a reduction in guaiacyl units, but a near total loss of syringyl units (Guo et al., 2001 in [123]). In the second, Guo et al. (2001) [123] generated transgenic alfalfa plants with strong down-regulation of the gene encoding CCoAOMT. This also led to a reduction in lignin levels, but the composition changes only showed a reduction in guaiacyl units, without a reduction in syringyl units [123]. This method of modifying lignin composition and increasing digestibility of forage crops is subject to a patent (International Publication Number WO 01/73090 A2 published 2001).

2.200 The alfalfa plants with down-regulated CCoAOMT were found to be more digestible than either the OMT down-regulated plants or the wild-type; thereby pointing to the


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down-regulation of CCoAOMT as a method of improving the digestibility of alfalfa forage. This was thought to be due to the combination of reduced lignin and increased syringyl:guaiacyl ratio in the lignin composition [123].

2.201 The transgenic plants with down-regulated CCoAOMT did not appear to have any significant changes in their vascular morphology compared with the wild-type, however, the authors urged further studies and field trials to be undertaken, in order to look at whether this genetic modification resulted in any negative impact on the yield or persistence of this forage crop [123].

2.202 Pinçon et al. (2001) [124] also showed that the inhibition of CCoAOMT and OMT gene expression either alone or together in transgenic tobacco affected lignin biosynthesis. CCoAOMT down-regulated plants were produced by A. tumefaciens mediated transformation. The gene construct used contained the antisense sequence for CCoAOMT under the control of a 35S RNA promoter and terminator. The transgenic plants showed a reduction in total lignin content, whereas the relative amounts of syringyl units stayed approximately the same as the wild-type. OMT down-regulated plants were also produced by A. tumefaciens mediated transformation. The gene construct used was the same as for CCoAOMT modified plants, but with the antisense sequence for OMT present instead of that for CCoAOMT. The transgenic plants showed changes in lignin composition, with a decrease in syringyl units. Transgenic tobacco plants were also produced through Agrobacterium mediated transformation with a double antisense construct, the same as for the single antisense gene constructs, but with both the CCoAOMT and OMT antisense cDNAs present. In these plants both the quantity and composition of lignin was affected, with a reduction in lignin content and a reduction in syringyl unit content. This was accompanied by an accumulation of the 5-hydroxyguaiacyl lignin subunit.

2.203 It was also found that the development of transgenic plants modified for altered levels of CCoAOMT, and CCoAOMT and OMT was affected, with a reduction in plant growth and an alteration in flowering activity being observed in those lines that were most inhibited. No such changes were observed in OMT altered transgenic plants. These changes were particularly prominent in the later stages of plant development. This experiment demonstrates the additive effects of the inhibition of the enzymes OMT and CCoAOMT [124]. This has important implications for the possible use of OMT and CCoAOMT modified plants in industry and agriculture in that it should be made sure that the plant is not adversely affected in its development. The discovery that these adverse effects were most prominent in the later stages of the plants� development indicated that longer term studies are required to investigate the effects of modification of these genes, particularly for longer-lived species such as trees.


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2.204 Any future work on the effect of genetic manipulation of OMT or CCoAOMt would need to bear in mind the potential effects of the manipulation on the survival of the plant in the longer term; field studies may be used as a suitable medium for such studies. The discovery that OMT is not rate-limiting for the lignin biosynthetic pathway may limit further work in this area; a number of different isoforms of CCoAOMT have been identified (at least six), the roles of each of these are unclear and therefore further work may also be carried out to elucidate this further.

Modification of F5H

2.205 F5H is a cytochrome P450-linked monooxygenase, but is distinct from C4H and is thought to be involved in aromatic hydroxylation that ultimately forms sinapyl alcohol [101, 98, 100]. F5H deficient Arabidopsis mutants have been found to contain only guaiacyl lignin units, whereas wild-type plants contain both syringyl and guaiacyl lignin units. This indicates that F5H is a determining step in the monolignol composition of lignin. This has potential to be used in the pulp and paper-making industry to manipulate the F5H levels in plants, therefore resulting in a manipulation in the proportion of syringyl units making up the lignin, with the aim of increasing the efficiency of the pulping process [93, 101, 95] Not unsurprisingly therefore the modification of lignin composition in gymnosperms through the manipulation of F5H is subject to a patent [125].

2.206 Initial studies found that the over-expression of F5H cDNA in transgenic Arabidopsis resulted in the production of a novel lignin in the plant, composed almost entirely of syringyl units (up to 95 percent syringyl units) (Meyer et al., 1997; cited by [101, 98]). This clearly demonstrates the potential importance of the manipulation of F5H levels in determining the lignin monomer composition.

2.207 Meyer et al. (1997, cited by [98]) also found that the use of a lignin associated promoter (Arabidopsis C4H promoter) was much more efficacious than a general promoter such as CaMV 35S (the promoter most commonly used in lignin genetic modification experiments). It has been suggested that it may be worth revisiting other lignin biosynthesis manipulation strategies using this more specific promoter to see if this is more effective than the general promoter [98].

2.208 In a recent example, Franke et al. (2000) [126] carried out further studies looking at the effects of the over-expression of the cDNA of F5H in tobacco and poplar as model woody plants that undergo secondary growth (unlike Arabidopsis).

2.209 Two different constructs were produced: one containing the cDNA of the F5H gene where the regulatory sequences have been replaced with the CaMV promoter sequence (35S-F5H), and the other where the regulatory sequences had been replaced with the Arabidopsis C4H promoter (C4H-F5H) [126].


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2.210 For the CaMV 35S promoter-driven F5H expression in tobacco, high levels of expression of the F5H gene were detected, but there was no increase in the syringyl content of the secondary xylem of the genetically modified tobacco plants, compared with the wild-type, despite increases in syringyl content of primary xylem in the GM plants. However, the expression of F5H driven by the C4H promoter led to an increase in the lignin syringyl monomer content in the secondary tissues. This was correlated positively with the amount of F5H mRNA abundance measured in the leaves of the plant. The total lignin content was the same for wild-type plants and the F5H transgenic plants with the CaMV 35S promoter. However, the C4H-F5H transgenic plants with the C4H promoter had a reduced amount of lignin present [126]

2.211 Transformation of poplar using the C4H-F5H construct also resulted in transformed plants with a higher proportion of syringyl units in the lignin than the wild-type and an apparent overall reduction in lignin content (although this may be due to the methodology used for lignin content measurement, rather than a reflection of the true lignin content and therefore merits further study) [126].

2.212 No obvious phenotypic differences were seen between the wild-type and genetically transformed tobacco lines. Therefore, the modification of lignin composition through the expression of F5H under the C4H promoter seems to provide a promising strategy for the benefit of industry and agriculture without consequences on plant viability that have been seen in other attempts at the genetic modification of this pathway [126]. However, further experiments need to be carried out to estimate the changes (weakening or strengthening) that may have occurred in the vascular apparatus, including microscopy to check that the integrity of the vascular structure remains [100].

2.213 The data reported by Franke et al. (2000) [126] also point towards a much more complex biosynthetic pathway than the approximately linear one described in the section above. Lignin monomer biosynthesis probably actually occurs by a cross-linked network of pathways, reinforcing the view that the lignin biosynthetic pathway is more complex than once was thought.

2.214 The apparent reduction in lignin content; with concomitant increase in S units in GM plants with genetically modified F5H would be of great benefit to the paper-making industry at least. Therefore we could expect to see further experiments investigating the effects of F5H manipulation further, and this may include field trials to establish the longer-term survival of such GM plants, as well as cellular level investigations into the effects of the genetic modification on cellular structure and function.


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Lignin-specific pathway

2.215 The two enzymes involved in this pathway, CCR and CAD have previously been considered to be specific to lignin biosynthesis, and therefore also considered to be a rate-limiting step. However, the monolignols produced as a result of the action of these enzymes are not only precursors to lignin, they are also precursors for other compounds (e.g. other related phenolics). The down-regulation of CAD and CCR could therefore also affect the synthesis of compounds other than lignin [93, 100]

2.216 Recently it has been proposed that an enzyme distinct from CAD operates to mediate the reduction of sinapaldehyde into syringyl monolignols in aspen; this is sinapyl alcohol dehydrogenase (SAD) [127] The use of the gene encoding SAD and the potential to modify crop species to produce syringyl-enriched lignin in the plants is subject to both a patent and patent application [128, 129].

2.217 The results show that the full picture of the biosynthesis of lignin is still incomplete. Further research should help to elucidate this, making it easier to manipulate the biosynthetic pathway and confidently predict how this will affect the content and composition of lignin within the plant.

Modification of CCR

2.218 A full-length cDNA for CCR was isolated in 1994 [93] There has been some discussion in the literature as to whether there are in fact three distinct isoforms of CCR that utilise different CoA esters of cinnamic acid. However, only one CCR gene has been found thus far in a number of different plant species; two were found in Arabidopsis but only one of these was thought to be involved in lignification [100].

2.219 It can be predicted that the down-regulation of CCR would have significant deleterious physiological effects on the plant, as the down-regulation of this enzyme would affect the biosynthesis of compounds other than lignin. It would also be expected that a severe reduction in CCR activity would result in a weakening of the vascular apparatus due to the lack of lignin present [100].

2.220 CCR down-regulation in transgenic tobacco was found to result in decreased lignin content, an increase in S:G ratio and an orange-brown colouration of the xylem (Piquemal et al., 1998; cited by [99]). Tensile testing of CCR-modified tobacco stem tissue supported the prediction that plant vasculature would be weakened (Hepworth et al., 1998; cited by [100]). Despite this, the modification of lignin synthesis through the down-regulation of CCR activity in cereal and forage crops is the subject of a US patent [130](suggesting some researchers view this modification as having commercial potential).


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2.221 Similar results to those discussed by Mellerowicz et al. (2001) [99] have been obtained more recently in CCR down-regulated transgenic tobacco where the most severely CCR-suppressed line had a significantly decreased lignin content, and an increase in S:G ratio. These changes would be likely to make lignin easier to extract in the paper pulping process, if CCR was down-regulated similarly in poplar [131]. However, supporting Anterola and Lewis� (2002) [100] prediction, the suppressed lines also showed adverse effects in their phenotypes including reduced growth compared with the wild-type.

2.222 Because of these adverse effects the authors [131] suggested that CCR-suppression will not be the optimal target for genetic manipulation of lignin content in plants in the future.

Modification of CAD

2.223 At least two isoforms of CAD have been found to occur in angiosperms whilst only a single CAD has been identified in gymnosperms. The most studied CAD isoform is the one classically associated with lignin biosynthesis, whereas the functional significance of the other isoform is unknown, although this too may give a supporting role in lignin biosynthesis [93, 100].

2.224 The first evidence of the possibility of the genetic manipulation of lignin in trees came from the identification of a null allele for the CAD gene in loblolly pine (MacKay et al., 1997 and Ralph et al., 1997; both in [114]). Although the total lignin content of the tree homozygous for this recessive allele was very similar to the wild-type tree, it was suggested that the lignin was made up of different subunits to the normal types. Subsequent studies found that this lignin did contain the same lignin subunit types as the wild-type, although the lignin were more easily removed in mild alkali treatments and soda pulping, suggesting that this could be used to the advantage of the pulping and paper-making industries (MacKay et al., 1999 in [114])(Ralph et al., 1998; cited by [100]). The implications of these findings are unclear, as additional mutations may be present within the plant (and not just in CAD) that cause the lignin differences in the mutant pine; it is likely that these will be studied further in the future [100].

2.225 CAD down-regulated plants have been produced in a number of different species. One of the first CAD down-regulated plants was produced through ectopic expression in tobacco of a homologous CAD cDNA antisense construct driven by the CaMV 35S promoter. The transgenic plants were found to have a very low CAD activity but the lignin content was found to be similar in the transgenic and control plants. However, it was found that the composition and structure of the lignin had been altered in the transgenic plants, making it easier to extract the lignin chemically. The lines that had the most severely repressed CAD activity also had reddish-brown coloured xylem, but showed no apparent changes in growth or development (Halpin


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et al., 1994; cited by [93] and [100]). Similar results were also found in the downregulation of CAD in alfalfa and poplar. Further investigation is required to determine whether the red coloration seen in down-regulated CAD and OMT plants can be attributed to similar biochemical alterations [118].

2.226 Halpin et al. (1994) [132] postulated that the improved chemical extractability of the CAD down-regulated plants had potential application in the paper-making industry, if applied successfully to forest trees. They also suggested it could be used in forage crops to make them more digestible.

2.227 CAD activity has been manipulated in transgenic poplars to test this hypothesis. CAD activity was reduced by up to 70 percent in the transgenic plants through the expression of an antisense CAD gene [133]. This resulted in a small reduction in lignin content and importantly, for the industrial sector, improved pulping characteristics (Lapierre, et al. 1999 in [114])(Lapierre, pers. comm; cited by [107]). These characteristics are hopeful for the pulping and paper-making industry as such trees would lead to savings in the amount of chemicals required for pulp production and the amount of energy used in the pulping process (Petit-Conil et al., 1999; cited by [95]). The modification of lignin biosynthesis through the down-regulation of CAD is subject to a patent (US Patent 6,066,780, granted 2000) whose aim is the improvement of plants by the modification of lignin biosynthesis, particularly, but not exclusively, the improvement of the digestibility of fodder crops.

2.228 CAD down-regulated plants have now been developed by a number of different research groups in a number of species (alfalfa, poplar and tobacco) [100] The total lignin content in all of the plants is not affected, although the changes in the composition of lignin result in a pink/red colour in the xylem along with an increased extractability of the lignins in the pulp production process used in paper-making (as indicated by a decrease in �Kappa� number) [107].

2.229 The pink/red colouration of the xylem is found to result in a change in the colour of the wood in such transgenic trees. Instead of the usual pale colour, the wood of many of the GM plants had a mottled or red-brown colouration due to the accumulation of coniferyl aldehyde residues. It has been proposed that this coloured wood could have advantages in furniture manufacture since it would not require any staining (Tsai et al. 1998 and Higuchi et al. 1994; cited by [12]).

2.230 The effects of CAD down-regulation on plant development and growth and therefore plant persistence and survival have also been investigated. Preliminary investigations on CAD down-regulated tobacco plants found that they had a reduced tensile stiffness in their secondary vasculature, compared with the wild-type. This could have adverse effects on the plants development and/or persistence [134, 100] However further study by Hepworth and Vincent (1999) [134] found that the


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genetically modified plants were able to compensate for this by increasing the thickness of the xylem tissue cylinder more than the wild-type plants did when exposed to mechanical stimulation (flexing of the stems).

2.231 Recent field trials have provided additional evidence that transgenic trees expressing CAD antisense genes suffer no adverse effects. It was found that after four years of growth at two different sites the GM trees (poplars) remained healthy throughout and showed no significant differences to the wild-type trees in growth or fitness [135]. The authors of this study also claimed that the interactions of the GM trees with insects were normal and no changes were detected in soil microbial communities beneath the GM plants.

2.232 These developments and their potential benefits for the paper-making industry make it more likely that such transgenic plants will proceed further down the path towards commercialisation. There are eight field trials of crops genetically modified for reduced lignin content in the USA [57]. These comprise the plants alfalfa, pine, Festuca sp. (grass), Paspalum sp. (grass), maize and potato. The modification of the grasses (Festuca sp. and Paspalum sp.), alfalfa, maize and potato is probably intended for increasing the digestability of forage crops for livestock, rather the paper-making industry.

Multiple gene manipulation and targeted gene manipulation

2.233 The genetic manipulation of lignin biosynthesis has to date (2002) been met with success for a number of different plant species. Up until very recently, all of the transgenic plants with modified plant lignins had been obtained through altering the expression levels of a single gene.

2.234 In further attempts to optimise lignin composition, for industrial and agricultural purposes, several research groups have been looking at developing approaches in which more than one transgene, from different parts of the lignin biosynthetic pathway, maybe manipulated in an individual plant. Two strategies have been developed and compared:

• the crossing of plants carrying single antisense genes; and

• the use of double constructs in which two transgenes are expressed through a single promoter [103]

2.235 The modification of lignin biosynthesis through the expression of more than one transgene in the plant, along with variations in the associated promoters is subject to at least two patents [136, 137].


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Modification of OMT and CCR

2.236 Pinçon et al. (2001) [124] produced two single-gene transgenic tobacco lines; one expressing antisense OMT cDNA and one expressing antisense CCR cDNA. These were crossed to produce a double down-regulated transgenic plant. The consequences for both plant development and lignin biosynthesis were investigated. It was discovered that the successful double transformants had a significant reduction in total lignin content, compared with the wild-type25 and an increase in the S:G ratio (as might be expected from previous single-transformant experiments). Some double transformants produced from the crossing did not have a reduction in the activity of one or other of the target enzymes. This was thought to be due to gene silencing [102].

2.237 Ultrastructural analysis found that the double transformants had a slight loosening of the cellular wall structure, but that this effect was much less than that seen in the single CCR transformant. The growth and development of the double transformants was always less adversely affected in the double transformants than in the single antisense CCR transformant. However, both the phenotypical differences and the biochemical differences between the wild-type and the double transgenic plants appeared to increase with increasing age and with exposure to non-laboratory growing conditions (i.e. field- and greenhouse-grown) [124] The reduction in size of the double transformants (both in size and leaf) along with the ultrastructural changes mean that such a plant would be less able to survive and persist in the environment than the wild-type. There would be no danger of the double-transformant being able to out-compete the wild-type.

2.238 The findings of Pinçon et al. (2001) [124] are particularly pertinent in the application of this technology to silviculture (i.e. longer-lived plants that are planted in field conditions seem to display more adverse effects of the double transformation as they develop). Therefore these double CCR and OMT double antisense transformants may not be suitable for silviculture despite the benefits of reduced lignin and increased S:R ratio, due to the implications for the longer term survival of the trees.

Modification of CCR and CAD

2.239 Chabannes et al. (2001) also obtained double transformants with CAD and CCR down-regulation in tobacco. This was achieved through the crossing of individual plants down-regulated for CCR or CAD. These double transformants showed up to 50 percent reduction in lignin content which was more than twice that obtained for either the CCR or CAD single gene down-regulated transgenic plants. This suggested that the effect of the down-regulation of the two genes was more than

25 The lignin content of the single transformants was not used as a comparison


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additive i.e. it had a synergistic effect. The double down-regulated transformants also showed an increase in the S:G ratio, compared with the wild-type. Their morphology and size appeared to be similar to wild-type. However, the xylem showed a brownish colouration (similar to single CCR down-regulated plants). The xylem vessels of the double transformant also appeared to not be significantly affected by the decrease in lignin content.

2.240 The fact that the double transgenic plants do not appear to be significantly different to the wild-type suggests that they would not have any selective advantage over the wild-type.

2.241 The authors suggested that other transgenic plants (e.g. woody species) that are down-regulated for both CAD and CCR should be evaluated further � in field experiments to see whether the effect of decreasing lignin content without the adverse effects seen in tobacco also applies in other species and in other conditions [138]

Modification of OMT, CCR and CAD

2.242 Abbott et al. (2002) [102] used a slightly different approach to the suppression of multiple genes in transgenic plants. Instead of crossing transgenic plants that suppressed single genes (as in the above example) they used a single chimaeric DNA construct that incorporated partial sense sequences of three genes to target suppression of three lignin biosynthetic enzymes (CCR, OMT and CAD).

2.243 The incorporation of this genetic construct into tobacco plants was successful, resulting in down-regulation of the expression of all three of these genes. However, although this resulted in plants with reduced lignin content, their phenotype was severely adversely affected � the transgenic plants being much smaller than the wild-type, having growth defects and deformed xylem vessels [102].

2.244 Although such a combination of genes would not be suitable for application in industry due to the adverse effects on plant growth and development (and therefore survival), this experiment usefully demonstrates the feasibility of using the chimaeric transgene system for the manipulation of combinations of genes, without the issues of gene silencing seen in transformants produced by crossing [102].

2.245 The only experiment identified from the scientific literature that has looked specifically at the wider ecological impact of the genetic manipulation of lignin biosynthesis in plants, particularly relating to the persistence of modified plant material in the environment, is one by Hopkins et al. (2001) [139] which considered whether the lignin genetic modifications in tobacco plants affected their decomposition rate. The tobacco plants used were either unmodified (wild-type) or had antisense or partial


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sense transgenes for one of three lignin biosynthetic enzymes (CAD, OMT or CCR). Solid-state C-13 nuclear magnetic resonance spectroscopy indicated that stem material from the unmodified plants, reduced CAD and reduced caffeic acid O-methyltransferase (COMT) plants all had similar amount of lignins, whereas stem material from the reduced CCR plants contained less lignin. Material from all of the modified plants decomposed more rapidly than material from the wild-type plants, with CCR-modified plants decomposing the most rapidly [139].

2.246 The increased decomposition rate of the CAD and COMT down-regulated plants was thought to be due primarily to differences in the lignin composition and conformation of these plants that offers a lower degree of protection from microbial attack to the polysaccharides and other relatively labile plant components than in the wild-type lignin. The greater rate of decomposition of reduced CCR plants compared with the wild-type plants was though to be due to their overall reduced lignin content [139]. However, CCR down-regulated plants have also been found to have altered S:G ratio (see CCR section above), therefore the reduced lignin content per se is unlikely to be the only reason for the increased decomposition rate.

2.247 The increased decomposition rate seen in plants that are down-regulated for these lignin biosynthetic enzymes means that the transgenic plants break down quicker, and therefore would persist for a shorter time in the environment than the wild-type plants. Conversely, it might be expected that an increase in lignin content would result in plant material that was more persistent in the environment.

Enzymes involved in the regulation, final production and deposition of lignin

2.248 It is also likely that the rate of lignin deposition is regulated, not only by monolignol synthesis, but also by the rate of transport, storage and mobilisation of monolignol precursors to the cell wall. It appears that complex and tight regulation of the deposition of lignins into different cell types of xylem and the different sub layers of the cell wall is involved [103, 107].

2.249 The final assembly of lignin is carried out by the coupling of radicals produced through the single-electron oxidation of lignin precursors (the monolignols). The oxidases involved in this final step are unknown, although two candidates that have been considered are peroxidases and laccases. The process is further complicated by the fact that these oxidases may be under the control of other plant chemicals, including hormones such as auxin (indole acetic acid (IAA)) and gibberellin [120, 97].

2.250 A patent has also recently (2002) been granted that claims to have discovered a group of genes in Arabidopsis that can act as �lignin regulatory factors� (US Patent 6,410,826 granted 2002). However, it is not clear at which stage in the lignification process the regulatory factors have their effect.


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Peroxidases

2.251 There are many genes encoding peroxidases (more than 60 have been identified from the Arabidopsis genome). This combined with the low substrate specificity of peroxidases makes it difficult to identify a peroxidase that is involved specifically in lignification. A cDNA coding for one possible peroxidase has been cloned and expressed in poplars. Despite an increase in peroxidase activity, there was no change in the amount of lignin or the composition of lignin (Christensen et al., unpublished; cited by [99]).

2.252 Sitbon et al. (1999) [140] looked at the effects of the overproduction of IAA in transgenic tobacco plants on peroxidase activity, the activity of other enzymes of the lignin biosynthetic pathway (CAD) and associated effects on lignin content and composition. It was found that the overproduction of IAA resulted in an increase in the total lignin content of the plant compared with the wild-type, and that there was a decrease in S:G ratio due to an increase in G subunits. This was associated with increased ethylene production in leaves and increased peroxidase activity. However, there was no difference in the activity of the CAD between the wild-type and transgenic plant. This suggests that IAA, through an increase in ethylene synthesis, increases the levels of peroxidase activity that are involved in the final production and deposition of lignin [140]. Further investigation is required to examine the hormonal control of the peroxidase activity, and hence lignin content. It is unclear from the study whether there are any potential adverse effects on the persistence or survival of the transgenic plant.

2.253 The overexpression of IAA gene and hence the increase in peroxidase activity in transgenic plants appear to have little application to industry as it results in an increase in lignin (rather than a decrease). Commercialisation of such crops is therefore not expected.

Gibberellins

2.254 The role of IAA and peroxidases is complicated by the fact that other plant hormones often act with IAA. For example, gibberellins which also stimulate meristematic activity and xylem fibre elongation when applied together with auxin [99].

2.255 Eriksson et al. (2000) [141] looked at the effect of overexpression of the gene encoding for gibberellin in hybrid aspen. These transgenic plants were found to have more numerous and longer xylem fibres (and hence probably higher lignin content, although this was not looked at specifically in this study). The transgenic plants also had an increased growth rate and plant biomass.


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2.256 This has implications for persistence and survival as transgenic plants that have an increased growth rate and biomass could, potentially, out-compete the wild-type plants. The lab-grown plants did show a poorer rooting efficiency which may be a disadvantage to such transgenic plants growing in the field [141]

2.257 No other studies have investigated the application of this genetic modification. This is probably because the roles of gibberellins and other plant hormones in lignin biosynthesis and deposition are still not fully understood.

Laccase

2.258 Other enzymes believed to be involved in the polymerisation of monolignols are the laccases, but their precise role is also unclear [99].

2.259 To investigate the role of laccases in lignification, Ranocha et al. (2002) [142] down-regulated laccase in transgenic poplars. The transgenic poplars had a reduction in laccase activity, and this did not appear to have any effects on the growth and development of the plant. Neither was lignin content nor composition significantly affected by the decrease in laccase activity. However, laccase does appear in some way to be required for normal cell wall structure and integrity in xylem fibres as the laccase suppression led to deformed xylem fibre cells and cell wall detachment in one laccase down-regulated line.

2.260 The lack of alteration in lignin content and composition despite a decrease in laccase could be that there is still sufficient residual laccase activity in the transgenic plants to allow lignification to proceed; that is plants normally produce far more laccase than they need for lignin biosynthesis [142]. It could be concluded from this that laccase is not a rate-limiting step in lignin deposition.

2.261 Alternatively, laccases may only be important in lignification in certain cells or under certain conditions. For example, Pilate et al. 2000 (in [142]) found that one laccase isozyme was significantly up-regulated in wood that was under tension, suggesting that laccase has a role in the formation of wood under stress. Alternatively, laccase may not be involved in lignification, but may be involved in determining cell wall structure in xylem fibres [142]. Laccase down-regulation in certain cases has implications for cell structure and function and therefore for the longer-term persistence and survival of the transgenic plant.

2.262 Clearly the role of laccase in lignification is unclear and may be subject to further study.


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Lignin regulatory factors

2.263 A group of Arabidopsis genes (AGAMOUS-LIKE8 (AGL8), AGL1, AGL5, and a specific transcription factor � R-LIKE bHLH) that have, up until now, been thought of as �floral organ identity genes� that specify the identity of the various floral organs, have been found to regulate the process of lignification (as claimed in US patent no. 6,410,826 and US patent application no. 20030005481, 2003). The authors suggest that this would be particularly useful in the reduction of lignin in woody plants such as pine or spruce or in leguminous or graminaceous forage crops. However, the examples discussed in the patent only look at the reduction of lignin in Arabidopsis. Also there is no discussion of how these genes might be affecting lignin biosynthesis, or the effect this may have on persistence or survival; presumably the down-regulation of such genes may also have an adverse effect on the development of the flowers of the plant.

2.264 This provides an interesting potential alternative to the control of lignin biosynthesis. Further study would be required to establish how these genes affect lignin biosynthesis and the effects that this would have on the persistence and survival of crop species.

Conclusions and final thoughts on genetic manipulation of lignin biosynthesis and deposition

2.265 Many of the attempts to manipulate enzymes described in this section have been introduced into the plants under the control of the �constitutive� promoter CaMV 35S. This results in the enzyme being expressed or repressed throughout the plant system, rather than its expression or repression being directed to the lignin-forming parts of the plant. This constitutive expression may therefore cause adverse effects in the development and/or growth of the GM plant for this reason [100]. To investigate the effects of the overexpression or repression of the genes of these enzymes without such effects, genes encoding for enzymes in the lignin biosynthetic pathway should be introduced to the plant (in the sense or antisense orientation) under the control of promoters that are specific to the lignin-forming parts of the plant (e.g. vascular bundles). An example of this is seen in the use of a lignin-specific promoter for the down-regulation of F5H by Franke et al. (2000) [126] in the F5H section above. It is likely that those enzymes that have been identified as �rate-limiting� will, at least, be subject to further experimentation using such lignin-specific promoters in plants of industrial and/or agricultural interest. The effects of these manipulations on the persistence and/or survival of the crop plant will vary according to the crop plant, the enzyme manipulated and, possibly, the promoter used and therefore should be considered carefully on a case-by-case basis.


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MODIFIED OIL AND FAT CONTENT

Biosynthesis of fatty acids in plants Note on fatty acid nomenclature

Genetic modification of fatty acid composition Limitations of single gene modifications Protein engineering Increasing fatty acid yield

Modified oil content for food and cooking applications Modified oil content for industrial applications

Wax production 2.266 The purpose of this section of the report is to review modifications to the fatty acid

content of plants that result in changes to their oil or fat content. The modifications presented in this section of the report include those that have been developed to alter the fatty acid composition in plants for both food/cooking and industrial applications. Alterations of fatty acid content for improved levels of �essential� fatty acids (nutraceutical applications) are presented in the section �Modified Micronutrient Content�.

2.267 Interest in the modification of the fatty acid content in plants is based on the diversity of applications for vegetable-based oils and their prominent position in the biological oil market. Vegetable oils make up approximately 85 percent of the global production of fats and oils from biotic sources [143], with soybean-derived oil accounting for 23 percent of the total; palm oil, 18 percent; and rapeseed, 13.5 percent [143].

2.268 For the purposes of this report, the modifications of plants for altered fatty acid content may be divided into two areas:

• for food and cooking applications � includes modifications to improve specific properties of the oil so that it offers greater health benefits for use in foods destined for human consumption; and

• for industrial applications � includes modifications to reduce the production costs of speciality oils that are produced naturally by tropical crops or those with poor agronomic traits. Applications to modify the oil content of plants are reported to be more diverse than those for food/cooking applications [144].

2.269 Because of the diversity of potential modifications, it is viewed as useful to review the biosynthesis of fatty acids in plants and to highlight the important points in the biosynthetic pathway at which genetic modification may be targeted to alter the fatty acid composition of the plant.


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2.270 In plants, fatty acids are present predominantly in membrane lipids or in specialised storage organs such as seeds. Plant membrane lipids (also described as structural fatty acids) are composed primarily of glycerolipids which have a relatively uniform structure, consisting of two fatty acids linked to a glycerol backbone at the sn-1 and sn-2 positions, with a polar headgroup at the sn-3 position [145]. The fatty acids present in glycerolipids consist primarily of 16-carbon (C16) and 18-carbon (C18) fatty acids containing up to three double bonds. They are largely conservative in composition with little variation in the degree of unsaturation or the types of functional side groups present [146].

2.271 The conservative structure of glycerolipids is designed to ensure that the fluidity and integrity of the membrane is maintained under the variety of environmental conditions to which the plant might be exposed. The ability of plants to survive variations in temperature or exposure to environmental stresses is due in part to their ability to alter the fluidity of their lipid membranes through slight changes to the fatty acids present. Greater changes to the fatty acids present are not desired, as these are likely to have a detrimental effect on membrane fluidity and the ability of plants to survive in the environment [143].

2.272 In contrast to the glycerolipids, the fatty acids present in seed oils have a much greater variation in structure. In excess of 300 naturally occurring fatty acids have been reported in seed oils [146], with the fatty acids present having between eight and twenty-four carbons with double bonds in novel positions and a variety of functional groups. As with membrane fatty acids, the seed fatty acids (also described as storage fatty acids) are esterified to a glycerol backbone, although all three positions on the glycerol are occupied by fatty acids. The resulting triacylglycerol (TAG) is a major form of lipid storage in seeds, and consequently the predominant constituent of all refined edible vegetable oils [145, 143].

Biosynthesis of fatty acids in plants

2.273 In plants de novo fatty acid biosynthesis occurs in the plastid, where acetate (C2) is elongated by the sequential addition of two carbon units while attached to a soluble acyl-carrier protein (ACP). The growing acyl chain is then cleaved from the ACP by an acyl-ACP thioesterase to leave a free fatty acid. This is exported to the cell cytoplasm (Figure 2.5) and assembled into glycerolipids or CoA at the endoplasmic reticulum (ER) [146, 147](Figure 2.6).

2.274 Typically, elongation of the carbon chain in the plastid continues until the fatty acid is between 16 and 18 carbons in length. Fatty acids with a chain length of <16 carbons (C8-C14) are described as medium chain fatty acids (MCFAs), and are formed in the plastid by the action of an additional acyl-ACP thioesterase which cleaves the acyl chain from the ACP before it reaches C16/C18 length [148]. Fatty acids with a chain


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length in excess of 18 carbons (designated very long chain fatty acids or VLCFAs) are synthesised in the ER through successive rounds of elongation of a C18 fatty acyl precursor, with the additional carbons originating from malonyl-CoA [146]

2.275 The triacylglycerols (TAGs) (Figure 2.4) are synthesised through a number of different processes [143]. After activation to CoA the fatty acids formed in the plastid are either esterified sequentially directly to glycerol-3-phosphate (G-3-P) to produce lysophosphatidic acid (LPA), phosphatidic acid (PA), diacylglycerol and triacylglycerol, or are fluxed through phosphatidylcholine (PC) where further desaturation and hydroxylation reactions occur [147].

Figure 2.4 � Structure of triacylglycerol molecule [149].

2.276 Whilst wax esters are formed in all plants, higher than average concentrations occur

in the seed oil of wild-type jojoba. Research on the alteration of plants to increase quantities of wax esters present has therefore focused on the modification the jojoba plant, or the incorporation of the relevant jojoba genes into other crop plants [147].

Note on fatty acid nomenclature

2.277 Fatty acids consist of a chain of carbon atoms with a carboxyl group at one end and a methyl group at the other. Fatty acids are described using the nomenclature A:B, where A is the number of carbon atoms in the chain, and B the number of double bonds. Fatty acid 18:2∆9,12 denotes a 18 carbon fatty acid with two double bonds located at carbons nine and twelve, counting from the carboxy (�front�) or delta (∆) terminus of the fatty acid. (The omega (ω) system of notation is used to describe double bond position from the methyl (�back�) end of the fatty acid).

CH2O

OCH

CH2O linolenic acid

(18:3)

O

O

O

stearic acid (18:0)

oleic acid (18:1)


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Figure 2.5 � Simplified scheme of plastid fatty acid synthesis (adapted from Thelen and Ohlrogge, 2002 [147]) (see Table 2.1 for key to number references)

acetyl-CoAATP, HCO3-

malonyl-CoANAD(P)HACP fatty acid synthase

4:0 ACP

10:0 ACPfatB (1)

12:0 ACPfatB (2)

14:0 ACP

16:1?4 ACP 16:0 ACPpalmitoyl-ACP ?4

desaturase (6)fatB (3)

18:1?6 ACP 18:0 ACP(4)

18:1?9 ACPfatA

CoA

SH, A

TP

AC

YL-C

oAs

FREE

FA

TTY

AC

IDS

stearoyl-ACP ?9 desaturase (5)


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Figure 2.6 � Simplified scheme of reactions for modification of fatty acids in oilseeds and their assembly into triacylglycerols (adapted from Thelen and Ohlrogge, 2002

[147]) (see Table 2.1 for key to number references)

glycerol-3-phosphate

18:1-CoA 16:0-CoA

18:0-CoA

lysophosphatidic acid 20:1-CoA

phosphatidylcholine phosphatidylcholine(mono-unsaturated) (di-unsaturated) 20:0-CoA

phosphatidic acid

22:1-CoA 20:1-CoA

alcohols

wax

diacylglycerol

triacylglycerol

acyl-CoA

Acetyl CoA pool

12

13

9

8

12

14

7


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Table 2.1 � Current understanding on the fatty acids, plants, enzymes and gene regulation available for modification to alter the fatty acid composition in plants

(adapted from Thelen and Ohlrogge, 2002 [147])

Target fatty acid

Modified plant Max. produced (mol %)

Enzyme involved (numbers refer to Figures

2.4 and 2.5)

Source of transgene

Gene regulation

Capric Brassica napus 38 Acyl-ACP thioesterase (1) Cuphea Up

Lauric Brassica napus 58 Acyl-ACP thioesterase (2) Calfornia bay Up

Lauric Arabidopsis 24 Acyl-ACP thioesterase (2) Calfornia bay Up

Palmitic Arabidopsis 39 Acyl-ACP thioesterase (3) Arabidopsis Up

Palmitic Brassica napus 34 Acyl-ACP thioesterase (3) Cuphea Up

Stearic Soybean 30 Stearoyl-ACP ∆9 desaturase(5)

Oleoyl-∆12 desaturase (7)

Soybean Down

Stearic Brassica napus 40 Stearoyl-ACP ∆9 desaturase (5)

Brassica Down

Stearic Cotton 38 Stearoyl-ACP ∆9 desaturase (5)

Cotton Down

Stearic Brassica napus 22 Acyl-ACP thioesterase (4) Mangosteen Up

Petroselenic Tobacco 4 Palmitoyl-ACP ∆4 desaturase (6)

Coriander Up

Oleic Soybean 86 oleoyl ∆12 desaturase (7) Soybean Down

Oleic Brassica napus 89 oleoyl ∆12 desaturase (7) Brassica Down

Oleic Cotton 77 oleoyl ∆12 desaturase (7) Cotton Down

Oleic Brassica juncea 73 oleoyl ∆12 desaturase (7) Brassica Down

Oleic Arabidopsis 54 oleoyl ∆12 desaturase (7) Arabidopsis Down

γ-linolenic Brassica napus 47 oleoyl ∆6 and ∆12 desaturase (7)

Mortierella apina

Up

γ-linolenic Tobacco 1 oleoyl ∆6 desaturase (7) Cyanobacteria Up

Eleostearic Soybean 17 Conjugase (11) Momordica Up

∆5 eicosenoic Soybean 18 β-ketoacyl-CoA synthase (8)

Acyl-CoA desaturase (9)

Meadowfoam Up

Hydroxyl fatty acids

Arabidopsis 30 Oleate-12-hydroxylase (10) Castor Up

Ricinoleic Arabidopsis 17 Oleate-12-hydroxylase (10) Castor Up

Acetylenic Arabidopsis 25 Acetylenase (11) Crepis Up

12,13-epoxy-cis-9-oleic

Arabidopsis 15 Epoxygenase (11) Crepis Up


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Wax esters Arabidopsis 70 β-ketoacyl synthase (12)

acyl-CoA reductase (13)

was synthase (14)

Jojoba up

Genetic modification of fatty acid composition

2.278 The genetic modification of fatty acids in plants is concerned largely with the alteration of the composition of seed fatty acids. Membrane fatty acids offer little potential for alteration as they have limited structural variation and any significant changes in structure are likely to have implications for the behaviour and survival of the plant in the environment.

2.279 In contrast, seed fatty acids exhibit a large variety of structural diversity and can be accumulated in some plants at high levels [143, 146]. Also, because they have no role in maintaining membrane fluidity, then changes to seed fatty acids are proposed to have no affect on the behaviour or survival of plants in the environment26.

2.280 The application of genetic modification techniques has been/is proposed to allow the alteration of the types and relative quantities of seed fatty acids that are expressed and accumulated by plants, and may have the potential to increase the total amounts of fatty acids produced [147]. In some cases, changes in the types of fatty acids accumulated can be achieved through the alteration of a single gene. For example, a single mutation of an elongase gene in canola inactivates the gene encoding a β-keto-acyl-ACP synthase. This synthase enzyme is involved in the elongation of C18 fatty acids and its inactivation prevents the accumulation of the long-chain fatty acid erucic acid (22:1n-9) which is normally present in high quantities in this plant27 [143].

2.281 Alteration of the enzymes involved in the synthesis of the fatty acids, such as the thioesterases and desaturases has been the main approach adopted to date for the modification of fatty acid composition. Enzyme activity can be modified through:

• introduction of new genes;

• overexpression of the relevant gene � increased levels of the required enzyme can be achieved by overexpression of the relevant gene by placing it under the control of a strong promoter. A possible limitation with this approach is that the increased level of mRNA may incur a defensive reaction from the plant and lead to a down-regulation of the expression of the target

26 Although this statement is correct for many of the modifications to seed fatty acid composition conducted to date, it is not applicable to all modifications (see Chapter 4 of this report).


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gene until the mRNA pool is reduced to below a critical threshold [150]. Increased production of novel fatty acids in particular has been reported to result in the induction of pathways28 by the GM plant to break them down [147];

• gene silencing and antisense � will reduce activity of the required enzyme; and

• protein engineering � means that modifications for fatty acid content in plants is not limited by the natural diversity of fatty acids. By redesigning the enzymes involved in the biosynthesis of fatty acids it is proposed to be possible to insert any functional group into a fatty acid of any chain length at any position on that chain [144].

2.282 The identification of a wide range of the genes involved in the expression of enzymes in fatty acid biosynthesis means that the basic genetic information required to control chain length, the degree of unsaturation, and positional specificity is now available for use [143]. The identification of the relevant genes is reported to no longer be a major limitation to research [143, 144]. The determination that the insertion of the same lipid-related transgenes into either soybean or rapeseed results in essentially the same modification is reported to mean that fatty acid modifications may be applicable to all oil-producing crops [144].

2.283 Examples of the various genes identified include those encoding:

• the four different fatty acyl desaturases that are responsible for the introduction of the double bonds required for the synthesis of α and γ linolenate [143];

• various desaturases that may allow fatty acid isomers that are not normally produced in common sources of edible oils, to be produced in such oils;

• the thioesterases responsible for the production of MCFAs. This has allowed the development of GM oilseed rape that accumulate MCFAs, such as caprylic acid (8:0), capric acid (10:0) and lauric acid (12:0) [148];

• the elongases required to convert oleate to fatty acids 20:1 and 22:1. Although each elongation step requires four enzymatic reactions

27 This mutation, which has been achieved by conventional (non-GM) breeding, is the basis for the distinction between canola and rapeseed (Broun et al., 1999). 28 Up to 40 percent of the lauric acid formed in transgenic canola modified to overexpress lauroyl-ACP thioesterase is recycled by the plant and not incorporated into TAG (Thelen and Ohlrogge, 2002).


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(condensation between an acyl precursor and the malonyl-CoA, a reduction, a dehydration and another reduction), studies with transgenic plants have found that expression of just the gene encoding the condensation reaction is sufficient for the synthesis of the VLCFAs. Levels of the other three enzymes required are therefore concluded to be present in the cells in sufficient quantities [146]; and

• the acyltransferases involved in the synthesis of diacylglycerol.

Limitations of single gene modifications

2.284 As discussed in the following sections, the modification of a single gene has resulted in significant changes to the fatty acid composition of crop plants. Examples of single gene alterations include the introduction of the gene encoding a lauroyl-ACP thioesterase from California bay into Brassica napus [148], and the antisense expression of a stearate desaturase gene in Brassica [151].

2.285 The modification of Brassica napus with the lauroyl-ACP thioesterase resulted in a significant increase in lauric acid (12:0) so that it accounted for 40 percent of the total seed fatty acid [148, 152]. However, further attempts to increase the lauric acid content of the oil to 70-90 percent using this single gene insertion were not successful [144]. Achieving this level of lauric acid content would make the rapeseed oil comparable with coconut and palm kernel oils [152].

2.286 Antisense expression of a stearate desaturase in Brassica sp. resulted in increasing the stearic acid content of the oil to ~40 percent. However, as with the lauric acid modification, further increases of the stearic acid content were not achieved through this single gene modification. Levels of up to 60 percent stearic acid have been achieved through the antisense expression of the desaturase and the overexpression of the fatA gene [149, 152]. However, achieving the more commercially viable level of 90 percent will require the modification of additional genes and metabolic processes [144].

2.287 The limitations of the single gene insertion approach in the modification of both the lauric acid and stearic acid content of plants highlights the importance of understanding as much of the fatty acid biosynthetic process as possible. The identification of non-GM plants such as Cuphea sp. that contain upwards of 80 percent lauric acid in their seeds [144] means that processes do occur naturally for the accumulation of the target high levels of lauric acid.

2.288 Improved levels of lauric acid in Brassica have been achieved by adopting a broader based approach to the modification. The high specificity of the lysophatidic acid acyltransferase (LPAAT), which mediates esterification of the TAG at the sn-2


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position, meant that the TAG only contained lauric acid at the sn-1 and sn-3 positions, thereby limiting levels of lauric acid in the oil. Therefore by modifying the Brassica with a less specific LPAAT gene from coconut [153] in addition to the lauroyl-ACP thioesterase, the GM crop accumulated up to 65 percent lauric acid [144].

2.289 Murphy (1999) [144] proposed that the incorporation of further genes, such as other lauric-specific acyltransferases, a lauric-specific acyl-CoA synthetase and enzymes such as phospholipases may allow even greater levels of lauric acid accumulation. One area of further modification that has been proposed is to increase the availability of short-chain acyl-ACP substrate [147]. The enzyme 3-ketoacyl-ACP synthase (KAS) from Cuphea sp. has a high specificity for short-chain fatty acids. The modification of plants with this enzyme is reported to increase the 10:0- and 12:0-acyl-ACP substrate reservoirs for the thioesterases and consequently increase the accumulation of medium-chain fatty acids [147].

2.290 Therefore, although single gene modifications can produce GM plants with increased accumulation of desired fatty acids, a more holistic approach involving additional genes and other metabolic pathways is likely to be required to increase levels to those required for the GM crops to be commercially viable.

Protein engineering

2.291 Protein engineering offers the potential to modify plants to produce a fatty acid of any configuration. The applications of protein engineering are most relevant to the development of GM plants for the production of industrial precursors and compounds for the paint, varnish and fine chemicals industries [144]. Novel engineered proteins may also be more effective than equivalent naturally occurring enzymes from wild plant species [147].

2.292 Possible areas of development include the design of novel desaturase enzymes that will generate double bonds at new points in the fatty acid chain. Naturally occurring desaturated fatty acids have double bonds only at the ∆6, ∆9, ∆12 and ∆15 positions. Protein engineering may allow the formation of double bonds at the ∆7 or ∆8 positions for example, and thereby produce fatty acids with novel properties [144]. Work with a ∆9 stearoyl desaturase from castor bean and a ∆6 palmitoyl desaturase from Thunbergia alata has shown that the modification of the proteins by swapping certain domains is able to produce desaturases that can introduce double bonds at novel positions [154]. Further developments in formation of novel proteins are likely to result in the production of fatty acids with new applications.


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Increasing fatty acid yield

2.293 Although the modification of single genes or groups of genes does result in changes to the fatty acid composition in the GM plant, the total amount of fatty acid produced remains unchanged. Therefore, in addition to altering the fatty acid composition, another proposed area of genetic modification of the fatty acids is the alteration of plants to increase their total oil content [147].

Modified oil content for food and cooking applications

2.294 Fatty acid composition in plants can be altered in a number of ways for food or cooking applications. Although some of the applications have been developed to provide health benefits they have been included in this section as the modifications involve basic changes to the accumulation and relative composition of �non-essential� fatty acids. Modifications involving �essential� fatty acids such as linoleic acid and linolenic acid are addressed in the section on �Modified Micronutrient Content�.

2.295 Widely reported targets for modification are the fatty acids palmitic acid (16:0) and stearic acid (18:0) present in the plant [155, 149, 143]. These two fatty acids are the principal saturated fatty acids present in vegetable oils [143], and interest in these compounds is a consequence of the known link between coronary heart disease and the consumption of saturated fats, and the effect that the refining of vegetable oils by hydrogenation has on the relative composition of fatty acids present [149].

2.296 One of the main risk factors for coronary heart disease is reported to be the concentration of cholesterol in the low density lipoprotein (LDL) fraction of the blood plasma. Whilst some saturated fatty acids such as stearic acid have a neutral (zero) effect on the plasma concentration of LDL-cholesterol29, other saturated fatty acids, primarily palmitic acid, myristic acid (14:0) and lauric acid (12:0) are reported to increase plasma LDL-cholesterol levels [149].

2.297 In contrast, unsaturated fatty acids have either a neutral or beneficial effect on reducing plasma LDL-cholesterol levels. Therefore, the objective of a number of plant modifications has been to substitute or reduce levels of saturated fats in vegetable oils. Two distinct objectives of the modifications are reported. For oils intended for use as salad oils the most desirable goal is the reduction of the total amount of saturated fatty acids to as close to zero as possible [149], whereas for oils that are processed for use in margarines and spreads the objective is to produce oils rich in stearic acid, low in palmitic acid and low in trans-unsaturated fatty acids [149].

29 Although stearic acid has been implicated in increased heart disease through other mechanisms (Kinney, 1996).


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2.298 The presence of high quantities of trans-unsaturated fatty acids in vegetable oils used for processing makes these oils oxidatively unstable and consequently unusable in their unmodified form. Therefore, for oils such as soybean oil, the stability of the oil and its suitability for use in food frying operations or baking, is improved chemically by selective hydrogenation. However, hydrogenation results in a decrease in the content of polyunsaturated fatty acids (PUFAs), principally linoleic acid and linolenic acid (important �essential� fatty acids)30, and an increase in monounsaturated fatty acids such as oleic acid (18:1) [149]. The development of oils with lower concentrations of trans-unsaturated fatty acids would therefore reduce the requirement for hydrogenation and its associated non-beneficial health effects on the composition of the resulting oil. The reduction of trans-unsaturated fatty acids is also reported by some nutritionists to be beneficial in terms of human health [156].

2.299 The modification of soybean through silencing the fatB gene, which confers the expression of a 16:0 ACP thioesterase, resulted in the soybean oil having a decreased saturated fat content (<4 percent) compared with 15 percent in current soybean processing oil [149]. Complete suppression of the fatB gene is reported to have the potential to reduce the saturated fat content of the oil to <3 percent. The results of other modifications are presented in Table 2.2.

Table 2.2 - Fatty acid composition of oil from soybean seeds of different transgenic lines (adapted from Kinney, 1996 [149])

Modification Fatty acid content (relative abundance of the fatty acid in the seed oil)

palmitic (16:0)

stearic (18:0)

oleic (18:1)

linoleic (18:2)

linolenic (18:3)

current commercial oil* 11 3 22 55 9

Transgenic lines:

fatB overexpressed 36 4 7 47 9

fatA overexpressed 16 10 12 54 9

HST1 mutant + fad2 silenced 5 30 60 1 4

fatB silenced 2 1 25 64 9

fad2 silenced 7 3 86 1 3

fad3 silenced 12 3 32 50 3 * commonly referred to as RBD (refined, bleached, deodorised)

2.300 The transgenic lines described in Table 2.2 represent just the basic steps that can be achieved through the modification of individual genes. Overexpression of fatB for example results in increased levels of palmitic acid, whereas the overexpression of

30 For further information see section on �Modified Micronutrient Content�


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fatA increases levels of stearic acid and reduces palmitic acid levels [157]. A combination of suppressing the fat2-1 gene and the fatB gene is proposed as allowing even greater reduction in the palmitic acid content of the oil [149].

2.301 Overexpression of the fad3 gene, which encodes or specifically controls a ∆15 desaturase converting linoleic (18:2∆9,12) to linolenic acid (18:3∆9,12,15), in canola resulted in an increased production of linolenic acid and a concomitant decrease in levels of linoleic acid [155]. Although specific data on the changes to the fatty acid content are not available, the importance of linolenic acid as an �essential� fatty acid means that the overexpression of fad3 gene has the potential to improve the micronutrient content of the resulting oil. Alteration of the linolenic acid content of oils

also has industrial applications (see following section) as this fatty acid is a significant component of linseed oil which has a diversity of industrial applications [155].

2.302 Combination of the high oleic line (generated by the silencing of the fad2 gene) with a line that has a mutation in one of the genes encoding delta-9 desaturase (used to convert 18:0 ACP to 18:1 ACP), produced seeds whose oil had a stearic acid content of ~30 percent, an oleic acid content of 60 percent and a polyunsaturated content of <5 percent. The stearic acid content may be increased further by the combination of this line with the fatA overexpression line. Oils from such lines are proposed to eventually replace chemically-hydrogenated oils in margarine and spreads [149]. Oils with a high oleic content have a ten-fold greater oxidative stability compared with the non-hydrogenated forms of current commercial oils, and a similar oxidative stability to the hydrogenated form.

2.303 Comparison of the soybean high oleic acid oil with those from GM canola and sunflower found that even where the fatty acid composition of the oils were similar, the oxidative stability of the soybean oil was three to four times greater than the other �GM oils�. The reasons for this are unknown, but indicate that other compounds such as tocopherols or cartenoids that confer some degree of oxidative stability on the oil [149]. Although genetic modification of tocopherol content of vegetable oils has been conducted [158], the effect of the modification on the oxidative stability of the oil was not addressed.

2.304 An alternative approach to altering the ratio of palmitic to stearic acid involves increasing the extent of unsaturation of the fatty acids through the introduction of novel desaturases [143]. In plants, the desaturation of stearate to oleate is conducted by the stearoyl-ACP desaturase, with desaturation only conducted in the plastid. Therefore, when the remaining stearate is exported from the plastid it is no longer a substrate for desaturation [143]. In contrast, in mammals and yeasts the stearate is desaturated in the ER by the stearoyl-CoA desaturase.


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2.305 The expression of a yeast or mammalian stearoyl-CoA desaturase in plants is therefore proposed to provide the plant an additional opportunity to further desaturase the stearate. In addition to reducing stearate levels, because palmitate is a much better substrate for the yeast or mammalian stearoyl-CoA desaturase than it is for the plant�s stearoyl-ACP desaturase, then the modified system is also able to decrease levels of palmitate by converting it to palmitoleate (16:1∆9) [143].

2.306 Modification of tobacco with a stearoyl-CoA desaturase gene isolated from a rat resulted in the increased accumulation of 16:1 and a reduction in levels of 16:0 and 18:0. Levels of 18:2 were also increased [159].

2.307 Although the modification of seed fatty acids is proposed not to affect the composition of membrane fatty acids, the alteration of some crops including oilseed rape and soybean with particular genes to improve their oleic acid content has resulted in changes to the fatty acid composition of root membranes. These changes have meant the GM plants have been unable to grow at low temperatures [160]. Further details on the reported changes to the behaviour and survival of plants modified for high oleic acid content are presented in Section 3 of this report (Assessment of the Effects of Compositional Changes on Persistence and Survival in the Environment).

2.308 Plants modified for altered oil content for food and cooking applications have already been grown in field trials and developed for commercialisation. Further developments may be expected in this field, particularly for modifications designed to impart various �nutraceutical� benefits.

Modified oil content for industrial applications

2.309 The purpose of this section of the report is to review applications where the fatty acid content of plants can be/has been altered to produce fatty acids for industrial applications. The modifications addressed in this section differ from those reviewed in the subsequent section �Production of speciality compounds for non-medical applications� in that all of the modifications reviewed in this current section deal with modified plant fatty acids.

2.310 As discussed in the previous section, the modification of fatty acids for food or cooking applications is concerned largely with the 18:0 and 16:0 fatty acids (and assorted desaturated variations). Although these fatty acids are used in industrial applications, substantial quantities of organic oils used by industry contain fatty acids that are either longer or shorter than C18 or C16. VLCFAs for example are used as lubricants and plasticisers [155].


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2.311 Of the C18 fatty acids used industrially, linolenic acid (18:3∆9,12,15) is particularly important as it is the major component of linseed oil which is used widely in paints, coatings, printing inks, automobile brake linings and as a curing additive in concrete [155]. Of the plants grown at a commercial scale, only flax (Linum usitatissmum L.) produces significant quantities of this fatty acid. Overexpression of the fad3 gene (which confers conversion of 18:2∆9,12 to 18:3∆9,12,15) in flax is reported to offer a 50 percent increase in levels of linolenic acid present in the oil [155].

2.312 Specific derivatives of oleic acid (18:1∆9), particularly those with acetylenic or epoxy groups present at the ∆12 position also have important industrial applications in the production of nylon, paints, varnishes and plasticisers [161]. The enzymes involved in the insertion of these acetylenic or epoxy groups are reported to be closely related to the ∆12 desaturase (FAD2) [162].

2.313 The addition of epoxy groups to high-oleic acid oil used for production of plasticisers is required to prevent cross-linking between the TAG and the plastic polymer. Epoxidation is currently achieved chemically, but as reported by Okuley et al. (1994) [162] it may be possible to produce epoxidised oils biologically. Such oils would provide a renewable alternative to petrochemically-derived plasticisers such as phthalates, which would not migrate from the plastic polymer [163].

2.314 The plant Vernonia galamensis is able to accumulate epoxy fatty acids, with these compounds accounting for 80 percent of total storage fatty acids [163]. The modification of soybean or other commercial oil crops with the gene responsible for the production of 12-epoxy fatty acid in Vernonia sp. has been proposed, although no further details are available [163].

2.315 Industrial applications of the medium chain fatty acids, especially lauric acid (12:0), are concerned largely with the synthesis of detergents [155, 164]. Lauric acid is currently produced on a commercial scale from coconut and palm kernel. However, because of large fluctuations in the value of oils from tropical plants and the consequent effect that has on the commercial viability of sourcing lauric acid from such crops, a number of studies have investigated the potential of developing a temperate plant as a source of lauric acid.

2.316 Several species of temperate herbaceous plants, including members of the genus Cuphea, produce lauric acid. However, strategies to cultivate these plants on a commercial scale have not been successful, and more recent studies have focused on the application of genetic modification to modify existing temperate crop plants to produce lauric acid [155, 143]. The modification of rapeseed to overexpress a thioesterase, encoded by the gene chfatb2 from Cuphea hookeriana resulted in the rapeseed accumulating significant quantities of 8:0 and 10:0 fatty acids [153].


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2.317 The genetic modification of Brassica napus with a medium-chain specific thioesterase from California bay31 (encoded by fatB) resulted in the accumulation of high levels of lauric acid (up to 65 percent in their seed TAG) [148, 152]. In addition to the accumulation of the target fatty acid, the lauric acid was specifically incorporated into the TAGs and not the membrane lipids in the transgenic oilseed rape plants. Although the reason for this �directed� accumulation was not known it does represent a significant result in the development of GM plants with modified oil content, in which the modification does not involve changes to the composition of membrane lipids and consequently has no deleterious effects on plant development and behaviour in the environment.

2.318 An alternative strategy for the production of lauric acid, is the development of plants able to accumulate petroselenic acid (18:1∆6). The chemical cleavage of a double bond in petroselenic acid results in the formation of both lauric acid and adipic acid (6:0 dicarboxylic)32 [165, 166]. As petroselenic acid is solid at room temperature, it also has applications in the production of margarines that do not require hydrogenation [156].

2.319 A desaturase enzyme that appeared to be responsible for the synthesis of petroselenic acid has been identified in coriander (Coriandrum sativum) [165]. The expression of this desaturase in transgenic tobacco however only resulted in the accumulation of low levels of the desired fatty acid. The limited production of petroselenic acid in the modified tobacco means that expression of the fatty acid is not solely under the control of the single acyl-ACP desaturase. Two other enzymes, a palmitoyl-ACP∆4 desaturase and a 3-ketoacyl-ACP synthase are also reported to be involved [155, 156]. No further developments in this area have been reported.

2.320 The most important VLCFA is reported to be erucic acid (22:1∆13) which is used for the production of precursors for the synthesis of nylon 13,13 [166]. Erucic acid is currently obtained from conventionally bred varieties of rapeseed which produce oil containing 56 percent of this fatty acid. A potential target of genetic modification is therefore the development of oilseed rape crops that produce a much greater proportion of erucic acid in their oil, with the ultimate objective being the development of a crop whose triacylglycerols contain essentially pure erucic acid.

2.321 However, the production of rapeseed with triacylglycerols containing just erucic acid is limited by the fact that in existing rapeseed cultivars, erucic acid is not found on the sn-2 position of the TGA [167]. However, the TGAs from other plants, such as meadowfoam (Limnanthes alba) do contain erucic acid at all three positions. The reasons for the differences between rapeseed and plants such as meadowfoam are proposed to be due to variations in the specificity of the lysophatidic acid

31 California bay (Umbellularia californica) produce a laurate-rich oil (Voelker, 1996).


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acyltransferase (LPA-AT) that mediates the esterification at the sn-2 position on the glycerol backbone of the TAG [168].

2.322 In the rapeseed, the LPA-AT is unable to utilise erucoyl-CoA [168], and consequently lead to the esterification of an erucic acid at the sn-2 position. The advantage of using the LPA-AT from Limnanthes sp. is that the enzyme from members of this genus positively selects for erucic acid at the sn-2 position [155, 168]. Studies are currently underway to identify further the gene encoding the sn-2-acyltransferase, with the ultimate intention of modifying the rapeseed pathway to increase yields of erucic acid [155]. Similar problems have been reported with the modification of plants to produce lauric acid at levels >60 percent of their total seed fatty acid [144, 147].

2.323 As with plants developed for modified oil content for food and cooking applications, field trials and commercial development of GM crops with altered oil content for industrial applications are well underway. There are for example 115 field trials of crops with modified �oil profile� and 63 with modified �oil quality underway or completed in the USA� [57]. Further developments are likely in this field, particularly where the modified crops offer the potential to produce fatty acids such as petroselenic acid which are currently difficult to produce on an agronomic scale.

Wax production

2.324 Waxes differ from TAGs in that the fatty acid chains are esterified to a long-chain alcohol. Waxes have many industrial applications, particularly as lubricants and as components of transmission fluids. The desert shrub jojoba, a native plant of the American Southwest is the only plant species known to accumulate waxes (up to 60 percent dry weight) rather than TAG as a seed storage compound [147]. Jojoba waxes are derived mostly from C20-C24 monounsaturated fatty acids and alcohols and are formed by the fatty acyl-CoA:fatty alcohol acyltransferase enzyme (also referred to as wax synthase) [169].

2.325 The modification of Arabidopsis with three genes; the reductase and acyltransferase from jojoba and a long-chain acyl-CoA from Lunaria annua, resulted in the GM Arabidopsis producing oil containing 70 percent jojoba wax. The high level of accumulation was reported to indicate that all of the genes necessary for the production of jojoba wax had been identified, and that the modification of commercial crop plants with these genes could provide an alternative source for this compound [147].

32 Used as a monomeric component in the synthesis of Nylon 6,6.


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2.326 The development of GM crops to produce wax offers a much cheaper alternative to harvesting the material from jojoba plants, especially if more widely cultivable plants such oil seed rape can be modified. Studies conducted to date have, as discussed, focused on the genes involved and not on the development of an agronomically viable crop. However, the development of such crops is viewed as having a commercial future.


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MODIFIED MICRONUTRIENT CONTENT

Modified amino acid content Modification of amino acid biosynthetic pathways Modification of endogenous seed storage proteins Modification using naturally occurring proteins Modification using synthetic proteins

Modified fatty acid content Modified flavonoid content Modified vitamin content

Vitamin E Vitamin A Vitamin C

Modified mineral content Iron

2.327 The purpose of this section is to review applications of genetic modification to alter

the composition of plants to improve their nutritional qualities, including the concentrations of �essential compounds�33 present. Modifications to improve nutritional properties have been termed �nutraceutical applications� [9], and include alterations to protein, fatty acid and carbohydrate content of a range of plants [170, 9, 7].

Modified amino acid content

2.328 A major target of both conventional and transgenic plant breeding programmes has been the alteration of amino acid composition of plant seed protein [170]. Because animals, including humans, are unable to produce ten out of the twenty amino acids required for protein synthesis, then they must obtain the remaining so-called �essential� amino acids from their diet [170, 171].

2.329 Plant proteins represent the primary source of food proteins for humans, with more than two thirds of these proteins being derived from cereal and legume seeds. However, due to the imbalance in the composition of particular amino acids in both cereals and legumes, the dominance of these plants in the human diet means that the actual nutritional value derived from plant protein is relatively low, and diets therefore require supplementation with animal proteins (eggs, meat, milk and fish) [172]. A low level of methionine in legume seeds for example restricts their biological value to between 55 and 75 percent of that of animal protein [172].

2.330 In developed countries, the availability of animal protein means that the compositional deficiency of cereal and legume seeds does not have any implications to human nutrition. However, in developing countries in situations where diets

33 Defined as those compounds that cannot be synthesised by animals (including humans) and must therefore be obtained through the diet (Charkraborty et al., 2000).


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depend strongly on seed protein, nutrition may be affected and in more extreme cases can retard physical and mental development in children under four years of age (Waterlow and Payne, 1975; cited by [172]). Although effects on human nutrition in developed countries are unlikely, the amino acid composition of plant protein fed to poultry and monogastric livestock (e.g. pigs) has implications for the level of nitrogen pollution generated by these animals. The better the balance in amino acid composition, with no amino acids being provided in excess, then the lower the level of nitrogen emissions [172].

2.331 Different plants contain different quantities of these �essential� amino acids. Particular targets for improvement are the lysine content of cereal grains and the methionine content of legume seeds, as these are the primary limiting �essential� amino acids in these crops, respectively. In terms of �essential� amino acids, the nutritional value of potato for example, is limited by low levels of lysine, tyrosine, methionine and cysteine [171]. Improvements in the biological value of both cereal and legume seed proteins by conventional breeding have not been successful. Although traditional breeding strategies have resulted in increases in levels of for example the sulphur rich legumin protein fraction, or the sulphur rich albumin protein fraction, in peas, an increase in one group occurs at the expense of levels of the other, resulting in no net gain in the sulphur containing amino acids (S-amino acids) [173].

2.332 Genetic modification however is reported to offer a number of different strategies, with several transgenic crops including lupins and clover modified for altered amino acid content currently undergoing experimental trials [170].

2.333 Four basic strategies have been reported for the modification of �essential� amino acids in seeds [172, 170]:

• modification of the seed�s amino acid biosynthesis. This increases the free amount of the respective amino acid, but not the fixed content;

• modification of endogenous storage proteins;

• the transfer of existing genes encoding high expression of the desired proteins into plants with good agronomic traits; and

• the creation of entirely synthetic gene sequences encoding artificial proteins with the desired amino acid content.

2.334 Much of the work reported to date has focused on the improvement of lysine and methionine content of cereals and legumes respectively. Genetic modification has


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been applied to the development of legume crops especially for improved amino acid content through each of the four basic strategies described above. Modification of pathways for amino acid biosynthesis has also been used to improve lysine content in soybean and oilseed rape [172].

2.335 The amino acids lysine and methionine are part of the aspartate family of amino acids which includes other nutritionally �essential� amino acids such as threonine and isoleucine [170]. Methionine is one of the S-amino acids. These compounds are �essential� amino acids, and animals require a daily dietary intake of 3-5 percent by weight of dietary protein [170]. For pigs for example, the daily dietary intake of protein must contain 3.5 percent by weight of S-amino acids of which 1.6 percent must be methionine34 with the remainder either methionine or cysteine [174].

Modification of amino acid biosynthetic pathways

2.336 Both methionine and lysine are produced biochemically by the plant from aspartate (see Figure 2.7). The key control points in this process are the steps catalysed by the enzymes aspartate kinase and dihydrodipicolinate synthase (DHPS). The activities of both enzymes are under the control of negative feedback loops with aspartate kinase feedback-inhibited by threonine or methionine, and DHPS by lysine.

2.337 The insertion of a bacterial aspartate kinase into tobacco which was feedback insensitive resulted in a 17-fold increase in levels of free threonine and a three-fold increase in the amount of methionine by the transgenic plant relative to the control [175]. The proportions of other amino acids, including lysine remained unchanged.

2.338 Although the modification of methionine biosynthesis in tobacco was found to be successful in increasing the free concentrations of the amino acid, the system was found to have a number of drawbacks that were proposed as limiting its application in other crop plants. Increasing the concentration of the free amino acid does not necessarily lead to an increase in the fixed content present, as the free amino acid may leach from the plant tissue, or be lost during post-harvest processing [171]. In the tobacco, the increase in free concentrations of methionine resulted in the activation of a process that degraded the excess amino acid [172].

2.339 However, the modification of soybean and oilseed rape with a feedback-insensitive DHPS enzyme, indicated that these problems could be overcome. Expression of both a feedback-insensitive DHPS and aspartate kinase resulted in a 25 percent and 100 percent increase in total seed lysine in soybean and oilseed rape respectively [176].

34 Because animals can convert methionine to cysteine, but not cysteine to methionine, then there is a specific dietary requirement for methionine.


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Figure 2.7 - Biosynthetic pathway of the aspartate family of amino acids (adapted from Tabe and Higgins (1998) [170]).

2.340 A number of studies have investigated the 14-3-3 protein family as a target for

modifying plant compositional traits [177, 178]. Results from in vivo and in vitro studies suggest that the 14-3-3 protein family fulfil the role of metabolism coordinator in plants, including amino acid synthesis. The protein is involved in the regulation of nitrogen fixation and carbohydrate metabolism through direct interaction with nitrate reductase and sucrose phosphate synthase, respectively [178]. Studies on tobacco with modified levels of 14-3-3 protein also suggested a role of this protein in adaptation of the plant to environmental stresses, particularly cold and salinity [179]. Such adaptations have implications as to persistence and survival of these plants in more temperate and saline environments.

2.341 Genetic modification of potatoes by antisense transformation resulted in the repression of the 14-3-3 protein. Analysis of amino acid content of the transgenic potatoes found a significant increase in methionine, proline and arginine levels during the four year field trial, and a consistent but non-significant increase in lysine content.

2.342 The modification of existing amino acid biosynthetic pathways has been found to offer a successful approach to the alteration of amino acid content of particular plants. Certain modifications appear to be more successful in some crops than others. The commercial development of this approach in a range of plants including soybean, potato and oilseed rape may be expected.

Aspartate

Isoleucine

Threonine

MethionineLysine

multiple enzyme steps

aspartate kinase

DHPS feedback inhibition feedback inhibition


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Modification of endogenous seed storage proteins

2.343 Modification of sulphur-poor endogenous seed storage genes so that their nucleotide sequence encodes a protein with an increased S-amino acid composition has been reported as another approach to increasing methionine levels. Possible target proteins include β-phaseolin from Paseolus vulgaris, glycinin from soybean and vicilin from Vicia faba [173].

2.344 However, due to problems with the stability of the modified protein (in the case of β-phaseolin and glycinin), and insufficient expression to cause a significant increase in the total S-amino acid content (in the case of vicilin) [173], this approach did not increase methionine levels significantly and is not viewed as a practical approach at a nutritionally relevant level [170].

2.345 The modification of endogenous seed proteins as a means to alter the amino acid content of crop plants is viewed as a less successful strategy than the other approaches addressed (modification of existing biosynthetic pathways and using naturally occurring or synthetic proteins). Although other approaches to modifying endogenous seed storage proteins may be developed, the strategies for soybean and pea reported here are unlikely to be developed further.

Modification using naturally occurring proteins

2.346 Strategies involving the use of naturally occurring proteins with high methionine levels have also been applied in the development of transgenic plants with improved methionine content. The most appropriate candidates reported so far are:

• from monocotyledonous plants - a 21 kilo Dalton (kDa) zein which contains 28 percent methionine residues [180] and a 10 kDa zein containing 23 percent methionine residues [181], both from maize, and a 10 kDa prolamin with 20 percent methionine residues identified in rice [182]; and

• from dicotyledonous plants � 2S seed albumin from Brazil nut (Bertholletia excelsa) (also referred to as Brazil nut albumin (BNA)) which contains 18 percent methionine residues [183], a 2S albumin from sunflower (Helianthus annuus) (sunflower seed albumin (SSA)) containing 16 percent methionine residues [184] and the AmA1 protein from Amaranthus hypochondriacus which contains a good balance of lysine, methionine, cysteine and tyrosine residues [171],.

2.347 The identification of candidates from both mono- and dicotyledonous plants allows subsequent modifications of both plant types, with methionine-rich proteins from


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monocotyledonous plants generally having been used for expression in transgenic monocotyledon seeds [170]. Of the plants modified to date with this technology, maize is the principal monocotyledon modified, whereas a variety of dicotyledonous plants have been modified, including soybean, lupin, narbon bean, oilseed rape and potato [170].

2.348 One effect reported with the modification of maize in this way is that the introduced proteins competed with the plant�s endogenous sulphur-rich proteins for limited sulphur reserves. Although similar results have been reported with other transgenic plants including soybean, no knock-on effects on other plant properties or agronomic characteristics of this finding have however been reported [170].

2.349 The use of BNA in the modification of a range of dicotyledonous plants including soybean, lupin and chickpea has been reported [170]. The expression of a chimaeric gene encoding BNA under the control of a promoter from Phaseolus vulgaris for example resulted in the production of the BNA protein at 10 percent total seed protein and a 50 percent increase in seed methionine content. This level of methionine production is sufficient to raise methionine levels from 1.2 percent (in non-transgenic soybean lines) to 1.8 percent by weight [170].

2.350 A similar modification of narbon bean35 involving the insertion of a chimaeric BNA gene controlled by a promoter of the LeB4 legumin gene from Vicia faba resulted in the accumulation of BNA at up to 4.8 percent of SDS-soluble seed protein [185]. Because the methionine content of both the transgenic soybean and narbon bean is within the range required by animals for optimal growth, then these modifications therefore have the potential for use agriculturally.

2.351 A chimaeric gene (designated ssa) encoding the SSA protein from sunflower has been used to increase methionine levels in the narrow leaf lupin (Lupinus angustifolius L.) [173], pea and chickpea [170]. Modification of the lupin with ssa under the control of a seed specific promoter from the pea vicilin gene resulted in 94 percent increase in methionine levels (and a 12 percent decrease in cysteine levels) in the seed compared with the non-transgenic control. Levels of other amino acids were similar in both lines [173].

2.352 In feeding studies with rats, the transgenic lupin resulted in statistically significant increases in live weight gain, true protein digestibility and biological value36 (15

35 Narbon bean (Vicia narbonensis L.) is a Mediterranean grain legume closely related to the field bean. 36 Biological value is defined as a measure of the efficiency of conversion of feed protein into body protein by an animal. Improved biological value indicates a better balance of the amino acids in the feed. Because biological value takes into account excreted urinary nitrogen corrected for endogenous


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percent increase) compared with the non-modified control [173]. The results of the trials were similar to those when rats were fed a non-transgenic lupin-based diet supplemented with pure methionine, indicating the potential for the transgenic lupins to replace the need for methionine supplements in animal feed.

2.353 Lupins have been selected for modification as a consequence of their year-round importance in feeds for the beef, pig and poultry industries, and as a supplementary summer feed for sheep when the feed quality of pastures is poor. In Australia, the lupin is the major grain legume grown, with in excess of 800,000 tons produced annually [173]. Transgenic lupins expressing SSA have undergone field trials in Australia [173, 170].

2.354 In contrast to the transgenic lupins that showed specific increases in methionine content as a percentage of total seed protein, the modified pea and chickpea expressing SSA had increased levels of total protein. Therefore, although levels of methionine were higher compared with the control, the total seed protein was not enriched with methionine when compared with the non-transgenic strains [170].

2.355 An advantage of modifications expressing the SSA protein is that as well as high levels of methionine and cysteine, it is resistant to degradation by ruminants (cattle and sheep) [173]. One of the reasons for supplementing the diet of these animals with methionine is the large loss of amino acids in feed protein that occurs during ruminant digestion. Sheep in particular have a high requirement for S-amino acids to produce wool, as this contains fibre proteins rich in cysteine [170]. Although the modified soybean described above would be suitable to provide livestock such as sheep with additional methionine, a more desirable approach agriculturally is to improve the methionine content in the vegetative parts of pasture crops such as clover and grasses [170]. Studies with clover (Trifolium subterraneum)37 expressing SSA have reported a sufficient increased protein content in the vegetative plant material to make such crops useful agriculturally as replacements for protein feed supplements [186].

2.356 As a consequence of its importance as a crop for food, animal feed and an industrial raw material38, the potato has been recognised as a good target for modification for improved nutritional value. Modification of potato with the AmA1 gene encoding the seed specific amaranth seed albumin protein (AmA1), resulted in increased total protein content including level of most �essential� amino acids, as well as improvements in the growth of the potato tubers [171]. Levels of lysine, methionine,

excreted nitrogen, it is viewed as the most sensitive parameter with which to address amino acid imbalance in food. 37 Widely cultivated pasture legume in Australia. 38 For the manufacture of starch, alcohol and other food products.


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cysteine and tyrosine were 2.5-4 times higher in the transgenic potatoes compared with the control [171].

2.357 In addition to improvements in amino acid content, the potatoes modified with AmA1 also showed a two-fold increase in total tuber number and a 3-3.5 fold increase in tuber yield (in terms of fresh weight) compared with the non-transgenic controls [171]. Because the tubers are food storage organs for the potato plant, then increases in tuber number and yield may improve the survival and/or persistence of these transgenic potatoes outside the agronomic environment. Potatoes modified to express AmA1 are reported to be undergoing field trials [171], presumably in India.

2.358 The AmA1 protein is reported to have great potential as a donor protein to improve the nutritional value of agronomic crops [171]. The advantages of AmA1 compared with most other proteins are that it has a well-balanced amino acid composition, it is encoded by a single gene (AmA1) and is non-allergenic in its purified form. Because AmA1 has a good balance of amino acids it is viewed as potentially more useful than 2S albumins from Brazil nut and sunflower discussed above. Although modification with these 2S albumins is reported to result in significant increases in methionine levels, such changes are concomitant with reductions in levels of other amino acids, such as cysteine [173].

2.359 The absence of allergenicity of the AmA1 protein means that it also is particularly suitable for use in the modification of crops for human consumption. Conversely, the high allergenicity of the BNA protein in both its pure form and in extracts of transgenic soybean is reported to be a significant limitation in the use of this protein for the modification of food or feed crops for nutritional improvements [173, 171].

2.360 As discussed, plants modified for altered amino acid content through the modification of naturally occurring proteins have and are undergoing field trials. Commercialisation of such systems may be expected. Crops identified as possible candidates for commercial development include lupins (already undergoing field trials and animal feeding studies) and potatoes. The potential for such crops to offer the alternative to protein feed supplements currently given to ruminants provides a further �driver� for the commercial development of such crops.

Modification using synthetic proteins

2.361 The generation of entirely synthetic gene sequences encoding artificial proteins with a high content of the desired amino acid has been identified as a potentially successful approach. Genes encoding novel proteins that contain large amounts of methionine, lysine and tryptophan (another �essential� amino acid) have been synthesised in the bacterium Escherichia coli and expressed in tobacco [170]. Modification of tobacco with one of the proteins (containing 31 percent lysine


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residues39 and 22 percent methionine residues) resulted in levels of methionine in the transgenic tobacco that were 20 percent higher than in the non-transgenic control. Such a concentration would be nutritionally significant if similar levels could be achieved in legume seeds [170].

2.362 A similar study expressing a synthetic protein (which included 13 percent methionine residues) in sweet potato (Ipomoea batatas) was reported to result in a nutritionally significant increase in levels of methionine in the storage roots of the plant (Prakash et al., cited by [170]). The agronomic performance of the transgenic sweet potato has undergone testing in field trials [170].

2.363 The modification of crops with synthetic proteins to alter the amino acid content is viewed as a potentially successful approach, and suitable for commercialisation. Field trials with sweet potatoes have been conducted, and trials with other crops may be expected.

Modified fatty acid content

2.364 The purpose of this section is to review applications where the fatty acid content of plants has been/may be altered to provide �essential� fatty acids.

2.365 The role of particular fatty acids, such as linoleic acid (18:2∆9,12), γ-linolenic acid (18:3∆6,9,12) and arachidonic acid40 (20:4∆5,8,11,14) as essential components of dietary intake in animals including humans has led to the identification of these compounds as potential targets for expression in GM plants [143, 9]. Both linoleic and linolenic acids are required by mammals for the production of a group of regulatory molecules called eicosanoids (includes prostaglandins, leukotrienes and thromboxanes) [161].

2.366 As well as being �essential� fatty acids linoleic acid and γ-linolenic acid are also potential targets for genetic modification as they are only produced naturally in a limited number of plant species, none of which are suitable for large-scale agronomic production [161]. Genetic modification has therefore been proposed as one mechanism to produce these compounds in crops more suited to large-scale cultivation.

2.367 The synthesis of γ-linolenic acid and arachidonic acid is reported to involve a group of microsomal fatty acid desaturase enzymes called �front-end� desaturases. This group of enzymes are characterised by their ability to insert a double bond between pre-existing double bonds and the carboxyl group at the ∆-end (front-end) of the fatty

39 Therefore 31 percent of the amino acid residues in the protein are lysine. 40 Arachidonic acid is produced in the pathway converting linoleic acid to eicosanoids, by the elongation of γ-linolenic acid to a C20 molecule.


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acid molecule [161]. Expression of a �front-end� desaturase from common borage (Borago officinalis) in tobacco resulted in the accumulation of high levels of ∆6-desaturated fatty acids, with γ-linolenic acid accounting for 13.2 percent of total fatty acids present [187].

2.368 The synthesis of arachidonic acid requires ∆5- and ∆6-destaurase enzymes. A ∆5-desaturase has been isolated from the filamentous fungi Mortierella alpina [188], and a ∆6-desaturase from the moss Physcomitrella patens [189]. Expression of the fungal ∆5-desaturase in canola resulted in the production of a number of unusual ∆5-desaturated 18 carbon fatty acids.

2.369 However, although the desaturases involved in the production of γ-linolenic and arachidonic acids have been identified, the development of GM crops able to produce and accumulate eicosanoid compounds has yet to be achieved [161]. The issue of the correct targeting of the fatty acid away from the membrane lipids will need to be addressed, before such GM crops can be developed for use at an agronomic scale. The involvement of α-linolenic acid in the response by plants to pathogens, and its essential role in pollen development [190] means that modifications involving α-linolenic acid may have implications to the persistence and survival of the modified plants in the environment. In studies with modified tobacco, over-accumulation of α-linolenic acid has however not been found to improve the tolerance of plants to either low temperatures or freezing [191].

2.370 As discussed, the development of crops modified to produce fatty acids with a nutraceutical application is currently in its infancy in terms of commercialisation. The relevant desaturase enzymes have been identified, but targeting issues remain to be solved before any agronomic developments can be made. However, the requirements of fatty acids such as linoleic acid and arachidonic acid as essential nutrients, and the absence of other non-GM plants suitable for the production of these compounds on an agronomic scale means that GM technology offers a potentially realistic option for the commercial production of these compounds at a relatively low price. The commercial development of crops modified to produce such fatty acids may therefore be expected (although may not occur in the immediate future).

Modified flavonoid content

2.371 Flavonoids consist of a large and diverse group of polyphenolic compounds that are ubiquitous in plants [7]. Interest in these compounds with respect to nutraceutical applications is a consequence of their antioxidant properties [7] which are thought to protect against cardiovascular disease and therefore are seen as being beneficial to human health.


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Figure 2.8 � Schematic overview of the flavonoid biosynthesis pathway (adapted from Sevenier, 2002 [7]).

phenylamine

phenylalanine-ammonia lyase

4-coumaryl-CoA malonyl-CoA

chalcone synthase (CHS)

Chalcones

chalcone isomerase (CHI)

Flavanones

flavone synthase (FNS) flavanone-3-hydroxylase (F3H) isoflavone synthase (IFS)

Flavones IsoflavonesDihydroflavonols

flavonol synthase (FLS) dihydroflavonol reductase (DFR)anthocyanidin synthase (ANS)

Flavonols Anthocyanins

2.372 The other roles of plant flavonoids in pathogen resistance and protection against UV

radiation [7] however mean that the modification of the quantities of these compounds in plants could have subsequent effects on the ability of the modified plant to persist or survive in the environment.


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2.373 Because many of the enzymes and biochemical pathways involved in the synthesis of flavonoids have been identified, and the encoding and relevant regulatory genes isolated (Figure 2.8) [7], then the information required to modify plants for altered flavonoid content is already available. Many of the studies conducted to date have focused on the tomato, which contains small quantities of flavenols in the peel of its fruit [7], and is also an important food crop worldwide.

2.374 A rate-limiting step in flavonoid biosynthesis in tomatoes has been identified as the production of chalcone isomerase (encoded by ch1). The overexpression of a ch1 gene from petunia in transgenic tomatoes was reported to result in the 70-fold increase in production of the flavenol quercetin glycoside [13].

2.375 Although many of the relevant genes and biochemical pathways have been identified, only limited studies with plants modified for altered flavonoid content have been reported to date. However, as a consequence of the reported health benefits of flavonoids then, as with other systems offering nutraceutical/health benefits, the continuing development of such plants towards commercialisation should be expected.

Modified vitamin content

2.376 The purpose of this section of the report is to review the application of GM technology to modify the vitamin content of plants. Plants produce a number of compounds that are �essential� components of the mammalian diet. Many of the applications of GM technology in this area are directed towards the modification of plants to increase their production of these �essential� compounds.

Vitamin E

2.377 The �essential� compound vitamin E consists of the four forms (α-, β-, δ- and γ-) of the lipid-soluble antioxidant tocopherol. These four tocopherols are produced in the chloroplasts of higher plants and are used by the plant as antioxidants and to stabilise polyunsaturated fatty acids from lipoxygenase attack [158]. Of the four tocopherols, α-tocopherol has the highest vitamin E activity, with a recommended daily intake in the human diet of 7-9 mg (10-13.4 international units (IU)) [158]. This compound is present in many plant oils, and the consumption of a balanced diet is reported to be sufficient to supply the recommended level of vitamin E. However, ingestion of vitamin E at levels of 100-1000 IU is reported to be beneficial in reducing the risk of cardiovascular disease and improving immune function [158]. Such levels however can only be achieved by taking vitamin supplements or by ingesting large quantities of foods enriched in vitamin E.


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2.378 In plants γ-tocopherol is the primary biosynthetic precursor for α-tocopherol, and is converted to the more bioactive form by the action of a methyltransferase encoded by the γ-TMT gene [158]. Although plants such as soybean produce both γ- and α-tocopherol in their seed oils, levels of the α- isomer are relatively low, indicating poor expression of the γ-TMT gene and consequently limited conversion of the γ- to the α- form.

2.379 Overexpression of the γ-TMT gene in Arabidopsis under the control of a seed specific DC3 carrot promoter resulted in an 80-fold increase in the production of α-tocopherol compared with the non-modified control. Although the ratio of the different tocopherol isomers was altered by the modification, the total amount of tocopherol produced by the GM plants remained unchanged [158]. Based on the findings with Arabidopsis, the overexpression of the γ-TMT gene in other plants, including soybean, maize and oilseed rape should elevate the level of α-tocopherol in the seed oil [158].

2.380 Work to date has only been reported for Arabidopsis which is of course not suitable for agronomic cultivation. However, if the findings from the Arabidopsis work can be applied to other more suitable �crop� plants then further developments may be expected in this area with the ultimate objective the production of GM plants as (probably cheaper) alternatives to existing vitamin E supplements.

Vitamin A

2.381 The alteration of plants to provide increased concentrations of vitamin A has been described as a key application offered by the genetic modification of plants. The particular modification that is often described is the �golden rice� that has been altered to express increased concentrations of provitamin A (also referred to as β-carotene and a precursor of vitamin A) (Gregor, 2000; cited by [192, 193]). It should be noted that the �golden rice� described in this section of the report is the genetically modified version. The term �golden rice� has also been applied to rice that is formed during a specific type of industrial milling of a conventional (non-GM) brown rice [194]41.

2.382 Vitamin A deficiency is the single most important cause of blindness amongst children in developing countries and is a substantial contributor to illness and death from other infectious diseases [192]. Mortality rates are reported to fall by as much as 54 percent in children given vitamin A supplements (either in their diet or injected).

41 This non-GM �golden rice� losses only 1 percent of its weight during milling compared with the 17 percent lost during the conversion of brown rice to white rice. The non-GM �golden rice� therefore has a greater essential nutrient content than white rice but has a comparable cooking time (Tainsh and Bursey, 1982).


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2.383 As a consequence of the prominence of the GM �golden rice� as an example of a GM plant with a modified compositional trait, then it is viewed as of relevance to this report to comment in some detail on both the development of this GM rice and on some of the limitations that have been reported that may affect the potential of the GM rice in fulfilling its objective to reduce vitamin A deficiency.

2.384 The development of the GM �golden rice� is presented as an example of the potential benefits offered by the genetic modification of plants for a number of reasons. Rice is of course a well-established crop and the major staple food for many people worldwide. It is generally consumed in its milled form (white rice42) with the outer layers of the rice seed (pericarp, tegmen and aleurone layers) removed. Although these outer layers are rich in essential nutrients, milling is required to remove the oil-rich aleurone layer which turns rancid during storage, especially in tropical and sub-tropical areas [193]. Because no rice cultivars are able to produce provitamin A in their endosperm, then the development of rice strains containing provitamin A in their endosperm through conventional breeding techniques has not been possible [193].

2.385 However, because the genetic modification of rice is well-established and the entire carotenoid biosynthetic pathway (including the formation of provitamin A has been identified, it was proposed that genetic modification techniques could be applied to introduce the complete provitamin A biosynthetic pathway into rice endosperm [193].

2.386 The formation of provitamin A (β-carotene) in plants involves the four enzymes phytoene synthase, phytoene desaturase, ζ-carotene desaturase and lycopene β-cyclase. Initial studies involved the introduction of the four respective genes into the rice embryos by particle bombardment. This proved unsuccessful so an A. tumefaciens mediated approach was employed in which the entire β-carotene pathway was installed into the rice endosperm in a single transformation event [193]. The construct used consisted of the psy and β-lcy genes from the daffodil (Narcissus pseudonarcissus) (encoding phytoene synthase and lycopene β-cyclase respectively) under the control of an endosperm-specific glutelin promoter, and the crtl gene from the soil bacterium Erwinia uredovora which was under the control of a constitutive CaMV 35S promoter. The crtl gene encodes a carotene desaturase enzyme capable of performing the roles of both the plant desaturases (phytoene desaturase, ζ-carotene desaturase) [193]

2.387 Further studies found that the β-lcy gene was not needed, and that the psy and crtl genes were sufficient for the production of β-carotene, and also other xanthophyll compounds such as lutein and zeaxanthin43 [193]. The highest levels of β-carotene

42 Consisting of just the endosperm, which although containing starch granules and protein bodies, lacks several essential nutrients, including those with provitamin A activity. 43 Although not of direct relevance to this modification, the alteration of levels of zeaxanthin may have implications to the expression of genes (e.g. aba2 whose products catalyse this compound to other


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reported in the modified rice strains were 1.6 µg carotenoid g-1 of dry rice endosperm. However, these lines are described by Beyer et al. (2002) [193] as only prototype lines and studies are currently underway to triple the levels of β-carotene present.

2.388 The production of the xanthophyll compounds has potential implications in reducing the occurrence of macular degeneration, which is another cause of blindness. Xanthophylls are present in the eye�s macula and their deficiency may contribute to macular degeneration (Landrum et al., 1997; cited by [193]).

2.389 The potential limitation of the GM �golden rice� to fulfil its objective of reducing vitamin A deficiency centres on the fact that the GM rice contains β-carotene (provitamin A), and not the vitamin A itself. In order for the provitamin A to become active (i.e. converted to vitamin A) it must be split by an enzyme present in the intestinal mucosa of the liver. However, as with vitamin A, provitamin A is fat soluble and requires the presence of dietary fat for absorption through the gut. Effective conversion therefore requires a functional digestive tract, adequate energy, fat and protein in the diet. Many children though who are deficient in vitamin A are also lacking in these requirements [192] and may therefore not be able to benefit from the GM �golden rice�, even if the bioavailable levels of provitamin A present are increased beyond their current ten percent level.

2.390 The GM �golden rice� was reported (2001) to being developed further by the International Rice Research Institute (IRRI) in the Philippines [195]. The work conducted by the IRRI will include increasing the levels of the β-carotene present in the rice. Statements by the IRRI indicate that further development of the rice will take at least three to four years before the crop is ready for field trials and another two years beyond that before it may be available to farmers [195].

2.391 There are no expected changes in the persistence or survival of this GM rice in the environment. Applications, in the form of the �golden rice� are examples of a crop plant modified for altered vitamin A content that has undergone field trials and is being developed for further commercialisation. Although limitations have been expressed as to the actual potential of the �golden rice� strain to reduce vitamin A deficiency, the development of this crop is viewed as an indication of the types of GM plants designed for improved vitamin content that may be expected in the future. Further commercial development of such crops may be expected. There are 16 field trials of tomatoes and one of maize genetically modified for altered carotenoid content underway (or completed) in the USA [57].

xanophylls). The gene aba2 is also involved in the biosynthesis of the plant hormone abscisic acid (ABA). The alteration of levels of ABA may change seed dormancy and germination levels, the accumulation of food storage reserves by the plant and tolerance to desiccation (Frey et al. 1999. Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression. Plant Molecular Biology 39, p1267-1274).


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Vitamin C

2.392 The US Patent Application (20020012979) [196] reported the modification of microalgae for increased biosynthesis of L-ascorbic acid. The production of L-ascorbic acid in microalgae such as Prototheca and Chlorella pyrenoidosa proceeds through the production of mannose intermediates to GDP-D-mannose, followed by the conversion of GDP-D-mannose to GDP-L-galactose by GDP-D-mannose:GDP-L-galactose epimerase. This is subsequently converted to L-galactose-1-P, L-galactose, (L-galactonic acid), L-galactono-γ-lactone and ultimately L-ascorbic acid.

2.393 Stages in the L-ascorbic acid biosynthetic pathway which may be targeted by genetic modification include [196]:

• enhanced production of GDP-D-mannose (conversion of a carbon source into GDP-D-mannose) � achieved through the overexpression of enzymes such as hexokinase, glucose phosphate isomerase, phosphomannose isomerase (PMI), phosphomannomutase (PMM) and/or GDP-D-mannose pyrophosphorylase (GMP);

• inhibition of pathways converting GDP-D-mannose into compounds other than GDP-L-galactose - achieved by modifications which inhibit polysaccharide synthesis, GDP-D-rhamnose synthesis, GDP-L-fructose synthesis and/or GDP-D-mannuronic acid synthesis;

• increased activity of GDP-D-mannose:GDP-L-galactose epimerase through the overexpression of the epimerase gene; and

• inhibition of pathways which compete for substrates involved in the production of any of the intermediates within the L-ascorbic acid production pathway. Inhibition may be achieved through the deletion or mutation of enzymes.

2.394 The genetic modification of plants for altered vitamin C content has to date only been reported for microalgae and tobacco [57]. The successful modification (and field trialling) of tobacco, which is often used as an experimental crop, indicates the potential to modify other crop plants for altered vitamin C content. The production of ascorbic acid in plants is the subject of a commercial patent [197]. The probable requirement to grow microalgae under contained conditions (bioreactor) means that producing vitamin C by this system would be more expensive than field grown crop plants.


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Modified mineral content

2.395 Potatoes modified by antisense transformation to cause the repression of 14-3-3 protein showed a significant increase in calcium content in the tubers. A slight increase in magnesium content was also recorded. No changes however were found in levels of potassium or phosphorus, the two main elements present in potato tubers [178].

Iron

2.396 Conventional plant breeding programmes have identified high iron and zinc traits in a range of crops. However, whilst high iron accumulating wheat and rice cultivars have been developed through conventional (i.e. non-GM) breeding, only a small fraction of the iron (20 percent in wheat and 5 percent in rice) is transported from the senescing leaves to the grain44 [198]. This of course limits the benefits (in terms of increased iron intake) offered by the high iron accumulating cultivars. The benefits are further limited by the almost exclusive storage of the iron in the husk, alerone and embryo parts of the grain. These parts are removed during milling and polishing of the grains (Welsh and Graham, 1999; cited by [198]).

2.397 Iron is stored in plants in vesicles of ferritin (which also serve as a phosphate storage molecule). The genetic modification of rice with an endosperm-specific ferritin gene from soybean [199] or Phaseolus vulgaris [200] resulted in a threefold increase or doubling, respectively, of the iron content of the seed. Such a system should therefore improve iron levels in the diet.

2.398 For salad crops such as lettuce, seed-specific accumulation of the iron is of course not important as it is the vegetative part of the plant that is eaten. The modification of lettuce with a ferritin gene resulted in the transgenic lettuce accumulating iron at levels ranging from 1.2-1.7 higher than those in the non-GM controls [201]. The GM lettuces also exhibited enhanced growth rate and superior photosynthetic rates to the non-GM plants, especially during the early developmental stages [201]. This may have implications to the persistence or survival of the GM lettuce in the environment.

2.399 Iron uptake can also be improved by overexpression of the cysteine-rich metallothionein-like proteins in the rice. Cysteine peptides are reported to be enhancers of iron absorption [202]. Such an approach may not be suitable for improving iron levels in grain crops for the human diet, if it does not involve increased iron levels in the grains.

44 With the high zinc cultivars >70 percent of the zinc is mobilised to the grain (Grusak et al. (1999) The physiology of micronutrient homeostasis in field crops. Field Crops Research 60, p.41-56).


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2.400 Improving levels of iron and other minerals (Mg, Zn and Cu) in non-ruminant animals (including humans) can also be achieved through improving the phytase activity in those animals. The issues associated with phytase and modification of plants to produce this compound is addressed in the review of the �Production of Speciality Compounds for Non-medical Applications�.

2.401 The modification of rice to reduce iron deficiency in diets is not sufficiently developed (2003) for implementation in human diets. Animal and human feeding trials will be required to determine the efficacy of the modified rice. However, the modified crop does potentially offer a very suitable approach to reducing iron deficiency, particularly if it is combined with alterations of phytase activity to increase bioavailability.


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PRODUCTION OF SPECIALITY COMPOUNDS FOR MEDICAL APPLICATIONS

Transgene expression strategies Vaccines

Enteric disease vaccines Hepatitis B virus Measles vaccine Malaria Protection against autoimmune diseases and responses HIV vaccine Lymphoma vaccine Caveat on production of vaccines in plants

Antibodies Cancer diagnosis and therapy Diagnostic antibody for crossmatching donors/receivers Diagnosis and treatment of alloimmunisation or haemolytic disease of the newborn Antibodies for the treatment of HIV

Biopharmaceutical Proteins Modification of protein glycosylation Anti-hypertensive protein Human plasma proteins Other medical proteins

2.402 The application of GM technology in the production of speciality compounds for

medical applications (also referred to as �molecular farming�45) [203] is identified as an area of significant recent expansion. The potential benefits of the availability of reliable and economic methods for the production of vaccines in plants have led to the establishment of a number of biotechnology companies targeting this area of research (e.g. Prodigene, Large Scale Biology and Ventria Bioscience). Such interest is viewed as likely to result in the development of a range of plants for the production of vaccines and other medical-related compounds [12, 14]. The purpose of this section of the report is to review the developments in this field, with the review divided into three sections: vaccines, antibodies and other biopharmaceutical proteins.

2.403 The use of plants for the production of medical-related compounds is viewed as an important area of development and research for a number of qualitative and quantitative reasons:

• plant products may present a lower level of risk to human health because of the likely absence of human or other animal pathogens, particular microbial toxins or oncogenic sequences. The absence of such compounds also reduces the costs of screening for viruses and bacteria and subsequently reduces potential production costs [14, 204-213];

45 Defined as the production of antibodies, biopharmaceuticals and edible vaccines in plants using genetic engineered modifications (Fischer et al. 1999).


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• plants have complex and sophisticated cellular machinery that allows them to produce functional complex compounds. The process of protein synthesis, secretion and post-translational modifications are similar in plant and animal cells, with only small differences in protein glycosylation (see section on biopharmaceutical proteins) [203, 205, 214, 207-212];

• plants can be manipulated so that the proteins are produced in intracellular compartments in which they are more stable [215];

• plants can be propagated endlessly and their production can be conducted at a large scale under controlled conditions. This means that the production of proteins for medical purposes by plants could easily be scaled up for mass production, at a lower cost than the systems currently used for medical protein production [207, 208, 211, 216, 215, 217]. One estimate is that production costs of recombinant proteins in plants could be between 10-50 times lower than those for producing the same protein in E. coli [209, 215];

• the technology for harvesting and processing plants and plant products is already available on a large scale through existing agricultural practices [204, 206, 215];

• any processing requirements can be eliminated when the plant tissue containing the recombinant protein can be ingested as food (see vaccines below) [204, 211, 212, 218]; and

• the use of transgenic plants would mean that the ethical problems associated with transgenic animals and the use of animal materials would be avoided [204, 216].

2.404 The potential of using plants as a production system for recombinant pharmaceuticals was established between 1986 and 1990 with a series of medical proteins being expressed in plants. These included human growth hormone fusion protein, an interferon and human serum albumin [205]. Many mammalian proteins for different disease targets have to date been successfully produced at the laboratory scale level in tobacco: the proteins include hormones, blood proteins, enzymes, cytokines, milk protein, structural proteins, antigens and antibodies [203, 219].

2.405 The most widely studied therapeutic proteins produced in plants are the monoclonal antibodies used for passive immunotherapy, and antigens for use in oral vaccines. Clinical trials have been conducted with plant-produced antibodies (termed �plantibodies�) and plant-produced vaccines [14, 211, 212, 220]. Recombinant


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antibodies, plasma proteins and diagnostic reagents are particular targets for expression in transgenic plants as their conventional production or purification is often expensive and can potentially be contaminated by pathogens [205]. Antibody expression in plants is a more established technology and serves as a model for the expression of other types of medical molecules [214].

2.406 The medical applications of transgenic plants have been reviewed extensively. Table 2.3 describes some examples of the work that has been carried out to date. This does not cover all of the work done in this area: the sheer volume of the examples available demonstrates the potential of plant-produced medical products.

Transgene expression strategies

2.407 Early studies used strong generic promoters e.g. CaMV 35S promoter, which resulted in the expression of medical protein throughout the transgenic plant. Early experiments tended to produce only low expression levels of the desired product. These levels needed to be increased in order to achieve the viable commercial production of such proteins. It is thought that at least one percent total soluble protein (TSP) is needed for the system to be commercially viable if the protein is to be purified [215].

2.408 The success and the level of expression of medical-related proteins in plants is related to a number of factors. Considerable increases in protein accumulation have been achieved through the careful choice of 3� polyadenylation signals (which affect mRNA stability). Changing the pattern of codon usage within a cDNA to match more closely those codons more commonly used by plants, thereby increasing the rate of translation, has also been used successfully.

2.409 More recent work has begun to focus on the use of promoters that direct the expression of the transgenic product to specific parts of the plant or to specific organelles within the plant cells in order to increase expression levels; expression levels have also been increased through the use of chloroplast genome transformation [211] The increased expression levels seen possibly result from the concentration and interaction of the subunits of the more complex proteins. In the case of chloroplasts, higher levels of expression of transgenes are thought to be due to multiple copies of chloroplasts within each cell [203, 205, 219, 208, 209, 216, 221]. Foreign genes that have been integrated into the tobacco chloroplast genome have resulted in the accumulation of recombinant proteins at up to 47 percent TSP [218]. Chloroplast transformation uses two flanking sequences that result in the insertion of a foreign DNA into the spacer region between the functional genes of the chloroplast genome [212]. This results in the targeting of the foreign gene to a precise location.


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Table 2.3 - Medical applications of transgenic plants

Medical Function

Recombinant Protein

Transgenic Plant

Application Company/reference

Antibody Streptococcus surface antigen SAI/II � sIgA/G (CaroRX)

Tobacco Therapeutic (topical) Planet Biotechnology, California, USA; [222]

Antibody Herpes simplex virus IgG

Soybean, rice Therapeutic (topical) Epicyte, San Diego, USA; Seitline et al., 1998 in [221]

Antigen scFv of immunoglobulin from the 38C13 mouse B cell lymphoma

Tobacco, Vaccine/therapeutic patient-specific non-Hodgkin�s lymphoma vaccine/treatment

Large Scale Biology Corporation, California, USA ; [223]

Antibody Human IgG (C5-1) Alfalfa Diagnostic [224]

Antibody scFv from monoclonal antibody T84.66 (Anti-carcinoembryonic antigen antibody)

Tobacco, rice, wheat, pea, tomato

Therapeutic and diagnostic cancer-specific antibodies

[225, 226]

Antibody Carcinoembryonic antigen diabody

Tobacco Therapeutic and diagnostic

Vaquero et al., 2002 (in press) in [221]

Antibody Clostridium difficile IgG

Corn Therapeutic (oral) Epicyte in [221]

Antibody Respiratory suncytial virus IgG

Corn Therapeutic (inhaled) Epicyte in [221]

Antibody Sperm IgG Corn Contraceptive (topical)

Epicyte in [221]

Antibody Various IgG Corn Therapeutic and diagnostic

Integrated Protein Technologies c/o Monsanto, MO and Baez et al., 2000 in [221]

Antibody Colon Cancer antigen IgG

Tobacco Therapeutic and Diagnostic

Verch et al., 1998 in [221] and [211]

Antibody Human creatinine kinase

Tobacco and Arabidopsis

Therapeutic De Neve et al., 1993 in [211]

Antigen Hepatitis B surface antigen

Tobacco, Lupin, Lettuce and Potato

Vaccine Mason et al., 1992 in [211]; Thanavala et al., 1995 in [227, 228, 220].

Prodigene, Texas, USA (patent)

Medical Protein

Growth Hormone Tobacco Therapeutic Staub et al., 2000 in [211]

Antigen Norwalk capsid protein

Tobacco and Potato

Vaccine; development of therapeutic antibodies

Mason et al., 1996 in [211]

Antigen E. coli heat labile toxin

Potato, maize Vaccine Tacket et al. 1998; Haq et al., 1995; Mason et al., 1998 all in [211, 229, 230](Prodigene); [231, 232]


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Antigen Cholera toxin Potato, tobacco, tomato

Vaccine [233]; Arakawa et al., 1997 in [211]; [218, 234]

Medical Function

Recombinant Protein

Transgenic Plant

Application Company/reference

Multiple antigens

Cholera, rotavirus and E.coli antigens

Potato Vaccine [235]

Antigen Measles H protein Tobacco, rice and lettuce

Vaccine [217]; [236]

Antigen Merozoite surface protein MSP 19 and MSP42

Tobacco Vaccine [237]; [238]

Antigen Glutamic acid decarboxylase

Tobacco and carrot

Vaccine (via induction of oral tolerance)

[239]; [240]

Antigen HIV-1 p24 nucleocapsid protein

Tobacco Vaccine [241, 242]

Antigen gp 120 protein from simian immunodeficiency virus

? Vaccine for HIV Prodigene

Antibody Human anti-Rhesus D antibody

Arabidopsis Therapeutic [243]

Antigen Protective Antigen Tobacco Vaccine for Anthrax Aziz et al., 2002

Antigen Rabies virus glycoprotein (G protein) and nucleoprotein (N protein)

Tobacco and spinach

Vaccine for rabies [244]

Antibody Various IgA antibodies for HIV treatment

? Therapeutic Epicyte

Medical protein

α-1 Antitrypsin Rice Therapeutic (treatment of congenital deficiency)

[245, 246] Ventria Bioscience, California, USA

Medical protein

α-galactosidase ? Therapeutic (treatment of Fabry�s disease)

Large Scale Biology Corporation, California, USA

Medical protein

Aprotinin ? Therapeutic (protein inhibitor used in surgery to reduce blood loss and inflammation)

Prodigene (patent)

Medical protein

Ribosome inactivating proteins

Tobacco Therapeutic (anti-viral)

[247]

Medical protein

Cytokines (human interferon-α, human tumour necrosis factor and interleukin-10)

Potato and tobacco

Therapeutic (anti-viral, anti-tumour and treatment for auto-immune disease including inflammatory bowel

[248-250]


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inflammatory bowel disease)

scFV = single chain antibody fragments; Ig = antibody

2.410 Chloroplast genetic engineering is also described as an environmentally friendly approach, minimising several environmental concerns, including the outcrossing of transgenic pollen to weeds or related crops. Engineering genes in the chloroplast genome has been shown to biologically contain transgenes effectively, as the transgenes will not be present in the pollen, although there are some exceptions where the chloroplast genome is inherited bi-parentally (e.g. pines) [218]. Several other gene containment methods are also under investigation, including apomixis, incompatible genomes, transgenic mitigation, control of seed dormancy or shattering, suicide genes, infertility barriers, male sterility and maternal inheritance [218]. These will not be considered in detail here as they are outside the scope of this report.

2.411 Expression levels can also depend on the selection of the correct crop species for production. Work has also been carried out on enhancing the stability of the medical product, particularly large complex molecules such as some antibodies [203, 205, 219, 209, 216, 221]. Another method of increasing expression levels is the simultaneous suppression of naturally occurring genes (e.g. for plant storage proteins) in the plant by antisense expression. This has been shown to increase the yield of pharmaceutical protein genes in seeds [205]. Experiments would need to confirm that the suppression of the natural plant protein did not have an adverse effect on its survival, and therefore its ability to produce the desired pharmaceutical protein in the longer term.

2.412 An alternative method to producing transgenic plants is to infect non-genetically modified plants with recombinant viruses that express transgenes during their replication in the host [206, 208]. Although this technology does not result strictly in the production of transgenic plants, as the transgene does not become integrated into the plant�s genome, the plant�s cell machinery is harnessed by GM organisms, resulting in the transient expression of genes in the plant. This method can be used to obtain large amounts of protein over a relatively short time period (e.g. up to 2 g kg-1 of plant tissue, 1-2 weeks after inoculation of the plant with the virus) [251].

2.413 Such methods could also be a useful tool for verifying that a gene product is functional, prior to large-scale production of transgenic plants [203, 205, 214]. When long-term production of a recombinant antibody is necessary, stable transgenic plants are possibly the most attractive strategy, as virus-infected plants are eventually overwhelmed by the infection and die, necessitating re-infection for every harvest of protein [220]. Transient production may be more attractive however for potentially damaging compounds, as the plant would not be able to pass on the genetically modified trait through pollination, for example. It would need to be made sure that other plants would not become infected with the same virus (some plant viruses have a wide host range), leading to recombinant gene transfer into other


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plant species, and also that any of the transgene product left in unharvested parts of the plant did not have any adverse effect on non-target systems [220].

2.414 Many of the experiments in the field of transgenic plants for medical production have used easily-transformable species such as tobacco or potato. Full-scale production will probably involve more traditional grain and oilseed crops such as maize, rice, wheat, soybeans and oilseed rape [206]. Crop species currently being used to express recombinant antibodies and vaccines include cereals, such as maize for antibodies and edible plants such as spinach for vaccines [208]. The choice of crop used will depend on a number of considerations such as the predicted production rate, the value and use of the product, the geographical area of production, processing methods and facilities, intellectual property rights, environmental safety considerations and economic considerations [221]. Crop based expression systems (wheat, rice, corn, legumes) are most likely to be used heavily as they have a lower content of toxic compounds, compared with tobacco for example, and there is also an existing infrastructure for their cultivation, harvest and processing [203]. Other companies are specialising in non-crop species such as the water plant Lemna (www.biolex.com/), being tested in a joint venture with Bayer for the production of human plasminogen, or the moss Physcomitrella (www.greenovation.com/). Escape of such species into the general environment may have ecological implications.

2.415 A small number of products produced by transgenic plants are already available on the market. These include two antibodies, an oral vaccine and pancreatic lipase [211]

Vaccines

2.416 A vaccine can be defined as �an antigenic preparation used to stimulate the production of antibodies and procure immunity from one or several diseases� (Oxford English Dictionary). The generation of vaccines through their expression in genetically modified plants and the concept of edible vaccines are popular approaches reported in the scientific literature (e.g. [220]). Edible vaccines involve the transgenic plant material containing the protein being eaten directly as an oral vaccine. Alternatively, the protein may be purified and delivered orally or parenterally (by injection) [230]. The production of edible vaccines offers the potential to allow mass vaccination at a reduced cost, and in a more effective and safe manner, and is reported to offer particular benefits to the developing world [205, 212, 252, 216].

2.417 Oral immunisation may be the most efficient means of immunisation to induce protection at the site where most pathogens initiate infection i.e. the mucosal surface. The first step in developing an effective vaccine is to identify the antigen (usually a protein or glycoprotein) that is involved in inducing the immune response. Once identified, the key immunogenic proteins of major pathogens may be produced

http://www.biolex.com/

http://www.greenovation.com/


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in transgenic plant tissues and then fed as edible subunit vaccines to humans. The proteins of microbial and viral pathogens were some of the first examples used to demonstrate the ability of plants to express such proteins that could have a medical use in humans [251, 252, 216, 215].

2.418 The concept of producing a subunit vaccine in transgenic plants was first described in 1992 when the DNA encoding for the hepatitis B major surface antigen was expressed in tobacco plants [252]. Other antigens that have been successfully produced in transgenic plants include: the capsid protein of Norwalk virus (causal agent of acute gastroenteritis), subunit B of the heat-labile toxin (LT) of enterotoxigenic Escherichia coli (causing diarrhoea) and subunit B of the cholera toxin [14].

2.419 Initial attempts at creating oral vaccines failed due to proteolysis of the antigenic protein in the gut. A number of methods have subsequently been developed to ensure that the oral vaccine elicits an immune response before it degrades. These include recombinant strains of attenuated microorganisms (Salmonella and Vibrio cholerae) and bioencapsulation vehicles such as liposomes or proteosomes, as well as higher doses of immunogen and/or other adjuvants than those administered via parenteral delivery. The delivery of the antigen within the transgenic plant material may also provide some protection due to the presence of the tough plant cell walls. The stability and immunogenicity of orally delivered antigens vary greatly, which necessitates further study on protein engineering to enhance mucosal delivery [252, 218, 215, 220, 253, 230].

2.420 The most attractive plant species for expressing subunit vaccine components are those with high levels of soluble protein that is stable during storage. Seed crops such as cereals are therefore particularly suitable. Some plant tissues will require processing before they are ingested, and it is important that any heat or pressure treatments involved do not destroy the antigen. The ultimate goal is to produce plant tissues containing vaccine proteins that can be consumed directly with little or no preparation [254, 229, 215, 220].

2.421 The quantity of plant tissue constituting a vaccine dose must be of a practical size for consumption. Achieving a high level of expression is therefore vital, and this has been one of the major limitations of the expression of recombinant antigens in plants, as expression levels are often low. The expression of vaccine components in plants has been increased by using a range of leader and polyadenylation signals and by optimising codon usage for plants [254, 251, 215]. Increases in protein yields have also been achieved using chloroplast transformation. This method can be applied to bacterial antigen expression, but is not suitable for the production of glycoproteins as these proteins are synthesised in the chloroplasts and therefore do not pass through the endoplasmic reticulum and the Golgi apparatus (where N-glycosylation takes


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place) [254, 255]. Experiments are also still required to determine the optimum doses and frequency of dose required to achieve the maximum efficacy of plant-produced vaccines [220].

2.422 To increase vaccination convenience further, transgenic plants could be developed to produce subunit vaccines that could include multiple antigen epitopes for protection against slightly different types of the same pathogen (multivalent vaccine) and/or a combination of antigens from several different species of pathogen (multicomponent vaccine) within the one plant [255, 220].

2.423 Recent advances in the use of plants for the production of vaccines include the development of plant based vaccines for a number of different diseases including enteric diseases, hepatitis B, measles, malaria, HIV and autoimmune diseases and have sparked a number of patent applications (e.g. US Patent 5,686,079 published 1997). Prodigene�s oral vaccine patent claims to cover all oral viral vaccines produced and/or delivered by plants (www.prodigene.com, 2002; US Patent 6,136,320 published 2000 and other previous related patents). A similar patent exists that covers the production of bacterial antigens and adjuvants in transgenic plants, particularly relating to cholera and Escherichia coli [256](and related patents). Note that the well-documented �dental caries vaccine� is in fact an antibody, and is therefore discussed in the next section on antibodies.

Enteric disease vaccines

2.424 Enterotoxic strains of Escherichia coli (ETEC) are responsible for over 650 million cases of diarrhoea, resulting in approximately 800,000 deaths each year in children under the age of five in developing countries. Approximately 20 percent of all visitors to developing countries also develop diarrhoea as a result of ETEC infection. A major disease agent of ETEC is the heat-labile toxin (LT). Approximately 66 percent of ETEC strains harbour LT [229].

2.425 The receptor-binding B subunits and the LT of ETEC have been expressed in plants and found to be effective as an oral vaccine in mice (Arakawa et al., 1998; Mason et al., 1998; both in [230]) and in humans (Tacket et al., 1998; cited by [230]). The first human clinical trials for a transgenic plant-derived antigen were carried out in 1997 when raw transgenic potatoes expressing LT-B subunit of LT were eaten by humans, resulting in an immune response (Tacket et al., 1998; cited by [251]).

2.426 Two different groups of researchers have since then looked, independently, at expressing the LT-B subunit in a more palatable crop that produces a large amount of seed, namely maize (Prodigene group - [230] and Iowa State University - [231, 232]. Streatfield et al. (2001, 2002) [229, 230] expressed the LT-B subunit of LT in maize to try and demonstrate its immunogenicity and efficacy when fed to mice, as a


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model for the delivery of an edible vaccine for ETEC for humans in a cereal crop. A synthetic version of the gene encoding for LT-B was synthesised so that codon usage could be optimised for highly expressed maize genes. A maize codon optimised version of the barley α-amylase signal sequence was also incorporated to provide a cell secretion signal. This resulted in high levels of protein accumulation in the cell wall. The synthetic genes were sub-cloned into a maize expression cassette within a transformation vector, and maize plants were transformed using A. tumefaciens mediated transformation [229, 230].

2.427 It was found that the LT-B produced in the transgenic maize induced an immune response when fed to mice, which appeared to protect the mice when exposed to the LT holotoxin [229]. The LT-B produced in the maize was also found to be stable within the maize seed for at least six months, when stored at room temperature [230]. The LT-B vaccine produced in plants by Prodigene is now subject to a Phase I study on the efficacy of this oral vaccine derived from transgenic corn (www.prodigene.com, 2002).

2.428 No consideration was given to the effect that the LT-B transgene may have on the persistence or survival of maize. Previous studies had found that levels of transgene expression were fairly low (typically 0.01-0.1 TSP). One study increased the expression levels of LT-B through the creation of a synthetic gene containing plant-preferred codons and eliminating potential mRNA processing signals and destabilising motifs in the native gene. However it was found that high levels of expression of the LT-B resulted in the stunted growth of transgenic plants. These adverse effects were overcome by directing the expression of the protein to the storage organs of the plants only (Mason et al., 1998; cited by [218]).

2.429 Studies by Chikwamba et al. [231, 232] found similar results, although they used slightly different methods. They used a synthetic LT-B gene with codons optimised for plant sequences; in some transgenic plants they also used the SEKDEL endoplasmic reticulum retention motif. Two different promoters were used: the CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels [231, 232]. The SEKDEL resulted in increased LT-B levels in the kernels when combined with the gamma zein promoter. Significant variation in gene expression was observed, however the highest expressing line third generation had a Total Aqueous Extractable Protein LT-B level of 3.7 percent (in the line carrying the gamma zein promoter/LT-B construct). SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter [232] They also found that mice fed with maize expressing LT-B were successfully immunised against the toxin [231]. The effects of the LT-B expression on plant growth and development were not considered.


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2.430 Extensive codon modification, such as that undertaken for the LT-B vaccine in both research groups discussed above, can be laborious and expensive. Levels of gene expression have also been increased via an alternative method of chloroplast transformation. One study reported using this method for the expression of the cholera toxin B subunit (CTB) in tobacco plants [218].

2.431 Cholera is another enteric disease that causes acute watery diarrhoea by colonising the small intestine and producing the enterotoxin, cholera toxin (CT). Because it is the cholera toxin B subunits (CTB) that bind to eukaryotic cell surfaces and elicit a mucosal immune response, then CTB could therefore potentially be used as the basis of an oral vaccine for cholera [218] The production of an oral vaccine in edible transgenic plants using genes encoding bacterial toxin subunits LT-A, LT-B or CT-A and CT-B is the subject of patent [256].

2.432 Daniell et al. (2001) [218] cloned the CTB gene into the chloroplast expression vector pLD-CtV2 along with the constitutive promoter of the rRNA operon and a marker gene, to form the chloroplast expression vector pLD-LH-CTB. This vector integrates the genes of interest into the spacer regions between functional genes of the chloroplast genome. Tobacco plants were subsequently transformed by microprojectile bombardment. Transgenic plants yielded CTB levels between 3.5-4.1 percent of TSP. These high levels of expression did not have any apparent effects on the growth rate, flowering or seed setting of the transgenic tobacco, compared with the wild-type, suggesting that the transgene would have no effect on the persistence or survival of the transgenic plant) [218]. The levels of expression in this experiment were thought to be suitable for commercial exploitation, but it was suggested that levels could be increased further (up to 50 percent TSP) through the insertion of a putative chaperonin) [218].

2.433 Another way of increasing the effect (rather than just increasing the amount) of the autoantigen is to combine them with adjuvants (immunostimulants). Many bacterial toxins have subunits that can act as adjuvants, and can also bind to specific receptors in the mucosal associated lymphoid tissue, including the gut. Cholera toxin (CT) has been used in fusion protein vaccines due to its highly immunogenic subunits that bind to the mucosal surface. Such adjuvants could also be used for a number of different disease pathogens. The concept of producing an oral vaccine against several enteric disease pathogens using adjuvants has been tested by one research group [235].

2.434 Yu and Langridge (2001) [235] have produced transgenic potato plants that synthesise cholera, rotavirus and E. coli antigens in the same plant. Two subunits of the cholera toxin, A2 (CTA2) and B (CTB), were used as oral adjuvants. Yu and Langridge (2001) [235] designed a construct which consisted of a rotavirus epitope (NSP4) fused to CTB and an E. coli antigen (CFA/I) fused to CTA2, to produce the


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plant expression vector pPCV701CFA/I-CTB-NSP4. Potato plants were transformed via A. tumefaciens mediated transformation. Transgenic potato plants expressing these fusion antigens were fed to mice, resulting in systemic and mucosal antibody responses against CTB, NSP4 and CFA/I. Directly linking small antigens with the CTB subunit is reported to not only result in specific targeting of the antigens to the mucosal immune system through specific enterocyte attachment, but also increases the local antigen concentration at the mucosal surface. This may explain the strength of the immune response directly against the CTB-NP4 fusion protein. The outputs of the study demonstrated the real potential of developing an oral vaccine against a number of different pathogens within the single plant. The use of this type of vaccine in clinical applications will however depend upon the licensing of oral adjuvants for human use [255, 235].

2.435 It has been suggested that future development of edible vaccines for humans could concentrate on the expression of the vaccine in the �appealing� and edible parts of the plant. It was suggested that since the efficiency of the translation of foreign genes has been found to be similar in chromoplasts to that of chloroplasts, it is feasible that vaccines could be expressed at high levels in the chromoplasts of transgenic fruit) [218].

2.436 A step in the direction of vaccine production in �appealing� edible plants has recently come about through the expression of the CTB cholera toxin subunit in tomato plants [234] Tomato plants were transformed using a gene encoding CTB, along with the endoplasmic reticulum retention signal (SEKDEL) under the control of the CaMV 35S promoter using A. tumefaciens mediated transformation. Tomato leaves and fruits expressed CTB at levels up to 0.02 and 0.04 percent of TSP, respectively [234]. It is unclear whether this expression level would be sufficient to prompt the desired immune response. No mention was made of any adverse impact on the growth and/or development of the plant, nor its persistence or survival.

2.437 The expression of cholera and Escherichia vaccines in edible plants has been proved. Expression levels of the antigen are likely to increase in the future, to ensure the immune response. No studies have yet been carried out that look specifically at the effect these modifications could have on persistence and/or survival of the plant.

2.438 The plant-produced E. coli vaccine and another plant-produced vaccine to protect against enteric disease are entering Phase I clinical trials (Charles Arntzen, communication during Royal Society Discussion Meeting, 10th February 2003).

2.439 Recently E. coli vaccine has been expressed in tomatoes (results not published) produced in highly controlled glasshouses. Future work is likely to focus on producing sterile vaccine-producing plants and in vaccine-producing plants that are easily recognisable (e.g. in colour). Groups are also considering the production of vaccines


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in non-crop plants (e.g. Lemna www.biolex.com) (Charles Arntzen and Julian Ma, communication during Royal Society Discussion Meeting, 10th February 2003).

Hepatitis B virus

2.440 Although a very effective and safe hepatitis B vaccine is in use in the industrialised world, the disease burden of hepatitis B in developing countries is still very high because of the high cost of the currently available vaccine. It is thought that GM technology could provide a cheaper vaccine that could be produced in plants for human consumption [228]. Such a vaccine would have a very large global market, hence one of the oral vaccines covered by Prodigene�s patent is that for an hepatitis B oral vaccine (www.prodigene .com and US Patent) [257].

2.441 Kapusta et al. (1999) [227] used the DNA fragment encoding a hepatitis B surface antigen (HBsAg) to transform lupins and lettuce via A. tumefaciens mediated transformation. Mice fed the transgenic lupin developed significant levels of HBsAg-specific antigen antibodies. Similarly, humans fed with the transgenic lettuce also developed a high HBsAg-specific immunoglobulin G response. A fairly large amount of lettuce was required (200 g, followed by 150 g dose) in order to obtain an immune response that was thought to be sufficiently protective [227].

2.442 In order to try and increase immune responses to the HBsAg an oral adjuvant, cholera toxin, was used by Kong et al. (2001) [228] They expressed the HBsAg gene in potatoes (via A. tumefaciens mediated transformation) and compared the efficacy of this edible vaccine produced in transgenic potatoes when they were cooked and when they were uncooked.

2.443 Mice fed with the transgenic potato were found to develop an immune response to HBsAg. This was much more effective with raw potato (rather than cooked) and when the potato was fed to the mice with the oral adjuvant (rather than without). The immune response developed in the mice fed with raw transgenic potato with oral adjuvant was found to be much greater than that thought to be required to be protective against the active virus.

2.444 The fact that raw potatoes are unpalatable for humans precludes the use of this plant as a delivery vehicle for the hepatitis B vaccine. However, the research group involved in this experiment is currently involved in further experiments where the HBsAg transgenic plants that can be eaten raw (e.g. tomatoes and bananas) [228].

2.445 Neither of the above experiments considered explicitly the effect of the genetic transformation of the plant species on its persistence or survival. No adverse effects were reported.


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2.446 If hepatitis B antigens can successfully be expressed in edible, palatable plants such as banana or tomato, this could become a commercial crop species that would be particularly useful in developing countries. The use of this type of vaccine in clinical applications will however depend upon the licensing of oral adjuvants for human use [255, 235] Although the potential for this has increased recently with the discovery of reduced toxicity mutant forms of the oral adjuvants [228].

Measles vaccine

2.447 Measles live-attenuated vaccine is destroyed by heat and has no oral tolerance [258, 217] Measles is therefore a target for production within plants as there are real issues with its distribution, storage and administration. An antigen, the H protein � a major surface protein of the measles virus, has been identified. The antibodies that are raised to this have been found to have a measles-neutralising activity and therefore give immunological protection against measles [217].

2.448 Webster et al. (2002) [217] introduced the measles H protein (MV-H) DNA sequence into the genome of the tobacco plant using Agrobacterium mediated transformation. This transgenic plant was fed to mice, which resulted in an immunological response in the mice. The antibodies produced were found to be able to neutralise wild-type measles virus in vitro. It was found that the immune response in mice was five times greater than that thought to be required in humans to gain protection against the measles virus.

2.449 Tobacco was chosen for these experiments as it is a good model species for evaluating the production of recombinant proteins. However, it contains toxic compounds that make it unsuitable for use in human vaccine delivery. Therefore other suitable crop species are likely to be the target for further development in this area [217] It is also likely that to obtain a consistent immune response will require vaccination with an oral adjuvant (e.g. cholera toxin), which enhances immune responses at mucosal surfaces (the site for measles attack) and reduces the oral dose required to induce an immune response. Delivery without such oral adjuvants will only be possible if antigen doses can be increased within the transgenic plant [236, 217].

2.450 The potential effect of the MV-H gene on the persistence and or survival of transgenic plants is currently unclear, although the gene is not expected to confer a selective advantage on transgenic plants [217].

2.451 Future development is likely to see the production of vaccine antigens for measles in crop species such as rice [217]. Experiments similar to the one above are currently been undertaken with the expression of the MV-H protein in rice and lettuce [236].


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Malaria

2.452 The first step towards the development of an edible vaccine against malaria has recently been made. This has been possible through the identification of a promising antigen candidate that is referred to as the merozoite surface protein (MSP1). During merozoite invasion this protein remains as a C-terminal cysteine-rich 19 kDa fragment (MSP119). The gene encoding for this fragment has been cloned in a vector containing plant-specific codons to ensure expression, the CaMV 35S promoter and the nopaline synthase terminator to produce the construct pPM121. Tobacco plants were transformed with this vector using A. tumefaciens mediated transformation. It was shown that this gene was successfully integrated into the genome of the tobacco plants and that the protein was successfully expressed in the plant. The highest level of expression produced 35 ng mg-1 of recombinant MSP119 of TSP (0.0035 percent) [237].

2.453 Although the authors did not look specifically at the potential effects this genetic transformation would have on the persistence or survival of the transgenic plant, they did note that although the plants flowered they did not set seed [237], suggesting that they have some developmental abnormality in flower/seed development or that they were sterile.

2.454 The stated eventual aim of this group was to express malarial antigens in edible plants [237]. However, tobacco was used in the first instance as a model system. Their proposed next step was to design new gene constructs with signals targeted to cellular compartments, or to use new plant transformation techniques (e.g. chloroplast transformation) to increase expression levels and thereby increase the amount of protein produced in the plant [237].

2.455 It is likely that the production of malarial vaccine in edible plants will be subject to future experimentation as this first experiment shows the feasibility of using plants to express malarial vaccine candidate antigens.

2.456 A separate group have looked at the expression of a second recombinant form of MSP1: a 42 kDa C-terminal portion of MSP1, in transgenic tobacco plants. This is subject to a patent application [259] It has been suggested that the (non-GM plant-produced) recombinant vaccine based on the 42 kDa C-terminal portion of MSP1 is more efficacious in producing an immune response than the 19 kDa form when used to vaccinate monkeys [238].

2.457 It is likely that the most successful vaccines against malaria will consist of a number of different antigens of malaria from the various life-stages of the parasite [260]


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2.458 With the technology available to express a number of different xenogenic proteins within a transgenic plant successfully, it is possible that plants could provide an ideal production system for such multi-antigenic malarial vaccines in the future.

Protection against autoimmune diseases and responses

2.459 Autoimmunity occurs when environmental and/or genetic factors predispose the immune system to recognise the body�s own proteins as foreign or threatening. Autoantigens trigger the immune system to cause tissue-specific or systemic damage through cytotoxic T-cell mediated destruction and macrophage-stimulated inflammatory responses. Low doses of antigen have been found to result in the immune system deliberately suppressing this autoimmune response (effectively immunising a patient against the autoimmune response). This is called �oral tolerance� as it results in the body�s ability to tolerate these proteins [261].

2.460 The most widespread of autoimmune diseases is diabetes mellitus. Insulin dependent diabetes mellitus (IDDM) is caused by the autoimmune destruction of insulin-secreting pancreatic β cells. This disease affects 0.2-0.3 percent of all humans and the requirement for life-long insulin treatment combined with complications that can occur later in life make this disease a target for the development of prevention strategies [12, 239, 220].

2.461 Large doses of antigen given orally to IDDM patients were required to suppress or delay disease onset; presumably because the protein is degraded in the stomach. The production of large quantities of this antigen was thought to be unfeasible due to the cost involved. Transgenic plants however offer the opportunity to resolve this problem [12].

2.462 A major autoantigen involved in IDDM is glutamic acid decarboxylase (GAD). The larger isoform of this protein (GAD67) expressed in transgenic plants and fed to mice induced oral immune tolerance. However, it is thought that the smaller isoform GAD65 plays the major role as the autoantigen in human IDDM [240, 12, 239, 220]. Since the concept had already been demonstrated in mice, Porceddu et al. (1999) [239] set out to see if transgenic plants expressing high levels of recombinant human GAD65 could be the source of food for oral administration of the autoantigen.

2.463 The cDNA encoding for human GAD65 was introduced into the plant binary vector pBin19, downstream from a CaMV 35S promoter and upstream from a 3�-untranslated region of the nopaline synthase terminator. Tobacco and carrot plants were transformed via A. tumefaciens mediated transformation [239]. The result of this genetic modification was the expression of recombinant human GAD65 in both tobacco and carrot. The transgenic product retained its correct enzymatic activity and was found to immunoreact with IDDM-associated autoantibodies in vitro.


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Expression levels in both carrot and tobacco were quite low (up to 0.04 percent TSP), and it was not certain whether these expression levels would be sufficient for the induction of oral tolerance in humans, if they were fed such plants. There are concerns regarding the optimal dose required to achieve maximum protective efficacy from plant-based vaccines, and too much of the autoantigen can be just as harmful as not enough. It was hoped that this study would lead eventually to future studies investigating whether oral tolerance could be induced by eating transgenic carrots expressing recombinant human GAD65 [239].

2.464 Expression levels of protein could be enhanced by the targeting of the production of GAD65 to the cytosol through site-directed mutagenesis of critical residues responsible for membrane interaction [239]. [240] obtained 10-fold higher levels of GAD67 in their transgenic plants; GAD67 is a cytosolic isoform of GAD.

2.465 Another way of increasing the effect of the autoantigen would be to combine them with adjuvants (immunostimulants), as discussed in the other vaccine sections above. Cholera toxin (CT) has been used in fusion protein vaccines due to its highly immunogenic subunits. Transgenic potatoes expressing a hybrid protein which linked insulin to the C-terminus of the cholera toxin B subunit (CTB) have been produced. This directs the production of the insulin to the gut-associated lymphoid tissues. Non-obese diabetic mice fed potato tuber tissue containing milligram amounts of this fusion protein showed a significant reduction in pancreatic inflammation and a delay in the onset of diabetic symptoms [233, 220]. Plants have also been produced that express both GAD65 and a fusion protein where GAD65 is linked to CTB. Both of these are subject to a US Patent Application [262].

2.466 The induction of oral tolerance through the exposure of patients to the autoantigen involved in their autoimmune disease also has potential for diseases other than IDDM including multiple sclerosis, rheumatoid arthritis, irritable bowel disease; it also has a potential application in inducing oral tolerance to antigens present within transplanted tissues. A patent [263] and patent application [264] have been issued that cover the methods for expressing a mammalian antigen in GM plants and providing a source of plant material for oral administration to produce tolerance to the antigen. A similar patent application [265] also covers the prevention and treatment of autoimmune disorders, using genetic material encoding at least a part of an autoreactive antigen that, upon administration to a subject, modulates the immune system response to reduce the effect of the autoreactive antigen.

2.467 None of the above experiments looked specifically at the effect of these modifications on the growth and or survival of the transgenic plants. However, it was noted in previous experiments expressing a modified petunia GAD in tobacco plants, that dramatic phenotypic variations were seen in the transgenic plant compared with the wild-type, with adverse effects observed on the development of the transgenic plant


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and consequently its long-term survival [239]. It was noted in the study reported above [239] that no phenotypic differences were found between human GAD65 expressing transgenic plants, or their progeny and the wild-type; suggesting that the GAD65 expression did not have an adverse effect on the transgenic plant�s development. No long-term studies on the effect of this transgene on persistence or survival of the plant were carried out.

2.468 The induction of oral tolerance to antigens expressed in transgenic plants holds real promise for autoimmune diseases that are traditionally treated with immunosuppressants (leading to increased rates of infection, malignancy and other side effects). However, the dosage required to produce this result and the long-term effects of this treatment are unclear. Similarly, it is unclear whether this would have any adverse effect on growth and development of the plant. It is likely that this field will be subject to further studies.

HIV Vaccine

2.469 An antigen that is an early marker of human immunodeficiency virus (HIV) infection has been used in the development of HIV vaccines; it is also used widely in blood screening, clinical diagnoses and HIV-related research. This antigen is the HIV-1 p24 nucleocapsid protein. The development of a cost-effective production system for this protein would be beneficial to all of these areas [241]. A research team has looked at developing such a production system in plants.

2.470 Zhang et al. (2000) [241] expressed HIV-1 p24 nucleocapsid protein in tobacco using a recombinant virus protein (tomato bushy stunt virus). Using such a plant virus as a vector for the expression of the HIV antigen means that the infection can be delayed until the plant has reached a sufficient size so as to maximise the yield of the protein. The HIV-1 p24 cDNA was introduced into a cloned cDNA copy of the tomato bushy stunt virus genome. When the tobacco plants were infected with this recombinant virus this led to the production of the antigen in infected tissues, which was able to react with the anti-p24 antibody. However, there was some difficulty with vector stability and the infection did not spread systemically to the apical parts of the plant.

2.471 This study shows the feasibility of using plant viruses as expression vectors of antigens for the production of vaccines, however, work needs to be carried out to improve vector stability, and therefore protein yield.

2.472 To improve the stability of the expression of the HIV-1 p24 protein, Zhang et al. (2002) [242] attempted a more traditional approach to the expression of the protein in the plant, using A. tumefaciens mediated transformation with the HIV-1 p24 cDNA under the control of the CaMV 35S promoter and nopaline synthase (NOS) terminator. The HIV-1 p24 protein was expressed successfully in the plant tissue. It


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was thought that there may have been slight differences in this plant produced protein due to the presence of glycol-residues; however, it was found that this protein was still immuno-reactive with the p24 specific antibody. Subsequent generations of the transgenic plants also expressed this protein, showing that it is expressed stably (Zhang et al., 2002) [242]

2.473 The American company Prodigene are reported to have looked at developing an HIV vaccine through the expression of the gp120 protein from simian immunodeficiency virus (SIV) in plants for use in acquired immunodeficiency syndrome (AIDS) research and as a potential oral delivery system for an AIDS vaccine. The gp120 protein is found on the surface of both SIV and HIV; it is thought that this protein may not be the only protein that will eventually form an effective AIDS vaccine, but this experiment proves that such proteins can successfully be produced in plants (www.prodigene.com, 2002).

2.474 None of these studies have however considered the effect of the production of HIV proteins on the plant�s ability to persist or survive in the environment.

2.475 Several groups are working on such a plant produced vaccine (e.g. Monash Medical School, Alfred Hospital, Australia; Department of Molecular and Cellular Biology, University of Cape Town, South Africa). The usefulness of the HIV-1 p24 in screening and diagnosis is clear, namely to function effectively as a vaccine it will probably be combined with a number of other HIV surface proteins. These early experiments provide a proof-of-concept rather than an immediately effective AIDS vaccine.

Lymphoma Vaccine

2.476 Lymphoma is a cancer of the white blood cells. Specifically, non-Hodgkin�s B-cell lymphoma is a cancer of the B-cells that is often incurable and is increasing in frequency in industrialised nations [223]. The cancerous B-cells proliferate at a much faster rate than the non-cancerous white blood cells and also undergo cell division prior to cell maturation so that the malignant B-cells cannot perform their normal duties in their role in the immune system. Scientists searching for a cure to this disease have identified a specific immunoglobulin (antibody) that occurs on the surface of each malignant B-cell. This holds great promise for the production of a patient-specific vaccine for non-Hodgkin�s lymphoma patients. The production of such a vaccine in plants has great potential as providing a safe, easily purified vaccine [223].

2.477 A vaccine of this type has been produced in tobacco plants through the expression of the antigen-binding site of the immunoglobulin, the single-chain variable region (scFv). This immunogenic binding site is also known as the idiotype. The scFv


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vaccines are able to elicit an anti-idiotype specific response in animals. Thus the anti-idiotype antibodies may generate a strong immune response to tumour antigens and be effective for treating and/or preventing cancers [223].

2.478 In order to speed up the process of production of such a vaccine, a transient plant expression system has been developed by Biosource (Vacaville, CA, USA) (now Large Scale Biology Corporation (LSBC)) and Stanford University. The method of expressing genes in plants through their infection with viruses containing the gene construct encoding for the scFv within the viral RNA is subject to a US Patent [266] and is called Geneware.

2.479 In this application, the Large Scale Biology team modified a tobacco mosaic virus vector so that it encoded the idiotype-specific scFv of the immunoglobulin from the 38C13 mouse B-cell lymphoma. Tobacco plants infected with this virus produced high levels of secreted scFv protein. Mice vaccinated with the purified scFv produced by the plant generated anti-idiotype immunoglobulin and were protected from challenge by a lethal dose of 38C13 tumour. The goal of such research is to develop a vaccine for the treatment of non-Hodgkin�s lymphoma and eventually to create antibodies customised for each patient that will recognise markers on the surface of malignant cells and target these cells for destruction [203, 223]

2.480 A Phase 1 clinical trial of personal vaccines for non-Hodgkin�s lymphoma has been undertaken using the scFv produced in tobacco via Geneware technology (www.lsbc.com). The results of the clinical trial have recently been presented (44th Annual Meeting and Exposition of the American Society of Haematology, December 2002).

2.481 These initial results were encouraging and Large Scale Biology proposes to proceed with larger efficacy trials once it has secured further funding (www.lsbc.com); it also plans to extend this type of research into the treatment of other types of cancer. The method of inducing an immunological response to tumours using such anti-idiotype vaccines, particularly relating to non-Hodgkin�s B-cell lymphoma, is subject to a US Patent [267].

2.482 Vaccines are also being developed against other types of cancer e.g. a vaccine produced in plants against the Papilloma virus, which is one of the major causes of cervical cancer (Julian Ma, communication during Royal Society Discussion Meeting, 10th February 2003).


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Caveat on production of vaccines in plants

2.483 It has been noted by Webster et al. (2002) [217] that although the technology for the production of vaccines in plants is very appealing, it is not a simple case of getting the plant to express the antigen; numerous other issues need to be resolved before it could truly be a viable option. These include: accumulation of sufficient antigen within the plant tissues; safety and oral tolerance (as noted above low doses of antigen given orally can induce oral tolerance so that the immune system no longer recognises an antigen as �foreign� � resulting in susceptibility to the disease, rather than protection). However, such a desensitisation of immune response has never been demonstrated for a subunit vaccine (Charles Arntzen and Julian Ma, communication during Royal Society Discussion Meeting, 10th February 2003). The potential benefits of this technology mean that there are many research groups that are willing to continue experimentation in this field, despite these potential problems that need to be investigated.

Antibodies

2.484 Antibodies are required for the diagnosis, management and treatment of contagious diseases and cancer [206, 209]. However, the widespread use of antibodies in medicine is prevented, mainly by the expense and difficulty of producing enough antibodies to meet the demand. The demand for safe, recombinant pharmaceutical proteins is rapidly expanding, both in terms of the amounts of protein required and the protein complexity [208]. Plant production systems are proposed as having the potential to meet that demand.

2.485 It was first demonstrated that functional antibodies could be produced in transgenic plants in 1989 [206, 211, 220, 221]. Recombinant antibodies that have been produced in transgenic plants since then include fully assembled whole complex immunoglobulins made up of a number of different proteins, including; antigen�binding fragments of immunoglobulins (Fab), chimaeric antibodies, single domain antibodies (dAb) and synthetic single-chain antibody fragments (scFv) [205, 214, 206, 215]. A number of patents exist that relate to the production of recombinant antibodies in plants including: [268, 269], both relating to the production of assembled secretory antibodies in transgenic plants. Another patent [270] relates to the production of immunoglobulins in plants. All of these patents are held by the American company Epicyte (www.Epicyte.com).

2.486 Various functional recombinant antibodies have been produced in plants (plantibodies) and have been successfully stored in dried leaves, seeds and potatoes, demonstrating the potential for long-term storage of both the proteins and their production system. There are some concerns regarding protein degradation linked to plant senescence, or long-term storage. However plantibodies that are


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stored in seed have been found to retain their antigen-binding activity for at least three years, after storage in a refrigerator [209, 211, 221]. Similarly, De Wilde et al. (2002) [271] found that transgenic potato tubers expressing full-size IgGs and Fab fragments could be stored for up to six months without significant loss of antibody amount, or activity.

2.487 The first reported clinical trials of plant-produced antibodies were carried out by Planet Biotechnology, Inc. (Mountain View, CA). Their drug CaroRxTM is based on tobacco-produced secretory immunoglobulin A (IgA) against Streptococcus mutans, a causal agent of tooth decay in humans. This is the most advanced plantibody in terms of its progress toward becoming a commercially viable product, with initial clinical studies in humans having provided favourable results [222, 12, 203, 209, 211, 215, 220, 221].

2.488 In addition to direct therapeutic applications, plant derived antigens/antibodies are also being tested for use in diagnostic systems. For example, the hepatitis B virus (HBV) core antigen has been expressed in tobacco, and the recombinant product used successfully (as successfully as the standard antigen produced via the E. coli production system) in the haem-agglutination test routinely conducted by the Japanese Blood Centre on blood samples from HBV positive donors (Tsuda et al., 1998; cited by [12]).

2.489 Antibodies have been most commonly produced from transgenic plants where the transgene has been inserted directly into the genome of the plant. The expression and deposition of the antibody, and its accumulation, have been manipulated through the use of specific targeting signals within the gene-construct that is inserted into the plant. In addition to nuclear gene transfer, transgenes have also been expressed in chloroplasts. Transient expression systems have also been demonstrated that use recombinant viruses to infect the plant, resulting in the production of the transgenic product transiently by the plant without direct manipulation of the plant�s genome [215, 221].

2.490 Recent examples of the potential application of this technology include the use of antibodies produced by transgenic plants in the diagnosis and/or treatment of cancer, HIV, haemolytic disease of the newborn and in the cross-matching of donors/receivers.

Cancer Diagnosis and Therapy

2.491 In cancer therapy the purpose of antibody administration is specific cancer-cell destruction or starving of tumours through the targeting of the tumour vasculature. New technologies in the panning of antibody libraries on intact cells have made it


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possible to isolate antibodies to novel and promising cancer-specific and cancer-associated antigens [272].

2.492 One study looked at the feasibility of producing cancer-specific antibodies in plants by expressing two recombinant antibodies specific for the human carcinoembryonic antigen (CEA). CEA is a cell surface glycoprotein that can be detected in almost all human colon cancers. Anti-CEA antibodies have applications in antibody-mediated cancer therapies and in tumour imaging [225] Both recombinant antibodies were successfully expressed in tobacco leaves following A. tumefaciens mediated transient gene expression [225].

2.493 The above study showed the feasibility of producing such antibodies in plants. A subsequent study looked at the production of antibodies for cancer diagnosis by expressing a scFv derived from the monoclonal antibody T84.66 in transgenic pea plants. Anti-CEA antibodies, of which T84.66 shows the highest specificity and affinity, are used widely for the in vitro diagnosis and in vivo imaging of human colorectal carcinomas and adenocarcinomas of the lung, breast and other organs [226]. The pea was selected as it is a protein-rich crop and therefore has the potential to produce high levels of stable foreign proteins. Also, the seeds produced by the pea are large and can be stored for several years (containing the scFv), and processing systems are already in place for this species [226].

2.494 The scFvT84.66 coding region was subcloned in a pGEM3zf vector carrying the tobacco mosaic virus (TMV) sequence; other elements were located upstream of the scFvT84.66 coding region in order to direct the protein into the secretory pathway. These sequences were codon-optimised to enhance plant expression. The vector also encoded a KDEL motif for endoplasmic reticulum retention of the protein, downstream from the scFvT84.66 coding region. This whole cassette was inserted into the plant expression vector pG229LegA, containing the LegA promoter and the NOS terminator. The LegA promoter controls the legumin A gene, which encodes a protein belonging to a highly abundant class of seed storage proteins in pea, therefore resulting in the production of the protein in the pea seed. The plants were transformed with this plasmid using A. tumefaciens mediated transformation [226].

2.495 It was found that the scFvT84.66 antibody is folded correctly when produced in pea seeds; demonstrating the suitability of pea seeds for the production of a therapeutic scFv that recognises a cell surface cancer antigen and can therefore be used in cancer diagnosis and treatment. The stability of the scFvT84.66 in stored pea seeds was in agreement with similar work expressing scFv in other plant species. The pea seeds were stored for several months at room temperature without degradation of the scFv [226].


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2.496 The amount of scFv produced in the pea seeds was equivalent to up to 9 µg g-1 fresh weight. Using current techniques for the purification of scFv, less than 2 kg of pea seeds would be required to provide the 10 mg dose of radio-labelled scFvT84.66 required for a single diagnostic or therapeutic application [226].

2.497 No comparison was made between the wild-type and the transgenic plant to try and assess if there would be any effect of this transgene on persistence or survival. However this was considered in the selection of the plant to be transformed: field peas are strictly self-pollinators, therefore this reduces concerns regarding the �loss� of transgenes through outcrossing [226].

2.498 A similar approach was used to express two recombinant antibodies specific for the human carcinoembryonic antigen (CEA). CEA is a cell surface glycoprotein that can be detected in almost all human colon cancers. Anti-CEA antibodies have application in antibody-mediated cancer therapies and in tumour imaging [225].

2.499 If similar tumour-specific antibodies can be identified and their DNA sequenced this technology holds promise for the treatment of cancers.

Diagnostic antibody for crossmatching donors/receivers

2.500 The demand for monoclonal antibodies has increased substantially; existing production systems such as murine hybridoma are often very expensive and the products are often unstable in long-term cultures. One high-affinity anti-human IgG is a monoclonal antibody (C5-1) secreted by a murine hybridoma cell line, which is suitable for use in anti-human globulin (AHG) reagents. AHG reagents are commonly used in blood banks for phenotyping and crossmatching red blood cells of receivers/donors. The C5-1, as a component of the AHG, allows the detection of incomplete blood group antibodies in human serum. C5-1 is usually produced by large-scale culture of B-cell hybridomas. These are reliable, but very costly. One gram of C5-1 antibody costs approximately $5000 [224].

2.501 Khoudi et al. (1999) [224] investigated the production of C5-1 in the perennial plant alfalfa; with the idea of investigating a cheaper production system for this antibody in a perennial plant that has the potential to become a stable, permanent source of recombinant molecules.

2.502 The cDNAs of the heavy (H) and light (L) chains of the C5-1 were cloned and modified. These fragments were then cloned into the binary expression vector pGA643 to generate pGA643-kappa and pGA643-gamma. Each cDNA was under the control of the CaMV 35S promoter and the T-DNA transcript 7 gene terminator. The plants were transformed using A. tumefaciens mediated transformation. The


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single transgenics were then crossed to produce double-transgenics expressing the cDNAs for the H and L components of C5-1 [224].

2.503 This resulted in the successful production of the antibody C5-1 by the transgenic plants. This was the first example of the production of a complex multimeric protein in a perennial plant. Double-transgenic plants expressed the C5-1 antibody at levels between 0.13 and 1 percent TSP. However, these were not intended to be representative of transgenic lines to be used for commercial production. The aim of the study was simply to demonstrate the possibility of using perennial plants to produce complex medical proteins and to confirm the feasibility of the approach. The study also confirmed that the plant-derived C5-1 could be used as efficiently as hybridoma-derived C5-1. The plant-produced C5-1 was also stable, both within the plant (even after harvest and drying) and once it had been extracted. The accumulation levels of C5-1 remained stable within the plant over 2.5 years of repeated harvesting. The plant-derived C5-1 was also as stable as the hybridoma-derived C5-1 in the bloodstream of mice; therefore demonstrating that it has the stability required for therapeutic use [224] It is unclear whether this line of experimentation was pursued further.

2.504 No consideration was made of the effect this gene had on the persistence or survivability of the transgenic plant. However, it obviously did not have a major adverse effect on the transgenic over the 2.5 year study period as the plant continued to grow even after being subject to repeated harvest.

Diagnosis and treatment of alloimmunisation or haemolytic disease of the newborn

2.505 Alloimmunisation or haemolytic disease of the newborn (HDN) is characterised by maternal immunoglobulins (IgG) that cross the placenta and cause haemolysis in the foetus. Alloimmunisation of a Rhesus D-ve (RhD-) mother carrying an RhD+ foetus is prevented by antenatal or perinatal injection of human anti RhD antibody. This antibody is currently supplied by previously alloimmunised mothers or by RhD- donors deliberately immunised with RhD+ red blood cells. This practice has ethical and practical limitations and therefore the supply of this treatment is very limited. An alternative production method could be to produce human monoclonal antibodies through hybridoma technology, but this would have to be scrutinised very carefully for contagious elements and could be very expensive [243].

2.506 The production of such antibodies in transgenic plants could provide a more useful alternative. The majority of immunoglobulins produced thus far in plants have been targeted against mucosa associated diseases. Bouquin et al. (2002) [243] attempted to demonstrate the production of the anti-RhD antibody in Arabidopsis. There are still challenges regarding the use of plantibodies produced for intravenous use. The efficacy of such antibodies is dependent upon a number of factors including its half-


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life in the bloodstream, its ability to be recognised by receptors on relevant cells and by other effector systems [243]. These characteristics are determined mainly by the glycosylation pattern (see section on glycosylation below).

2.507 The cDNAs of the light chain and heavy chain of the anti-RhD antibody were isolated. Three different approaches were used to generate transgenic plants producing both chains of the RhD IgG1 antibody. Firstly, lines expressing individual immunoglobulin chains under control of the CaMV 35S promoter were crossed to obtain plants producing intact antibody. Secondly, plants were produced expressing both immunoglobulin chains, each driven by a separate CaMV 35S promoter, situated on the same T-DNA vector. Thirdly, plants were generated that expressed both chains in a head-to-head orientation from the mas1�2� dual promoter of A. tumefaciens [243].

2.508 The transformation efficiencies of the three methods varied, but all resulted in the production of a transgenic plant that successfully expressed the anti-RhD antibody in A. thaliana.

2.509 This plant-produced IgG was also found to be able to trigger erythrophagocytosis by monocytes, indicating that the post-translational protein modification in Arabidopsis, in particular the addition and composition of complex glycans, is sufficient to produce functional anti-human RhD antibody, indicating the potential of this technology in this application [243]. Slight differences did occur in the glycan chains produced by the transgenic plant, it was thought that this could affect the half-life of the IgG, and also the potential immunogenicity of the plant-derived antibody (see section below on glycosylation) [243].

2.510 Once again, there was no consideration on the effect of the transgene on the persistence or survival of the transgenic plant.

Antibodies for the Treatment of HIV

2.511 Several human monoclonal antibodies have been found to act in synergy to neutralise the HIV-1 [273, 274] These antibodies are being investigated for their potential as passive anti-HIV immunotherapy. The group that has been carrying out this research has recently joined forces with the biotech company Epicyte (based in San Diego, USA). In a recent press statement, Epicyte announced that it was planning to produce these antibodies (IgA antibodies: 2G12, 4E10 and 2F5) in plants (www.Epicyte.com).

Biopharmaceutical Proteins

2.512 In addition to vaccines and antibodies, various other pharmaceutical proteins have been produced in transgenic plants, including two of the world�s most expensive

http://www.epicyte.com/


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drugs glucocerebrosidase and granulocyte-macrophyte colony stimulating factor. Other applications using plants to produce compounds for medical use include the development of pharmaceutically-active enzymes such as pancreatic lipase-colipase complexes (required by patients with defective pancreatic activity), mammalian growth factors, human haemoglobin and other pharmaceutical products [12, 209].

2.513 The production of correctly processed human somatotrophin (a growth hormone) in transgenic tobacco plants has been reported with an expression level of up to seven percent TSP. Somatotrophin was one of the first proteins to become available from transgenic E. coli. However transgenic plants have an advantage over the E. coli production system in that they need no post-extraction chemical processing [209].

2.514 In this section of the report a number of advances in the area of biopharmaceutical proteins are reviewed, including:

• the production of transgenic plants that can modify the glycosylation of plant-produced proteins (the significance of this in relation to plant-produced medical proteins is discussed below);

• the identification of anti-hypertensive proteins for expression in plant systems;

• the production of human plasma proteins; and

• the production of a number of other proteins that have medical applications in plants.

Modification of protein glycosylation

2.515 There are some concerns reported in the literature regarding the potential immunogenicity and/or allergenicity of plantibodies that could be used in human health applications. This is a consequence of slight differences in the glycan structure (due to slight differences in the way the plant system produces the protein, either during transcription or in post-translational modifications) and the presence of plant glycans (e.g. α1-3 fructose and β1-2 xylose residues). Although differences in glycosylation may not affect the activity of the protein, other properties such as the folding, stability, solubility, susceptibility to proteases, blood clearance rate and antigenicity could be affected. Where the plant is eaten it is unlikely that this would present a problem as plant glycoproteins are ingested normally in the human diet with no ill effects [204, 206, 275, 255, 209, 212, 215, 220, 221].

2.516 Recent studies have shown that topically applied plantibodies are safe [211]. However, studies have not yet managed to address the potential toxicity of


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plantibody glycans in humans [211, 212]. Glycosylation is not an issue where non-glycosylated proteins and peptides are concerned, such as those used as antigens in some vaccine applications, and may only be a problem for therapeutic glycoproteins that are administered to patients via injection [204, 205]. Where differences in glycosylation present a problem through immunogenicity, it may be possible to modify plants with altered protein maturation pathways [206].

2.517 One approach to avoid the potential for plant glycans to cause an allergic reaction in humans was investigated by Bakker et al. (2001) [276], in which an attempt was made to generate transgenic plants that provided �humanised� plant glycans. This approach was proposed as having the potential to reduce the possible allergenicity of the plant-produced proteins and in generating plant glycoproteins that require mammalian-type glycans for their activity or antigenicity [255]. The most important enzyme for the conversion of typical plant glycans into those more typical of mammals is β1,4-galactosyltransferase (GalT). cDNAs encoding for this enzyme have been identified from several different mammalian species [276].

2.518 Bakker et al. (2001) [276] have expressed GaIT successfully in tobacco, using a plant transformation vector containing human GalT, the CaMV 35S promoter and NOS terminator. The plants were transformed using an A. tumefaciens mediated transfer method [276]. The successfully transformed plants that expressed high levels of GalT were then crossed with a transgenic tobacco expressing a mouse antibody Mgr-48. The expression of the enzyme in the transgenic plants was found to �humanise� partially the glycans of the plant glycoproteins as well as those of the transgenic mammalian antibodies. This is an important development in the transgenic plant production of antibodies with fully humanised glycans as it would eliminate the possibility of potential immunogenic reactions and potentially allow their use in human medicine [276, 212]. Further studies are being carried out in order to optimise the galactosylation levels in GalT transformants and to investigate the effects of modified GalT targeting within the plant cell [276].

2.519 No obvious physiological or phenotypical differences were observed between the transgenic and wild-type plants, despite these changes in the glycosylation of non-transgenic plant proteins [276]. However, no field trials have compared how these plants grow in non-laboratory conditions. Further studies that will change the galactosylation of natural proteins even more may find that this has an adverse effect on the physiology of the plant. However, the fact that the transgenic plants seemed to be unaffected by the major changes in the glycans of natural plant proteins in the example of GaIT suggests that adverse changes to physiological or phenotypic characteristics may not be realised.


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Anti-hypertensive protein

2.520 A protein that has not yet been expressed in plants, but that has been highlighted as one that could potentially be introduced and produced using a GM crop system, is the modified soybean protein RPLKPW. This protein has been shown to reduce the blood pressure of spontaneously hypertensive rats, without affecting the blood pressure of �normo-tensive� ones. RPLKPW was also found to have an effect at oral administration doses comparable to those of synthetically produced anti-hypertensive drugs that are already in use [277]. Rice and soybean have been identified as candidate crops that could be used to produce the RPLKPW protein, although further investigations with regard to the allergenicity and chronic toxicity of the protein, as well as the effect that the transgene may have on the GM plant itself, would need to be carried out [277].

Human plasma proteins

2.521 Congenital deficiencies of numerous plasma proteins of the blood involved in clotting exist, with haemophilia type A and B being the most frequent. haemophilia type A has been treated for over ten years using recombinant factor VIII products that are produced in vivo using mammalian cells. The limitations of these products are related to their cost and availability and the theoretical risk of transmitting disease e.g. mammalian viruses that are present in the plasma [278].

2.522 The possession of the gene for the protein and a plant system in which to express it provides an opportunity to manipulate the sequence of that protein to improve its characteristics or to eliminate undesirable ones. Changes can also be introduced to improve the efficiency with which the recombinant protein can be produced [278].

2.523 Another plasma protein, whose congenital deficiency results in increased risk of liver disease in children, and pulmonary emphysema, haemorrhages and a variety of skin disorders in adults, is α-1-proteinase inhibitor (API) (also known as α1-antitrypsin (AAT)). AAT is a glycoprotein that inhibits the activity of the serine protease neutrophil elastase, thereby protecting tissues from being digested by enzymes released from inflammatory cells [245, 206, 278, 279].

2.524 A research team at Ventria Bioscience has expressed AAT successfully in transgenic rice cell culture, using the Amy3D promoter and signal peptide via microprojectile bombardment. The use of this promoter and signal peptide means that the target protein is only induced as a result of sugar depletion. The signal peptide of the protein was found to be processed correctly, although the transgenic protein was less glycosylated than that derived from human plasma. This aspect of the work is reported to require further investigation in order to achieve an efficient production system [245].


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2.525 A similar experiment following on from this altered the AAT gene slightly to optimise the codons for plant expression, so that the codons within the AAT gene corresponded with those found in highly-expressed rice genes. This achieved expression levels of 20 percent of TSPs within the rice embryonic tissue suspension cell culture [246].

2.526 The use of plant cell culture systems as described above can be a useful stage in the development of a successful transgenic plant protein production system, and provides a link between the theoretical application and large-scale open-field cultivation and harvesting of recombinant proteins from transgenic plants [245].

2.527 The production of large quantities of AAT would be very useful as treatment for those patients afflicted with AAT deficiency (intravenous or inhalation administration) [279]. These experiments hold promise for this in the future.

2.528 The impact of the gene on the persistence and survival of the species is not an issue in this case as it is proposed that plant cell cultures within a lab would be used to produce the protein, rather than the protein being produced in plants grown in the field.

Other medical proteins

2.529 There are numerous other medical proteins that have also been expressed in plants including drugs that could be used in the treatment and diagnosis of cancer, autoimmune diseases and genetic enzyme deficiencies. Examples are considered below:

♦ Alpha-galactosidase

2.530 Fabry�s disease is a genetic disease resulting in a deficiency in a key enzyme (alpha-galactosidase). This is a serious genetic disorder, resulting in microvascular disease of the kidneys, heart and brain; causing pain, kidney failure, stroke and heart disease [280, 281].

2.531 Two biotechnology companies, Transkaryotic Therapies Inc. and Genzyme General, both produce this enzyme through traditional techniques; this drug has been approved for sale in Europe, the companies are awaiting approval from the US. The cost of this effective drug treatment is very high � an annual cost of $175, 000 per year, per patient [281].

2.532 Aiming to produce this enzyme at a lower cost, the company LSBC has been investigating the production of this enzyme in plants using Geneware, a system that


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uses viruses containing the cDNA encoding the desired enzyme to produce the enzyme in the plant (www.lsbc.com). The recombinant alpha-galactosidase produced by the transgenic plants has recently been granted �orphan status� by the US Food and Drug Administration. This grants LSBC market exclusivity to this plant-produced product for seven years. LSBC plan to start early-stage clinical trials in the near future with this plant-produced enzyme (www.lsbc.com [281].

♦ Aprotinin

2.533 Aprotinin is a human therapeutic protein that is a protein inhibitor. It is commonly used in surgery, particularly cardiac surgery to control blood loss; but is also used in wound healing and as a component in pharmaceutical manufacturing. It is also known to reduce the inflammatory response generated during cardiopulmonary bypass (www.med.ub.es [282]. Aprotinin is extracted traditionally from bovine lungs. Transgenic plant technology therefore holds the opportunity to produce this protein in plants that is equivalent to the bovine aprotinin, but that is not limited by the supply of bovine lungs (www.prodigene.com). Prodigene (based in Texas, USA) holds the patent for exclusive commercial production of aprotinin in all plants [283]. Prodigene started pre-clinical trials with the GM plant- produced aprotinin in autumn 2002 (www.prodigene.com).

♦ Ribosome inactivating proteins

2.534 Plants naturally contain proteins that irreversibly inactivate eukaryotic ribosomes, and hence can inhibit protein synthesis. These are known as ribosome inactivating proteins (RIPs), and are one of the most active inhibitors known to science. RIPs act catalytically to promote the inactivation of ribosomes [247].

2.535 RIPs have been identified as having two potential uses in medicine. Firstly, the production of �immunotoxins� used to treat cancer. These are tumour-specific monoclonal antibodies linked to a RIP (e.g. ricin). Due to their mode of action these immunotoxins are extremely toxic [247]. Secondly, some RIPs have also been shown to display a powerful anti-viral action against viruses such as HIV (e.g. Pokeweed anti-viral protein, Trichosanthin and Momordica anti-viral protein) and HSV (herpes simplex virus) (e.g. gelonin, Dianthin 32 and Pokeweed anti-viral protein from seeds) [247].

2.536 Transgenic plants have been produced that express RIP genes. This has resulted in the transgenic plant expressing very strong antipathogenic properties, and also has applications for the control of plant diseases and provides an important tool for the biological control of pathogen-dependent spoilage of crop production. For example, the Pokeweed anti-viral protein (PAP) cDNA was inserted into the transformation

http://www.lsbc.com/

http://www.lsbc.com/

http://www.med.ub.es/

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plasmids pMON8443 (with the 35S RNA promoter from CaMV) and pMON8484 (with the 35S RNA promoter from figwort mosaic virus) and expressed in tobacco by A. tumefaciens mediated transformation. The transgenic tobacco was very resistant to infection by potato viruses. However, the PAP was also active on some of the potato ribosomes. This resulted in local inhibition of plant protein synthesis and hence local plant development; leading to local rolling up of leaves at the site of infection [247].

2.537 Although similar studies have been reported for the development of RIPs for use against HIV and cancers, such plants have not yet been produced. This is due mainly to the fact that these proteins are very toxic and until their use can be focused effectively against the target virus or eukaryotic cell, their use is assessed to be of too great a risk, both to the long-term survival of the transgenic plant and to the potential recipient of such therapy.

2.538 If transgenic plants were successfully produced with such proteins, as a consequence of the highly toxic nature of the proteins, any growth or release of such plants would have to contain stringent management strategies to ensure effective containment of the transgene(s).

♦ Cytokines

2.539 Cytokines are proteins closely linked to the function of the immune system. They can have a role in the regulation of the immune response and can have immunological effects [248, 249]. These proteins have great potential as pharmaceutical products, particularly if they could be produced efficiently and safely through plant expression systems.

2.540 One example of the development of a GM plant system expressing a cytokine is the generation of human interferon-α (IFN-α) in potato plants. IFN-α is an anti-viral cytokine that exhibits a wide range of immunological effects, including anti-tumour activity. Such IFNs (termed type I IFNs) are thought to be a critical link between innate and adaptive immunity, providing a scope of clinical applications for a wide range of infectious diseases, other than viral ones [249].

2.541 Ohya and colleagues expressed two sub-types of human IFN-α (Hu IFN-α2b and Hu IFN-α8) in potato plants using A. tumefaciens mediated transformation. The transgene constructs were made using plasmids pHSG398 containing cDNA encoding Hu IFN-α2b or Hu IFN-α8. Each cDNA was separately cloned into pBluescript KS(+)[249].

2.542 Successful production of biologically active Hu IFN-α8 was demonstrated, and although a larger mRNA was detected than expected for Hu IFN-α2b,


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immunologically detectable levels of Hu IFN-α2b were produced in the transgenic plants. This was the first report to demonstrate the potential for transgenic plant production systems to produce human cytokines. There were substances that were toxic to animal cells present, but these were naturally occurring plant toxins, rather than a trait of the transgenic potato [249]. The expression levels of Hu IFN-α in the transgenic plants were very low (210-560 IU g-1 plant tissue). However, doses of IFN-α as low as 10 IU kg-1 body weight have been shown to have a positive effect such as an improvement in autoimmune diseases such as Sjögren�s syndrome [249].

2.543 Ohya et al. (2002) [250] have also investigated the expression of another cytokine in transgenic potato plants. The human tumour necrosis factor-α (Hu TNF-α) is a known and essential cytokine that mediates host defence against tumours and infectious diseases, and it has also proven to be useful in the treatment of autoimmune diseases such as rheumatoid arthritis. Systemic injection of TNF-α induces severe side-effects, limiting its clinical uses. However, it has been used directly and successfully against certain cancers, and when delivered orally it has been shown to enhance the immune response against certain bacteria [250]:

• one gene construct (chimaeric Hu TNF-α) contained a nucleotide sequence encoding an endoplasmic reticulum retention signal (SEKDEL) and a signal sequence for a seed storage protein (legumin); and

• the other contained just the cDNA for Hu TNF-α.

2.544 The expression levels for the transgenic plants expressing Hu TNF-α were higher than in previous experiments with Hu IFN-α. The expression levels with the chimaeric Hu TNF-α were insignificant, despite the use of the legumin and SEKDEL genes, which have previously been shown to enhance the expression of recombinant products. However the expression levels within the Hu TNF-α transgenic plant would probably provide sufficient Hu TNF-α for its purpose as cytokines are known to have positive effects at very low dose levels [250].

2.545 There was no discussion in the previous two examples of the effect the transgene for the cytokine may have on the persistence or survival of the transgenic plant. No comparison was made between the wild-type and transgenic plant.

2.546 Concerns have been raised regarding the production of drugs such as cytokines in transgenic plants as these compounds can be highly toxic to non-target recipients or cells. The effects of acute or chronic exposure to these proteins or their escape into the environment are not known, therefore practical issues such as safety and containment need to be considered [248].


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2.547 The issue of safety and containment has been addressed to some extent by investigations that have been conducted in the production of contained recombinant plant production systems. Systems have been developed that are based on the transgene being induced by certain environmental conditions, or by the transgenic plant producing inactive proteins that are only activated after harvest and purification. Such systems reduce potential environmental damage or contamination by the transgene product, but they do not affect the persistence or survival of the transgene in the wider environment e.g. through its spread via pollen or seed [248].

2.548 Menassa et al. (2001) [248] have investigated this by producing transgenic tobacco plants expressing the gene encoding human interleukin-10 (phIL-10). Interleukin phIL-10 is a cytokine with the potential to treat inflammatory bowel disease and autoimmune diseases. The gene constructs were produced using the cDNA for phIL-10, with three different constructs being generated.

2.549 Homozygous transgenic plants were then crossed with low-alkaloid tobacco plants. The resultant plants expressed the transgene and were male sterile. The plants were harvested prior to flowering. The combination of the harvest time and the male sterility is reported to ensure the containment of the transgene [248]. In small scale field trials, the overall phenotype of hybrids was not discernible from either the homozygous transgenic or the non-transgenic control; indicating that neither male sterility nor the transgene product had an effect on growth or vigour of the plant. Accumulation of phIL-10 was similar for the hybrids (721-996 mg ha-1) and the homozygous transgenics (780 mg ha-1) [248]. Further investigation in this area is reported to involve the efficacy of phIL-10 in an animal model of Crohn�s disease, although no results have been published to date [248].


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PRODUCTION OF SPECIALITY COMPOUNDS FOR NON-MEDICAL APPLICATIONS

Use of Plants for the Production of Enzymes for Agronomic Applications Production of glucanase Production of phytase Modification of pest resistance

Use of Plants for the Production of Polyhydroxyalkonates (PHAs) Biosynthesis of PHB in plant cytoplasm Biosynthesis of PHB in the chloroplast Biosynthesis of PHB in the leucoplast Biosynthesis of PHBV in plants

Use of Plants for the Production of Spider Silk Protein Use of Plants for the Production of Industrial Enzymes

Cellulase Xylanase Production of beer and spirits

Use of Plants for the Production of Collagen Use of Plants for the Production of Squalene Production of Trehalose as a Stabilising Agent Use of Plants for the Production of Biofuels

2.550 The purpose of this section of the report is to review the reported application of GM

plants to produce speciality compounds for non-medical applications. As with the previous section, the modifications are concerned largely with the production of non-plant materials. These are:

• the production of enzymes for agronomic applications, such as glucanase and phytase;

• the production of collagen;

• the production of polyhydroxyalkanoates (PHAs) for the synthesis of biodegradable plastics;

• the production of spider silk proteins;

• the production of enzymes for industrial processing (cellulase and xylanase), and enzymes produced for the production of beer and spirits;

• squalene (used in the cosmetics industry and sourced traditionally from shark�s liver);

• the production of trehalose as a stabilising agent; and


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• the production of biofuels.

2.551 Plant systems have been shown to be able to express xenogenic proteins with a wide range of conformations and molecular weights, including some proteins that have not been expressed successfully using other systems. Plants also have many advantages as protein producers including low cost of production, stability of the protein products in storage tissues, (such as seeds), the ease and speed of scale-up and the possibility of direct addition of plant material to industrial processes [213].

Use of Plants for the Production of Compounds with Agronomic Applications

Production of glucanase

2.552 Armstrong et al. (2002) [284] looked at the possibility of using plant systems for the large-scale, cost-efficient production of heterologous protein products, such as the enzymes used in the livestock industry. A specific part of the study investigated the use of the potato as an expression system for the enzyme 1,3-1,4-β-glucanase. This enzyme catalyses the hydrolysis of mixed linkage glucans that are a dominant component of endosperm walls in oats (Avena sativa) and barley (Hordeum vulgare). Poultry lack the ability to produce 1,3-1,4-β-glucanase and the provision of supplements of this enzyme in barley diets have been shown to reduce digestive problems in poultry. However, currently the use of such enzyme supplements is prohibitive in terms of cost and so the development of GM potatoes expressing high levels of 1,3-1,4-β-glucanase was investigated to determine whether such a system could potentially provide a cheaper source of the enzyme [284].

2.553 The 1,3-1,4-β-glucanase mature peptide coding region, was inserted in the sense orientation relative to a constitutive promoter (CaMV 35S). The β-glucanase cDNA was altered slightly to allow for easier translation by the plant. The terminator sequence used was nopaline synthase (NOS) terminator. Agrobacterium tumefaciens mediated transformation was used to produce the transgenic plants [284].

2.554 Analysis of the transgenic potatoes detected recombinant protein in both leaf and tuber tissue at a concentration that would result in the cost effective production of this enzyme at the levels required by poultry in their feed. However, it was found that although high levels of 1,3-1,4-β-glucanase were expressed, tuber yields in the GM potatoes were substantially reduced compared with the non-transgenic control. Furthermore, microscopic observations of the transformed plants revealed defects in cell wall morphology within the transgenic potato lines, with the greatest effects detected in those plants exhibiting the highest levels of expression [284].


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2.555 The substrate of 1,3-1,4-β-glucanase consists of mixed linkage glucans that are typically present in type I cell walls of grasses. Type II cell walls that are characteristic of most dicotyledonous plants, including potato, do not contain such mixed linkage glucans. Therefore the changes in the cell wall morphology of the GM potato was suggested to be due to either the presence of type I links in its cell wall, or that the 1,3-1,4-β-glucanase may affect other aspects of cell wall formation [284]. Such changes may have implications for the persistence and survival of the GM potato in the environment, in that defects in cell wall morphology could adversely affect cell structure and function and therefore plant survival as a whole.

2.556 Armstrong et al. (2002) [284] suggested that alternative promoters could be used so that the 1,3-1,4-β-glucanase was only expressed in certain parts of the plant, or a promoter could be used that is not active under field conditions while the plant is growing, but becomes active during storage. The inactive 1,3-1,4-β-glucanase genes would be expected to have less impact during the growing season. After harvest, an inducible promoter could then be activated and β-glucanase would subsequently accumulate in tubers. Cold-inducible promoters are one example that could be used. These promoters have been proved to stimulate high levels of expression of heterologous gene products within days of exposure to cold temperatures [284, 213]. The GM potatoes could however have reduced persistence and survival in the wild, compared with the wild-type in areas where frost could affect the plants, otherwise the gene would not give the plant any obvious selective advantage or disadvantage over the wild-type, if the inactive 1,3-1,4β-glucanase genes did not have any affect on the growth and/or development of such GM plants.

2.557 These GM plants may be subject to field trials in order to establish the effect of the modified compositional trait on yield and to see if this method of producing agronomic enzyme would be cost effective.

Production of phytase

2.558 As mentioned in the Modified Micronutrient chapter, the alteration of phytase activity offers a strategy to improve the bioavailability of iron and other minerals for non-ruminant animals (pigs, poultry, humans for example). The basis of the approach is that in cereal grains, the compound phytic acid which contains most of the phosphate present in the grains, also readily forms complexes with a range of minerals (including iron, zinc and calcium) [198]. Non-ruminant animals are unable to digest phytic acid as they have negligible phytase activity. Consequently the phytic acid is excreted from the animals along with the complexed minerals [198].


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2.559 Strategies to minimise the problem with phytic acid include:

• the development of crop cultivars, through mutagensis, that have a reduced level of phytic acid. This strategy has been successful with diploid crops such as maize, barley and rice [198]. However because of the additional role of phytic acid as a plant protection mechanism against oxidative damage during storage and from moulds [92], then such changes may have implications to the persistence or survival of such plants in the environment;

• the addition of microbially-derived phytase to animal feed. This approach is already used in animal husbandry (particularly poultry and pigs)46 to improve the uptake of phosphorus and minerals from feed [285-287]. The advantages of improved phosphorus uptake are twofold, with improvements in the growth rate and feeding efficiency of the animals, as well as reduced levels of phosphorus and other nutrients in the manure (thereby reducing the adverse environmental effects of poultry and pig manure47) [288, 286];

• the genetic modification of animals to produce phytase themselves. Pigs for example have been modified to produce phytase in their saliva, thereby negating a requirement for phytase supplements in their diet [289]; and

• the genetic modification of plants used in animal feeds to produce phytase, as an alternative to the use of microbially-derived supplements [200, 198].

2.560 GM tobacco, soybean, oilseed rape, alfalfa and wheat have been developed to date (2002) which express the phytase gene (phyA) from Aspergillus niger [285, 290, 198]. As with other applications discussed in this report, where plants are used as alternatives for the production of microbially-derived compounds, plant-based systems offer a cheaper product [291]. The high cost of existing sources of supplemental phytase is reported to limit its use in the poultry industry [287].

2.561 The use of GM plants to produce phytase has been described as probably the most advanced example (in terms of commercial application) of a project aimed at improving animal feed diets [27]. Initial studies involved the modification of tobacco to express phytase from A. niger [288]. This construct uses the same gene to express the phytase as in the fungal Natuphos® system [286], and despite differences in glycosylation, the specific activities of the tobacco and fungal phytase are identical [292, 293]. The phytase is stably accumulated in the leaves of the transgenic tobacco during plant growth [292]. However, as tobacco is not a normal

46 Marketed commercially as Natuphos® for example. 47 Phosphorus is described as the single most important pollutant in pig manure.


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constituent of poultry or pig diets, the system has since been applied to soybean [285], oilseed rape (trade name Phytaseed®) [286, 294] and wheat [290, 198].

2.562 One limitation with the system is that phytases from A. niger are inactivated at temperatures >60°C and are therefore not suitable for transgenic cereals destined for cooking or other high temperature processing [198]. A phytase from Aspergillus fumigatus is able to sustain boiling at 100 °C for 20 min (Wyss et al., 1998; cited by [198]). Lucca et al. (2001) reported the development of a transgenic rice modified to express a phytase from A. fumigatus [200]. Although the GM rice retained some phytase activity after cooking, the enzyme was found to be less heat stabile than the fungal-derived version. The reason for this was not reported. The phytase from A. fumigatus is known to undergo denaturation and inactivation during heating, but is able to refold into an active form when the temperature is reduced [198]. It has been proposed that differences in the cellular environment of the rice endosperm compared to the fungus accounts for the poor heat stability of the plant-derived version of the enzyme. Other microbial phytases may therefore be required for the strategy to be applied to GM phytase-producing plants that undergo some sort of high temperature processing [198].

2.563 No information is available on the effect that phytase production has on the persistence and survival of the GM plants. With respect to the future development of transgenic phytase plants, such plants have already undergone field trials and the resulting material marketed as a commercial dietary supplement for monogastric livestock. The advantages of plant-derived phytase in terms of its low cost, improved returns on livestock and reduced environmental impact of manure mean that GM phytase crops are expected to be increasingly used as alternatives to existing phytase supplements.

Modification of pest resistance

2.564 The modification of plants to express various compounds to improve their resistance to agricultural pests (insects and other parasites, fungi, bacteria and viruses) is a key area in which GM technology has been applied to agriculture. Plants modified for improved pest resistance do of course have an altered composition as they are modified to express particular proteins or other compounds that have an adverse effect on a particular pest or group of pests (insects, nematodes, fungi, bacteria and viruses for example).

2.565 However, under the definition of �input� and �output� traits discussed in the Introduction section to this report, modifications to pest resistance fit better in the group of modified �input� traits as the primary purpose of the modifications are to reduce the susceptibility of the plants to pests and thereby reduce the inputs of pesticides and/or manual labour necessary to protect equivalent non-GM varieties


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from the same pests. For this reason, traits designed to modify the susceptibility of plants to pests, such as the expression of the insecticidal Bt toxin48, are not reviewed in detail in this report.

2.566 Some discussion on modified pest resistance is included because of the obvious selective advantage offered by such modifications and the subsequent effects that advantage may have on the persistence and/or survival of the transgenic plant in the environment, compared with the wild-type. Field studies with sugar beet modified for improved resistance to beet necrotic yellow vein virus for example found that the GM beet had a small ecological advantage over the non-GM (and consequently non-resistant) varieties. However, this advantage was not transferred to wild beet populations and should therefore not have an effect on persistence and survival of beet outside the field environment [295].

2.567 In addition to the occurrence of direct advantages of improved pest resistance, a number of secondary more indirect effects have also been observed.

2.568 In Bt-modified GM maize it has been noted that maize expressing the Cry 1 Ab protein also has a higher lignin content (33-97 percent higher) than the non-Bt isolines [296]. It was speculated by the authors that these significant alterations in lignin content may have ecological implications for this crop. Such plants may not be suitable for field cultivation, if such ecological effects are found to be extremely adverse (e.g. plant material containing Bt toxin becoming recalcitrant to decay due to the high lignin content). Lignin modification as a target effect of GM plants is discussed in more detail in a separate section, along with the potential effects this may have on persistence and survival.

2.569 This study appears to be the only one that specifically looks at the effect of modification for pesticide resistence on the secondary effects of the modification on the persistence and/or survival of the plant.

2.570 Some recent work has looked at the manipulation of naturally occurring plant-based insecticides using GM technology [297] This would involve the perturbation of the levels of these natural insecticides within the plant, using GM technology, in cell culture or in the field. The production of such natural insecticides is seen as desirable as such pesticides do not have unwanted effects on non-target mammals, but are very effective against target insects [297].

48 Transgenic plants, including cotton, maize and soybean expressing this toxin are currently under cultivation in a number of countries worldwide. The toxin is produced following the expression of the cry group of genes that were isolated from the soil bacterium Bacillus thuringiensis.


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2.571 This application of GM technology is in the early days of development as the mechanism for the biosynthesis of these naturally occurring plant insecticides (e.g. pyrethrin) are not known precisely [297] However, recently an enzyme and part of its cDNA have been identified and isolated from the pyrethrin biosynthetic pathway in chrysanthemum. It is unclear whether the genetic manipulation of the biosynthesis of plant insecticides � through the up-regulation of the enzymes involved in their biosynthesis would have any adverse effects on plant persistence and survival.

Use of Plants for the Production of Polyhydroxyalkonates (PHAs)

2.572 Polyhydroxyalkonates (PHAs) are polyesters of hydroxyacids that are synthesised naturally by a wide variety of bacteria for use as carbon reserves or electron sinks [298]. Because these compounds are used by microorganisms as a carbon source they may be expected to be biodegradable in the environment [216]. Interest in these compounds is a consequence of them having thermoplastic and elastomeric properties similar to existing plastic polymers, particularly polypropylene [299], and that they may therefore have applications as biodegradable, renewable and environmentally-friendly plastics [298].

2.573 To date over one hundred different PHA monomers have been identified in bacteria, and the metabolic pathways involved in the synthesis of the polymers have been determined in part [298]. The chemical diversity of the monomers identified means that PHA-based plastics might be designed for use as stiff or soft plastics, as elastomers, rubbers and glues [298].

2.574 The use of transgenic plants to produce PHA-based plastics offers a much cheaper alternative to producing these compounds by bacterial fermentation, although for the production of PHA by plants to be viable commercially, the PHA must be synthesised in plants to a level of approximately 15-40 percent dry weight. At this level, production costs are reported to be similar to those of vegetable oil and would be competitive with petroleum-derived plastics [300].

2.575 In addition to PHA, two other PHA-type polymers, polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV)49, have been produced to date in transgenic plants. The PHA polymers that have been produced are described as MCL-PHA (medium chain length PHAs) and contain C6-C16 chain length monomers [298]. Modification of plants to produce MCL-PHAs has been achieved through the addition of the phaC1 gene from Pseudomonas aeruginosa, although levels of only 0.4 percent (dry weight) have been achieved to date [216, 298].

49 Trade name BiopolTM.


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2.576 The synthesis of PHB and PHBV is similar, differing only in the initial step of the biosynthetic pathway [216]. Both polymers are synthesised naturally from acetyl-CoA by bacteria. Because this precursor is present in plants in the cytoplasm, plastid, mitochondrian and peroxisome then it has been proposed that both polymers could be produced in any of these sub-cellular compartments in the modified plant [298]. Initial studies focused on the formation of PHB.

Biosynthesis of PHB in plant cytoplasm

2.577 The advantage of using the plant�s cytoplasm as the site for production of PHB is that the bacterial enzymes can be expressed in this compartment without any modification of the proteins [298]. Production of PHB in plants requires the addition of two bacterial enzymes, the acetoacetyl-CoA reductase and the PHA synthase (encoded respectively by the bacterial genes phaB and phaC50). The modification of Arabidopsis with both of these genes resulted in the formation of PHB in the cytoplasm at a level of 0.1 percent (dry weight) [298]. A similar modification of Brassica napus resulted in the accumulation of 0.02 � 0.1 percent PHB, although the expression of phaA51, phaB and phaC in cotton achieved a PHB level of 0.3 percent [299].

2.578 The modification of cotton to express the pha genes was performed for different reasons than the Arabidopsis and Brassica. Modification of cotton to produce PHB has been found to improve the thermal characteristics of the cotton fibres and result in better insulating properties [299].

2.579 The modification of Arabidopsis and Brassica sp. with the pha genes was found to have a significant effect on growth and size of the modified plants [298]. Plants expressing high levels of acetoacetyl-CoA reductase were approximately five times smaller than wild-type strains, and further growth reductions were reported for plants expressing both the acetoacetyl-CoA reductase and the PHB synthase enzymes (although expression of just PHB synthase had no effect on growth). Similar effects on seed production by the modified plants have also been reported [298].

2.580 Although the reason for the adverse effect on plant growth is not known, it has been proposed that because the transgenes are using the plant�s acetyl-CoA and acetoacetyl-CoA, then the reservoirs of these compounds necessary for the plant�s isoprenoid and flavonoid pathways may be depleted with consequent effects on these endogenous pathways [298]. Because the isoprenoid pathway is itself a precursor for the production of plant hormones such as cytokinins and gibberellins

50 Isolated from the soil bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus). 51 Encodes a 3-ketothiolase that catalyses the reversible condensation reaction of acetyl-CoA to acetoacetyl-CoA.


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[298] then it is likely that any affect on the isoprenoid pathway will alter the growth of the plant, and consequently its survival in the environment.

Biosynthesis of PHB in the chloroplast

2.581 Because the chloroplast has a higher flux through of acetyl-CoA than the cytoplasm, then it was proposed that by locating the biosynthesis of PHB in the plastid then the problems encountered with limited supplies of acetyl-CoA (and the subsequent knock-on effects on plant growth) could be avoided [298]. Biosynthesis of PHB in the plastid is also reported not to affect other sub-cellular structures as the PHB is retained within the plastid [298].

2.582 Modification of plants to produce PHB in the chloroplast requires the insertion of the phaA, phaB and phaC genes from Ralstonia eutropha. The chloroplasts do not have an endogenous 3-ketothiolase capability and therefore require the phaA gene to allow PHB synthesis [301].

2.583 The expression of these genes in transgenic Arabidopsis resulted in the formation of PHB in the chloroplasts, with a maximum concentration of 14 percent (dry weight) (equivalent to 10 mg g-1 fresh weight). The level of PHB in the leaves was found to be correlated positively with the age of the leaf, with the highest concentrations being recorded in pre-senescent leaves. No adverse effects on plant growth were identified, but some leaf chlorosis did occur in leaves containing PHB at levels >3 mg g-1 (fresh weight) [301]. The results obtained with the transgenic Arabidopsis suggested that although biosynthesis of the PHB in the plastid had no direct effect on plant growth, some chloroplast function was impaired [301]. Adverse effects to chloroplast function may be expected to have a knock-on effect on the survival of the plant in the environment.

2.584 Further studies using a single vector approach in which all three of the pha genes are inserted into the recipient plant on a single vector, achieved PHB levels in the transgenic Arabidopsis of between 3 � 40 percent (dry weight) [301]. However, although the previous studies reported no adverse effects on plant growth, the transgenic lines that accumulated >30 percent PHB were dwarfed and also produced no seeds. All transgenic lines producing >3 percent PHB showed some leaf chlorosis [301].

2.585 Studies on the effects of PHB accumulation in the chloroplast on other plant biochemical pathways found no effect on fatty acid synthesis, a negative effect on levels of isocitrate and fumarate and a positive effect on concentrations of the sugars mannitol, glucose, fructose and sucrose (Bohmert et al., 2000; cited by [298]). In combination these effects are reported to indicate that a high level of biosynthesis of PHB in the chloroplast has a negative effect on plant metabolism [298], and


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consequently on the persistence and survival of the modified plant in the environment.

2.586 Maize and alfalfa has also been modified with the pha genes to produce PHB in the chloroplasts in the leaves and stalk [298, 302]. Similar findings were reported to the Arabidopsis work with PHB levels increasing with the age of the plant tissue, and chlorosis occurring in tissue containing higher quantities of PHB. However, in contrast to the modified Arabidopsis lines, the maximum PHB levels achieved in the transgenic maize were only 5.7 percent (dw).

Biosynthesis of PHB in the leucoplast

2.587 The limitations described for the production of PHB in the chloroplasts and cytoplasm of green tissue, have led to studies investigating the biosynthesis of PHB in the leucoplast of seeds of an oil crop such as oilseed rape [298].

2.588 The modification of Brassica napus with the phaA, phaB and phaC genes, under the control of a fatty acid hydroxylase promoter from Lesquerella fendeleri, resulted in strong expression in the developing seed. PHB levels of 7.7 percent (fresh weight) were reported in the modified plants.

2.589 The seeds from the modified plants showed no differences in terms of appearance or germination rate compared with the non-modified seeds. However, as studies to increase the PHB content beyond eight percent were not conducted, it is not known whether higher levels of PHB production would have an affect on the seed�s characteristics.

Biosynthesis of PHBV in plants

2.590 PHBV offers better thermoplastic and elastomeric properties than PHB [216], specifically greater flexibility and reduced brittleness [298]. The more attractive properties of PHBV also mean that it is more commercially valuable than PHB, and therefore production levels in plants may not need to be as high to make the crop commercially viable. The biosynthetic pathway for PHBV is illustrated in Figure 2.9.

2.591 Biosynthesis of PHBV is similar to that for PHB except for the first step which requires the addition of the ilvA gene from E. coli which encodes a threonine deaminase and consequently boosts the availability of 2-ketobutyrate. The 2-ketobutyrate is converted to propionyl-CoA by a plant�s endogenous pyruvate dehydrogenase complex (PDC).


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2.592 Modification of Arabidopsis with the ilvA, phaB, phaC and btkB (encodes a novel 3-thiolase with high affinity for both acetyl-CoA and propionyl-CoA) genes under the control of a constitutive promoter resulted in the accumulation of PHBV in the plastid at levels of 0.1-1.6 percent (dw).

2.593 The modification of B. napus with the same genes, but under the control of a seed-specific promoter achieved PHBV levels of 0.7-2.3 percent (dw) in the leucoplast. No adverse effects on other metabolic pathways in either of the transgenic plants were reported [298].

Figure 2.9 - Biosynthetic pathway for polyhydroxybutyrate-co-hydroxyvalerate (PHBV) in transgenic plants (adapted from Poirier, 2002 [298]).

threonine

ilvA aminotransferase

2-aminobutyrate 2-ketobutyrate

ketoglutarate glutamate isoleucinePDC

acetyl-CoA propionyl-CoA

btkBphaBphaC

PHBV

2.594 The use of plants for the biosynthesis of polyhydroxyalkonates is currently in the early stages of development. Work to date has focused on the development of the system in Arabidopsis, although some studies have been reported in oilseed rape. The major limitation with approaches to date has been the level of production of PHAs achieved in the GM plants. This level needs to reach 15-40 percent dry weight for the system to be viable commercially. However, if such a figure can be achieved in crop plants then commercialisation may be expected. From the information presented it appears that transgenic plants where the modification is directed at the leucoplast may offer the most successful approach in developing this technology.


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Use of Plants for the Production of Spider Silk Protein

2.595 Spider silk proteins (also known as spidroins) consist largely of glycine and alanine. Silks are secreted from specialised glands by insects and spiders and once outside the glands, are processed into a fibre. Spider silks are of particular interest as they combine strength, toughness and enough elasticity to avoid breaking when flying insects are caught on the web.

2.596 Silk proteins and their genes isolated from native silkworm moth Bombyx mori exhibit variability in size, which have been put down to recombination events. If spidroins are to be grown in yeast or bacteria, a vast amount of these amino acids must be provided in the growth medium for their synthesis. Also, in bacterial production, genetic instability from recombination can become a problem due to the large proportion of repetitive genes encoding the spidroins. For these reasons, plants have been proposed for the production of synthetic spidroins. In several cases where plants have been used to produce transgenic products, stable accumulation of functional proteins at high levels has occurred by retention at the endoplasmic reticulum (ER). This method of accumulation was employed for spidroins in tobacco and potato.

2.597 Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. For these constructs, the repetitive units were patterned using model sequences identified from native dragline silk. The sequences were reverse-translated to DNA sequences with a codon selected to maximise expression levels in E. coli. These fragments were used to build upon, to form larger multimers by using a �head-to-tail� construction strategy. This meant that two enzymes of identical cohesive ends were self condensed to form large concatemers, which were subsequently digested by those same enzymes. Multimers of a modest size (4-6 repeats) were inserted into plasmids containing other multimers and all sized variants were generated.

2.598 Two particular cDNA sequences were reported in N. clavipes dragline silk, namely spidroin I and spidroin II. Much of both of these identified sequences are repetitive in nature.

2.599 From 18 oligodeoxyribonucleotides, six short gene fragments were created. These fragments were then used to construct synthetic spidroin genes that matched the natural spidroin sequences to a considerable extent. These units were inserted into a plasmid and cloned in E. coli. Fragments were digested with specific restriction endonucleases and then used in a series of ligation steps to construct gradually larger segments of the synthetic spidroin1 gene. Finally, a 1.8kb cDNA synthetic homologue of the N. clavipes spidroin was generated, lacking the non-repetitive part at the 3� terminus.


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2.600 A synthetic homologue of a segment of the part of a gene in B. mori was created by a similar method. Combined with spider silk, it would be possible to investigate the feasibility of engineering silks with new properties.

2.601 Spider silk genes were cloned in an expression vector and transferred into plants under the control of CaMV 35S promoter. A signal peptide at the N terminus of the spider silk protein, together with the KDEL signal at the C terminus, provide ER retention of the transgenic proteins in plant cells. So that the silk protein could be detected, a tag was fused to the C terminus of the protein for western blot analysis.

2.602 High amounts of recombinant spider silk protein accumulated in 31-69 percent of the transformed plants (potato and tobacco). All transgenic plants showed normal growth and morphology (Scheller et al. 2001), demonstrating the feasibility of the method. The amounts of spider silk protein accumulated in the plant were determined by comparing defined standards of antibody molecules carrying the a c-myc tag with the c-myc tag amounts in the plant tissues.

2.603 The modification of plants such as potato and tobacco to produce spider silk protein at relatively high levels suggests that this technology is ready for commercial development.

Use of Plants for the Production of Industrial Enzymes

Cellulase

2.604 Plants have been modified for the production of cellulase for two reasons:

• as a cheaper alternative to microbial fermentation for use in subsequent industrial processes; and

• for post-harvest degradation of the plant�s own cellulose to produce fermentable sugars.

2.605 Arabidopsis has been modified to express a thermostable bacterial cellulase (endo-1,4-β-D-glucanase) at levels up to 25 percent of total soluble leaf protein. Because the enzyme is only activated at greater than ambient temperatures (optimum of 81 °C), then expression of the enzyme in the transgenic plant has no detrimental effect on the plant�s own cellulose. The application of this approach to crop plants is viewed as offering a cost effective approach for producing a thermostable cellulase for use in industrial processes.


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2.606 Plants have also been modified to express a cellulase for use post-harvest in the degradation of the plant�s own cellulose [303]. The objective of the modification is that post-harvest processing releases the novel cellulase from the plant, which then degrades the plant�s cellulose to produce fermentable sugars. The work described by US Patent Application 20020138878 [303] includes the modification of a diverse range of plants including maize, wheat, barley, rye, hops, hemp, rice, potato, soybean, sorghum, sugarcane, clover, tobacco, alfalfa, a coniferous tree and a deciduous tree (both trees not specified). The cellulase enzymes are microbial in origin and have been inserted under the control of a leaf-specific promoter.

2.607 The US Patent Application 20020138878 [303] also included the modification of the plants to express a ligninase from the white rot fungus Phanerochaete chrysosporium that was also designed to degrade the plant�s lignin post-harvest.

2.608 The modification of crop plants for post-harvest degradation of cellulose and lignin offers potential benefits to a range of industrial processes, including the biofuel industry (production of fermentable sugars for the manufacture of ethanol), and also as a means to reduce processing costs for the pulp and paper industry. These modifications are therefore viewed as having future development potential.

Xylanase

2.609 Another example of a cell-wall degrading enzyme produced in transgenic plants is a fungal xylanase. The gene encoding this enzyme was isolated from the yeast Cryptococcus albidus and inserted into tobacco plants by A. tumefaciens mediated transfer under the transcriptional control the CaMV 35S promoter.

2.610 This enzyme was also produced in Canola by Lui et al. (1997) and was fused to an oleosin protein so that it would be immobilised on oil bodies. This way, the oil system allowed for the recycling of the enzyme for re-use. The enzyme produced was similar in its activity to the one produced in E. coli. However, the expression levels in the transgenic plants were too low (300-2000 units/kg seed) for production to be commercially viable [304].

2.611 Xylanase is being produced in large quantities in maize by Prodigene and will probably be commercialised soon [305]. The system reported by Hood and Jilka (1999) [304] is viewed as not suitable for commercialisation due to the low levels of xylanase produced.

Production of beer and spirits

2.612 The application of GM technology to the production of beer and spirits involves the development of cereal grains containing the enzymes necessary for the beer


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production process. This application is reported to offer the potential to make classic malt beer but with a minimum use of malt [306].

2.613 In conventional beer and whisky manufacture, malt is used to impart flavour and colour of the drink and also to provide a source of fermentable sugar and enzymes for the brewing process. However the enzyme level in malt is variable and can therefore provide an inconsistent or unpredictable result to the brewing process. High flavour and colour requirements may also be deleterious for enzyme activity of the malt [307].

2.614 Enzyme supplements are reported to be used by the brewing industry to improve the consistency of their products and reduce the unpredictability of the brewing process. Such supplements are currently obtained from microorganisms produced in biofermentation systems. As discussed the production of microbial enzymes in fermenters, even on an industrial scale, is relatively expensive. The US Patent Application (20020164399) [306] describes the development of transgenic plants to express the enzymes required, as a cheaper alternative to production by microbial fermentation.

2.615 Modified plants described in the patent application include barley, corn, rice, wheat, sorghum, millet, oats and cassava [306].

2.616 The work presented in US Patent Application (20020164399) [306] was developed with a commercial application in mind. Therefore, if the reported modifications are successful and can offer a cheaper alternative to existing microbially produced enzyme supplements, then it is likely that such transgenic crops may be grown commercially. However, the adoption of GMOs by some of the more �traditional� parts of the brewing industry may have a bearing on the future use of this technology. Other areas of potential commercial development for GMOs with food or drink applications include decaffeinated coffee where the coffee plant is genetically modified for reduced caffeine levels, rather than the caffeine being removed chemically post-harvest, and introduction of protein sweeteners.

Use of Plants for the Production of Collagen

2.617 Collagen is an animal protein representing 30 percent of total body protein in mammals [308]. It is used directly in medical, cosmetic and therapeutic applications, and also as gelatine (denatured collagen) in food [308]. Collagen is currently produced following purification from animal sources, and its extraction from potentially infected animals has raised concerns (of unquantifiable proportion) [309] of risks from viral or bacterial agents, and from new variant Creutzfeldt-Jakob Disease (nvCJD) [308]. The expression of the protein in plants would negate this risk [309].


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2.618 Fibrillar collagen (the most abundant collagen in mammals) has to date (2002) been expressed in mammalian, insect and yeast cells [308], and human collagen has been produced in transgenic tobacco plants [308]. The use of plants to produce this protein not only negates the potential risk posed by purification from animal sources, but may also offer an alternative more cost-effective source for the large scale production of this protein [308].

2.619 The tobacco plants were modified with cDNA encoding the human proα1(I) chain to enable them to express collagen I. The spontaneous processing of the precursor collagen (procollagen) to mature collagen by the plant avoids the use of pepsin to cleave the N- and C-terminal extensions which generally persist in recombinant collagen molecules produced by other expression systems [308]. Therefore, the use of plants to produce collagen offers a potentially more efficient system than other expression systems, including transgenic animals [308].

2.620 Although tobacco has been used in this case as an experimental system for the production of collagen, the success of the process in producing mature collagen, means that further developments in this field should be expected. No information is available on the potential of this modification to alter the persistence or survival of the transgenic tobacco. However, because the plants are altered to produce a xenogenic (non plant) protein then it is unlikely that the modification would have an affect on other plant processes, and consequently on its behaviour in the environment.

Use of Plants for the Production of Squalene

2.621 Squalene is used widely in the cosmetics industry (market value of $125 million)(Kaiya, 1990; cited by [310]). Although this compound occurs in small quantities in vegetable oils it is sourced primarily from shark�s liver. Interest in the use of plants as a larger source of squalene is due to the declining supply of shark liver and the requirement to use a more environmentally friendly means of production.

2.622 Current levels of squalene in vegetable oils are in the 0.1-3 percent range. In order for plants to offer a commercially viable source of squalene then levels in excess of five percent are required [310].

2.623 Studies with plant cell cultures have shown that squalene is naturally converted to the epoxide (and ultimately plant sterols) by the action of squalene epoxidase. In the presence of inhibitors of squalene epoxidase, e.g. allylamines such as terbinafine, degradation is blocked and squalene levels increase [311, 310].


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2.624 Genes encoding squalene epoxidase have been identified in Arabidopsis and Brassica napus. Down-regulation of the genes encoding the epoxidase was achieved through antisense expression or ribozyme technology (reduction of mRNA levels) and achieved squalene levels of ≤12.38 µg g-1 (dw) [310].

2.625 As a consequence of the market value for squalene and the fact that squalene derived from GM plants could be marketed as a more environmentally friendly alternative to that obtained from shark�s liver, then the commercial prospects of squalene producing GM plants are viewed as very promising. Although the technology is in the early stages of development, some success has been reported (and patented) for the expression of squalene in Brassica napus. The use of such a crop is viewed as ideal for the production of squalene in plants at an agronomic scale.

Production of Trehalose as a Stabilising Agent

2.626 The sugar trehalose is reported to have a possible commercial application as a stabilising agent in pharmaceuticals and other products [312]. Whilst information on the potential size of market involved is limited, the importance of trehalose as an osmoprotectant compound conferring increased tolerance to drought, means that any plants designed to express improved levels of this compound may be expected to have a selective advantage in the environment, especially in areas with low rainfall.

2.627 The modification of plants to express trehalose is discussed in more detail in the following chapter �Modified Plant Growth�. Developments in this field are however still in their infancy.

Use of Plants for the Production of Biofuels

2.628 Plant biomass is already used widely for the production of biofuels (usually ethanol) [313]. Estimates in 2001 indicated that 1.8 billion gallons of fuel ethanol are produced annually in the USA [313]. Much of the ethanol currently produced is obtained through the fermentation of sugar derived from maize starch, although there is increased interest in the use of lignocellulosic feedstocks such as softwoods and hardwoods. The limitation of these materials is that because it is cellulose rather than starch that is being used as the starting material then cellulase enzymes are required to degrade the cellulose. It is the cost of the cellulase enzymes that is one of the factors limiting the use of lignocellulosic feedstocks for the production of biofuels [313].

2.629 The developments reported in the US Patent Application (20020138878) [303] in which plants have been modified to express cellulase and ligninase to degrade plant


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cellulose and lignin, post-harvest, may offer significant benefits to the biofuel industry.

2.630 Although the development of GM plants for biofuel production has not been identified, plants modified for altered cellulose and/or lignin content (see relevant chapters) may have applications in this field. If these plants are suitable then the commercial �drivers� for the development of biofuels as cheaper (and renewable) alternatives to petroleum-based fuels should see this technology developed further.


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MODIFIED PLANT GROWTH

Introduction Modified tolerance to drought stress

Improved drought tolerance using osmoprotectants Osmoprotectants � modification of glycine betaine production Osmoprotectants � modification of trehalose production Osmoprotectants � modification of fructan production Osmoprotectants � modification of D-ononitol production

Modified tolerance to salt stress Improving salt tolerance through detoxification Improving salt tolerance through homeostasis Improving salt tolerance through growth regulation

Modified tolerance to cold stress CBF pathway Modification of cold-induced genes LEA (late-embryogenesis abundant) proteins Plant antifreeze proteins

2.631 The purpose of this section is to review the modifications designed to alter a plant�s ability to respond to the stresses imposed by reduced water availability, increased salt levels and lower temperatures. Whilst some of the modifications reviewed in previous sections of this report have been found to alter the ability of the plant to respond to abiotic stresses, these changes are an indirect effect of the modification. Examples include the modification of plants for altered levels of RFOs (raffinose family of oligosaccharides) and trehalose (see Chapter �Modified Carbohydrate Content�), and various fatty acids (see Chapter �Modified Oil and Fat Content�).

2.632 The focus of this section is to review the modifications whose primary role is to alter the plant�s tolerance to the key abiotic stresses of water availability, salt content and cold or freezing temperature. In the environment these stresses have a significant effect on plant growth and subsequent yields [314, 315], and whilst the effects can be minimised through efficient crop (use of plant varieties with some degree of natural tolerance) and land management (good irrigation and drainage), the increasing demand for food and water resources means that in many areas of the world such stresses cannot be avoided. Whilst issues such as poor water availability are often associated with developing countries, many developed countries are also experiencing the adverse effects caused by a scarcity of water and high salt levels. Twenty-five percent of the United States for example is subject to drought, and globally 20 percent of cultivated land (and 50 percent of irrigated land) is affected by high salt levels [316, 312].

2.633 Because plants are sessile and poikilothermic52 [317, 318], they are unable (except through seed dispersal) to avoid exposure to adverse environmental conditions by

52 they assume the temperature of their environment


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moving to a different area, or in the case of temperature by regulating their internal temperature. Plants exposed to environmental stresses have therefore evolved to express a range of capabilities with which to address them. However, not all plants naturally possess or express these capabilities. Maize and tobacco for example do not possess a functional cold-acclimation pathway and are therefore unable to tolerate exposure to low or freezing temperatures [319]. This of course limits their cultivation to non-frost prone environments. The potential of GM technology in this area is therefore in the:

• adaptation of plants to express stress tolerance traits that they do not possess naturally; and

• improvement of the expression of existing stress tolerance traits so the plants can withstand greater levels of salt or water deficiency or freezing.

2.634 However, to date such potential has yet to be realised with most reported studies focusing on understanding the biochemical processes involved, and working with model plants such as Arabidopsis thaliana.

2.635 Changes to a plant�s tolerance to environmental stress will of course provide the plant with a significant selective advantage in environments where that stress is realised. Such a selective advantage is therefore likely to alter the ability of the modified plant to persist or survive in the environment. These changes are reviewed in Chapter 3 of this report.

2.636 Whilst this section of the report is structured according to the purpose of the altered trait, reviewing modifications for altered tolerance to drought, salt levels and temperature extremes in turn; the commonality of physiological effects caused means that many of the modifications affect the plant�s tolerance to more than one of the three environmental stresses. This is true particularly for modifications designed to limit the effects caused by dehydration as these confer improved tolerance to both drought and high salt levels [320, 316].


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Modified tolerance to drought stress

2.637 Drought stress is defined as the inadequacy of water availability (precipitation and soil-moisture storage capacity) that restricts the expression of the full genetic potential of the plant. Drought stress is a significant agricultural issue as it has the potential to limit the genetically determined maximum yield of crop plants [321]. Plants respond naturally to drought stress through three basic mechanisms [321]:

• drought escape � defined as the ability of the plant to complete its life cycle before serious soil and plant water deficits develop. Plants that exhibit drought escape strategies are therefore characterised by rapid phonological development (early flowering and early maturity), developmental plasticity (variation in growth patterns in response to water availability) and the remobilisation of preanthesis assimilates to grain;

• drought avoidance � defined as the ability of plants to maintain a relatively high tissue water potential through strategies such as increased rooting depth, increased hydraulic conductance, reduced epidermal conductance, reduced absorption of radiation (through leaf rolling or folding) and reduced foliar evaporation (through production of smaller leaves); and

• drought tolerance � defined as the ability of plants to respond to water-deficit by reducing the water potential of their tissues. This is achieved by varying the osmotic potential across the cell membrane through the accumulation of osmoprotectant compounds, as well as a reduction in cell size.

2.638 However, whilst some plants possess strategies to counter drought stress, the challenge to agriculture has been the development of crop plants to express drought tolerant traits without a concomitant reduction in yield. Drought escape, drought avoidance and drought tolerance strategies are all reported to cause some reduction in yield [321]. However, whilst the yield from a drought stressed plant with some natural tolerance to drought might be lower than from the same plant in a non-stressed environments, the yield is still likely to be higher than from plants unable to express any drought resistance strategies that are exposed to drought stress.

2.639 The application of genetic modification to improving the responses of crop plants to drought stress has focused on the drought tolerance strategy of increased accumulation of osmoprotectants. However, changes to any of the properties highlighted in the bullet points above (production of smaller leaves for example) that are brought about through genetic modification of the plant may have an effect on the tolerance of that plant to reduced water availability.


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Improved drought tolerance using osmoprotectants

2.640 Osmoprotectants are compounds produced naturally by many plants (and other organisms) in response to osmotic stress. They are all small, electrically neutral molecules that are non-toxic at molar concentrations. Osmoprotectants act by stabilising proteins and membranes against the denaturing effect(s) of high concentrations of salts and other harmful solutes that may occur during periods of low water availability or when the plant is exposed to high salt levels.

2.641 Whilst some plants are able to express significant quantities of osmoprotectants, many crop plants (including rice and tobacco) are unable to produce them [314, 312]. Genetic modification is particularly suitable for improving the production of these compounds as many osmoprotectants can be synthesised through the expression of single genes, and the action and beneficial effects conferred are not species-specific (due in part to their non-toxic nature). This enables osmoprotectants to be inserted into other un-related plants [322, 312].

2.642 At a chemical level osmoprotectants can be divided into three groups [314, 312]:

• betaines (fully N-methylated amino acid derivatives) and related compounds such as dimethylsulfoniopropionate (DMSP) and choline-O-sulfate. Betaines (particularly glycine betaine) occur widely in plants (e.g. spinach and sugar beet), whilst DMSP and choline-O-sulfate only occur rarely. Increased production of these compounds in planta does improve tolerance to a range of abiotic stresses;

• certain amino acids such as proline (produced widely in plants) and ectoine (found only in bacteria); and

• polyols and non-reducing sugars such as trehalose (occurs rarely in plants).

2.643 In plants that produce them naturally, osmoprotectants reach concentrations of between 5-50 µmol g-1 fresh weight (fw), with the highest concentrations occurring when the plant is exposed to the environmental stress [312].

2.644 To date (2002), whilst some of the genes and enzymes involved in the formation of osmoprotectants have been identified, the genetic modification of plants has focused on model plants (Arabidopsis and tobacco) with only limited modification of crop plants (just rice, oilseed rape and potato reported) (Table 2.4) [312]. The majority of the transgenic plants produced have been modified to express only a single gene under the control of a constitutive promoter. Whilst the level of osmoprotectant produced is lower than that occurring in non-transgenic plants that produce these


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compounds naturally, the transgenic plants did demonstrate improved stress tolerance (under laboratory conditions) relative to the non-transgenic controls [312]. The genetic modification of plants for altered osmoprotectant content has only been reported for a limited number of osmoprotectants (Table 2.4). Should other modifications be developed that alter the content of any compound with osmoprotectant capability may also be expected to enhance the tolerance of the plant to drought (and possibly high salt).

Table 2.4 � Examples of genetic modification to express osmoprotectants in higher plants (adapted from [312])

Osmoprotectant Host organism Enzyme or gene involved

Recipient organism

Product level (%) a

glycine betaine E. coli CDH b tobacco ND

E. coli CDH + BADH tobacco 1

Arthrobacter COX Arabidopsis 5

Arthrobacter COX rice 5-25

Arthrobacter COX canola 5-10

spinach CMO tobacco 1

spinach, beet CMO + BADH tobacco 1

spinach, beet CMO + BADH + PEAMT

tobacco cells 1-5

proline mothbean b P5CS tobacco 100-200

ectoine Halomonas ectA + ectB + ectC Arabidopsis 1

mannitol E. coli MtlD tobacco 16

sorbitol apple Stpd1 tobacco 1-260

D-ononitol ice plant c Imtl tobacco 10-70

trehalose E. coli TPS or TPS + TPP potato tuber 1-24

yeast TPS1 tobacco 5-19

yeast TPS1 tobacco 1 a defined as the level of osmoprotectant synthesised in osmotically stressed GM plants as a percentage of the level found in a representative organism that accumulates the osmoprotectant naturally under similar conditions. b mothbean (Vigna aconitifolia) is a drought hardy crop grown extensively in India. c Mesembryanthemum crystallinum CDH - choline dehydrogenase; COX � choline oxidase; BADH � betaine aldehyde dehydrogenase; CMO � choline monoxygenase;


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Osmoprotectants - modification of glycine betaine production

2.645 The modification of plants for increased production of glycine betaine (GlyBet) is significant as this compound confers enhanced tolerance to a range of abiotic stresses, including drought, chilling, salt, heat and strong light [314]. The importance of glycine betaine in enhancing tolerance to a range of abiotic stresses is reflected in a greater number of investigations being conducted with this osmoprotectant than any others [322].

2.646 The genes encoding the enzymes involved in the production of glycine betaine have been cloned [314]. Studies investigating the modification of plants to produce glycine betaine have reported that its formation in the chloroplast is severely limited by internal choline supply, with the import of choline into the chloroplast a major flux-controlling step [314, 312]. Increasing the conversion of choline (Cho) to betaine aldehyde (Bet Ald) has however been found to have little effect on improving the accumulation of glycine betaine. Expression of the gene for choline monooxygenase (CMO) from spinach in tobacco only achieved glycine betaine concentrations of 0.43 µmol g-1 fw, and levels were five-fold less when the gene was expressed in the chloroplast53 [314].

2.647 Improving the conversion of choline → betaine aldehyde → glycine betaine therefore offers limited benefit on its own to increasing glycine betaine levels in the plant. However, the addition of 5 mM choline to the growth medium achieved a 30-fold increase in glycine betaine levels [312], supporting reports that it is the availability of choline that offers the key mechanism to improve the production of glycine betaine.

2.648 In tobacco and other plants choline is produced mainly via a cytosolic pathway in which phospho-ethanolamine (P-EA) is methylated three times to form phosphocholine (P-Cho) from which choline is released (Figure 2.10). Whilst the pathway illustrated in Figure 2.10 exists naturally in both tobacco and spinach, levels of glycine betaine synthesis are significantly higher in spinach. This is due in part to the poor activity of the phosphoethanolamine N-methyltransferase (PEAMT) enzyme in tobacco, with activity only 1-3 percent of that in spinach (Nuccio et al. 1998; cited by [312]). Overexpression of spinach PEAMT in tobacco resulted in increased levels of free choline (50-fold) and glycine betaine (30-fold) in the transgenic plants. No changes in growth were reported.

53 Expression of the transgenes in the chloroplast rather than constitutively throughout the plant does though provide a biological containment barrier preventing transfer of the transgenes to other plants through pollen dispersal. Containment of the inserted genes is a significant issue for GM plants modified for improved tolerance to environmental stress, due to the strong selective advantages conferred by such traits.


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2.649 Increased production of ethanolamine (EA) should improve the flux to glycine betaine still further [312]. The recent cloning of serine decarboxylase (SDC) may provide the means to do this as serine is the main source of ethanolamine in plants (Rontein et al., 2001; cited by [312]).

Figure 2.10 - Synthesis of glycine betaine and its precursors in plants (adapted from Rontein et al., 2002) [312]. This pathway applies to almost all biological systems, although Arthrobacter sp. produce GlyBet through the N-methylation of glycine.

2.650 The highest levels of glycine betaine accumulated in transgenic Arabidopsis,

Brassica napus and tobacco (5 µmol g-1 fw) have though been achieved through a different system, involving the expression of a modified betA gene from E. coli in the plant�s mitachondria or the codA gene from Arthrobacter globiformis in their cytosol [314]. The modified plants exhibited improved tolerance to salinity and freezing as well as drought, although tolerance varied between the species [322].

2.651 Formation of glycine betaine through the expression of codA (and formation of choline oxidase (designated COX or COD)) from the bacterium Arthrobacter does though result in the creation of hydrogen peroxide which may have deleterious consequences in the plant. Whilst no adverse effects to the plant have been reported (possibly due to the protective effects conferred by the production of the osmoprotectant), the high expression levels required in transgenic plants may produce excessively high amounts of H2O2 [312].

2.652 Improving drought tolerance of crop plants through increased accumulation of glycine betaine has to date (2004) focused on understanding the processes involved through work with model plants (Arabidopsis and tobacco), with only limited modification of crop plants (oilseed rape). However, the significant role of glycine betaine in conferring tolerance to a range of environmental stresses means that further work is


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expected in this area. Several field trials have been conducted in the USA involving various so-called �amenity� grasses54 (Bermuda grass, perennial ryegrass, Kentucky blue grass and creeping bent grass) modified to express betaine aldehyde dehydrogenase. The modified grasses are reported to be both drought tolerant and have improved salt tolerance [323].

Osmoprotectants - modification of trehalose production

2.653 Natural accumulation of trehalose in plants is limited to a few extremely desiccation-tolerant plants (the �resurrection plants�), although it does occur in many bacteria and fungi. The characteristics of trehalose mean that it is able to protect biological molecules from desiccation-induced damage [324] and is consequently accumulated by microorganisms such as yeast in response to abiotic stress (heat, cold and drought) [325]. Trehalose is reported to be superior to other sugars at conferring such protection [312].

2.654 Trehalose is synthesised in two steps (Figure 2.11) with the first stage mediated by trehalose phosphate synthase (TPS) and the second by trehalose-6-phosphate phosphatase (TPP) [312].

Figure 2.11 - Synthesis and metabolism of trehalose (adapted from Rontein et al., 2002) [312]

2.655 A number of approaches have been proposed for the modification of plants to produce trehalose. As levels of both UDP-glucose and glucose-6-phosphate are high in non-trehalose producing plants then initial studies have focused on the insertion of a cytosolic TPS. However, the modification of plants to express the TPS gene from E. coli (otsA) or yeast (TPS1) constitutively, resulted in little accumulation of trehalose (≤0.5 µmol g-1 fw) as well as stunted growth (poor leaf and root development), altered sugar metabolism and altered fertility [326, 314, 324]. The modified plants did though exhibit enhanced drought tolerance, with a positive correlation between severity of the morphological damage and drought tolerance [324]. The use of a leaf-specific promoter achieved slightly greater trehalose accumulation in tobacco, although a similar approach in potato with a tuber-specific promoter generated only limited levels. The potato did though exhibit enhanced tolerance to drought (Holmstrom et al., 1996; cited by [312]) [314].

54 Used to cover areas such as golf courses, front lawns and parks.


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2.656 The modification of tobacco to express otsB from E. coli (encoding TPP) achieved a trehalose level of 90 µg g-1 fw, compared to 8 µg g-1 fw for an equivalent study using the otsA gene (encoding TPS). In both cases the transgenic tobacco, although only producing low levels of trehalose, demonstrated improved drought tolerance relative to the controls. The modified plants did however exhibit substantial changes in morphology and accumulated higher levels of non-structural carbohydrates [314].

2.657 The poor accumulation of trehalose reported for many of these approaches was found to be due to its degradation by trehalase to 2-glucose (Figure 2.11). Trehalase activity is common in plants and has been demonstrated in both tobacco leaves and potato tubers [312]. Blocking trehalase activity with the specific inhibitor validamycin A improved trehalose levels in potato tubers to 12 µmol g-1 (fw). A small number of trials were conducted in 1998 involving tomato modified to express either a sense or antisense version of the trehalase enzyme from potato. The modified plants exhibited increased yield, although any effect on drought tolerance was not reported [327].

2.658 The modification of rice with a bifunctional fusion gene (comprising otsA and otsB) under the control of a stress-inducible promoter (abscisic acid-inducible abscisic acid) resulted in the rice accumulating trehalose under both drought (3-9 fold increase) and salt (2.5-3 fold increase compared to the control) stress conditions. Improved tolerance to cold was also reported. Significantly, the transgenic plants also did not exhibit the adverse growth effects seen in previous studies investigating trehalose modification. The GM rice displayed vigorous growth under stressed conditions, and also exhibited improved photosystem function and increased photosynthetic capacity under non-stressed conditions. The non-transgenic control plants did not survive [325]. The changes reported in the transgenic plants mean that these plants would have a selective advantage over wild type plants under both stressed and non-stressed conditions. This is different to the other GM plants described in this chapter where the selective advantage is only realised in the presence of the environmental stress (i.e. under drought conditions for example). Such a selective advantage is expected to confer increased persistence and survival of the GM plant in the environment.

2.659 With respect to the future development of GM plants expressing trehalose, most of the studies conducted to date have focused on understanding the biochemical pathways involved in model plants such as Arabidopsis and tobacco. Whilst the modification does confer improved drought tolerance, the detrimental effects on plant growth and development that occur following the accumulation of this compound may limit the commercialisation of trehalose-accumulating GM crops. However, the results from the latest studies with transgenic rice offer a possible route for further development in which adverse effects on growth do not occur.


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Osmoprotectants � modification of fructan production

2.660 Tobacco modified to express increased levels of fructan demonstrated better performance under drought conditions with a 55 percent more rapid growth rate, 33 percent greater fresh weight and 59 percent greater dry weight, compared to non-transgenic controls [328]. The tobacco plants were transformed with a construct containing the sacB gene for levansucrase (generates fructan from fructose) from the bacterium Bacillus subtilis under the control of the constitutive CaMV 35S promoter.

2.661 Under drought conditions the GM tobacco accumulated fructan to concentrations as high as 0.35 mg g-1 fw. Fructan concentrations under non-drought conditions were ~0.05 mg g-1 fw [328]. A similar modification has been reported for sugar beet, with the transgenic beet accumulating fructan to ~0.5 percent of their dry weight in both roots and shoots. The modified beet grew significantly better under drought conditions compared to the non-transgenic controls [79]. The potential effects of reducing fructose levels in the GM tobacco (as the transgene confers the conversion of fructose to fructan) on the persistence and survival of the plant in the environment were however not addressed. The role of fructose as an energy storage compound means that a reduction of levels of fructose in the plant may have implications to its growth and survival in the environment.

2.662 The modifications of sugar beet with the 1-sst and 1-fft genes from Helianthus described in the �Modified Carbohydrate Content� section of this report may also be expected to improve drought tolerance of the transgenic plants. The levels of fructans in these GM beet were significantly higher (4.8 � 37.2 mg g-1 fw) [78] than those reported for the sacB+ tobacco [328].

2.663 The modification of sugar beet to express enhanced drought tolerance indicates the potential for this trait in crop plants. The absence of the adverse morphological traits observed following accumulation of other osmoprotectants such as trehalose, means that further work with fructan modification may be expected.

Osmoprotectants � modification of D-ononitol production

2.664 D-ononitol has a role improving plant tolerance to both drought and salt [314]. The modification of tobacco to express a cDNA encoding D-myo-inositol methyltransferase from the ice plant Mesembryanthemum crystallinum resulted in the accumulation of 35 µmol g-1 fw of D-ononitol in the cytosol when the plants were exposed to either drought or salt stress [329]. Photosynthetic fixation of CO2 was inhibited to a lesser extent in the transgenic plants under drought or salt stress.

2.665 Accumulation of D-ononitol offers another approach to improving drought (and salt) tolerance in plants. Studies comparing the level of drought tolerance conferred by


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each approach have not been conducted, and therefore it is not possible to determine which system is the most viable commercially. This though is likely to be that which confers the greatest drought tolerance with the least adverse effect(s) on the modified plant. The absence of any adverse effects reported for D-ononitol accumulation suggests that this approach might be more suitable than trehalose accumulation for example.

Modified tolerance to salt stress

2.666 Environmental stress caused by high levels of salt is a serious factor affecting the productivity of agricultural crops [330, 316, 331]. Many crop plants are sensitive to high salt concentrations, and whilst good drainage and irrigation with high quality water can alleviate or avoid high salt levels in agricultural land, such measures are costly and often not feasible. High salt levels have a detrimental impact on plants through water deficiency leading to osmotic stress, and the effects caused by high concentrations of sodium ions on biochemical processes [330]. As discussed, the osmotic stress caused by high salt levels is similar to that caused by drought. Therefore plant mechanisms designed to deal with drought stress are likely to also apply to salt stress.

2.667 Improving salt tolerance in crop plants through conventional breeding with salt-tolerant relatives is reported to have achieved only limited success [322, 332]. Some crops are naturally relatively tolerant of salt stress. Barley for example is able to survive NaCl concentrations of up to 250 mM [333]. In contrast, lupin (one of the most salt-sensitive crops) is killed by salt concentrations ≥100 mM NaCl [333]. GM technology is being applied to improve salt tolerance in three main ways [316]:

• detoxification - prevention or alleviation of the damage caused;

• homeostasis � re-establishment of homeostatic conditions; and

• growth regulation.

2.668 However, whilst genetic modification has been proposed as one option to improve salt tolerance in crop plants, the genetic and physiological complexity of salt tolerance means that it is unlikely to be achieved through the transfer of one or two genes. Such single or several gene modifications have been reported (and are discussed below), but it should be noted when assessing such modifications that the experimental conditions may not represent those likely to be encountered by the modified plant in the environment [331]. The ability of the transgenic plants to survive in the absence of saline conditions may be important in the environment, and the modification, whilst conferring improved salt tolerance relative to the wild type


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may actually reduce survival in non-saline conditions. In the recent review of salt tolerance in crop plants Flowers (2004) concluded that after ten years of research using GM technology to improve salt tolerance, the value of this approach to the field has yet to be established [331].

Improving salt tolerance through detoxification

2.669 The basis of this approach involves the identification of the points and mechanisms through which high salt concentrations inflict damage on the plants, and the subsequent development of approaches to prevent or alleviate this damage [316]. Most of the improvements in salt tolerance conferred using genetic modification have been achieved through this detoxification strategy [316]. However, because many detoxification strategies are non-specific then changes in tolerance to other abiotic stresses (drought, cold and possibly heat shock) as well as salt must be expected [316].

2.670 Transgenic plants overexpressing enzymes involved in oxidative protection such as glutathione peroxidase, superoxide dismutase, ascorbate peroxidases and glutathione reductases have all been reported to improve the modified plant�s tolerance to abiotic stress [316]. Tobacco modified to overexpress the enzymes for glutathione S-transferases (GST) and glutathione peroxidases (GPX) demonstrated improved salt tolerance for example [322]. The oxidation detoxification role of such enzymes has also been demonstrated in Arabidopsis plants expressing a mutant version of pst1 (a negative regulator of oxidative stress responses). Arabidopsis pst1 mutants were found to be more resistant to high salt concentrations [334].

2.671 As discussed in the previous section on drought tolerance, osmoprotectant compounds will also confer improved tolerance to salt stress (and temperature stress; both hot and cold). Osmoprotectants such as mannitol, fructans, trehalose, ononitol, proline, sorbitol, glycine betaine and ectoine are proposed as having a dual role in improving stress tolerance as they are able to both buffer against osmotic stress, and act in a similar way to the peroxidase enzymes and scavenge reactive oxygen species (ROS) [316, 322]. The efficacy of the modification was found to be improved when expression of the osmoprotectants was targeted to the chloroplasts as these are the primary site of ROS production [316]. The role of osmoprotectants as scavengers of ROS is significant as it means that plants can be modified to accumulate only slightly more osmoprotectant and still exhibit improved stress tolerance. Such slight changes are unlikely to be sufficient to have an osmotic effect but have been shown to improve oxidative detoxification [322].


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♦ Modification of mannitol production

2.672 Although increased production of mannitol has been identified as likely to improve tolerance to a range of abiotic stresses [316], specific studies have been conducted with tobacco to improve the tolerance of this plant to high salt levels (Tarczynski et al., 1992; cited by [314]). Wild type tobacco does not produce mannitol. Expression of the gene mt1D from E. coli which encodes mannitol-1-phoshate dehydrogenase resulted in the accumulation of 6 µmol g-1 fw mannitol in the leaves of the transgenic plants and an enhanced tolerance to high salinity (150 mM NaCl). Targeted expression of the gene to the chloroplast achieved higher mannitol concentrations of up to 7 µmol g-1 fw. However, under non-stressed conditions the modified tobacco plants were 20-25 percent smaller than the wild type plants.

2.673 Whilst the protection conferred by mannitol accumulation was proposed originally to be a consequence of the compound acting as an osmoprotectant (Tarczynski et al., 1993; cited by [332]), further studies have suggested that the levels accumulated were insufficient to provide osmotic protection and that mannitol may act as an antioxidant rather than an osmoprotectant (Karakas et al., 1997; cited by [332]).

2.674 Whilst mannitol accumulation has been found to confer improved salt tolerance, the adverse effects on plant size in non-stressed environments mean that this modification is most suited to soils with an existing high saline content and not those with a fluctuating salt content where non-stressing levels of salt could occur. The current focus of the work on tobacco means that the studies are at an earlyish stage of development. However, further work might be expected, but no immediate commercialisation.

♦ Modification of proline production

2.675 The modification of plants to accumulate higher levels of proline has been found to confer increased tolerance to salt (200 mM NaCl (mothbean (Vigna aconitifolia)) and 600 mM NaCl (Arabidopsis)) and cold (-7 °C for two days (Arabidopsis)) [314]. Both the studies used modifications that blocked naturally occurring proline inhibition or degradation pathways, thereby increasing the levels of proline. No adverse effects on the phenotype of the transgenic plants were reported.

2.676 As illustrated in Table 2.4, increased proline accumulation also confers enhanced drought tolerance [321, 312]. Overproduction of proline in GM tobacco through the expression of the P5CS gene from mothbean (encoding the enzyme pyrroline-5-carboxylate synthetase) resulted in enhanced root biomass and flower development compared to the wild type [321].


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♦ Modification of sorbitol production

2.677 Sorbitol is another osmoprotectant compound reported to confer improved tolerance to salt stress. The modification of tobacco to express a cDNA for sorbitol-6-phosphate dehydrogenase (S6PDH) from apple resulted in the modified plants expressing sorbitol at levels of between 0.2 and 130 µmol g-1 fw [335]. However, whilst the tobacco plants that accumulated up to 2-3 µmol g-1 fw were phenotypically normal, those plants that accumulated higher levels of sorbitol developed necrotic lesions on their leaves, infertility, and/or the inability to regenerate roots [335].

2.678 Modifications to improve salt tolerance through detoxification strategies are at an early stage of development with work focusing on model plants such as Arabidopsis and tobacco. All of the modifications described improve tolerance to high salt levels (and other abiotic stresses). However, as with the methods to improve drought tolerance, no studies have been conducted comparing the various approaches. The absence of significant adverse phenotypic effects under stressed conditions suggests that any of the modifications described might be suitable candidates for further study.

Improving salt tolerance through homeostasis

2.679 Salt tolerance can be improved by assisting plants in the re-establishment of homeostasis (ionic and osmotic) in stressful environments [316]. Osmotic homeostasis is assisted through the accumulation of a range of osmoprotectant compounds (see discussions in previous sections). Ionic homeostasis is determined in part by the activity of various ion transporters. Whilst only limited modification of plants in this area have been reported, blocking the influx of Na+ ions into the cells, or improving the removal of Na+ ions from the cells should confer greater salt tolerance [316, 322]. The identification of AtSOS1 in Arabidopsis which encodes a plasma membrane Na+/H+ antiport may offer a system to improve the efflux of Na+ ions from cells. Transgenic Arabidopsis modified to overexpress AtSOS1 displayed enhanced salt tolerance (Shi and Zhu, cited by [322]).

2.680 If the plant can be designed to store the Na+ ions in the vacuole rather than export them out of the cell, then the Na+ ions will act as an osmolyte in the vacuole and thereby assist the plant in achieving osmotic homeostasis. This is the strategy employed by many halophytic plants, and overexpression of the vacuolar transporter AtNHX1 from Arabidopsis (in Arabidopsis) conferred a substantial improvement in salt tolerance (Apse et al., 1999; cited by [316]). The same modification has also been reported for tomato and oilseed rape, with the transgenic plants able to grow in the presence of 200 mM NaCl [322]. No adverse effects on plant growth or phenotype were reported.


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2.681 The HAL1 gene from the yeast Saccharomyces cerevisiae is reported to have a role in ion homeostasis by increasing intracellular K+ and decreasing Na+, albeit by an as yet (2003) unknown mechanism [336]. Overexpression of this gene in watermelon and tomato resulted in enhanced salt tolerance in the transgenic plants [336]. Expression of the same gene in tomato resulted in the modified plants exhibiting less fruit loss compared to the wild type when exposed to salt stress, under greenhouse conditions. However, when grown in the absence of salt the modified tomato plants exhibited only half the shoot dry weight on the wild type (Gisbert et al. 2000; cited by [331]).

2.682 Greater fruit production relative to the wild type is expected to confer a selective advantage to the modified plants, and would therefore provide a positive change to the persistence/survival of the plants should they be released into environments where salt stress exists. However, if the plants are released into an environment with epheremal saline conditions then survival may in fact be lower relative to wild type.

2.683 The modification of salt tolerance through altering homeostasis systems is at an early stage of development. Whilst a number of genes have been identified whose modification has an effect on salt tolerance, much of the actual mechanism(s) involved have yet to be elucidated. Such systems will need to be worked out before any commercialisation or significant research field trials can take place.

Improving salt tolerance through growth regulation

2.684 The adverse effects of abiotic stresses on plant growth are one of the key reasons for investigating mechanisms that may improve the tolerance of plants to stress. A reduction in growth rate is a key adaptive feature exhibited by plants when exposed to abiotic stresses [321]. Because cell wall properties such as water permeability and elasticity are involved in the maintenance of cell growth during salt stress, then cell wall alterations may be crucial to conferring stress tolerance [332]. Such changes may though have other phenotypic effects. Overexpression of the cell wall peroxidase (TPX2) from tomato in tobacco resulted in a significant increase in germination rate under conditions of salt stress [332]. There is also theoretical evidence that root morphology also influences a plant�s tolerance to salt stress [337]. Genes that affect root growth independently of shoot growth for example are reported to have a large impact on salt loading and growth in saline environments [337]. A number of patents have been registered claiming benefits of altered root growth or morphology. These include the use of cytokinin oxidase to stimulate root growth and thereby improve tolerance to stress conditions [338].

2.685 However, with some plants the reduction in growth rate may be much higher than that warranted by the level of stress, suggesting that the plant has �panicked� in response to a relatively mild level of stress. Conversely, other plants do not respond


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fast enough and therefore risk incurring serious adverse effects [316]. Therefore modifying plant growth may provide an indirect approach to improving stress tolerance by removing or ameliorating these over- or under-responses of the plant(s) to stress. The application of this approach to improving salt tolerance has not been reported, although connections between stress tolerance and growth regulation have been made with respect to drought and cold stresses (see section on CBF pathway).

2.686 With respect to the further development of salt tolerance in plants through genetic modification and the potential for the eventual commercialisation of salt tolerant crop plants, significant further investigation is still required. Much of the work to date has focused on the identification of particular genes or enzymes whose expression or production confers some degree of salt tolerance. However, the occurrence of undesirable pleiotrophic effects in plants where the transgenes are expressed constitutively means that the identification of stress inducible promoters represents a key area of future work. Greater improvements may in fact be realised by applying GM technology to improving plant yields rather than salt tolerance, as it is the effect of saline conditions on yield that is the ultimate driver behind the research. A high-yielding genotype which loses 50 percent of its yield under saline conditions may still outperform the salt tolerant varieties if those plants are intrinsically low-yielding, and would therefore still be the crop grown commercially (or for subsistence) in saline environments .

2.687 However, salinity levels in many agricultural soils are on the increase, and therefore demand for crop plants with salt tolerant traits may also be expected to increase, especially if they can compete in yield against non-tolerant varieties. Further developments in this area are therefore expected, although large scale field trials or commercial plantings are not likely in the near future.

Modified tolerance to cold stress

2.688 Plants vary greatly in their ability to survive low or freezing temperatures [339]. Herbaceous plants from temperate regions for example can generally survive freezing temperatures between -5 °C to -30 °C, whilst perennials in the boreal forests are able to survive routine winter temperatures below -30 °C. However, in all cases tolerance to freezing temperatures is not a constitutive trait, but is induced in response to low non-freezing temperatures of <10 °C. This process of induction is described as �cold acclimation� [320]. For a crop such as wheat, cold acclimation enables the plant to lower its freezing tolerance from -5 °C to -20 °C [339].

2.689 However, whilst many temperate plants (including crop plants) already have a mechanism that allows them to tolerate freezing temperatures, the stresses caused by cold still limit the geographical locations where certain crops can be grown and


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periodically cause significant losses in plant productivity (especially when freezing temperatures occur without a preceding cold spell) [320, 340, 341].

2.690 Knowledge of the molecular basis of freezing tolerance means that genetic modification may offer the means to improve cold tolerance in plants. The limited success of traditional breeding techniques to advance freezing tolerance from levels achieved by the end of the 19th century mean that further development through conventional breeding are likely to be small [339].

2.691 Freezing injury is caused by ice forming in the intercellular spaces of plant tissues, leading to the physical disruption of cells and tissues [339]. Some of the disruption is caused by the formation of adhesions between the intercellular ice and the cell walls and membranes. However, the majority of the damage is caused as a consequence of the severe cellular dehydration55 that occurs with freezing [339].

2.692 Freezing causes cellular dehydration through the creation of an osmotic gradient across the cell membrane. As the chemical potential of ice is less than water, the formation of ice in the intercellular spaces results in a decrease in water potential outside the cell, causing movement of unfrozen water out of the cell. At a temperature of -10 °C for example, the osmotic gradient is sufficient to cause more than 90 percent of the osmotically active water to move out of the cells into the intercellular spaces [339].

2.693 In plants that are not cold-acclimated, the effects of freeze-induced dehydration on plant cell membranes vary with temperature [339]:

• between -2 °C and -4 °C the predominant injury is �expansion-induced lysis� caused by the osmotic contraction and expansion cycle that occurs through repeated freezing and thawing;

• between -4 °C and -10 °C the predominant injury is �freeze-induced lamellar-to-hexagonal II phase transitions�, an interbilayer event involving the fusion of cellular membranes; and

• at temperatures lower than -10 °C severe membrane damage occurs, including �fracture jump lesions�.

55 As exposure to high salt levels or low water availability will also result in cellular dehydration then many of the plant strategies for dealing with cold stress are also applicable to salt and drought stress. Therefore any modifications designed to improve plant tolerance to cold stress are also likely to improve tolerance to salt and/or drought.


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2.694 Whilst changes to the fatty acid content of the plant cell membrane (increasing the ratio of saturated:unsaturated, and cis:trans unsaturated fatty acids) are known to contribute to tolerance to lower temperatures through increasing the fluidity of the membrane [342, 320], they will only have a limited effect and are unlikely to confer tolerance to freezing temperatures. The fad8 gene of Arabidopsis encodes a fatty acid desaturase and is induced in response to low temperature [339]. The expression of this gene may therefore contribute to freezing tolerance by altering lipid composition.

2.695 Other genes proposed as having a possible role in improving cold tolerance in plants include those that encode [339]:

• molecular chaperones that act by stabilising certain proteins against freeze-induced denaturation. Examples include the spinach hsp70 gene, and the Brassica napus hsp90 gene;

• signal transduction and regulatory proteins;

• mitogen-activated protein kinases;

• calcium-dependent protein kinases;

• 14-3-3 proteins; and

• antifreeze proteins (AFPs).

2.696 Much of the current level (2003) of understanding of the mechanisms used by plants to tolerate low temperatures and freezing has been developed from studies with Arabidopsis [343]. Guy et al. (1985) first established that changes in gene expression occur with cold acclimation. In Arabidopsis for example a total of 306 genes have been identified as cold-responsive [344]. Studies on cold-regulated gene expression in Arabidopsis have identified a cold response pathway (CBF regulon) responsible for the activation of a range of cold-regulated genes conferring such properties as membrane stabilising functions and osmoprotectants [345, 343]. The CBF regulon is one of a number of as yet unidentified cold-regulatory pathways present in plants [344], and therefore represents one of a number of strategies by which genetic modification could be used to improve cold tolerance. Many of these have yet to be developed, although any of the various components of the pathway could provide a basis for improving cold tolerance in plants.


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CBF pathway

2.697 The basic mechanism of the CBF (C-repeat/dehydration-responsive element binding factor) pathway is presented in Figure 2.12.

2.698 The onset of low temperatures leads to the rapid induction of the CBF/DREB1 transcriptional activators (!), which in turn results in the expression of the CBF regulon of CRT/DRE-regulated genes56 ("). It is the expression of the CBF regulon, which includes COR (cold-regulated)57, ERD (early dehydration-inducible) and other as yet unidentified cold-regulated genes (designated �XYZ�) that result in the increase in freezing tolerance of the plant [345, 343].

2.699 The mechanism involving the initial expression of the CBF/DREB1 transcriptional activators is unknown but is proposed to involve the action of a regulatory protein (designated ICE or inducer of cold expression) that is present in the plant in warm temperatures. The onset of low temperature is proposed to either activate the ICE protein or other protein(s) with which it interacts (#). Such activation may involve alterations in protein phosphorylation caused by a cold-induced influx of calcium (Ca2+) ions. The SFR6 protein is reported to act between transcription of CBF/DREB1 and induction of CRT/DRE-regulated genes whereas HOS1 appears to act upstream of CBF transcription [343].

2.700 The CBF transcription factors are members of the AP2/EREBP family of DNA-binding proteins [345]. They recognise the CRT/DRE DNA regulatory elements ($), which have a conserved 5-bp core sequence of CCGAC and are present in the promoter regions of many cold- and dehydration-responsive genes in Arabidopsis [345, 315].

2.701 The modification of Arabidopsis to express CBF constitutively results in the induction of freezing tolerance without a low temperature stimulus [345]. However the use of a CaMV promoter to achieve this caused a severely stunted phenotype, a decrease in seed yield and a delay in flowering (Gilmour et al., 2000; cited by [345]). A possible reason for these adverse effects may be that some of the gene products or downstream target molecules probably actively feed into cell division and cell expansion processes and act to inhibit growth [316]. The gene products are therefore the �stress signals� used by the plant to inform other biochemical pathways of the environmental stress.

2.702 The modification of crop plants in a similar way would confer resistance to sudden cold snaps that occur without a prior reduction in temperature. The presence of CRT/DRE elements in the promoters of both cold- and drought-responsive genes in

56 CRT (C-repeat); DRE (dehydration-responsive element).


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Arabidopsis suggests that any changes in the induction of these elements is likely to have an effect on the plant�s tolerance to drought as well as low temperature. In fact, the modification of plants to overproduce CBF/DREB proteins has been found to confer improved tolerance to drought and salinity stress, as well as freezing (Liu et al., 1998; cited by [316]).

Figure 2.12 - CBF cold-acclimation pathway (adapted from Thomashow 2001) [343]

2.703 The use of a stress-inducible promoter has been found to avoid the deleterious

effects encountered with a constitutive promoter such as CaMV 35S [315]. The expression of the CBF3(DREB1a) gene in Arabidopsis, under the control of RD29a (a cold- and dehydration-inducible promoter) resulted in the modified Arabidopsis displaying enhanced tolerance to freezing and drought tolerance with only limited adverse effects on plant phenotype [346].

2.704 As discussed, research on improving cold tolerance in plants has focused on Arabidopsis. However, a CBF (or CBF-like) cold-response pathway has also been identified in oilseed rape, wheat, rye and tomato [345]. The overexpression of Arabidopsis CBF genes in oilseed rape has been found to increase freezing tolerance in both cold-acclimated and non-acclimated plants [345].

57 COR genes may also be designated LTI (low temperature induced), KIN (cold-inducible) and RD (responsive to desiccation).

COLD

CBF

ICE

ICE CBF

CBF

CBF

CBF

SFR6

HOS1 COR

ERD

�XYZ�

CBF Regulon

CRT/DRE

CRT/DRE

CRT/DRE

# activation of ICE ! induction of the CBF/DREB1 transcription activators $ binding of CBF to the CRT/DRE element " expression of the CRT/DRE-regulated genes

# !

"

$

�XYZ�

COR

ERD Increased freezing tolerance


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2.705 The identification of a CBF-like pathway in tomato is particularly significant as this plant does not naturally cold-acclimate [345]. Research into the modification of tomatoes to confer cold tolerance has however not been identified.

Modification of cold-induced genes

2.706 In addition to attempting to improve basic cold acclimation of plants through constitutive or enhanced expression of the CBF (or CBF-like) regulon, various studies have also investigated increasing expression of particular cold-induced genes [339].

2.707 The genes expressed as a result of the activation of the CBF regulon encode a range of extremely hydrophilic proteins classified according to amino acid sequence [339]. The COR15a gene of Arabidopsis for example encodes a polypeptide targeted to the chloroplasts. Increased expression of COR15a in Arabidopsis improved freezing tolerance of the chloroplasts by 1-2°C in non-acclimated plants. Significantly, overexpression of COR15a only increased freezing tolerance of the chloroplasts over a temperature range of -4 °C to -8 °C, with a slight decrease in survival between -2 °C and -4 °C [339]. This suggests that the product of COR15a has a role in deferring the incidence of freeze-induced formation of hexagonal II phase lipids to a lower temperature, but have little or no effect on the occurrence of expansion-induced lysis that occurs at just below freezing.

2.708 Whilst changes to the expression of COR15a do improve freezing tolerance at both the organelle (chloroplast) and cellular (protoplast) levels, the effects are not sufficient to increasing freezing survival of the whole plant (Jaglo-Ottosen et al., 1998; cited by [339]). Therefore, focusing on improving expression of the CBF regulon may offer a more effective approach in altering cold tolerance of crop plants.

LEA (late-embryogenesis abundant) proteins

2.709 LEA (late-embryogenesis abundant) proteins are expressed during cold acclimation, and in seedlings in response to abscisic acid (ABA) and water stress [339]. Whilst it is known that the HVA1 gene of barley (which encodes a LEA group II protein (also designated LEA D7)) confers tolerance to drought stress, it is not known whether it contributes directly to cold tolerance. The modification of rice with HVA1 resulted in increased tolerance to both drought and salt stress (Xu et al., 1996; cited by [339]), whilst the modification of wheat to express the barley LEA protein is claimed to improve drought tolerance to the transgenic plant. The GM wheat has been grown as part of several field trials in the USA [347].

2.710 Other LEA proteins have been shown to have a role in cold tolerance. The expression of the Le25 gene (encodes protein LEA D113) from tomato in yeast


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conferred increases in both freezing and salt tolerance (Imai et al., 1996; cited by [339]).

Plant antifreeze proteins

2.711 Antifreeze proteins (AFPs) are produced by a wide range of organisms, including certain plants, insects and fish [348]. AFPs are characterised by their ability (in vitro)58 to decrease the temperature at which ice is formed, but without affecting the melting point of the solution [340]. This characteristic, known as thermal hysteresis, results from the AFP binding to the surface of the ice nuclei and inhibiting ice crystal growth. AFPs are also potent inhibitors of ice recrystallisation, as they inhibit the coalescing of small ice crystals into large ice crystals [319, 318]. Thermal hysteresis and inhibition of ice recrystallisation are separate properties, with inhibition requiring significantly less (100-500 times less) AFP [318]. Studies with winter rye have suggested that it is the inhibition of recrystallisation rather than the thermal hysteresis characteristic that enables AFPs to confer cold tolerance [339]. Pearce (2001) [340] suggested that the thermal hysteresis property of AFPs may even not prevent freezing in the plants as it would only lower the temperature at which ice is formed by 0.3 °C.

2.712 AFPs have been isolated and characterised from winter rye, kale, peach, carrot and winter wheat [318]. However, whilst they accumulate in the apoplastic fluid of these crops during cold-acclimation they have not been identified in all crops that undergo cold-acclimation, such as spinach and spring oilseed rape, and have been detected at only very low levels in winter oilseed rape and kale [319]. Crops such as maize and tobacco that do not undergo cold-acclimation also do not produce AFPs [319].

2.713 The modification of plants to express AFPs is very limited to date (2003) [318], with no conclusions made on whether such modifications could be used to improve cold tolerance in crop plants. Whilst the modification of plants to express AFPs from fish [349, 350] or insects [351] has resulted in the transgenic plants accumulating AFPs in their apoplastic fluid, none of the studies reported any improved ability of the modified plants to survive freezing relative to the wild type. The only published study based on field results reported to date (2004), found that the transgenic tobacco (modified to express Type II fish AFP) did not demonstrate improved frost resistance [352].

2.714 With respect to the effect of such modifications on the persistence and survival of the transgenic crop in the environment, the plants modified for improved cold tolerance should demonstrate enhanced survival in regions where freezing temperatures occur. Whilst such improvements may not be achieved through the expression of AFPs (on

58 The same characteristic is not necessarily displayed in vivo.


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the basis of results reported to date (2003) [318]), the expression of AFPs does have a further indirect effect.

2.715 The similarity of many AFPs to pathogenesis-related proteins such as glucanase-like (GLPs), chitinase-like (CLPs) and thaumatin-like proteins (TLPs) [318], means that plants expressing high levels of AFPs may also exhibit enhanced resistance to pathogens. Winter cereals for example have been reported to be more resistant to fungal diseases after undergoing cold acclimation [319], although the mechanisms that confer disease resistance during this process are largely unknown [353]. The gene Tad1 for example is induced in winter wheat during cold-acclimation, and confers resistance to the phytopathogenic bacterium Pseudomonas cichorii [353]. The structure of the 23 kDa polypeptide encoded by Tad1 is similar to γ-thionin compounds which have a known pathogen-defensive function in plants. Because Tad1 is induced by low temperature and not by major defence signalling processes its function is thought to be in the wheat�s low-temperature resistance to pathogens during winter hardening [353].

2.716 Improved disease resistance should of course also improve the persistence/survival of the plants in the environment (where the relevant pathogens are present), and may confer an advantage to the modified plants in environments where freezing temperatures are unlikely.

2.717 The specific modification of plants to improve tolerance to cold, either through the alteration of the cold-acclimation pathways, or specific cold tolerance or anti-freeze proteins is very limited to date (2003). The studies reported so far have focused on understanding the systems that occur naturally in plants and how these may be altered through genetic modification. Modifications conducted to date have been unsuccessful, and therefore commercialisation or large scale field trialling is unlikely in the near future.


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3. ASSESSMENT OF THE EFFECTS OF COMPOSITIONAL CHANGES ON PERSISTENCE AND SURVIVAL IN THE ENVIRONMENT

Modified Protein Content 1,3-1,4-β-glucanase Vegetative storage proteins (VSPs)

Modified Carbohydrate Content Modified starch or sugar content

General effects of starch or sugar modification Sugars as signalling compounds Changes to AGPase Changes to triose-phosphate phosphate translocator (TPT) Increasing starch accumulation by reducing starch degradation Modification of starch branching enzyme (SBE) Modification of grain hardness (pinA and pinB) Modification of invertase enzymes Modification of sucrose synthase activity Modification of fructan content

Modified cellulose Modification of pectic polysaccharides Modification of raffinose family oligosaccharides

Modified Lignin Content Modified Oil and Fat Content

Fatty acid modification and altered sensitivity to cold Fatty acid modification and altered seed germination

Modified Micronutrient Content Modified α-linoleic acid content Modified flavonoid content

Modification for Speciality Medical Applications Persistence of the products of the genetic modification

Modification for Speciality Industrial (non-medical) Applications Modified pest resistance Production of biodegradable plastics Production of trehalose as a stabilising agent

Modified Plant Growth Improved drought tolerance through accumulation of osmoprotectants Improved salt tolerance

Detoxification Homeostasis

Improved cold tolerance Conclusions


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3.1 The purpose of this section of the report is to identify and assess the effects or likely

effects (if any) that the changes in the compositional traits identified in the previous section may have on the persistence and/or survival of the GM crop in the environment.

3.2 As with the review of modifications, the identification and assessment of the effects is presented according to the purpose of the intended modification. The effects reviewed include both direct and indirect effects. Direct effects are those that are related to the intended characteristics of the modified compositional trait, and include for example the increased survival of GM Bt-expressing plants due to reduced attack from lepidopteran pests. Indirect effects are less easy to identify, and are those that are not related directly to the purpose of the modification. Indirect effects are most likely to occur where the modification involves the alteration of enzymes or other regulatory systems that have a number of roles throughout the plant, rather than the specific role targeted by the modification.

3.3 The key traits that are likely to have an effect on the survival or persistence of the plant in the environment are outlined below. Any modification that affects one of these traits, either directly or indirectly should be expected to alter the survival or persistence of the transgenic plant in the environment:

• growth rate � increased growth rate will confer a selective advantage on the plant by enabling it to out-compete others for nutrients and sunlight. High growth rates during the early stages of plant development are viewed as particularly advantageous. However, larger/faster growing plants may be more susceptible to pests or herbivores, thereby meaning improvements in this trait having a negative effect on persistence or survival;

• seed production � an increase in the number or viability of the seeds produced should confer a selective advantage on the plant to produce more progeny, or to disseminate its progeny across a wider area;

• germination time � variations in germination time may provide a competitive advantage for nutrients and sunlight if the modified seed germinate earlier than other plants in the same environment. However, as with improved growth rate, earlier germination may make the plants more susceptible to pests or herbivory, thereby negating any advantage;

• seed dormancy � alteration of seed dormancy will affect the persistence of the seeds in the environment. More dormant seeds will persist longer in field seed banks. However, increased dormancy does not necessarily confer


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greater persistence of the GM plant, as without the correct conditions to initiate germination seed dormancy may actually be just one step removed from seed (and plant) death;

• resistance to pests � improved pest resistance will confer a competitive advantage, but only in environments containing a high level of the relevant pests (invertebrates, fungi, bacteria or viruses);

• resistance to decay � this refers to the resistance of the dead plant to decay by organisms such as fungi. Improved resistance to decay has only been identified in one instance as the specific purpose of a modification (for cellulose) [84], but may also occur for plants modified for increased lignin content;

• herbivory � changes to the plant to make it less palatable to herbivores should confer improved survival of the plant in the environment. Modifications to both lignin or cellulose content have been identified as having a direct effect on potential herbivory;

• flowering time � changes in flowering time are assessed to have either a positive or negative effect on survival of the GM plant, depending on the timing of the flowering and pollination mechanism used by the plant. If the flowers are insect pollinated for example then early flower formation may reduce the level of pollination (and consequently long-term survival) if the necessary insects are not available;

• development of storage structures � modifications that alter the structure, size or composition of structures such as tubers or bulbs that are used by the plant as food or energy stores are assessed to affect the survival of the plant in the environment. The potential for longer term survival (more than one growing season) is likely to be most affected as plants such as potatoes use their tubers as food stores to fuel early season growth;

• overwintering survival � any reduction in overwintering survival has implications for the long-term survival of biennial or perennial plant species. For annual plants, overwintering survival is only relevant for their seeds. Changes therefore to seed structure, composition and production of energy storage structures (tubers and bulbs) will have an effect, as will changes that alter the physical strength of the plant. Because adverse weather effects (wind and flooding) are more likely to occur during the winter then changes to lignin or cellulose content for example may reduce overwintering survival. Plants with reduced lignin levels have been reported to exhibit reduced


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overwintering capability, although this has also been proposed as a consequence of linkages between the genes controlling lignin synthesis and those involved in winter survival traits [1]. Changes to the plant�s ability to withstand cold/low temperatures will also affect overwintering survival; and

• tolerance to drought or high salt levels � improved tolerance to either of these abiotic stresses is likely to improve the ability of the plant to persist and/or survive in the environment. However, improved persistence or survival is not always guaranteed as the changes may have serious adverse effects to the plant�s phenotype. Improved persistence may also only occur in the presence of the particular stress. In some cases survival is reduced in the absence of the environmental stress.

3.4 Many of the effects on persistence and/or survival reported in the scientific literature have already been presented in this report as part of the review of the modifications. The purpose of this section is therefore to summarise the effects identified and to review the particular modifications which appear to have a greater effect on persistence and or survival of the GM crop in the environment. Modifications discussed in the review section that have not been addressed further here are assessed as not expected to have an effect on the survival and/or persistence of the transgenic plant in the environment.

3.5 In many cases there is little information on the persistence or survival of the GM plant as the research to date has not addressed this, focusing instead on the modification of the desired trait. Evaluation of agronomic traits (and persistence or survival) of the GM plant is often done at a later stage in the development programme. Therefore, where information is not available, details have been sought from studies with non GM plants where changes to compositional traits have been achieved through traditional breeding (although this of course only applies to a minority of the modifications addressed in this report). Examples include the alteration of starch content in potatoes and in peas.

3.6 With respect to the persistence and survival of GM plants in general, the consensus from many of the reports reviewed is that the GM plants are not inherently more persistent or likely to survive in the environment than non-GM varieties [354-356]. Survival and/or persistence will only be improved if the modified trait provides a selective advantage (either directly or indirectly), and if the plants are present in an environment where the relevant selective pressures exist. For example, plants modified for improved resistance to pests may be expected to exhibit greater survival in the environment compared to the non-GM (less pest-resistant) and therefore more susceptible varieties. However, the GM plants will only demonstrate a greater level of survival if the environment contains high levels of the relevant pest. In the


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absence of the pest they are likely to exhibit no greater survival than the non-modified plants [357].

MODIFIED PROTEIN CONTENT

3.7 Two modifications for altered protein content have been identified with reported effects on persistence and/or survival of the transgenic plant in the environment. The limited number may however be a consequence of the small number of modifications reviewed as part of this section. No adverse effects on survival or persistence have been reported or are assessed to occur following the modification of wheat proteins for altered dough characteristics, or following the removal of allergens. The alteration of allergenic properties in plants such as peanut is however at an early stage of development, with any effect on plant survival yet to be studied.

3.8 Potential changes to survival or persistence have been reported following the increased production of 1,3-1,4-β-glucanase in potatoes and the alteration of vegetative storage proteins in soybean.

1,3-1,4-β-glucanase

3.9 The modification of potatoes for the increased production of the enzyme 1,3-1,4-β-glucanase was found to have an adverse effect on tuber yields and cell wall morphology of the potato plants [284]. As discussed in the introduction to this section, changes in tuber yield (size or number) may be expected to have an effect on the survival of subsequent generations of the modified potato in the environment (because of the importance of the tubers as food storage organs). Changes in cell wall morphology may also be expected to have an adverse effect on the survival of the parent potato plant.

3.10 Both of the reported effects following increased production of 1,3-1,4-β-glucanase are assessed to reduce the persistence and survival of the GM potato in the environment. The level of reduction is not known and will depend on the size of the fall in tuber yield and number.

Vegetative storage proteins (VSPs)

3.11 The role of vegetative storage proteins (VSPs) when the plant is exposed to conditions of environmental stress (such as low water availability), means that the modification of these proteins may have an effect on the persistence and/or survival of the plant in the environment. Although the alteration of VSP levels in soybean (through antisense modification) was found to have no effect on the productivity of the plant [28], the effect on survival or persistence was not investigated. However, the modification of any plant property that is involved in the plant�s response to


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environmental stress should be expected to affect the survival of that plant in the environment.

MODIFIED CARBOHYDRATE CONTENT

3.12 A number of modifications designed to alter the carbohydrate content of crop plants have been identified as altering the survival or persistence of the plants in the environment. The changes to persistence and survival are a consequence of the roles of various carbohydrates as storage, signalling or structural compounds within the plant. The role of sugars as signalling molecules means that changes to these compounds may be expected to have a number of indirect effects.

Modified starch or sugar content

3.13 The role of starches and sugars as both food storage/energy supplying compounds, and signalling compounds, for plants means that any modification of these products may have an effect on the ability of the plant to survive in the environment. For example, as discussed in the previous section with potato tubers, the reduction of starch content in plants is likely to have an adverse effect on that plant�s survival as it will have reduced food and energy reserves. The key role of sugars in the regulation of cellular functions, including shifting cellular metabolism from a net synthesis of energy reserves to a net degradation [358], further highlights the potential changes that the modification of sugar levels may have on the persistence or survival of the plant in the environment.

General effects of starch or sugar modification

3.14 Changes to end products (such as starch or sucrose) are reported to have a particular effect on growth at low temperatures or growth under conditions of elevated CO2 content [42]. The modification of tomatoes with maize sucrose-phosphate synthase (SPS) resulted in the GM tomatoes having yields 70-80 percent greater than wild-type when grown under normal and elevated CO2 levels. Increased yields may confer improved survival in the environment due to the production of more progeny. However, such improvements may incur greater herbivory and consequently a reduction in survival.

3.15 The modification of the relative partitioning of sucrose and starch between the leaves and storage organs also has an effect on plant growth and development [359]. The effect of partitioning is of particular relevance to this review, because for many modifications in which the alteration is intended to increase carbohydrate levels, the levels are increased in the leaves rather than the storage organs. Therefore, although total plant starch levels have increased, accumulation and poor export of


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starch from the leaves means limited supply to actively growing parts of the plant and restricted growth and development.

Sugars as signalling compounds

3.16 Sugar signalling has been defined as the interaction between a sugar molecule and a sensor protein that generates a signal. The signal initiates signal transduction cascades that ultimately result in altered gene expression or changes in enzyme activities [66]. The role of sugars as signalling compounds in plants means that changes to plant sugars are assessed to have implications to all stages of a plant�s lifecycle, from seed germination and vegetative growth to reproductive development and seed formation [66, 358].

3.17 Although any neutral sugar or glycolytic intermediate is reported as having the potential to have a signalling function, to date such a role has only been described for hexose and sucrose [66]. Modifications involving either of these two sugars should therefore include a specific requirement to assess for any effect on signalling.

3.18 One of the examples identified was the constitutive expression of the gene pall (from the bacterium Erwinia rhapontici and encoding sucrose isomerase) in tobacco. Sucrose isomerase catalyses the conversion of sucrose to palatinose and trehalose [65], and therefore expression of pall will reduce sucrose levels in the plant. The modified plants exhibited a severe retardation in growth [75]. Such an adverse outcome is viewed as an indirect effect of sucrose modification. Similar effects (in this case severe impairment of tuber sprouting) were observed in potatoes following the blocking of sucrose transport through phloem (by expression of a phloem-specific cytostolic invertase) [358]. The potential for such changes should be considered as part of any field release of a modified plant.

3.19 An important consideration though is the level of targeting of the transgene expression. In the modifications described above, the transgenes were either not targeted to a particular area of the plant (tobacco), or targeted so that they could affect a major plant process (phloem transport in the potato). Where the tobacco work was repeated in potatoes, but with the pall under the control of a tuber specific promoter, the GM potatoes exhibited no adverse growth or development changes [65].

Changes to AGPase

3.20 Modification of the enzyme ADP-glucose pyrophosphorylase (AGPase) has been reported as a mechanism to modify starch quantity in crop plants. However, the alteration of AGPase activity (increased or decreased) has been found to have an effect on both the phenotype of the modified plant and on seed development. Such


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changes will of course alter the ability of the plants to persist or survive in the environment.

3.21 Increased AGPase activity is reported to improve seed development and overall plant productivity [47, 50]. In plants such as wheat and rice where levels of seed abortion are high, then an improvement in seed development is likely to have a positive effect on the long-term survival of the GM plant (over more than one generation) in the environment. Reduced seed abortion for example will result in more progeny being produced per plant. The modification of wheat or rice with gene Sh2r6hs (encoding the AGP large subunit from maize) increased the amount of seeds taken to maturity on the plants. Total seed weight of the transgenic wheat and rice was 31 percent and 23 percent (respectively) higher than the non-GM controls. There was no difference in the weights of individual seeds of the GM compared to the non-GM wheat or rice [50].

3.22 In addition to improving seed development, the persistence/survival of the transgenic wheat or rice in the environment is also increased as expression of Sh2r6hs also alters CO2 fixation and consequently improves photosynthetic output and ultimately growth rate [50].

3.23 Conversely, decreased AGPase activity is reported to reduce plant growth and flower development. In potatoes these changes are characterised by the formation of smaller sized but a greater number of tubers and reduced flower development [51]. The cause of the increase in tuber number is not known [360], although elevated sucrose concentrations (which would occur if AGPase activity was reduced) have been reported to promote tuberisation in potato plantlets [361]. It is not clear what effect such a change in phenotype would have on survival of the plant, if the total yield of tuber per plant remained unchanged. Smaller tubers may for example be more prone to damage by frost or pests.

3.24 The effect on flower development is a consequence of the role of sucrose in triggering the onset of flowering. Plants modified for high inhibition of AGPase activity (and therefore higher sucrose content as less sucrose is converted to starch) demonstrated altered flower development (due to greater anthocyanin accumulation). Flower formation occurred 2-4 weeks earlier compared with the wild-type [51]. Such a difference is assessed to have either a positive or negative effect on survival of the GM plant, depending on the timing of the flowering and pollination mechanism used by the plant.

Changes to triose-phosphate phosphate translocator (TPT)

3.25 The TPT acts as the major gateway for the export of triose-phosphates from the chloroplast during photosynthesis. Antisense inhibition of this gateway results in less


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triose-phosphates being available for the synthesis of sucrose in the cytosol during the day [359].

3.26 The modification of TPT activity was found to have a significant effect on the diurnal growth pattern of the plant. Compared with both the wild-type potatoes and the transgenic lines expressing antisense AGPase, the antisense TPT lines had lower levels of soluble sugars in their mesophyll cells and phloem during the light period (day) compared with the dark period (night). Although the changes in soluble sugar levels had no effect on the growth rate of the antisense TPT lines during the light period relative to the wild-type and antisense AGPase, the growth rate during the dark period was approximately doubled, resulting in a significant shift in diurnal growth pattern [359].

3.27 The absence of any significant effect on growth pattern in the AGPase lines and the TPT lines (during the light period) compared with the wild-type, was reported to be due to the changes in carbohydrate availability caused by the modifications being within the plant�s tolerance parameters. These must be relatively large otherwise the plant would end up responding to the changes in photosynthesis (and consequently sucrose availability) caused by a cloudy day for example [359].

3.28 The consequence of these changes, most notably the doubling of growth rate during the dark period, on persistence and survival of the modified plant was not reported. However, the changes are assessed as likely to provide the plant with an advantage over wild-type strains if the higher growth rate can be sustained.

Increasing starch accumulation by reducing starch degradation

3.29 Increased accumulation of starch by reducing starch degradation has been attempted in potatoes by modifying the plants with a yeast invertase, a bacterial glucokinase or a bacterial sucrose phosphorylase. Each of the modifications attempted resulted in an induction of glycolysis and a massive partitioning of the increased carbon into respiration, with no increase in starch accumulation. The addition of the sucrose phosphorylase also resulted in increased levels of a range of amino acids [52]. The changes exhibited are assessed to reduce the long-term survival of the GM plants in the environment.

3.30 The adverse effects reported with modifications aimed at increasing starch accumulation through reducing starch degradation are indicative of the potential problems that must be considered when modifying carbon flux in plants.


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Modification of starch branching enzyme

3.31 Modification of potatoes to increase the amylose content of the starch through inhibition of the starch branching enzyme (SBE) has been reported to have a significant reduction in tuber yield and total starch content. As has already been discussed such changes are likely to reduce the long-term survival of the modified potatoes in the environment.

Modification of grain hardness

3.32 The modification of grain hardness, achieved through altering the adhesion between the starch granules and the surrounding protein matrix, is assessed to have both direct and indirect effects on survival of the GM plant. Any changes to grain hardness that affect herbivory of the seeds are assessed to have a direct positive effect on survival. The indirect effects are a consequence of the antimicrobial properties of the proteins (PINA and PINB) that are involved in determining the level of adhesion.

3.33 The proteins PINA and PINB are both puroindolines which are a group of compounds with known in vivo activity against several fungal plant pathogens (e.g. Magnaporthe grisea and Rhizoctonia solani). Therefore the modification of plants such as maize and rice that do not naturally contain pinA and pinB is likely to confer some degree of fungal resistance to these plants. This would of course have a positive effect on their persistence and survival in environments containing these fungi.

Modification of invertase enzymes

3.34 Invertases such as β-fructosidase are a group of related enzymes that hydrolyse sucrose to glucose and fructose [68]. The modification of tomato with the TIV1 gene, which was conducted to achieve increased accumulation of sucrose, was found to have an indirect effect that has implications for plant survival. Antisense expression of TIV1 resulted in a 30 percent reduction in fruit size and an increase in ethylene production by the plant. Increased ethylene production will have an effect on fruit ripening, with the modified tomatoes likely to ripen faster than those present on wild-type plants.

3.35 Reduced fruit size is assessed to have implications to possible herbivory and also the potential for less dissemination of the seeds (although both may be of less relevance to glasshouse grown crops such as tomatoes). Smaller fruits will be less attractive to herbivores. If this is the case then fewer seeds will be taken from the plant and disseminated to other areas in the herbivore�s faeces. This is viewed as potentially reducing the competitive ability of the GM tomato in the environment, and consequently reducing its ability to survive or persist compared to wild type varieties.


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Modification of sucrose synthase activity

3.36 The reduction (through antisense inhibition) in fruit specific sucrose synthase in tomatoes has been found to reduce fruit setting in the modified plants [71]. A reduction in fruit setting will mean both a decrease in the number of seeds produced per plant and a reduction in the ability of the tomato to disseminate its seeds away from the parent. As discussed above, both of these changes are assessed to reduce the survival and persistence of the GM plant.

Modification of fructan content

3.37 The role of fructan as an osmoprotectant means that any modification that results in increased content of fructan in the plant may also confer increased tolerance to drought (see sections on �Modified Plant Growth� for further details).

Modified cellulose

3.38 There are hypothetical advantages of altering cellulose content, namely improved decay resistance or altered digestibility of fodder crops. However, due to the important role of cellulose as a structural material in plant cells then it may be expected that any alteration of cellulose content will have an effect on cell morphology and strength, and therefore on the phenotype of the plant and its persistence and survival in the environment. Indeed, such are the likely adverse effects on plant survival of cellulose modification that it is surprising that any studies have been conducted into this area.

3.39 A patent [84] has however been granted covering the development of plants with modified cellulose content, for the purposes of improving the digestability of fodder crops (reduced cellulose), or improving the innate decay resistance in timber (increased cellulose content). Such transgenic plants should be assessed for changes in persistence and/or survival. Any reduction in cellulose content may be expected to reduce plant fitness and ability to persist and survive in the environment as it would make the plant physically weaker, more attractive (and therefore more susceptible) to herbivores, and less resistant to decay. An increase in cellulose content may be expected to have the opposite effect, assuming that it didn�t have any indirect effect on other plant processes such as xylem or phloem transport.

Modification of pectic polysaccharides

3.40 The roles of pectic polysaccharides such as homogalacturonan and rhamnogalacturonan in the development of the cell walls and as plant signalling molecules mean that any modification of levels of these compounds in the plant may have implications to the persistence and/or survival of the plant in the environment.


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3.41 The reported modification of these polysaccharides is limited (as of 2002), but of the studies conducted, adverse effects on plant phenotype have been reported, for example the occurrence of smaller and more wrinkled tubers in potatoes modified for altered rhamnogalacturonan content [86]. Such a change in phenotype is assessed to reduce survival of the GM potato in the environment, especially over a period of several growing seasons. The pollen from the GM potatoes described exhibited reduced fertility [86]. This is also likely to reduce the potential survival of the modified plants in the environment.

Modification of raffinose family oligosaccharides

3.42 The role of raffinose family oligosaccharides (RFOs) as osmoprotectants in plants means that any reduction in levels of these compounds in planta may reduce the ability of the modified plants to respond to cold and drought stresses [89]. The likelihood of a reduction in plant fitness occurring in response to a reduction in levels of RFOs has not been reported. However, studies with non-GM cultivars of crops such as alfalfa found that cultivars that accumulated RFOs in their roots in October demonstrated higher survival rate over the winter than cultivars which accumulated RFOs later in the year (December) [362]. The reverse situation (reduced overwintering ability) is assumed for plants modified for reduced RFO levels.

MODIFIED LIGNIN CONTENT

3.43 As discussed for cellulose, the important role of lignin in ensuring the correct structure and morphology of plants, particularly woody shrubs and trees means that the modification of lignin is likely to have some effect on plant phenotype. Changes to lignin content will also have implications to the level of herbivory, attack by boreing invertebrates (and other animals), resistance to weather events (wind and rain) and erosion, and speed of decay once the plant has died. Lignin also confers more general disease resistance on the plant in its role in restricting the spread of pathogens through the plant [363]. The presence of lignin in seed casings confers resistance to microbial invasion and therefore improves the survival of the seed once it has fallen from the plant [364].

3.44 The objective of most commercially-orientated studies of lignin modification reported to date (2002) is to reduce lignin content59. This enables easier harvesting of the plant (or tree), easier and more energy efficient processing (especially true for paper production where removal of the lignin content of the wood is very energy intensive relative to the rest of the process), and improved digestibility if the plant is a fodder crop for livestock.

59 Although increased lignin content may be of benefit for the production of trees/shrubs for use as firewood or in bioenergy production.


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3.45 The reduction in lignin content is in general likely to reduce the persistence and/or survival of the GM plant in the environment. Lignin-reduced trees for example are likely to be structurally compromised, leading to them being more prone to herbivore damage or fungal disease, so that productivity, fecundity and life-span might be shortened relative to wild trees [108].

3.46 As reviewed in the section on Lignin Modification, the biosynthetic pathways involved in the formation and deposition of lignin in plants are extremely complex, and are linked to other biosynthetic pathways within the plant. This is particularly the case for the shikimate and phenylpropanoid pathways which form the initial two thirds of the lignin biosynthetic pathway (Figure 2.3). The modification of any aspect of these two pathways is therefore assessed to affect traits other than lignin biosynthesis. Changes to these two pathways should be designated as non-specific for lignin modification and (indirect) alterations of other traits should be expected.

3.47 Examples of adverse effects on plant morphology which may have an effect on persistence or survival, that have been reported following the modification of part of the shikimate or phenylpropanoid pathways include:

• inhibition of phenylalanine ammonia lyase (PAL). This resulted in a reduction of other phenolic compounds within the plant, and may reduce survival as phenolic compounds have a role in pest resistance;

• inhibition of hydroxycinnamoyl CoA ligase (4CL). Results from studies have reported this modification causing collapsed cell walls and stunted growth in Arabidopsis, but increased growth in aspen. All studies reported reduced lignin levels;

• downregulation of caffeoyl CoA O-methyltransferase (CCoAOMT). Studies in poplars reported reduced cross-linking in the lignin compared with the wild-type. This is assessed to reduce the strength of the wood;

• alteration of CCoAOMT and O-methyltransferase (OMT). Found to cause reduced plant growth and altered flowering activity. Changes were however not observed in plants where just OMT was altered. Changes were particularly prominent in the later stages of plant development; and

• downregulation of hydroxycinnamoyl CoA reductase (CCR). Although this enzyme is described as part of the lignin-specific pathway (Figure 2.3), the role of this enzyme in the formation of other compounds as well as lignin mean that changes to this enzyme are expected to have adverse effects on other plant traits.


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3.48 Adverse effects following changes to other aspects of the lignin specific pathway (other than CCR), such as hydroxycinnamoyl alcohol dehydrogenase (CAD) for example have not been reported. Increased decomposition of plants modified for altered CAD, CCR or OMT has been reported, with CCR-modified plants decomposing the quickest. This will of course reduce the persistence of the modified plant in the environment. Whether such changes would have an adverse affect on populations of saprophytic organisms such as fungi (because of the reduced time the modified wood remained in the environment) is not known (and is outside the scope of this report).

3.49 Reduced plant fitness, particularly reduced survival overwinter and lower plant biomass has been observed in plants with reduced lignin content [1] and will limit the persistence and survival of the plants in the environment. These changes have been reported for plants whose lignin content has been altered through genetic modification or conventional breeding. Because the changes in plant fitness could be moderated by selection and recombination, they were proposed as not being linked directly to the reduced lignin content. They are therefore more likely to have occurred as an indirect effect resulting from some linkage between the genes controlling lignin synthesis and those involved in winter survival traits [1]. The conclusions for this study were that plants genetically modified for reduced lignin content should be evaluated in a variety of environments before wider scale releases occur. These should include testing for overwintering capability.

3.50 An important consideration in assessing the potential effects to persistence and survival of plants with modified lignin (and indeed other modified traits), is the choice of promoter sequence used. With lignin modification many of the early studies employed a constitutive promoter (usually CaMV 35S). This of course results in the relevant enzyme being expressed or repressed throughout the plant, rather than being directed to the lignin-forming parts of the plant. Many of the adverse effects reported may be due to this non-targeted expression [100]. Fewer effects, and consequently fewer indirect effects to persistence and/or survival may therefore be expected if the gene(s) are under the control of xylem-specific promoters for example that are specific to the lignin-forming parts of the plant [103].

MODIFIED OIL AND FAT CONTENT

3.51 The important points to consider in the assessment of the potential for modified oil or fat content to alter the persistence or survival of the plant, are whether the modification has any effect on the type or amount of membrane fatty acids present, or if the changes affect the location within the plant where the modified fatty acids are accumulated. The important role of membrane fatty acids in responding to temperature changes and ensuring maintenance of membrane fluidity means that changes to membrane fatty acids are likely to have an adverse effect on the


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persistence and survival of the GM plant in the environment. Modification of membrane fatty acids may also result in a reduced level of seed germination with its concomitant reduced effect on the long-term survival of the plant.

3.52 As discussed in the review section, plants are able to tolerate high levels of unusual fatty acids in their seeds. One of the reasons for this is that these fatty acids have no structural function and are stored separately in designated storage lipids (seeds), away from the membrane lipids. Separation of the storage fatty acids from plant membranes is necessary to avoid excessive structural variation occurring in membrane lipids and any consequent adverse effects on normal plant growth and development. The use of seed specific promoters in combination with the transgene may assist in this segregation. Modifications that do not use seed specific promoters to target the altered fatty acid away from the membrane lipids should therefore be assessed as having the potential to reduce the survival of the GM plant in the environment.

3.53 Another possible scenario in which the modification of seed fatty acids has an effect on membrane fatty acids is where the enzymes involved in the biosynthesis of seed fatty acids are structurally related to those involved in the metabolism of membrane lipids. Shanklin and Cahoon (1998) [365] reported that the structural similarities between the enzymes was a consequence of them being expressed from a number of so-called �housekeeping genes� that have been recruited and subsequently specialised for the production of particular fatty acids.

3.54 The existence of similar enzymes in the biosynthesis of both membrane and seed fatty acids may result in incorrect sequestration of modified seed fatty acids to the plant�s membranes, and is of particular relevance for plants modified to express �novel� fatty acids that are not produced by the unmodified plant, as the correct sequestration pathways may not exist. Such modifications may therefore require the alteration of other enzymes (such as acyltransferases and phospholipases) in order to achieve the correct targeting of the fatty acid and its exclusion from the cell membranes, and this process may need to be considered as part of the risk assessment process. The development of GM plants designed to produce and accumulate only moderate amounts of a single fatty acid is however less likely to be affected by such problems.

3.55 The review of plants altered for modified oil or fat content identified two areas that have implications to the persistence and/or survival of the GM plant in the environment. These effects are associated with plants modified to express increased levels of oleic acid (18:1) and stearic acid (18:0) respectively:

• altered sensitivity to cold; and


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• altered seed germination.

Fatty acid modification and altered sensitivity to cold

3.56 Stress caused by changes in temperature is the most typical form of plant stress [366]. Altered sensitivity to changes in temperature, particularly a reduction in temperature, has been identified as one of the key characteristics associated with a modification in the fatty acid content of plant membranes. As with most other organisms, plants must maintain a fluid state to their membranes as a prerequisite for unimpaired survival at low temperatures (Lyons, 1973; cited by [367]).

3.57 Although altered sensitivity to temperature changes has been described as a trait likely to be changed by the modification of membrane fatty acids, changes to some storage fatty acids, principally oleic acid, is also reported to alter the sensitivity of the GM plant to cold.

3.58 As discussed in the review part of this report, oleic acid content in plants is increased by reducing expression of the fad2 gene that encodes a 18:1 desaturase. The effect of the modification on cold sensitivity appears to be plant specific, with no effect reported for high oleic sunflowers, whereas the alteration of Arabidopsis to express constitutively an antisense form of fad2 caused the GM Arabidopsis to be unable to survive at <6 ºC (growth at 22 ºC was unaffected). Similar results to those reported for Arabidopsis are expected in crops such as soybean and oilseed rape [368].

3.59 The absence of any effects in the sunflower is thought to be due to the different biochemistry and physiology of this plant compared with Arabidopsis. Membrane lipids in sunflower seeds are significantly less unsaturated than those in the other crops. The purpose of fad2 silencing is to reduce the overall level of polyunsaturation. Because sunflowers naturally have a lower level of unsaturation in their lipids, then they may be more adapted to the lower levels of membrane lipid unsaturation caused by reduced expression of the fad2 gene [368].

3.60 Studies with Arabidopsis with a mutated fad2 gene have demonstrated that vegetative tissues require the activity of the 18:1 desaturase (encoded by fad2) and that therefore the constitutive antisense expression of fad2 results in adverse effects to membrane lipids and plant death at <6 ºC. Similar results may be expected in oilseed rape and soybean.

3.61 One approach to preventing low temperature effects on high oleic crops is therefore linking the expression of the antisense fad2 gene to a seed specific promoter. A more sophisticated approach is to engineer low-temperature induced expression of the fad2 gene, although this would reduce the oleic acid content of the plant.


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3.62 It should be noted that although high oleic GM oilseed rape and soybean may be susceptible to low temperatures this trait is not expressed in all GM crops modified for antisense expression of a fad2 gene. Field trials of soybeans modified for antisense expression of the fad2-1 gene (a leaky allele of fad2) that were conducted in the USA (1995-1997) found that the GM varieties were competitive in terms of yield with the non-GM lines, with neither the transformation process nor the fatty acid phenotype having a detrimental effect on the yield [163]. Variations in environmental conditions between the different trial sites also had no effect on the fatty acid composition of the seed oils in the transgenic soybeans [163].

3.63 The difference between the soybeans expressing antisense fad2-1 and Arabidopsis expressing antisense fad2 was due to the antisense fad2-1 gene not resulting in complete inhibition of expression of the 18:1 desaturase. Therefore the antisense expression of fad2-1 is assessed to be sufficiently ineffective to allow some production of the 18:1 desaturase and enable to survival of the transgenic soybeans in the environment to be no different to the non-modified strains, including their ability to grow at low temperatures.

Fatty acid modification and altered seed germination

3.64 The modification of oilseed rape for the production of high levels of stearic (18:0) and lauric (12:0) acid is reported to affect the germination characteristics of the plants under some limited environmental conditions [369]. Changes to germination are likely to alter the survival and persistence of the GM plants in the environment.

3.65 In order to make a full assessment of the effect that the modification of fatty acid content may have on crop plants such as oilseed rape it is viewed as beneficial to review some of the basic processes involved in the germination of such plants. Crops such as oilseed rape are annuals, derived originally from weedy ephemeral plants. These progenitors of the current crops persist in the environment by germinating in disturbed habitats that would otherwise be occupied by perennial species. In order for the weedy plant to be successful it needs to be able to transfer its seed to newly disturbed habitats, or to remain dormant in an environment and wait for disturbance (and removal of the perennial competitors) to occur [369].

3.66 Therefore if the weedy plants do not possess long-range dispersion mechanisms then they must adopt a waiting strategy and be able to detect temporal changes indicating disturbance in their own environment before initiating germination [369]. Temporal monitoring of disturbance requires the seeds to be able to:

• remain dormant in the presence of perennial competitors;


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• possess sufficient seed longevity to allow survival between periods of disturbance; and

• cue their germination process when environmental factors such as temperature, nutrient levels and light quality indicate disturbance and the removal of perennial competitors.

3.67 The traditional breeding of most crops has however selected against seed dormancy and germination cueing mechanisms to ensure high uniform rates of germination shortly after sowing. This selection means that both conventional crops and their GM derivatives are unlikely to establish persistent feral populations in the environment, unless their dormancy or germination cueing properties are altered to become more similar to its weedy relatives [369].

3.68 The importance and relevance of these points to transgenic crops modified for altered oil content in their seeds, is that such modifications change the composition of the carbon stores that are the only energy sources available to the germinating seedlings prior to the initiation of photosynthesis [370]. Changing the oil content of the plants may therefore cause changes in the proportion of seeds surviving in the soil, the proportion of seeds emerging following germination and the timing of emergence and seedling vigour [370]

3.69 Effects on germination have been observed for Arabidopsis thaliana [368] as well as for high laurate and high stearate oilseed rape [369].

High stearate oilseed rape � reported altered effects on seed performance

3.70 Although Linder (1998) [369] reported altered seed performance for oilseed rape modified to produce high levels of stearic acid (18:0) that may increase its persistence or survival in the environment, the alterations observed only occurred under limited environmental conditions. Under conditions of high temperature, high nutrients and full light; or low nutrients and darkness, the GM seeds exhibited reduced germination and greater induced dormancy compared to the wild-type [369].

3.71 However, under conditions of low and optimum temperatures, or at high temperatures in low light conditions, the seed performance (germination and dormancy) of the GM seeds were identical to the non-GM parent lines [369].

3.72 Lower germination levels are unlikely to increase persistence in the environment as they should result in lower establishment densities. However, increased induced dormancy of the GM seeds relative to the non-GM parent lines may increase the


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survival of the GM seeds in the environment by improving their capacity to form a larger and more persistent seed bank [369, 371]

3.73 The level of risk associated with the observed changes is of course dependent on the necessary environmental conditions (high temperature (35 °C), high nutrients and full light, or low nutrients and darkness). Without these conditions the induced dormancy is probably only a step away from seed death and is consequently of negligible importance in terms of increased survival of the GM plant [369].

3.74 The required conditions do though occur during late summer at higher latitudes. Seed shatter during harvesting at these times may release a significant quantity of seeds onto the soil, and result in a dormant seed bank that may persist [369].

High laurate oilseed rape � reported altered effects on seed performance

3.75 High laurate oilseed rape is also reported to demonstrate altered germination and dormancy relative to the non-GM parent lines under certain environmental conditions [369], coupled with a higher relative growth rate 2-4 weeks post-emergence [370]. Under conditions of low temperature (10 °C), the GM seeds germinated later and with a lower success rate than the non-GM lines, with the differences enhanced in low nutrient soils. These factors do not favour greater persistence of the GM plants relative to the wild-type [369]. This conclusion assumes that the greater growth rate 2-4 weeks post-emergence is not sufficient to allow the GM plants catch up with the earlier germinating non-GM varieties.

3.76 However, under optimal temperatures, germination times are reported to be more similar, and therefore the increased growth rate 2-4 weeks post-emergence may give the GM plants an advantage over non-GM varieties [369] and increase their survival as part of a feral population.

♦ Implications to the growth of high laurate or high stearate transgenic crops

3.77 Although the findings reported by Linder (1998) [369] and Linder and Schmidt (1995) [370] found that oilseed rape genetically modified to produce higher levels of saturated fats demonstrated altered characteristics, that could, under certain conditions improve its potential to persist in the environment relative to the wild-type, the same effects were not found in all plants [369]. Effects were very different with seeds from plants with different genetic backgrounds [369].

3.78 The modification of plants to express high levels of stearic acid is also only reported to have a deleterious effect on normal seed development where the modification is


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sufficient to achieve a 20-fold increase in stearic acid levels. A lower level of inhibition in B. napus had no effect on seed viability [151, 156].

3.79 The results therefore reinforce the requirement for releases of such crops to be assessed on a case-by-case basis. The observed changes were attributed to a combination of the modified trait and the genetic background of the parent lines [369]. The transformation process itself has been assessed not to have an effect, with oilseed rape modified for tolerance to the herbicide glufosinate ammonium for example demonstrating no greater persistence in the environment than wild-type varieties [356]. The GM lines in this study did not survive in the environment for more than two years [356].

MODIFIED MICRONUTRIENT CONTENT

3.80 The potential for modifications to micronutrient content in plants to affect their persistence and/or survival in the environment is relatively high as the compounds involved often have a range of roles in the plant. Many of the changes to persistence or survival are therefore viewed as indirect effects of the modification(s).

3.81 Of the modifications reviewed in the Modified Micronutrient Content section of the report, three areas have been identified as having potential effects on persistence and survival:

• the modification of the fatty acid α-linoleic acid;

• the modification of flavonoid content; and

• the modification of iron content.

Modified α-linoleic acid content

3.82 The fatty acid α-linoleic acid (18:2∆9,12) is involved in the response by plants to pathogens and is essential in pollen development. These factors therefore need to be considered when assessing plants modified for altered levels of this compound. The role of α-linoleic acid in response to pathogens suggests that any reduction in the levels of this compound in plants may confer increased susceptibility to pathogens and a reduced ability of the plant to persist and survive in the environment.

3.83 Reduction in α-linoleic acid content will also have an adverse effect on pollen development and consequently reduce the long-term survival of the modified plant in the environment.


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Modified flavonoid content

3.84 Flavonoids consist of a large and diverse group of polyphenolic compounds that are ubiquitous in plants. The roles of plant flavonoids in pathogen resistance and protection against UV radiation however mean that the modification of the quantities of these compounds in plants could have subsequent effects on the ability of the modified plant to persist or survive in the environment.

3.85 Any modification involving an alteration of flavonoid content may therefore be expected to exhibit indirect changes leading to reduced persistence and/or survival of the plant in the environment.

Modified iron content

3.86 As discussed in the previous chapter, iron content in plants has been improved through genetically modifying the plant to express a ferritin gene. Transgenic lettuce expressing this gene exhibited iron levels between 1.2 and 1.7 higher than the control plants [201].

3.87 However, in addition to higher iron levels, the transgenic plants also showed enhanced growth during early development, with a 27-42 percent greater biomass than the non-GM controls within three months of germination. This was proposed to be due to a superior rate of photosynthesis in the transgenic plants [201]. The higher growth rate demonstrated by the GM lettuces, means that these plants would be expected to out-compete the non-GM varieties following germination. The implications of this in terms of persistence and survival though are not straightforward. Whilst the faster growing GM lettuces may out-compete the non-GM lettuces, their greater size may result in them being more attractive to herbivores, thereby limiting their survival in the environment.

MODIFICATION FOR SPECIALITY MEDICAL APPLICATIONS

3.88 Information on the effects posed by modifying plants to express speciality compounds for medical applications on their persistence and/or survival in the environment is limited. This is a consequence (in many cases) of the studies not addressing persistence and survival, as the research has focused on the correct production of the target compound. Also, because many of the crops developed with such modifications are grown in glasshouses then the issue of altered persistence/survival is of less relevance than in the field situation as the release can be more easily contained.


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3.89 However, thought is already being given to growing plants modified to express medical compounds60 in the field environment, and in such cases their persistence and/or survival relative to the wild-type will need to be addressed. Such plants have already been grown in several field trials (25 examples in APHIS records)61.

3.90 The review of such �pharmaceutical plants� only identified the following modifications which are assessed as having a potential effect on persistence and/or survival:

• tobacco plants modified to produce the antigen merozoite surface protein (MSP1) as part of a study to develop an edible vaccine to malaria, flowered but did not set seed. This of course would reduce the long-term survival of the GM plant; and

• plants modified to express ribosome inactivating proteins (RIPs). These proteins are described as one of the most active inhibitors known. Expression of an RIP from Pokeweed (Pokeweed antiviral protein (PAP)) in potato caused the inhibition of some protein synthesis in the potato, and caused rolling up of the leaves at the site of infection. This is assessed to reduce survival, should such plants be released into the environment.

Persistence of the products of the genetic modification

3.91 The persistence of the products of the genetic modification in the environment and their effect on non-target organisms may need to be considered as part of the assessment of �pharmaceutical plants�. Although this is slightly outside the scope of this report it is included here for completeness.

3.92 Biopharmaceuticals usually elicit responses at low concentrations, and may be toxic at higher levels. Many have physicochemical properties conferring environmental persistence and lipophilic characteristics that might cause them to persist in the environment or bio accumulate in living organisms, possibly damaging non-target organisms. Various biological containment systems have been reported including:

• the induction of the relevant transgenes post-harvest, thereby preventing environmental exposure to the pharmaceuticals;

• the activation of the product only once it has been purified from harvested plant tissue;

60 Also referred to as �pharmaceutical plants�. 61 In one case there was a significant problem caused by cross-pollination of conventional maize by GM crops grown by Prodigene (www.biomedcentral.com/news/20021120/03/). This lead to new


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• the use of RNA virus to express the foreign protein within the plant. In a �normal� transgenic plant, the transgene(s) is stably incorporated into the genome of the plant, transcribed through the nuclear apparatus and inherited by the next generations. In contrast plant RNA viruses provide a temporary expression system within the plant. The viruses are engineered to carry and replicate foreign genes within susceptible host plants. The sequences delivered by the viruses into the infected plant cell remain part of the viral RNA and do not become part of the plant genome, therefore they are not inherited [210]. Such viral vectors may have advantages over plant genomic engineering, including short cycle time, ease of scale-up and a wide host range that allows for the expression of the gene within different plant species using the same construct [210]; and

• use of plant tissue culture instead of whole plants (although this does have higher cost implications and therefore may be less desirable commercially.

MODIFICATION FOR SPECIALITY INDUSTRIAL (NON-MEDICAL) APPLICATIONS

3.93 Of the modifications addressed in the review section of this report that are designed to produce compounds for non-medical/industrial applications, only the modification of plants to synthesise biodegradable plastics, and the production of pest resistant compounds, have identified any adverse effects to the persistence and/or survival of the GM plant.

3.94 One issue associated with phytic acid has also been identified, although this is not linked directly to the genetic modifications currently in use and development. As discussed in the section on �Production of phytase�, plants such as maize and barley have been developed by conventional (non-GM) breeding for reduced phytic acid content. This approach is different from transgenic plants modified to express phytase, which have no reduction in phytic acid content.

3.95 However, should any GM plants be developed where phytic acid levels are reduced (to improve phosphate availability to non-ruminant livestock for example), then the role of phytic acid as a compound produced by plants as a protective agent against damage by moulds and oxidative damage during storage may need to be addressed. Reduced phytic acid content in the plants may reduce their potential to persist in the environment.

regulations governing isolation distances for non-food maize (www.aphis.usda.gov/lpa/news/2003/03/gepermits_brs).


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Modification of pest resistance

3.96 As discussed in the review of modifications, the alteration of plants to improve their resistance to pests is likely to have a direct effect on increasing their ability to persist and survive in the field environment. However, such advantages do not necessarily apply outside the field environment, especially during periods where the particular pest(s) is not present. This has been found to be the case for transgenic x wild type hybrids as well as the transgenic crops [357, 372].

3.97 A study with hybrids of sugar beet (modified to express resistance to beet necrotic yellow vein virus (BNYVV)) and Swiss chard (a close cultivated relative of beet) for example, found that the virus-resistant hybrids only demonstrated enhanced survival as feral plants during periods of high infestations of BNYVV [357]. The establishment of feral populations of the transgenic hybrids was therefore dependent on high levels of the virus. Under conditions of low background infestation of BNYVV the transgenic hybrids produced consistently lower levels of biomass, and this would reduce their ability to compete with non-GM hybrids (or feral Swiss chard and sugar beet). Competitive ability is also likely to be limited by the significantly lower rate of bolting of the transgenic hybrids [357].

3.98 Hybrids of wild type sunflowers and cultivated varieties genetically modified to express the Bt toxin and wild type have been found to exhibit greater fecundity (in terms of seed production) in the field environment [372]. Such an increase, which was as much as 50 percent in some plants, may be expected to improve the survival and persistence of the modified plants in the environment, although the study did not investigate this [372].

3.99 The important finding from the sunflower study was that the increase in fecundity observed in the field environment was not replicated when the same hybrids were grown in greenhouses in the absence of the lepidopteran pests [372]. This finding meant that the effect on fecundity was not a direct consequence of the expression of the cry1ac gene (and production of the Bt toxin). It was proposed that the observed effects observed were a response by the Bt+ plant to reduced attack and herbivory from lepidopteran pests and were an indirect effect of the genetic modification.

3.100 On the basis of this study, the expression of the Bt toxin (and possibly any other pest resistance trait) may therefore be viewed as improving the persistence and survival of the GM plant at two levels:

• a direct effect � expression of pest resistance will improve survival through reduced attack and damage by the relevant pest(s); and


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• an indirect effect � a reduction in attack and damage by the pest(s) may allow the plant to devote more resources to other traits, such as increased growth or seed production.

3.101 However, the modification of maize to express the Cry 1 Ab protein has also been reported to increase the plant�s lignin content. This is assessed as an indirect effect of the modification with implications to the persistence and survival of the GM plants. This will potentially improve the persistence and survival of the modified maize in the environment through:

• altered and therefore possibly reduced herbivory;

• altered and therefore possibly reduced attack by boreing invertebrates not susceptible to the Bt toxin;

• improved resistance to wind and rain; and

• improved resistance to decay once the plant has died.

Production of biodegradable plastics

3.102 A reason for the numerous adverse effects reported for PHA modified plants in the review section of the report, is in part due to the review charting the development of plants for the production of polyhydroxyalkonates (PHAs), rather than presenting the most recent developments. Many of the early transgenic plants developed in this area, such as those where accumulation of PHB was targeted to the cytoplasm, were discarded because of the adverse effects on the growth and development of the modified plants.

3.103 The modification of oilseed rape to express PHB in its leucoplast, which represents a later stage in the development of this modification, was reported to have no effect on plant phenotype or germination rate compared with the wild-type strains. However, the plants were only modified to express PHB at a level of 7.7 percent (fw). Higher levels are required for the crops to be commercially viable and it is possible that adverse effects may arise as the levels are increased.

Production of trehalose as a stabilising agent

3.104 The role of trehalose as an osmoprotectant compound means that any modification involving trehalose will have implications to plant�s tolerance to drought. Increased trehalose levels are likely to improve drought tolerance, and therefore potentially persistence and survival in the environment. However, the adverse effects on plant


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morphology identified in plants modified for enhanced accumulation of trehalose means that improved survival may not be realised.

MODIFIED PLANT GROWTH

3.105 The purpose of the modifications reviewed in Chapter 2 �Modified Plant Growth� was to improve the tolerance of the plants to the key abiotic stresses of drought, high salt levels and low/freezing temperatures. These modifications are designed specifically to improve the ability of the plants to survive stresses that might otherwise reduce the yields or even the survival of the crop in the field environment. Such modifications should therefore, by design, have a positive effect on the persistence or survival of the transgenic crop in the environment.

3.106 However, enhancement of the plant�s tolerance to a particular stress may not always improve the persistence and/or survival of the plant in the environment, and some stress tolerance modifications have been found not to improve the persistence or survival of the plant. With respect to the risk assessment process it may be appropriate to commence the assessment assuming that persistence is increased, and then evaluate these three key points to determine whether improved persistence/survival will occur:

• the potential for the modification to cause adverse phenotypic effects, and the severity of the effects caused;

• the behaviour of the modified plants in the absence of the abiotic stress; and

• the stress(es) to which tolerance may be conferred.

3.107 Some of the modifications have been reported to cause serious phenotypic deformities in the transgenic plants which are likely to reduce the plant�s survival in the environment to some degree even if it has enhanced stress tolerance. However, if the effects caused by the genetic modification are still less significant than those caused by the abiotic stress to non-tolerant plants, then the GM plants will still have a competitive advantage and persist in the environment relative to non-tolerant or wild type plants.

3.108 For the majority of the modifications, the plants only have a selective advantage over wild type plants during the periods when the stress occurs. Competitiveness in a non-stressed may be of equal importance. Therefore if, as a consequence of the modification, the transgenic plant is less competitive than the wild type under non-stressed conditions and the stress is only realised for a limited period of time then the modification may not have an overall positive effect on the persistence or survival of


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the crop in the environment. Linking expression of the transgenes to a stress-inducible promoter is likely to limit any lack of competitiveness under non-stressed conditions.

3.109 A further consideration in assessing the effect of stress tolerance modifications on the persistence of the plant in the environment is the potential for non-specific (indirect) effects on the tolerance to other abiotic stresses. Where a modification confers enhanced tolerance to more than one stress, for example drought and salinity then the selective advantage relative to wild type plants will be greater. Unless this advantage is negated by adverse phenotypic effects then the plant is likely to be more persistent in the environment. Enhanced tolerance to more than one stress increases the period in which the plant has a competitive advantage over other (non-GM) plants in the environment, and reduces any impact of poor performance under non-stressed conditions.

3.110 The major limitation in assessing the effect of modified stress tolerance traits on the persistence or survival of the GM plant in the environment is that most of the studies conducted to date (2003) have focused on the development of the GM plant. Only a limited number of studies have investigated the potential for indirect effects or the competitiveness of the plant under non-stressed conditions.

Improved drought tolerance through accumulation of osmoprotectants

3.111 As discussed, improvements to a plant�s ability to tolerate low water availability (drought) will by definition confer an increased ability of the plant to survive in the environment under drought conditions. The key considerations are the ability of the plant to survive in non-stressed environments, whether the modification has any adverse phenotypic effects and the potential for indirect effects conferring tolerance to other abiotic stresses.

3.112 To date (2003) improvements in drought tolerance through genetic modification have been achieved by altering the accumulation of various osmoprotectant compounds within the plant. Enhanced accumulation of glycine betaine, D-ononitol, fructan or trehalose has been found to improve drought tolerance in the modified plants relative to wild type.

3.113 The modification of plants to accumulate fructan or D-ononitol was found to have no adverse effect on the plant�s phenotype and should therefore have a positive effect on the persistence of the plant in environments where low water availability was likely. On the information available the modified plants are assessed to be no less competitive than wild type in the absence of drought stress. Although the sacB gene conferring fructan accumulating in the GM tobacco was under the control of a constitutive promoter fructan accumulation was very low under non-drought


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conditions (~0.05 mg g-1 fw, compared to 0.35 mg g-1 fw under drought conditions). Therefore under non-drought conditions the GM tobacco should be similar in composition to the wild type and should therefore be of similar competitiveness. Modification of plants to accumulate fructan is not reported to have any adverse phenotypic effects, but because of its role as an osmoprotectant may confer improved tolerance to saline soils.

3.114 One issue not addressed by studies with fructan accumulation is the effect on the plant of reductions in fructose levels. The role of fructose as an energy storage compound means that a reduction in fructose (caused by a proportion being converted to fructan) may have implications to the growth and survival of the plant in the environment. The significance of this has not been addressed by studies conducted to date.

3.115 The improved drought (and salt) tolerance in tobacco modified to accumulate D-ononitol was reported to be due to reduced inhibition of the photosynthetic fixation of CO2 under drought (or salt) stress. Whilst not reported by the study, if photosynthetic fixation of CO2 was unchanged under non-stressed conditions (relative to wild type) then the GM plants would be expected to be as competitive as the wild type. In this case the modification would be expected to confer a positive effect on the persistence and survival of the GM plant in the environment.

3.116 The modification of plants to accumulate glycine betaine is assessed to have a predominantly positive effect on the potential for the plant to survive in the environment. Glycine betaine accumulation has been achieved by several different mechanisms. The modification of plants to overexpress spinach PEAMT, betA, or codA genes is reported to achieve the greatest accumulation of glycine betaine to date. No adverse effects on growth were reported. The significance of glycine betaine accumulation with respect to stress tolerance is that it confers enhanced tolerance to drought, chilling, salt, heat and strong light. Therefore any plant modified to accumulate this osmoprotectant would have a selective advantage under a wide range of environmental conditions. Therefore, although the behaviour of the plant under non-stressed conditions has not been reported, the number of competitive advantages offered by the modification suggests that the transgenic plants will demonstrate enhanced persistence or survival in the environment.

3.117 Accumulation of trehalose is however only likely to confer a positive effect on persistence if the relevant transgene(s) is under the control of a stress-inducible promoter. Under a constitutive promoter system the plant exhibits severe phenotypic deformities (although it is still drought tolerant). Rice modified to accumulate trehalose under stress conditions (stress-inducible promoter) displayed vigorous growth under stress conditions, and significantly, improved photosystem function and photosynthetic capacity under non-stressed conditions. Such a modification is


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therefore assessed to have a positive effect on persistence/survival of the plant in the environment. Trehalose is also reported to be superior to other sugars as an osmoprotectant and offers enhanced tolerance to salt and cold as well as drought.


3.118 GM-based improvements in salt tolerance have been developed through two mechanisms (detoxification and homeostasis). Improving a plant�s ability to tolerate soils with a high salt content will by definition confer an increased ability of the plant to survive in more saline soils. Whilst the key considerations in assessing the effect on persistence and survival are similar to those for drought and cold tolerance (occurrence of adverse phenotypic effects and the potential for indirect effects), the issue of competitiveness under non-stressed conditions is less significant. High salt content in agricultural soils is the result of the particular mineral content of the soil or irrigation and subsequent evaporation of the water. Therefore the salt content of soils is likely to fluctuate less than stresses such as drought and cold during the growing season. Plants grown in high salt soils will not need to be competitive in low salt soils if such conditions are not encountered during the growing season.

Detoxification

3.119 Detoxification strategies involve the overexpression of enzymes involved in oxidative protection or the low level accumulation of osmoprotectants such as mannitol, sorbitol and proline. The only adverse effects reported were for high levels of sorbitol accumulation (>3 µmol g-1 fw) where the plants developed necrotic lesions on their leaves, infertility and/or the inability to regenerate roots. Tobacco plants overexpressing mannitol were 20-25 percent smaller under non-stressed conditions, although this may not be significant for the reasons discussed above.

3.120 All of the other detoxification-based salt tolerance strategies reviewed in Chapter 2 are therefore likely to confer a positive effect on the persistence and survival of the plant in the environment. The modifications are likely to confer tolerance to both salt and drought. The enhanced root biomass and flower development exhibited by plants modified to overproduce proline are assessed to confer further selective advantages to the plant and should further improve the persistence and survival of the GM plant in the environment.

Homeostasis

3.121 Homeostasis-based modifications to improve salt tolerance are limited to date (2003). No adverse effects on the plant�s phenotype have been reported, with the modification of tomato to express the HLA gene resulting in reduced fruit loss in addition to enhanced salt tolerance. All of the modifications reviewed in Chapter 2


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are therefore assessed to have a positive effect on persistence/survival in soils with a high salt content. Persistence in low saline soils (non-stressed) has not been reported, but may not be an issue. GM plants designed to be tolerant to high salt levels are unlikely to be cultivated in soils that do not have a high salt content.

Improved cold tolerance

3.122 Improvements in cold tolerance offer the potential for plants to withstand lower (often sub-zero) temperatures. In temperate environments sub-zero temperatures are likely to occur at some point during winter, and therefore any improvement in cold tolerance will improve the survival of the plant in the UK environment.

3.123 The genetic modification of plants to improve cold tolerance is at an early stage of development. Genetic modification strategies have focused on:

• improving the plant�s cold acclimation pathway wholesale. This is also reported to enhance the plant�s tolerance to drought and salt;

• modifying expression of specific genes within the cold-acclimation pathway. This though has been reported to have little effect at the whole plant level; and

• the accumulation of antifreeze proteins.

3.124 The alteration of plants to express their cold-acclimation pathway constitutively, and thereby separate expression from a low temperature stimulus, has been found to cause deleterious effects to the plant�s phenotype. However if the overexpression is controlled by a stress-inducible promoter (cold or dehydration) then the adverse effects are not realised. This modification is likely to have a positive effect on the persistence of the transgenic crops in a temperate environment as cold tolerance is improved, suggesting that the plants can withstand lower temperatures and therefore overwinter at higher latitudes or altitudes relative to the wild type.

3.125 The expression of AFPs has been found to have limited effect on improving the cold tolerance of the plant. However, because of the similarity of AFPs to some pathogenesis-related proteins, then the expression of AFPs may have an indirect effect on the disease resistance of the plant. Such an effect will have a positive effect on the persistence/survival of the plant in environments where the pest is present.


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CONCLUSIONS

3.126 The assessment of the effects of modified compositional and key stress tolerance traits has identified modifications that have both direct and indirect effects on the persistence and survival of the plants in the environment. The assessment is though limited by the fact that for many of the modifications the effect on persistence and/or survival has not been addressed. The effects that have been identified have been summarised in Table 3.1 at the end of this section.

3.127 Direct effects are those that occur as a direct consequence of the compositional modification, for example changes in tuber size or yield in potatoes modified for altered starch characteristics. Although such direct effects have been identified in this report, it is assessed that GM plants with characteristics that have an adverse effect on their fitness in the field (i.e. reduced ability to persist and survive) are unlikely to be released as part of a commercial development programme. The only exception to this is trees with reduced lignin content.

3.128 With the exception of the modification designed to improve tolerance to abiotic stress, very few of the direct effects identified have been assessed as having the potential to increase the persistence or survival of the modified plant in the environment.

3.129 A greater number of changes to persistence or survival of the GM plants are concluded to occur as a consequence of indirect effects. These arise as a consequence of the modification of biosynthetic pathways or compounds that have a number of roles in the plant. Examples of such modifications include changes to flavonoid levels (reported as a means to improve nutraceutical properties of the plant, but with the potential to alter resistance to UV radiation), puroindoline content (reported as a means to modify grain hardness, but with the potential to alter the resistance to fungal pathogens), and expression of the Bt toxin (conducted as a means to improve pest resistance, but also found to alter lignin content in maize). As a consequence of the effects identified in this report, it is concluded that indirect effects on persistence and/or survival are likely to occur with any modification that affects biosynthetic pathways or compounds that have a multitude of roles within the plant.


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Table 3.1 � Summary of effects of modified compositional traits on the persistence or survival of crop plants in the environment General purpose of

the modification Gene or enzyme modified Crop62 Effect observed Assessed effect on persistence or survival in the field

Protein content Increased production of 1,3-1,4-β-glucanase

Potato Reduced tuber yield and adverse cell wall morphology.

Negative (i.e. the modification will reduce the ability of the plant to persist or survive in the environment). In this case the reduction is due to diminished energy stores.

Protein content Reduced formation of vegetative storage proteins (VSPs)

Soybean No effects looked for, and therefore none reported.

Proposed to cause a negative effect on plant persistence or survival.

Starch or sugar content

Sucrose-phosphate synthase Tomato Increased yield. Positive (competitive advantage) or negative (greater exposure to herbivory).

Sugar content Increased sucrose isomerase (resulting in reduced sucrose levels). Crops modified using constitutive and phloem-specific promoter.

Tobacco and potato

Sever retardation in growth (tobacco) and severe impairment in tuber sprouting (potato).

Negative (reduced growth and development).

Sugar content Increased sucrose isomerase. Tuber specific promoter.

potato No adverse effects observed Negative due to reduction in starch levels. This is assessed to reduce survival of the plant due to the importance of starch as an energy storage material.

Sugar content Increased fructan accumulation through expression of sacB gene from B. subtilis

Sugar beet Improved drought tolerance Positive effect in areas with low water availability. No significant effect under non-drought conditions.

Starch content Expression of Sh2r6hs (increased AGPase activity)

Wheat and rice Improved CO2 fixation Positive due to improved photosynthesis and therefore improved plant growth.

Starch content Increased AGPase activity Wheat and rice Reduced seed abortion Positive due to greater numbers of seeds produced per plant.

62 Refers to the crop in which the modification has been reported. Depending on the trait(s) altered the modification may be applicable to other crops. Where a crop is not listed (for example as in the case of lignin modification), then the modification and its associated reported effect is applicable to a wide range of plants.


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survival in the field Starch content Decreased AGPase activity Potato Smaller but greater number of

tubers and earlier flower formation

Negative or positive. The effects on survival depend whether early flowering will provide a competitive advantage, and whether a reduction in tuber size will reduce overwintering survival of the tubers (and therefore next season�s growth).

Sucrose modification Decreased activity of triose-phosphate translocator

Potato Increased growth rate during the dark period

Positive or negative. Increased growth rate may confer a competitive advantage but may also make the plant more prone to herbivory.

Starch content Expression of yeast invertase, bacterial glucokinase or bacterial sucrose phosphorylase

Induction of glycolysis and greater partitioning of carbon into respiration. No increase in starch accumulation


Starch content Expression of inorganic pyrophosphatase enzyme

Potato Accelerated sprouting Positive or negative. If sprouting occurs too early in the season then conditions may be too cold or wet for the potatoes to survive. Otherwise, the modification should improve survival as it will provide a competitive advantage.

Starch quality Inhibition of starch branching enzyme

Potato Reduced tuber yield and decreased total starch content


Starch quality Increased expression of pinA and pinB

Maize and rice Increased grain hardness (direct effect), and improved resistance to specific fungal pathogens (indirect effect)

Positive (reduced herbivory and greater fungal resistance).

Sucrose content Reduced expression of the invertase enzyme β-fructosidase

Tomato Reduced fruit size and increased ethylene production (causing faster fruit ripening)

Negative (smaller fruits and shorter time for the fruits to remain on the plant. Therefore seed dispersal by herbivores will be reduced).

Sucrose content Inhibition of sucrose synthase Tomato Reduced fruit setting Negative � modification assessed to reduce seed development.


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survival in the field Cellulose content Not reported Increased or decreased

cellulose content Positive if cellulose increased (due to improved resistance to decay). Negative if cellulose decreased (due to reduced plant strength and greater herbivory).

Pectic polysaccharide content

Expression of eRGL (confers overproduction of rhamnogalacturonan lyase

Potato Reduced pollen fertility and formation of smaller, more wrinkled tubers

Negative due to reduced ability of the plant to produce fertile pollen and a reduction in energy stores (smaller tubers).

Raffinose family oligosaccharide content

Reduced production of raffinose family oligosaccharides (RFOs)

alfalfa Reduced ability to deal with cold and drought

Negative due to reduced resistance to environmental stresses (cold and drought).


Reduction in lignin content (direct effect)

Negative (reduced resistance to adverse weather, attack by pests and herbivory).


Reduction in lignin content (indirect effect)

Negative due to reduction in overwintering capability because of possible link between the genes controlling lignin synthesis and those involved in winter survival traits [1]

Lignin modification Inhibition of phenylalanine ammonia lyase (PAL)

Reduction in levels of phenolic compounds

Negative (due to the role of phenolics in pest resistance and therefore greater susceptibility of the plant to pests).

Lignin modification Inhibition of CCoAOMT and O-methyltransferase (OMT)

Reduction in plant growth and flowering activity

Negative due to reduced plant growth.

Lignin modification Alteration of levels of CAD, CCR or OMT

Increased decay. Greatest effect observed with OMT

Negative (plant will persist for less time in the environment once it has died).

Lignin modification Inhibition of hydroxycinnamoyl CoA ligase (4CL)

Arabidopsis thaliana and aspen

Collapsed cell walls and stunted growth in Arabidopsis but elevated growth rate in aspen. Only a limited number of plants were used in the aspen study [2] and the results should be viewed with caution.

Negative (in the Arabidopsis) but positive in the aspen.


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survival in the field Lignin modification Downregulation of caffeoyl CoA

O-methyltransferase (CCoAOMT)

Tobacco and poplar

Reduced cross-linking in the lignin

Negative due to reduced strength of the lignin. This would make the plant more susceptible to adverse weather events, and possibly also to decay, pest attack and herbivory.

Lignin modification Downregulation of hydroxycinnamoyl CoA reductase (CCR)

Indirect effects likely to occur. Positive or negative depending on the nature of the indirect effect. Due to the involvement of CCR in a range of plant processes a number of effects could occur.

Oil or fat modification Any alteration of membrane fatty acids

Reduced level of seed germination. Altered membrane fatty acids will also affect the plant�s response to temperature change (and other membrane transport processes, thereby causing a range of adverse effects).

Negative due to reduction in seed germination and therefore development of the plant.

Oil or fat modification Production of a fatty acid not normally produced by the plant

Poor sequestration of the novel fatty acid into membrane lipids

Negative due to reduced ability to deal with changes in environmental conditions (temperature and water availability).

Oil or fat modification Increased production of lauric acid (12:0)

Oilseed rape Delayed and a reduced level of germination. Once germinated the plants exhibit increased growth rate 2-4 weeks post emergence. Effects only observed under conditions of low temperature (10°C).

Negative. This assumes that the increased growth rate is not sufficient to compensate for the reduced germination levels.

Oil or fat modification Reduced expression of fad2 leading to increased production of oleic acid (18:1)

Arabidopsis sp., soybean and oilseed rape. Similar effects were not seen in sunflower

No survival at temperature <6°C. The effect though is plant and gene specific, and depends on the level of inhibition that occurs.

Negative if the plant is to survive overwinter. The effect is only of relevance to winter sown or perennial crop plants.


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survival in the field Oil or fat modification Increased production of stearic

acid (18:0) Oilseed rape Greater induced dormancy and

reduced germination of the seeds. Effects only observed under conditions of high temperature, high nutrients and full light; or low nutrients and darkness.

Negative due to reduction in germination, but positive if the conditions are sufficient to cause an increase in induced dormancy.

Modified micronutrient (linoleic acid) content

Reduction in level of fatty acid α-linoleic acid (18:2∆9,12)

Reduced resistance of the plant to plant pathogens and reduced pollen development.

Negative.

Modified micronutrient (flavonoid) content

Reduced flavonoid content Reduced resistance to plant pathogens and decreased UV protection.

Negative.

Modified micronutrient (iron) content

Expression of ferritin gene Lettuce Enhanced growth during early development.

Positive unless the larger plants are more attractive to herbivores.


Production of the antigen merozoite surface protein (MSP1)

Tobacco The plant flowered but did not set seed.

Negative due to reduced seed formation.


Expression of ribosome inactivating proteins (RIPs)

Potato Inhibition of protein synthesis and leaf rolling at the point of infection.

Negative due to impaired leaf development.


Expression of resistance to beet necrotic yellow vein virus (BNYVV)

Sugar beet Reduced rate of bolting Negative. This assumes that reduced bolting will lead to reduced seed dispersion from the plant.


Expression of Bt toxin Sunflower Increased seed production (up to 50 percent). Effect only observed in the field environment in the presence of lepidopteran pests.

Positive. The observed effect is an indirect knock-on effect of the reduction in pest attack. It was not observed in a glasshouse environment (in the absence of the pest) and is therefore not a genetic consequence of the modification.


Expression of any pest resistant characteristic

Improved pest resistance (direct effect).

Positive, but only of relevance in environments containing the pest(s).


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survival in the field Modified pest resistance

Expression of Bt toxin Maize Increased lignin content Positive. This is also an indirect effect of the production of the Bt toxin. Unlike the previous example it is probably due to expression of the cry gene affecting other plant processes and is therefore viewed as a genetic consequence of the modification.

Production of biodegradable plastic

Expression of polyhydroxyalkonates (PHAs)

Reduced growth and development. However, the effects have only been observed during early stages of the modification process. The most recently developed plant exhibited no adverse phenotypic characteristics.

Negative where impaired growth occurred.

Improved drought tolerance through accumulation of any osmoprotectant

Any gene or enzyme leading to increased accumulation of an osmoprotectant

Improved tolerance to drought and high salt levels. May confer further tolerance to other environmental stresses such as low temperature.

Positive if the environmental stress is realised. Zero or possibly negative effect in environments where the stress is not realised. If the trait is expressed constitutively then the likelihood of a negative effect is higher in non-stressed environments.


Expression of the Arthrobacter enzyme COX

Production of hydrogen peroxide Negative if the H2O2 has a deleterious effect on the plant. Such effects were however not reported by the study.


Expression of betA from E. coli or codA from Arthrobacter

Oilseed rape and tobacco

Accumulation of glycine betaine, and improved tolerance to salinity, freezing and drought.

Positive effects in environments where drought, high salt or freezing temperatures are encountered.



tobacco Some accumulation of trehalose, but also stunted growth (poor leaf and root development), altered sugar metabolism and altered fertility.

Increased tolerance to drought, but the adverse morphological effects mean that persistence/survival is likely to be reduced.


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survival in the field Improved drought tolerance through accumulation of trehalose


Tobacco Better accumulation of trehalose than with expression of TPS, but adverse morphological effects still realised.

Negative, although the stunted plants did exhibit greater drought tolerance.


Constitutive expression of the otsB gene from E. coli (encoding TPP enzyme)

Tobacco Low level accumulation of trehalose, but heightened drought tolerance. Plants exhibited substantial changes in morphology and accumulated higher levels of non-structural carbohydrates.

Negative due to changes in plant morphology.

Improved drought tolerance through accumulation of fructan

Constitutive expression of sacB gene from Bacillus subtilis.

Tobacco and sugar beet

Increased fructan accumulation and improved drought tolerance. Plants exhibited more rapid growth rate.

Positive under drought conditions due to great drought tolerance. Increased growth rate is also likely to confer a selective advantage over other plants. Not known whether any changes are expressed when no drought stresses are imposed.

Improved drought and salt tolerance through accumulation of D-ononitol

Expression of cDNA encoding D-myo-inositol methyltransferase.

Tobacco Increased accumulation of D-ononitol. Photosynthetic fixation of CO2 was inhibited to a lesser extent under conditions of drought or salt stress.

Positive under conditions of drought or salt stress.


Expression of mutant version of pst1 gene

Arabidopsis


Positive under conditions of high salt levels. Plants likely to have improved tolerance to other abiotic stresses (including drought).


Overexpression of AtSOS1 Arabidopsis


Positive under conditions of high salt levels.


Overexpression of AtNHX1 Arabidopsis, tomato and oilseed rape

Improved tolerance to salt. fruit loss when exposed to salt stress.

Positive under conditions of high salt levels. No adverse effects on plant growth or phenotype reported.


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survival in the field Improved salt tolerance

Overexpression of HLA gene from yeast

Watermelon and tomato

Improved salt tolerance. Modified plants expressed less fruit loss when exposed to salt stress.

Positive under conditions of high salt levels. Greater fruit production is assessed to confer a selective advantage to the long-term survival (over several generations) of the plant. Greater fruit production may of course result in increased herbivory and therefore result in reduced survival of individual plants in the environment.

Improved salt tolerance through accumulation of mannitol

Expression of mt1D gene from E. coli in the chloroplast

Tobacco Increased accumulation of mannitol and improved tolerance to salt. Under non-stressed conditions the plants were 20-25 percent smaller than wild type.

Positive under conditions of high salinity, but probably negative under non-stressed conditions due to the smaller size of the plants.

Improved stress tolerance through accumulation of proline

Modification of natural proline inhibition systems

Mothbean and Arabidopsis

Increased accumulation of proline. Plants exhibited greater tolerance to salt and also cold. No adverse phenotypic effects reported.

Positive under conditions of high salt or low temperature.

Improved drought tolerance through accumulation of proline

Expression of P5CS gene from mothbean

Tobacco Overproduction of proline, and also enhanced root biomass and flower development.

Positive under drought conditions. The enhanced root growth and flower development are also likely to confer a selective advantage to the modified plants.

Improved salt tolerance through accumulation of sorbitol

Expression of a cDNA for sorbitol-6-phosphate dehydrogenase (S6PDH) from apple

Tobacco Increased accumulation of sorbitol (0.2-130 µmol g-1 fw). Plants expressing >3 µmol g-1 fw developed necrotic lesions on their leaves, infertility, and/or the inability to regenerate roots.

Positive under conditions of salt stress if the level of expression is restricted to <3 µmol g-1 fw. Higher levels of expression are likely to have a negative effect on persistence/survival.

Cold tolerance in the absence of low temperature stimulus

Constitutive overexpression of CBF/DREB

Arabidopsis Severe stunting of phenotype, decrease in seed yield and a delay in flowering.

Negative effect on persistence or survival.


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4. COMMERCIALISATION OF GM CROPS WITH ALTERED COMPOSITIONAL TRAITS

4.1 The purpose of this section of the report is to provide an indication of the likelihood of GM crops, modified for the types of compositional traits discussed in the previous sections of this report, to be field trialled or marketed for commercial use.

4.2 Information on the likelihood of field trials or commercialisation of individual crops has been provided as part of the reviews presented in the previous sections of this report. Much of the information presented in this section is more generic in nature and has been taken from a �Review of GMOs under research and development and in the pipeline in Europe� published by the European Science and Technology Observatory and the European Commission�s Joint Research Centre [4]. This report reviews GM plants in the research and development �pipeline� and identifies the types of modified traits that are likely to be seen in field trials or commercial applications in the next five years, between five and ten years, and more than ten years in the future.

4.3 The information from the review of GMOs in the pipeline [4] is designed to provide a more general assessment of the relative progress of crops with altered compositional traits towards commercialisation, in addition to the specific details presented in Section 2 of this document.

4.4 With respect to GM crops with altered compositional traits the report made the following conclusions63.

Current situation in Europe

4.5 At the current time (2003) there is only one GM crop modified for an altered compositional trait(s)64 that has received favourable approval from the EC�s Scientific Committee on Plants pending authorisation under the old Directive EC 90/220/EEC. This is the Amylogene potato (C/SE/96/350165) modified for altered starch content

63 Conclusions based on information obtained from a review of the scientific literature and existing GMO databases, as well as a survey of ongoing R&D projects in laboratories in Europe, analysis of the EU SNIF database, EU import needs, and GMOs in Central and Eastern European Countries. 64 Also referred to as a modified �output� trait, as the modification is designed to alter or provide a particular substance or product. 65 Identification number designated by the EC for this potato.


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(high amylopectin levels). No other GM crops modified for altered compositional traits have been submitted to date (February, 2003) within the EU.

4.6 Evaluation of the marketing applications submitted to date shows that the focus to date (February, 2003) has been on the development of GM crops with modified �input� or agronomic traits, such as pest resistance or tolerance to specific herbicides66 [9, 4]. This preponderance towards plants with modified �input� traits is also reflected in the types of GM crops undergoing experimental field trials within the EU, in which 77 percent of GM crops express modified �input� traits [4]. If a timescale of 5-10 years is taken as the time required from experimental field testing to commercialisation [4], then the focus on GM crops with modified �input� traits may be expected to remain for the next five years.

Within the next five years

4.7 GM crops with altered compositional traits reported to reach commercialisation within the next five years are listed below [4]. However, as a consequence of the focus to date (2003) on modified �input� traits then the crops listed below may be expected to appear in the EU as imported products, rather than cultivated crops:

• potatoes, soybeans and oilseed rape modified for altered starch or fatty acid content; and

• tomatoes modified for altered fruit ripening.

In five to ten years

4.8 GM crops with altered compositional traits reported to reach commercialisation in five to ten years are [4]:

• potatoes and maize modified for altered starch content;

• soybeans and oilseed rape modified for altered fatty acid content, including high erucic acid content oilseed rape; and

• oilseed rape, maize and potatoes modified for altered protein content.

4.9 Analysis of the EU SNIF67 database (which contains details of GM crops undergoing experimental field trials within the EU), showed that GM crops with modified �input�

66 See �Introduction chapter� to this report for further details. 67 Acronym stands for Summary Notification Information Format.


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traits are more prevalent in field trials than those with �output� traits (77 versus 19 percent) [4].

4.10 The relatively low numbers of field trials involving plants with modified �output� or quality traits (19 percent of total) is described by Lheureux et al. (2003) [4] as indicating that such crops are still in an early phase of research. Modified nutrient content (starch and oil for example) is reported to be the main �output� trait currently undergoing field trials [4]. This trait accounts for 11.7 percent of the total (i.e. >50 percent of all field trials involving �output� traits).

4.11 The potential for GM crops to produce compounds for medical-related applications is, on the basis of the diverse applications identified in Section 2 of this report, very large. However, such plants are to date (2003) almost absent from EU field trials (only 16 field trials conducted between 1991-2001), although they do account for 11 percent of laboratory phase trials [4]. The relative absence of such crops from experimental field trials is viewed as indicative of the early stage of development of such crops within the EU [4].

Figure 4.1 � Number of permits issued by USDA (and distribution of crops) for experimental field trials in the USA involving transgenic crops modified for the production of medical-related products (1991 to June 2002). Data taken from

Lheureux et al. (2003) [4].

0102030405060708090

100110120130

maize

soyb

ean

tobac

co rice

alfalf

aba

rley

wheat

oilse

ed ra

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suga

rcane

tomato

safflo

wer


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4.12 However, if the situation in the USA is used as an indication of future developments within the EU, then field trials of crops modified to produce medical-related compounds may be expected in the EU. In the USA between 1991-2002, 198 permits/acknowledgements were issued by the USDA for such crops. This corresponded to 315 out-of-doors field trials of on average two hectares in area, with most of the trials conducted between 1999-2002 [4]. The trials involved a range of crops (Figure 4.1), although maize accounted for the bulk (68 percent) of the permits issued.

4.13 The commercialisation of such crops is expected in the USA within the next 10-15 years [4].

Beyond ten years

4.14 GM crops with altered compositional traits reported to reach commercialisation beyond ten years are [4]:

• a broad variety of crops modified for increased yields, in particular cereals, grasses and potatoes;

• tobacco, maize, potato and tomato modified for the production of medical-related compounds;

• rice and vegetable (carrots and tomatoes) crops modified for the production of �nutraceutical� compounds;

• trees modified for altered lignin content; and

• crops such as soybean modified for reduced allergenic effects.


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5. RECOMMENDATIONS FOR FUTURE WORK

5.1 The focus of the recommendation for future work in this study is the assessment of the effects the described compositional or stress tolerance traits have on the persistence and/or survival of the GM crops in the field environment. A general conclusion from the review of the modifications that already have been, or are under development is that information on environmental behaviour is limited. In most cases this is a consequence of the relatively early stage of development that many of the crops described have reached. Evaluation of agronomic performance is likely to come after a successful and stable modification has been achieved.

5.2 Further information on the effects of many of the modifications addressed on the persistence and survival of the plant is still needed in order to assess the likely changes in persistence and survival in the environment.

5.3 In terms of specific issues identified in this report that would benefit from further investigation, three areas have been identified:

• to determine whether certain effects have a positive or negative effect on the persistence or survival of the plant in the environment;

• the further investigation of the indirect effects that may be caused following the modification of broad acting metabolic pathways, such as the initial stages of the lignin biosynthetic pathway, or the modification of cell division68; and

• indirect effects on lignin levels reported in maize modified to express the Bt toxin.

5.4 In Table 3.1 (Summary of effects on persistence and survival), two observed effects (increased growth rate and reduction in tuber size) are proposed as having either a positive or negative effect on the fate of the plant in the environment. Increased growth rate will of course result in the formation of a larger plant, and as such the plant will be more competitive for sunlight and nutrients relative to the other non-modified plants in its vicinity. Such a competitive advantage is proposed to confer


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increased survival of the GM plant in the environment, and the modification is assessed to have a positive effect. However, it is also proposed that an increase in growth rate may have a negative effect on plant survival if the larger plant is more of a target to herbivores. Further work may be of use to determine whether greater herbivory would occur and whether this would be sufficient to outweigh the advantage posed by the increased growth rate.

5.5 A reduction in tuber size in potatoes has been assessed to have a negative effect on the survival of the plant as it will result in a reduction in the level of starch (and therefore energy) reserves available for the plant to initiate the next seasons growth. Smaller tubers may also be less able to survive overwinter as their smaller size will make them less resistant to frost or freezing. Further work or information would though be useful to show this. If smaller tubers demonstrate no difference in overwintering survival compared to larger tubers then modifications that result in smaller but more numerous tubers per plant are likely to have a positive effect on the survival of the plant in the environment.

5.6 Whilst changes to cell division may not be designed to have a direct effect on a plant�s compositional traits or tolerance to environmental stress, they have been reported to confer an increase in seed size in the modified plants. As discussed with other modifications reported to alter seed characteristics, increased seed size may have both a positive or negative effect on the ability of the plant to survive in the environment. Whilst larger seeds could confer greater survival if they contain larger energy stores for the developing seedling to utilise, larger seeds may also be more attractive to herbivores thereby reducing the long term survival of the plant in the environment.

The final comment is that the information presented in this report represents the current state of the science at the time of writing. Some of the areas addressed, particularly the review of the use of plants to produce speciality compounds for medical applications, are developing rapidly with an ongoing development of new modifications. Many of the modifications identified as representing an early stage of the developmental process are likely to be superceded by new constructs with differing potential to persist or survive in the environment. Therefore a key area of future work is the periodic updating of modified compositional and stress tolerance traits with respect to potential effects on the persistence and/or survival of the plant in the environment.

68 The US company Targeted Growth Inc. have proprietary technology based on a gene controlling plant cell division. This is claimed to cause increased seed size.


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6. REFERENCES

[1] Casler, MD, DR Buxton and KP Vogel (2002). Genetic modification of lignin concentration affects fitness of perennial herbaceous plants. Theoretical and Applied Genetics, 104(1): 127-131.

[2] Hu, W, S Harding, J Lung, J Popko, J Ralph, D Stokke, C Tsai and V Chiang (1999). Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nature Biotechnology, 17(8): 808-812.

[3] Dunwell, JM (2000). Transgenic approaches to crop improvement. Journal of Experimental Botany, 51: 487-496.

[4] Lheureux K, Libeau-Dulos M, Nilsagard H, Rodriguez Cerezo E, Menrad K, Menrad M and Vorgrimler D, Review of GMOs under research and development and in the pipeline in Europe. 2003, Report produced for the European Science and Technology Observatory, and the European Commision's Joint Research Centre.

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