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Technologies for Biological

Containment of GM and Non-GM Crops

Defra Contract CPEC 47
Final Report

J.M. Dunwell and C.S. Ford

School of Biological Sciences, University of Reading, UK
November 2005

DEFRA Contract CPEC 47

Contents
List of Tables

vii

List of Figures

Glossary

Executive Summary

xiv

Introduction

1.1

Potential benefits of GM crops

1.2

Potential risks of GM crops

1.3

Risk assessment principles and gene flow

European Agriculture and GM Crops

2.1
2.2

2.3

Review of global GM field trials

2.1.1

Summary of global GM field trials

Review of European crop production

2.2.1

Summary of European field crops

2.2.2

Overseas departments

2.2.3

Forestry

Potential gene flow: recipient species and wild relatives of

European crops

2.3.1

Crops posing less risk of transgene spread

2.3.2

Major crops species

2.3.3

Forage and fodder crops

2.3.3.1 Medicago

2.3.3.2 Trifolium

2.3.3.3 Lupin

2.3.3.4 Summary of forage and fodder crops

Forestry species

2.3.4.1 Poplar

2.3.4.2 Birch

2.3.4

DEFRA Contract CPEC 47

2.3.5

2.3.6

2.3.7

2.3.8

2.3.4.3 Chestnut

2.3.4.4 Spruce

2.3.4.5 Pine

2.3.4.6 Eucalyptus

2.3.4.7 Elm

2.3.4.8 Oak

2.3.4.9 Summary of forestry species

Orchard crops

2.3.5.1 Olives

2.3.5.2 Apples

2.3.5.3 Plums and cherries

2.3.5.4 Pear

2.3.5.5 Walnut

2.3.5.6 Citrus

2.3.5.7 Avocado

2.3.5.8 Papaya

2.3.5.9 Summary of orchard crops

Grasses

2.3.6.1 Agrostis

2.3.6.2 Festuca and Lolium

2.3.6.3 Other grasses

2.3.6.4 Summary of grasses

Ornamentals

2.3.7.1 Rose

2.3.7.2 Carnation

2.3.7.3 Statice

2.3.7.4 Other ornamentals

2.3.7.4 Summary of ornamentals

Summary of European crops with potential for

transgene escape

Review of Containment Methods of Conventional Crop Species

3.1

Physical separation

3.1.1

3.2

Summary of physical separation

Biological containment

3.2.1

Natural genetic containment

3.2.2

Plastid transformation

3.2.2.1 Plastid biology

3.2.2.2 Plastid transformation methodology

iii

DEFRA Contract CPEC 47

3.2.3

3.2.4
3.3

3.4

3.2.2.3 Commercial applications

3.2.2.4 European perspective

3.2.2.5 Other species

Genetically engineered gene containment

3.2.3.1 Conditional lethality

3.2.3.2 Inducible promoters

3.2.3.3 Engineered male sterility

3.2.3.4 Gene Use Restriction Technologies (GURTs)

3.2.3.5 Apomixis

3.2.3.6 Cleistogamy

3.2.3.7 Transgene mitigation

3.2.3.8 Recoverable block of function

3.2.3.9 Inteins

3.2.3.10 Auxotrophy

3.2.3.11 Transgene excision

Summary of containment strategies

Containment issues relating specifically to trees

3.3.1

Concerns of GM forest trees

3.3.2

Containment options

101

3.3.3

Summary of containment issues relating to trees

104

Containment issues relating specifically to grasses

105

3.4.1

Concerns of GM grasses

105

3.4.2

Containment options

106

3.4.3

Summary of containment issues relating to grasses

107

Review of Transgenic Methodologies in the Bio-Pharming of

Pharmaceutical Products

108

4.1

Conventional crop species

110

4.1.1

Pharmed products on the market

110

4.1.2

Pharmed products close to the market

114

4.1.3

Tobacco

115

4.1.4

Alfalfa

122

4.1.5

Sugarcane

124

4.1.6

Lettuce

125

4.1.7

Potato

126

4.1.8

Other species

128

4.1.9

Identity preservation

130

DEFRA Contract CPEC 47

144

4.3.2.2 Multicellular species

150

4.3.3

Moss

151

4.3.4

Summary of non-crop species production

154

4.3.2

4.4

131

Other protein expression techniques

155

4.4.1

Suspension and callus cell cultures

155

4.4.2

Root culture, hairy roots and root exudates

156

4.4.3

Guttation fluid

158

4.4.4

Summary of other techniques for protein expression

160

Conclusions

161

Recommendations

169

Acknowledgements

170

Additional important note

170

References

171

Appendices

222

Appendix I

GM Field Trial Applications: online databases

Appendix II

Overseas Departments of Europe: online sources of

agricultural data

Appendix III

222
223

Patents and patent applications relating to GM crops and

their containment

223

China

223

DEFRA Contract CPEC 47

Appendix IV

Europe

223

United States

223

World

237

Field trial applications of GM crops expressing

pharmaceutical and industrial products in the US

243

Appendix V

Deregulated GM crops in the US

256

Appendix VI

GM crops with petition pending for deregulation in the US 259

DEFRA Contract CPEC 47

List of Tables
1.1

Transgenic maize varieties inscribed in the Commercial Varieties' Register

of Spain

2.1

Territories classified according to level of inclusion in the European Union

2.2

Global GM field trial applications

2.3

Phenotype categories of GM field trial applications in the US

2.4

Phenotype categories of deregulated, commercial GM crops in the US

2.5

Range of US deregulated GM crops according to phenotype category

2.6

Crops Grown in Europe

2.7

Summary of European field crops and the development of GM varieties

2.8

Crops of Overseas Departments of Europe

2.9

Dominant forest species of Europe

2.10

Crops from GM field trials with wild relatives and hybridisation potential in
Europe

2.11

Summary of genetic modification in forest trees in China

2.12

Florigene patent portfolio

3.1

Expression of vaccine antigens and biopharmaceutical proteins in the plastid

3.2

Chlorogen patent portfolio

3.3

Icon Genetics patent portfolio

3.4

Summary of GM containment strategies

4.1

Comparison of the features of recombinant protein production for the various

bioreactor systems currently available

4.2

109

Plant-derived pharmaceutical proteins that are closest to commercialization for

the treatment of human diseases

114

4.3

The Large Scale Biology Corporation patent portfolio

117

4.4

US field trials of GM tobacco expressing pharmaceutical products

118

4.5

European field trials of tobacco expressing pharmaceutical products

119

4.6

US field trials of GM alfalfa expressing pharmaceutical products

123

4.7

Large Scale Biology Corporation products generated via viral transient

expression

135

4.8

The LEX SystemTM of bio-pharming

140

4.9

Patent portfolio of the Biolex LEX SystemTM

140

vii

DEFRA Contract CPEC 47

4.10

List of patents issued to PlantibodiesTM

4.11

Companies currently selling microalgal-based products or developing microalgae

as bioreactors for protein production

141
145

viii

DEFRA Contract CPEC 47

List of Figures
2.1

Position of the two Spanish enclaves on the coast of Morocco

2.2

Field test applications for GM forest trees in the USA

2.3

Transgenic research on trees according to genus

2.4

Transgenic research on poplar according to country

2.5

Transgenic research on poplar according to gene of interest

2.6

Transgenic American elm

2.7

Method for production of blue rose

2.8

Transgenic blue roses

2.9

Transgenic carnations with modified flower colours

3.1

Underground growth facilities of Prairie Plant Systems Inc.

3.2

Terminator technology consists of three genes

3.3

Terminator technology: production of viable seeds

3.4

Terminator technology: inhibition of seed viability

4.1

Forecast for the total North American market for biopharmed products for
pharmaceutical use

108
TM

4.2

Working hypothesis for mode of action of CaroRx

4.3

The tobacco-related Nicotiana crop, producing new materials for medical and

115

other applications

118

4.4

Transgenic lettuce

126

4.5

Lettuce transformation

126

4.6

Potato plants in vitro

128

4.7

Growth of Spirodela oligorrhiza in culture

144

4.8

Microalgae Haematococcus pluvialis as seen under the microscope

147

4.9

Algal Photobioreactor

147

4.10

Protonema of Physcomitrella patens. Protein is released into the medium

152

4.11

Tube reactor filled with protein-producing moss

153

4.12

Fermentors for the production of hairy roots

158

DEFRA Contract CPEC 47

Glossary
allele

alternative form of a gene

angiosperm

flowering plants with enclosed seeds

anther

pollen sac of the stamen of angiosperms, in which pollen grains

develop

APHIS

Animal & Plant Health Inspection Service (United States)

apomixis

the production of seed in the absence of sexual fertilisation

ARS

Agricultural Research Service, of the USDA

backcrossing

crossing of hybrids to one it is parents

BBC

British Broadcasting Corporation

Bacillus thuringiensis

cecropin

basic polypeptides with antibacterial activity

chloroplast

organelle of plant cells

cleistogamy

self fertilisation within a closed flower

clones

individual of the same genetic constitution as its progenitor

CSIRO

Commonwealth Scientific and Industrial Research Organisation, of

Australia

cytoplasm

entire components of a cell, excluding the nucleus

cytotoxic

causing cell death

DEFRA

Department of the Environment, Food and Rural Affairs (United

Kingdom)

DNA

di-deoxy nucleic acid

east

European Community

egg cells

the female gamete containing a haploid nucleus, located in the ovary

embryo

the structure that develops from a zygote following fertilisation, upto

the time of germination

Spain

European Union

EU25

European Union, 25 member states

DEFRA Contract CPEC 47

the first hybrid offspring following cross-fertilisation

FAO

Food & Agriculture Organisation of the United Nations

feral

having escaped cultivation or domestication and reverted to a wild

state

fertilisation

fusion of male and female gametes to form a zygote

flora

collective term for all plants characteristic of a place or period

France

gene

functional unity of heredity; DNA sequence that encodes polypeptides

gene flow

the loss or gain of alleles from a population due to the movement of

fertile individuals or the transfer of gametes

genome

the complete compliment of an organisms genes

GFP

green fluorescent protein

glufosinate

a systemically acting, non-selective herbicide, present in Basta, Rely,

Finale, Challenge and Liberty Link

glycoprotein

any protein containing convalently bound carbohydrate

glyphosate

a systemically acting, non-selective herbicide, present in Roundup and

Tumbleweed

genetically modified

GMO

genetically modified organism

GURT

genetic use restriction technology

guttation fluid

exudates from leaves and roots in plants

gymnosperm

vascular plants bearing naked seed not enclosed in any specialised

chambers

gynoecium

the female organ of plants comprising stigma, style and ovary

hectares

heterozygous

having two different alleles for a given gene or genetic character

homozygous

having two identical alleles for a given gene or genetic character

herbicide tolerant

in planta

within the plant, as grown in ordinary conditions

in vitro

grown under aseptic conditions

intein

internal peptide sequence of an protein precursor that is spliced out by

transpeptidation during processing to form a mature protein

introgression

the transplantation of genes between species as a result of crosspollination

ISB

Information Systems for Biotechnology (Virginia Tech)

KTRDC

Kentucky Tobacco Research & Development Center

LSBC

Large Scale Biology Corporation

DEFRA Contract CPEC 47

magainin

antibiotic peptides from Xenopus that act against bacterial membranes

mycorrhizae

mutualistic association of plant roots and fungi

north

NEPA

National Environmental Policy Act; the environmental policy of the US

Government

nucleus

the chromosome containing organelle of a eukaryotic cell

OECD

Organisation for Economic Co-operation and Development

out-crossing

the crossing of two individuals to form a hybrid

perennial

a plant that lives for several years

phytoremediation

the use of plants to reduce levels of pollutants

plastid

plant organelles including chloroplasts, chromoplasts amd amyloplasts

pollen

the male gametophyte of plants that develops within the anthers of

stamens

pollen cells

individual male gametophytes of plants

pollination

the placement of pollen on to the stigma of flowers as a precursor to

fertilisation

protease

an enzyme that hydrolyses one or more peptide bonds in a protein,

leading to degradation and non-function

proteins

a biological polymer consisting of amino acids, encoded by DNA

sequences

Portugal

recombinant

resulting from several different origins; recombinant proteins are

encoding within cells from DNA sequences that have been inserted
from another organism

recombinase

an enzyme that recognises specific sites in a nucleotide sequence and

binds sites on different DNA molecules

rhizomes

underground stem of a plant, often horizontal, non-green and root-like

in appearance

ribonuclease

a group of enzymes that cleave phosphodiester bonds of RNA and

leading to degradation and non-function

RNAi

RNA interference

Roundup

a systemic non-selective herbicide

south

self-compatible

capable of the generation of fertile offspring from fertilisation of pollen

and egg cells of the same individual plant

sp.

species

sperm cells

the male gamete, enclosed in pollen cells in plants

xii

DEFRA Contract CPEC 47

splicing

the linkage of two strands of duplex DNA at complementary singlestranded terminations by means of DNA ligase

spp.

species (plural)

sq Km

square kilometers

ssp.

sub-species

stolons

horizontally growing underground stem that forms a new plant at the

end

symbiotic

ecological relationship between two different species that co-exist in

direct contact

terminator

term adopted to describe a genetic containment system that

generates sterile seeds

transgene mitigation

TMV

tobacco mosaic virus

transgene

a functioning gene inserted inserted into another organism to modify

its performance

United Kingdom

United States

USDA

United States Department of Agriculture

vector

organism that carries an organism or gene to another species host

west

WHO

World Health Organisation

zygote

the diploid product following the union of haploid gametes; a fertilised

egg

xiii

DEFRA Contract CPEC 47

Executive Summary
International Perspective
The development of GM technology continues to expand into increasing numbers of crops
and conferred traits. Inevitably, the focus remains on the major field crops of soybean,
maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance
and/or pest resistance. Although there are comparatively few GM crops that have been
commercialised to date, GM versions of 172 plant species have been grown in field trials in
31 countries.
European Crops with Containment Issues
Of the 20 main crops in the EU there are four for which GM varieties are commercially
available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM
varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar
beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in
development (rye, triticale). Many of these crops have hybridisation potential with wild and
weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats,
sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate
fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have
varieties in field trials and these too may hybridise with wild relatives in the European flora
(alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm,
Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice
and rose). All these crops will require containment strategies to be in place if it is deemed
necessary to prevent transgene movement to wild relatives and non-GM crops.
Current Containment Strategies
A wide variety of GM containment strategies are currently under development, with a
particular focus on crops expressing pharmaceutical products. Physical containment in
greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for
research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and
duckweed) expressing pharmaceutical products have been adopted by some biotechnology
companies. There are obvious limitations of the scale of physical containment strategies,
addressed in part by the development of large underground facilities in the US and Canada.
The additional resources required to grow plants underground incurs high costs that in the
long term may negate any advantage of GM for commercial production.

xiv

DEFRA Contract CPEC 47

Natural genetic containment has been adopted by some companies through the selection of
either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms
with no wild relatives present in the local flora (safflower in the Americas). The expression of
pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth
and harvesting prior to and in the absence of flowering.
Transgenically controlled containment strategies range in their approach and degree of
development. Plastid transformation is relatively well developed but is not suited to all traits
or crops and does not offer complete containment. Male sterility is well developed across a
range of plants but has limitations in its application for fruit/seed bearing crops. It has been
adopted in some commercial lines of oilseed rape despite not preventing escape via seed.
Conditional lethality can be used to prevent flowering or seed development following the
application of a chemical inducer, but requires 100% induction of the trait and sufficient
application of the inducer to all plants. Equally, inducible expression of the GM trait requires
equally stringent application conditions. Such a method will contain the trait but will allow the
escape of a non-functioning transgene. Seed lethality (terminator technology) is the only
strategy at present that prevents transgene movement via seed, but due to public opinion
against the concept it has never been trialled in the field and is no longer under commercial
development.
Methods to control flowering and fruit development such as apomixis and cleistogamy will
prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are
complex and leaky. None of the genes controlling these traits have as yet been identified or
characterised and therefore have not been transgenically introduced into crop species.
Neither of these strategies will prevent transgene escape via seed and any feral apomicts
that form are arguably more likely to become invasives.
Transgene mitigation reduces the fitness of initial hybrids and so prevents stable
introgression of transgenes into wild populations. However, it does not prevent initial
formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild
populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents
persistence of escapes and hybrids containing the transgene in an uncontrolled
environment, but does not prevent transgene movement from the crop.
Recoverable block of function, intein trans-splicing and transgene excision all use
recombinases to modify the transgene in planta either to induce expression or to prevent it.
All require optimal conditions and 100% accuracy to function and none have been tested
under field conditions as yet. All will contain the GM trait but all will allow some non-native
DNA to escape to wild populations or to non-GM crops.

DEFRA Contract CPEC 47

There are particular issues with GM trees and grasses as both are largely undomesticated,
wind pollinated and perennial, thus providing many opportunities for hybridisation. Some
species of both trees and grass are also capable of vegetative propagation without sexual
reproduction. There are additional concerns regarding the weedy nature of many grass
species and the long-term stability of GM traits across the life span of trees. Transgene
stability and conferred sterility are difficult to trial in trees as most field trials are only
conducted during the juvenile phase of tree growth.
Bio-pharming of pharmaceutical and industrial compounds in plants
Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive
alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigenes avidin, -glucuronidase, trypsin
generated in GM maize; Ventrias lactoferrin generated in GM rice). Numerous products are
in clinical trials (collagen, antibodies against tooth decay and non-Hodgkins lymphoma from
tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize;
Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary
supplements

from

Arabidopsis).

The

initial

production

platforms

for

plant-based

pharmaceuticals were selected from conventional crops, largely because an established

knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and
spinach are widely used as leaves can be harvested and no flowering is required. Many of
these crops can be grown in contained greenhouses. Potato is also widely used and can
also be grown in contained conditions. The introduction of morphological markers may aid in
the recognition and traceability of crops expressing pharmaceutical products.
Plant cells or plant parts may be transformed and maintained in culture to produce
recombinant products in a contained environment. Plant cells in suspension or in vitro, roots,
root cells and guttation fluid from leaves may be engineered to secrete proteins that may be
harvested in a continuous, non-destructive manner. Most strategies in this category remain
developmental and have not been commercially adopted at present.
Transient expression produces GM compounds from non-GM plants via the utilisation of
bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole
plants or plant parts, but do not insert them into the heritable genome. There are some
limitations of scale and the field release of such crops will require the regulation of the
vector. However, several companies have several transiently expressed products in clinical
and pre-clinical trials from crops raised in physical containment.

xvi

DEFRA Contract CPEC 47

1. Introduction
Genetically modified (GM) crops were first grown commercially on a small scale in
1995 but have since consistently increased the global area under cultivation.
Specifically, between 1996 and 2004 the amount of land assigned to GM crops has
expanded by more than 47-fold, from 1.7 Mha to 81 Mha, whilst the number of
countries involved has similarly increased from 4 in 1996 to 17 in 2004 (James
2005). Consumer and political resistance to the technology mean that this trend has
not been followed in Europe (Dunwell 2005; Tencalla 2005). For this reason, the
global acreage of GM crops is highly skewed, with USA (Runge & Ryan 2004),
Canada, Argentina, China, Brazil and South Africa collectively accounting for >99%
of all GM crops grown. Whilst there has been a de facto moratorium on the
cultivation of GM crops within the UK since 1999, elsewhere in Europe, there has
been limited commercialisation of GM Bt corn and GM HT soybean, particularly in
Spain (Table 1.1).
Table 1.1 Transgenic Maize Varieties Inscribed in the Commercial Varieties' Register of Spain.
(Data obtained from USDA 2005)
Event

Strain

Company

Date

Bt-176
(SYN-EV176-9)

Compa CB
Jordi CB
Brama
Escobar
Alican BT
Aristis BT
DKC 6575
PR33P67
Campero Bt
Cuartal Bt
DKC 6550
Bambier Bt
Jaral Bt
PR 32 P76
Portect
Elgina, Olimpica
Bolsa, Levina
Novelis
DK 513

Syngenta

26 March 1998
26 March 1998
11 March 2003
16 February 2004
11 March 2003
11 March 2003
11 March 2003
11 March 2003
16 February 2004
16 February 2004
16 February 2004
16 February 2004
16 February 2004
16 February 2004
16 February 2004
EU catalogue
17 September 2004
17 September 2004
17 September 2004

MON-810
(MON-00810-6)

Limagrain
Nickson Sur
Monsanto
Pioneer Hi-Bred
Advanta
Arlesa
Monsanto
Nickson Sur
Semillas Fit
Pioneer Hi-Bred
Koipesol
Pioneer Hi-Bred
Pioneer Hi-Bred
Coop de Pau
Monsanto

DEFRA Contract CPEC 47

1.1

Potential benefits of GM crops

GM crops offer a range of potential benefits that may be environmentally

advantageous. There is a global need to move towards the sustainable generation of
the food, feed, fuel and fibre needs of an ever expanding population on limited and
degrading land resources (Nickson 2005). The global population has more than
doubled in the last 50 years from 2.5 billion in 1950 to over 6 billion at present.
Population pressure is reducing land and water resources by development, industry
and inappropriate agricultural practices. There are also insecurities regarding the
future of agriculture in climatically marginal areas due to the uncertainties of climate
change. In addition, there are pressures on maintaining areas for conservation and
to sequester carbon from the atmosphere. The development of sustainable
agriculture in the face of such pressure on resources is a challenge for this and
future generations.
The adoption of GM technology has the potential to reduce the inputs required in
agricultural systems (Bennett et al. 2004a,b). Crops engineered with resistance to
insects (Bates et al. 2005) and to herbicides require substantially simplified
management practices compared to conventional varieties and reduction in losses
leading to increased yields (Pray et al. 2002; Nickson 2005). Crops that require less
in-the-field management result in lower on-farm fuel consumption. In addition, fields
require less tillage between crops to manage weeds and as a result, no-tillage and
conservation tillage practices may reduce soil erosion (Fawcett & Towery 2003;
Nickson 2005). The cultivation of crops expressing insect resistance has resulted in
a reduction of pesticide applications on crops and thereby reduced operator
exposure (Hossein et al. 2004; Huang et al. 2005). This results in fewer residues in
food and feed crops, less chemical release into the environment and a potential
increase in on-farm diversity in insects and pollinators (Nickson 2005). Thus there is
a subsequent increase in efficiency and a potential reduction in the negative
environmental footprint of agriculture.

DEFRA Contract CPEC 47

occur. Risk can be deemed acceptable even when the consequence (hazard) is
deemed high, provided that the exposure is negligible. Equally, a risk can be
deemed acceptable when the exposure is high but the hazard (the consequence) is
considered of minor importance. Such decisions invariably involve a degree of
subjectivity. In the case of gene flow from GM crops (e.g. Anon 2002a; Anon 2003)
the risks relate to a variable number of potential ecological and economic hazards
(Smyth et al. 2002; Strategy Unit 2003) and depend on the features of both the crop
and the transgene. The resulting risk assessment procedure is complex. It breaks
down into the initial process of identifying and evaluating the hazards, and an
evaluation of exposure for hazards deemed to have significance. An extensive
volume outlining this risk assessment process has been published recently (Poppy &
Wilkinson 2005).

DEFRA Contract CPEC 47

2. European Agriculture and GM Crops

The present review is designed to inform DEFRA about GM crops in a broad
European context, and it is therefore necessary to consider the precise definition of
Europe. In addition to the 25 states that are now full members of the EU, it is
important not to overlook the other overseas territories administered by various EU
governments. These regions have a variety of legal relationships with the EU and
may only be subject to some areas of EU law (Table 2.1).
Two parts of the treaty of the European Union deal with special relationships: Article
299 sets out the territories to which the treaty applies, supplemented by the
accession treaties; and Articles 182-188 and Annex II on association with the nonEuropean countries and territories that have special relations with the member
states. For example, according to Article 299 2, Canary Islands (Spain), the Azores
and Madeira (Portugal), constitute outermost regions of the EU; provisions of the
EC treaty apply there while derogations are allowed. The four French overseas
departments (Guadeloupe, Martinique, French Guyana and Runion) have a similar
status in being fully integrated parts of the French state, whereas the other French
Overseas Territories and associated dependencies have varying degrees of self
government.

The two Spanish enclaves, Ceuta and Melilla are costal towns in Morocco (Figure
2.1). They have a combined area of 32 sq km (12 sq miles). Spain also controls a
number of scattered islands along the North African coast, including uninhabited
Perejil, which was at the centre of a dispute in 2002 when Moroccan soldiers
occupied it before being removed by the Spanish army.

DEFRA Contract CPEC 47

Table 2.1 Territories classified according to level of inclusion in the European Union
This list classifies territories under sovereignty of a EU member state by level of enforceability of EC
law in this territory.
EU law fully in force
The part of Cyprus under control of the Republic of Cyprus
Denmark, excluding Faroe Islands and Greenland
Finland, excluding land Islands
Metropolitan France
The Netherlands, that is, the European part of the Kingdom of the Netherlands
Portugal, excluding the Azores and Madeira
Spain, excluding Canary Islands, Ceuta and Melilla
The United Kingdom but no other territory under British sovereignty
The totality of the other 17 member states: Austria, Belgium, the Czech Republic, Estonia, Germany,
Greece, Hungary, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Poland, Slovakia, Slovenia,
and Sweden
EU law in force, with some exemptions
land Islands (Finland)
Guadeloupe, French Guiana, Martinique, Runion (France)
The Azores, Madeira (Portugal)
Canary Islands, Ceuta, Melilla (Spain)
Gibraltar (UK)
EU law not in force, but some chapters apply
Channel Islands and the Isle of Man (citizens of UK descent are EU citizens; residents do not vote at
EU elections)
UK sovereign bases in Cyprus (locals are mainly EU citizens by having Cypriot nationality)
EU law not in force but statute of association with the EU
Greenland (Denmark; locals are EU citizens, but do not vote at EU elections)
French Polynesia, French Southern Territories, Mayotte, New Caledonia, Saint-Pierre and Miquelon,
Wallis and Futuna (France; EURATOM Treaty does apply; locals are EU citizens, and do vote at EU
elections)
Aruba and Netherlands Antilles (The Netherlands; locals are EU citizens, but do not vote at EU
elections)
Anguilla, Bermuda, British Antarctic Territory, British Indian Ocean Territory, British Virgin Islands,
Cayman Islands, Falkland Islands,Montserrat, Pitcairn Islands, Saint Helena and Dependencies,
South Georgia and the South Sandwich Islands, Turks and Caicos Islands (UK; since 21 May 2002
locals are British citizens and hence EU citizens, however do not vote in EU elections)
EU law not enforceable at all
The part of Cyprus under de facto control of the Turkish Republic of Northern Cyprus (locals with
Republic of Cyprus nationality are EU citizens, and are entitled to vote at EU elections)
Faroe Islands (Denmark; locals are not EU citizens)
Unclear
Buffer zone in Cyprus
Scattered Islands in the Indian Ocean, Clipperton Island (France; unpopulated)

DEFRA Contract CPEC 47

Figure 2.1 Positions of the two Spanish enclaves on the coast of Morocco
(Source: BBC)

Full EU law applies in Ceuta and Melilla, but some exceptions have been made by a
protocol annexed to the treaty of accession of Spain to the Communities (notably
agricultural policy and fisheries policy do not apply there).
The relevance of these territories to the present review is that they greatly extend the
range of climatic conditions within Europe and therefore significantly increase the
diversity of crops to be included in the analysis.

DEFRA Contract CPEC 47

2.1

Review of global GM Field Trials

In order to evaluate the current and future status of GM technology of European

crops it is necessary to assess the development and trialling of new GM crop lines.
Online databases of field trial applications on a global scale have been examined to
determine the current level of commercial interest in a crop and the likelihood of
release in the next 5-10 years.
Table 2.2 lists 172 crops for which field trial applications have been made in 31
countries. The data show that most applications (>6000) have been made to trial
maize/corn (Zea mays). Potato ranks second (1223 applications) with oilseed rape
coming a close third (1217), followed by soybean (1022) and cotton (962).
Tomatoes, wheat, alfalfa/lucerne, tobacco and rice are also in the top ten, based on
applications. US-based biotech industries dominate the majority of applications, with
the private sector tending to focus on annual crops rather than perennials. The public
sector (Universities and Research Institutes) appears to have an equal if not greater
interest in perennials and more specifically tree crops (Table 2.2).

DEFRA Contract CPEC 47

Table 2.2 Global GM field trial applications

Species are listed alphabetically. Information on the source of the application, i.e. the public or the
private sector, is made where possible. Applicant details were not always available (No data). Private
sector refers to biotech companies, Public Sector to Universities and Research Institutes. Sources of
funding for applications were not analysed; thus the division into Public and Private may not be wholly
accurate.
Field Trial Applications

Private Sector

Public Sector

No details

No. of countries

Location

Kiwi

Actinidia deliciosa

Italy, New Zealand

Velvet bentgrass

Agrostis canina

USA

Bentgrass

Agrostis spp.

Japan

Creeping
bentgrass

Agrostis stolonifera

169

102

Canada, USA

cicla

Germany

Sea beet

Beta vulgaris subsp.

maritima

Germany

Sugar beet

Beta vulgaris subsp.

vulgaris

284

269

Argentina, Belgium, Canada, Chile, Czech Republic,

Denmark, Finland, France, Germany, Greece,
Hungary, Ireland, Italy, Japan, Netherlands, New
Zealand, Spain, Sweden, UK, USA

Beet

Beta vulgaris subsp.

vulgaris

177

176

France, Greece, Netherlands, Spain, Sweden, UK,

USA

Fodder beet

Beta vulgaris subsp.

vulgaris

Belgium, Denmark, France, Italy, Spain

Silver birch

Betula pendula

Finland

Birch

Betula spp.

China

Ethiopian mustard

Brassica carinata

Canada

Indian mustard

Brassica juncea

Australia, Belgium, Canada, USA

Canola/Oilseed
rape

Brassica napus

1217

1202

Australia, Belgium, Canada, Chile, Czech Republic,

Denmark, Finland, France, Germany, Italy, Ireland,
Mexico, Netherlands, New Zealand, Spain, Sweden,
UK, USA

Swede

Brassica napus

Netherlands

Canola

Brassica napus/rapa?

India, Italy, Mexico, South Africa

Brown mustard

Brassica nigra

Canada, India

B. oleracea

Brassica oleracea

USA

Cauliflower

Brassica oleracea

Belgium, Canada, Finland, India, Japan

Broccoli

Brassica oleracea

Canada, Finland, Japan, New Zealand

DEFRA Contract CPEC 47

Table 2.2 continued

Field Trial Applications

Private Sector

Public Sector

No details

No. of countries

Location

Cabbage

Brassica oleracea

Finland, India, Netherlands

Canola/Turnip
rape

Brassica rapa

Australia, Canada, Sweden

B. rapa

Brassica rapa

USA

Chinese
cabbage

Brassica rapa

China

Turnip

Brassica rapa

Hungary

Brassica

Brassica spp.

India, USA

Sweet pepper

Capsicum annuum

China, India, Thailand, USA

Chilli

Capsicum annuum

China, India, Korean Republic, Mexico

Papaya

Carica papaya

Australia, Brazil, China, Cuba, Japan, Mexico, USA

Safflower

Carthamus tinctorius

Mexico, USA

American
chestnut

Castanea dentata

USA

Sweet chestnut

Castanea sativa

France

Chrysanthemum

Chrysanthemum spp.

Australia, Japan, Netherlands, USA

Chicory

Cichorium intybus

Belgium, France, Italy, Netherlands, UK, USA

Watermelon

Citrullus lanatus

Italy, USA

Lime

Citrus aurantiifolia

USA (Hawaii)

Lemon

Citrus limon

Italy, Mexico

Sweet orange

Citrus sinensis

Spain

Citrange

Citrus sinensis X Poncirus

trifoliata

USA

Citrus

Citrus spp.

Spain

Grapefruit

Citrus x paradisi

USA

Coffee

Coffea arabica

USA (Hawaii)

Robusta coffee

Coffea canephora

France (Guyane)

Cucurbit

Cucumis melo

108

USA

Melon

Cucumis melo

Egypt, France, Italy, Japan, Mexico, Spain

Cantaloupe

Cucumis melo

Egypt, Spain

Cucurbit

Cucumis melo / Cucurbita

pepo

USA

Cucurbit

Cucumis melo / Cucurbita

pepo / Lycopersicon
esculentum

USA

Cucumber

Cucumis sativus

Egypt, Japan, USA

Cucurbit/Squash

Cucurbita pepo

Egypt, France, Italy, Mexico, Spain

Courgette

Cucurbita pepo

Mexico

Squash

Cucurbita texana

USA

Bermudagrass

Cynodon dactylon

USA

Tamarillo/Tree
tomato

Cyphomandra betacea

New Zealand

Carrot

Daucus carota

Brazil, Netherlands, USA

Orchid

Dendrobium spp.

USA (Hawaii)

Carnation

Dianthus caryophyllus

Australia, Japan, Mexico, Netherlands

Persimmon

Diospyros virginiana

USA

Murray red gum

Eucalyptus camaldulensis

Spain, UK, USA

Crop
Total

DEFRA Contract CPEC 47

Table 2.2 continued

Field Trial Applications

Private Sector

Public Sector

China, Costa Rica, Dominican Republic, France,
Germany, Indonesia, Italy, Japan, Mexico, Puerto
Rico, Romania, South Africa, Spain, USA, Uruguay

Cotton

Gossypium hirsutum

962

942

Argentina, Australia, Belize, Bolivia, Brazil, Canada,

China, Colombia, Costa Rica, France, Ghana, Greece,
India, Indonesia, Japan, Mexico, Pakistan, South
Africa, Thailand, USA, Zimbabwe

Sunflower

Helianthus annuus

Argentina, France, Netherlands, Spain, USA

Barley

Hordeum vulgare

Australia, Canada, Finland, Hungary, Iceland, New

Zealand, UK, USA

Sweet potato

Ipomoea batatus

Cuba, Kenya, USA, Venezuela

Walnut

Juglans spp.

USA

Lettuce

Lactuca sativa

Australia, France, Italy, Japan, USA

Lentils

Lens culinaris

Canada

Lily

Lilium longiflorum

Belgium

Statice

Limonium spp.

Italy

Flax

Linum usitatissimum

Canada, Mexico, Sweden

Sweetgum

Liquidambar styraciflua

USA

Italian ryegrass

Lolium multiflorum

USA

Perennial
ryegrass

Lolium perenne

Netherlands, USA

Narrow-leaved
lupin

Lupinus angustifolius

Australia

Tomato

Lycopersicon esculentum

744

717

Argentina, Australia, Brazil, Canada, Chile, China,

Egypt, France, Greece, Guatemala, India, Italy, Japan,
Mexico, Netherlands, Portugal, Spain, Thailand, UK,
USA

Apple

Malus domestica

Argentina, Belgium, Germany, Netherlands, New

Zealand, Sweden, UK, USA

Paradise apple

Malus pumila

Netherlands, UK

100

Cassava

Manihot esculentum

USA

101

Alfalfa/Lucerne

Medicago sativa

417

392

Argentina, Belgium, Bulgaria, Canada, Mexico, Spain,

USA

102

Barrel clover

Medicago truncatula

USA

103

Peppermint

Mentha x piperita

USA

104

Banana /
plantain

Musa spp.

Mexico, Philippines, USA

105

Watercress

Nasturtium officinale

USA

Crop

Total

DEFRA Contract CPEC 47

Table 2.2 continued

Field Trial Applications

Total

Private Sector

Public Sector

No details

No. of countries

Location

Nicotiana attenuata

USA

Crop

106

Nicotiana

107

Tobacco

Nicotiana benthamiana

USA

108

Tobacco

Nicotiana sylvestris

USA

109

Tobacco

Nicotiana tabacum

374

344

Australia, Brazil, Bulgaria, Canada, Finland, France,

Germany, Hungary, India, Italy, Japan, Korean
Republic, Mexico, Spain, UK, USA

110

Olive

Olea europea

Italy

111

Rice

Oryza sativa

323

295

Argentina, Brazil, Canada, China, France, India, Italy,

Japan, Mexico, Philippines, Spain, Thailand, USA

112

Marigold

Osteospermum ecklonis

Italy, USA

113

Poppy - oilseed

Papaver somniferum

Australia

114

Guayule

Parthenium argentatum

USA

115

Bahia grass

Paspalum notatum

USA

116

Scented
Pelargonium

Pelargonium
odoratissimum

Italy

117

Pelargonium

Pelargonium spp.

Italy, USA

118

Avocado

Persea americana

USA

119

Petunia

Petunia petunia X
P. hybrida

Germany, USA

120

Petunia

Petunia spp.

Japan, New Zealand

121

Canary grass

Phalaris canariensis

Canada

122

Norway spruce

Picea abies

Finland, New Zealand

123

Spruce

Picea spp.

USA

124

Pine

Pinus radiata

New Zealand

125

Pine

Pinus spp.

New Zealand, USA

126

Scots pine

Pinus sylvestris

Finland

127

Pea

Pisum sativum

Australia, Canada, Germany, New Zealand, UK, USA

128

Kentucky
bluegrass

Poa pratensis

USA

129

Meadow grass

Poa pratensis X Poa

arachnifera

USA

130

Grey poplar

France, Spain, UK

131

European
aspen

Populus alba X P.
tremula
Populus alba X P.
tremula

132

Poplar

Populus deltoides

France, Germany, Norway

143

Canada, France, Germany, UK, USA

133

Black poplar

Populus nigra

China

134

Poplar

Populus spp.

Belgium, China

135

Poplar/Spruce

Populus spp. / Picea spp.

USA

136

Hybrid aspen

Populus tremula X
P. tremuloides

Sweden

137

Sweet cherry

Prunus avium

Italy

138

European plum

Prunus domestica

USA

139

Cherry / Plum

Prunus domestica

Italy, Spain

140

Russian wildrye

Psathyrostachys juncea
(Elymus junceus)

USA

141

Pear

Pyrus communis

Sweden, USA

DEFRA Contract CPEC 47

Table 2.2 continued

Field Trial Applications

Total

Private Sector

Public Sector

No details

No. of countries

Location

France

Crop

142

Wild radish

Raphanus raphanistrum

143

Rhododendron

Rhododendron spp.

USA

144

Rose

Rosa hybrida

Australia, USA

145

Raspberry

Rubus idaeus

Italy, USA

146

Sugarcane

Saccharum officinarum

Australia, Brazil, Cuba, Egypt, South Africa, USA

147

African violet

Saintpaulia ionantha

Netherlands

149

White mustard

Sinapis alba

Canada

150

Eggplant/Brinjal/
Aubergine

Solanum melongena

India, Italy, USA

151

Black
nightshade

Solanum nigrum

Germany

1223

1083

113

Argentina, Australia, Austria, Belgium, Bolivia, Brazil,

Canada, China, Cuba, Czech Republic, Denmark,
Egypt, Finland, France, Germany, Hungary, India,
Italy, Japan, Mexico, Netherlands, New Zealand,
Peru, Poland, Portugal, South Africa, Spain, Sweden,
Sw

Sorghum bicolor

USA

Stenotaphrum
secundatum

USA

African/Cape
marigold

Tagetes erecta

Italy

156

Torenia

Tourenia fournieri

Japan

157

Hares foot
clover

Trifolium arvense

New Zealand

158

White clover

Trifolium repens

Australia

159

Subterranean
clover

Trifolium subterraneaum

Canada, New Zealand

160

Wheat

Triticum aestivum

451

429

Argentina, Australia, Belgium, Canada, Egypt,

Germany, Hungary, Italy, Japan, Mexico, Spain, UK,
USA

161

Wheat, durum

Triticum turgidum ssp

durum

Italy

162

American elm

Ulmus americana

USA

163

Blueberry

Vaccinium spp.

USA

164

Cranberry

Vaccinium spp.

USA

165

Adzuki bean

Vigna angularis

Japan

166

Grape

Vitis berlandieri X
V. riparia

France

167

Grape

Vitis berlandieri X
V. rupestris

France

168

Grape - sand
grape

Vitis rupestris

France

169

Grape

Vitis vinifera

Australia, Canada, France, Germany, Italy, USA

Grape

Vitis vinifera X
V. berlandieri

France

Potato

Solanum tuberosum

153

Sorghum

154

St. Augustine
grass

155

152

170

171

Corn/maize

Zea mays

172

Zoysiagrass

Zoysia matrella/japonica

6106

6066

Argentina, Austria, Belgium, Brazil, Canada, Chile,

China, Costa Rica, Czech Republic, Denmark, Egypt,
France, Germany, Greece, Honduras, Hungary,
Indonesia, Italy, Japan, Mexico, Netherlands, New
Zealand, Philippines, Portugal, Puerto Rica, Serbia &
Mont, UK

Japan

DEFRA Contract CPEC 47

The Information Systems for Biotechnology (ISB) database of US field trials and
commercialised, deregulated crops at Virginia Tech (http://www.isb.vt.edu) provides
information on the transgene phenotype in each application. Thus it is possible to
make some assessment of the more commonly trialled and commercialised
transgenic traits in the US.
Table 2.3 Phenotype categories of GM field trial applications in the US
*Other category includes fertility/sterility traits; novel/pharmaceutical protein production; altered gene
expression, growth habit or flowering time; tolerance to stress, drought or heavy metals.
Source: Information Systems for Biotechnology Database, Virginia Tech.
Phenotype category
Herbicide tolerance
Insect resistance
Product quality
Agronomic properties
Virus resistance
Other*
Fungal resistance
Marker gene
Bacterial resistance
Nematode resistance

Number of applications
3820
3250
2497
1099
886
673
661
627
115
32

The numbers of applications made in each phenotype category listed in the ISB
database clearly demonstrates the prevalence of herbicide tolerance and insect
resistance in the development of GM crops (Table 2.3). Agronomic properties and
product quality are third and fourth in the ranking.
Table 2.4 Phenotype categories of deregulated, commercial GM crops in the US
Source: Information Systems for Biotechnology Database, Virginia Tech.
Phenotype category
Herbicide tolerance
Insect resistance
Product quality
Virus resistance
Agronomic properties
Other
Fungal resistance
Marker gene
Bacterial resistance
Nematode resistance

Applications
45
36
20
11
8
1
0
0
0
0

Approved
32
23
13
6
6
1

Withdrawn
12
11
3
3
2

Pending
1
2
2
2

Void

DEFRA Contract CPEC 47

The small numbers of trials for crops with bacterial and nematode resistance will
relate to the specificity of these problems to some crops. For example, applications
for trials of material expressing nematode resistance have only been made for
soybean, pineapple, carrot, tomato, walnut, grape and tobacco.
Of the commercialised GM crops in the US, i.e. those crops deregulated and no
longer under the control of APHIS, the same pattern of transgenic traits is apparent
(Table 2.4). Equally, the range of commercialised crops in each phenotype category
reflects the focus on herbicide tolerance. This trait has been engineered into seven
commercially available lines, insect resistance, product quality, agronomic traits into
four lines, and virus resistance into only three.
Table 2.5 Range of US deregulated GM crops according to phenotype category
Source: Information Systems for Biotechnology Database, Virginia Tech.
Phenotype category
Herbicide tolerance

Insect resistance

Product quality

Agronomic properties

Virus resistance
Other

Deregulated cultivars
Maize 11
Oilseed rape 7
Cotton 5
Soybean 4
Beet 3
Rice 1
Alfalfa 1
Maize 12
Cotton 5
Potato 5
Tomato 1
Tomato 10
Tobacco 1
Soybean 1
Oilseed rape 1
Maize 3
Flax 1
Oilseed rape 1
Chicory 1
Potato 3
Squash 2
Papaya 1
Oilseed rape 1

DEFRA Contract CPEC 47

2.1.1

Summary of global GM field trials

Nearly 15,500 field trial applications have been made for 172 crops across 31
countries. The highest-ranking crops, based on applications are:
1.

Maize/corn (6106)

Potato (1223)

Oilseed rape (1217)

Soybean (1022)

Cotton (962)

Tomatoes (744)

Wheat (451)

Alfalfa/Lucerne (417)

Tobacco (374)

10.

Rice (323)

DEFRA Contract CPEC 47

2.2

Review of European crop production

Data for the 2004 harvest have been obtained from the EU Statistics database
Eurostat (http://epp.eurostat.cec.eu.int/portal/page?_pageid=1090,1&_dad=portal&_
schema=PORTAL). Data on individual countries have been consolidated into a list of
133 crops and groups of crops. These have been ranked in order of area under
production in both Europe as a geographical and political entity i.e. mainland Europe
and the EU25 (Table 2.6).
These data identify common wheat, barley, maize, oilseed rape and durum wheat as
the top ranking crops in Europe in terms of acreage. Pasture and meadows also rank
very highly in terms of area, indicative of the mixed nature of European agriculture.
Of the top twenty crop species, only GM rye have not reached field trials (Table 2.6),
although GM rye has been grown in greenhouse tests (Wieser et al. 2005).
Specific definitions for the terms used in Eurostat, with regard to pasture and fodder
crops, have been sought from Eurostat in London and Luxembourg but without
result. Similarly, unsuccessful enquiries were also made with regard to the groupings
of minority crops (e.g. Other oilseeds). Such groups occur at an artificially high
position in the rank order as individual acreages have been combined. The
combination of data in this way therefore does not provide an accurate list of crops
under cultivation in individual countries. Without complete knowledge of which crops
are being grown in which countries, identifying potential gene escape to the native
flora becomes more difficult.

DEFRA Contract CPEC 47

Table 2.6 Crops grown in Europe

Ranking is based on acreage. Definitions: EU25 Belgium, Czech Republic, Denmark, Germany,
Estonia, Greece, Spain, France, Ireland, Italy, Cyprus, Latvia, Lithuania, Luxembourg, Hungary,
Malta, Netherlands, Austria, Poland, Portugal, Slovenia, Slovakia, Finland, Sweden and UK. Europe
geographical Europe (EU25 plus data for Bulgaria, Croatia, Romania, Turkey, Iceland, Norway,
Albania, Bosnia & Herzegovina and Macedonia where available). * GM field trial application(s) made
for this crop; (*) GM trial application(s) made for a closely related species.
GM
Field
Trials
*

Europewide
Totals
1000ha

Crop

Wheat

Triticum aestivum

Permanent pasture
*

Barley

Hordeum vulgare

Permanent meadows
*

Grain maize

Zea mays

25673.540

Europe
rank

EU25
Totals
1000ha

EU25
rank

23276.271

23221.136

19154.573

13406.994

12937.664

11038.173

9558.198

9761.463

6489.338

Perennial green fodder

9514.773

9141.306

Annual green fodder

5090.719

4910.824

Forage maize

Zea mays

4675.841

4639.589

Rape

Brassica napus

4487.458

4436.689

4214.163

Temporary grasses
*

Durum wheat

Triticum turgidum

4048.791

4044.749

Sunflower seed

Helianthus annuus

3184.321

2201.892

Oats

Avena sativa

2904.393

2673.019

Rye

Secale cereale

2754.255

2727.872

2481.834

2453.905

Potatoes

Triticale
Solanum tuberosum

2464.768

2174.258

Sugar beet

Beta vulgaris subsp. vulgaris

2223.095

2200.721

Wine grapes (juice and/or wine making)

Vitis vinifera

2196.548

1962.113

Lucerne

Medicago sativa

2071.942

1836.550

Mixed grain other than maslin

Total olives (table and oil)

Olea europea

Other annual green fodder

*
*

1590.117

1568.289

1039.067

895.424

Field peas

Pisum sativum

733.920

715.481

Clover and mixtures

Trifolium spp.

725.360

671.797

689.241

641.146

600.090

472.505

477.115

476.730

463.900

Other oil seeds (poppy, mustard, safflower,

cotton, earth almond, sesame, groundnut)
*

Apples (including cider apples)

Cotton seed

Malus domestica

Other dried pulses

Gossypium hirsutum

Other legumes (sainfoin, sweet clover)

431.423

367.926

Rice

Oryza sativa

427.919

423.831

Broad and field beans

Vicia faba

406.832

391.876

Soya bean

Glycine max

395.025

273.358

370.922

Temporary grazings
*

Tomatoes

Lycopersicon esculentum

311.360

270.306

Kidney beans
Buckwheat, millet, canary seed (other
cereals)

Phaseolus vulgaris

224.978

69.808

217.314

213.854

201.145

158.947

199.404

111.469

198.861

198.616

Other industrial crops

Carrots

Daucus carota

Other dried pulses (e.g. lathyrus)

DEFRA Contract CPEC 47

Table 2.6 continued

GM
Field
Trials

Crop

Europewide
Totals
1000ha

Plums

Prunus domestica

195.076

Onions

Allium cepa

172.494

157.820

Cherries, including sour cherries

Prunus avium

158.650

135.366

Other root crops

Europe
rank

EU25
Totals
1000ha
78.182

EU25
rank

157.597

157.197

Vetches

Vicia spp.

153.241

Fodder beet

Beta vulgaris subsp. vulgaris

144.622

104.283

Cauliflower and broccoli

Brassica oleracea

143.956

142.146

Linum usitatissimum

135.705

135.410

Flax (straw)
Peas other than field peas (including chick
peas)

133.267

126.087

Prunus dulcis

130.407

128.495

Almonds
Tobacco raw (including seedlings
enclosures)

Nicotiana tabacum

128.819

103.692

Oranges

Citrus sinensis

127.059

118.945

109.848

Pyrus communis

110.351

104.608

Officinal herbs, aromatic plants, plants for

seasoning (e.g. thyme)
*
*

Pears (including perry pears

Table grapes

Vitis vinifera

107.406

89.650

Peaches

Prunus persica

105.511

96.621

Sorghum

Sorghum bicolor

103.384

94.708

Cabbage (white)

Brassica oleracea

103.131

74.065

Water melons

Citrullus lanatus

101.493

60.842

Lettuce

Lactuca sativa

100.647

99.078

Oil flax

99.834

98.427

Maslin

Linum usitatissimum
Triticum aestivum and Secale
cereale

96.131

Strawberries

Fragaria ananassa

95.428

91.794

Melons

Cucumis melo

95.288

89.940

Globe artichokes

Cynara scolymus

82.037

Lupins

Lupinus angustifolius

76.064

(*)

Turnip rape

Brassica rapa

75.984

Hazelnuts

Corylus avellana

73.359

73.253

Red pepper, capsicum

Capsicum annuum

68.241

48.483

Other permanent crops (carob-tree,

mulberry-tree, tea, coffee)

64.267

63.780

60.124

Industrial crops (rye-straw, fullers teasel,

lavender, hybrid lavender)

57.536

24.435

Other root crops (Jerusalem artichoke, sweet

potatoes, fodder parsnips, yams, cassava)

54.800

54.400

54.128

42.509

Carobs

Ceratonia siliqua

Apricots

Prunus armenaica

Asparagus

Asparagus officinalis

53.279

Black currants

Ribes nigrum

48.721

48.657

Lentils

Lens culinaris

45.709

45.569

Nectarines

Prunus persica var. nectarina

41.545

41.221

39.152

Allium sativum

40.311

33.691

Chicory

Cichorium intybus

39.802

Cucumbers

Cucumis sativus

38.339

30.912

Chestnuts

Castanea sativa

37.875

37.850

Other leafy or stalked vegetables

Garlic
*
*

DEFRA Contract CPEC 47

Table 2.6 continued

GM
Field
Trials
*

Europewide
Totals
1000ha

Crop

Walnuts

Europe
rank

EU25
Totals
1000ha

EU25
rank

Juglans regia

37.732

27.223

Hops

Humulus lupulus

36.543

36.493

Other brassicas

Brassica spp

34.098

33.795

Lemons

Citrus limon

31.676

(*)

Gourds

Cucurbitaceae

30.856

28.758

30.848

30.766

Other pulses
*

Kiwi

Actinidia deliciosa

28.944

28.920

Fodder kale

Brassica oleracea

28.004

Spinach

Spinacia oleracea

27.671

27.216

Beetroot

Beta vulgaris subsp. vulgaris

27.241

27.101

Marrows, courgettes

Cucurbita pepo

25.961

25.027

Clementines

Citrus reticulata cv. Clementine

24.985

Leeks

Allium ampeloprasum

23.536

22.267

Egg-plant

Solanum melongena

23.363

16.012

100

Raspberries

Rubus idaeus

22.007

20.328

Chicory for inulin

Cichorium intybus

20.861

Endive

Cichorium endivia

19.790

Caraway

Carum carvi

17.199

100

17.199

Brussels sprouts

Brassica oleracea

13.878

101

13.867

101

Hemp (straw)

Cannabis sativa

13.877

102

12.682

103

Red currants

Ribes rubrum

13.247

103

13.246

102

11.736

104

11.422

104

Other soft fruit

Turnips

Brassica rapa

10.962

105

10.962

105

Mandarins

Citrus reticulata

10.325

106

10.325

106

Other root and tuber vegetables

*
(*)
*

10.112

107

8.611

108

Chicory

Cichorium intybus

8.803

108

8.803

107

Radishes

Raphanus sativus

8.770

109

7.929

109

Turnips for stockfeeding

Brassica rapa

7.691

110

7.691

110

Celeriac

Apium graveolens var. rapaceum

6.712

111

6.712

111

Celery

Apium graveolens var. dulce

6.507

112

6.161

112

Gherkins

Cucumis sativus

5.351

113

4.394

114

Figs

Ficus carica

5.219

114

5.218

113

4.422

115

3.816

116

Other fruit

Gooseberries

Ribes uva-crispa

4.340

116

4.340

115

Kohl-Rabi

Brassica oleracea

3.196

117

3.136

118

Salsify and scorzonera

Tragopogon porrifolius and

Scorzonera hispanica

3.145

118

3.145

117

Shallots

Allium cepa

2.487

119

2.487

119

2.267

120

1.853

120

Other vegetables cultivated for fruit

Grapefruit

Citrus x paradisi

1.075

121

1.075

121

Quinces

Cydonia oblonga

0.696

122

0.571

122

Other stone fruit

*
*

0.292

123

0.292

123

Carrots for stockfeeding

Daucus carota

0.149

124

0.149

124

Avocados

Persea americana

0.139

125

0.139

125

0.120

126

0.120

126

Other nuts

DEFRA Contract CPEC 47

Table 2.6 continued

GM
Field
Trials

Europewide
Totals
1000ha

Crop

EU25
Totals
1000ha

EU25
rank

Cultivated mushrooms

Agaricus bisporus

0.088

127

0.088

127

Swedes
Wild products (truffles, other mushrooms,
grape hyacinth-bulbs)

Brassica napus

0.023

128

0.023

128

0.000

129

0.000

129

Satsumas

Citrus reticulata

0.000

130

0.000

130

Raisins (in fresh weight)

Vitis vinifera

0.000

131

0.000

131

0.000

132

0.000

132

0.000

133

0.000

133

Other citrus fruit

Europe
rank

Citrus aurantiifolia

Limes

2.2.1

Summary of European field crops

The highest-ranking crops based on area under cultivation (excluding

aggregates of species) are listed in Table 2.7.
Table 2.7 Summary of European field crops and the development of GM varieties

GM varieties
GM varieties
commercially
in field trials
available

Rank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

wheat
barley
grain maize
forage maize
oilseed rape
durum wheat
sunflowers
oats
rye
triticale
potatoes
sugar beet
grapes
alfalfa/lucerne
olives
field peas
clover
apples
cotton
rice

No GM
varieties in
field trials

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

DEFRA Contract CPEC 47

2.2.2

Overseas departments

Crop production data for the overseas departments of the EU have been more
difficult to source, as separate data are not available through Eurostat. Detailed
production data have therefore not been obtained, although a list of crop species has
been compiled from a variety of web sources (Appendix II) for the following
territories: Azores-PT, Madeira-PT, Canary Islands-ES, Guadeloupe-FR, GuyaneFR, Martinique-FR and Runion-FR. This list contains 55 different crops of which 12
do not occur on the mainland list of crops and thus appear to be unique to the
overseas territories (Table 2.8). These include cocoa, coconuts, dates, custard
apples, inhame, lychees, mangoes, papaya, passion fruit, pineapples, tea and
vanilla. These crops may not be entirely absent from mainland Europe as exhaustive
crop data for the mainland have not yet been obtained. Indeed, a small tea crop is
now being produced in the UK. Crops such as cocoa in Guyane however, are
restricted to the tropics and are unlikely to be cultivated in mainland Europe. Of
these 12 additional crops, only two have been through GM field trials: papaya and
pineapple. The remaining ten crops, with the addition of figs, rye, tangerines, tea and
vanilla do not have transgenic varieties that have reached field trials (Table 2.8).
Trialling of GM crops has not taken place on a large scale in the overseas territories,
although a recent report describes the first long-term field trial of GM coffee in
Guyane (Perthuis et al. 2005). Clones of Robusta coffee (Coffea canephora) were
transformed for resistance to the lepidopteran coffee leaf miner Leucoptera coffeella
by the insertion of a synthetic cry1ac Bt gene using A. tumefaciens. Over a 4-year
period six releases of L. coffeella were performed and the results showed that the
majority of the independent transformed clones displayed much less insect damage
than the control.
C. canephora is an insect pollinated, out-crossing crop that will hybridise with related
wild species. It is, however, native to central Africa (Zaire, Sudan, Uganda,
Tanzania) and all its wild relatives are indigenous to Africa, Madagascar and the

DEFRA Contract CPEC 47

Table 2.8 Crops of Overseas Departments of Europe

Figures where present represent area under cultivation in ha; + indicates that cultivation of a crop
occurs on some scale; * indicates that GM field trial applications(s) have been made for this crop.
GM Field
Crop
Trials

Guadeloupe Martinique Reunion Guyane Azores Madeira

kiwi

Actinidia deliciosa

onions

Allium cepa

pineapples

Ananas comosus

custard apple

Anona spp

oats

Avena sativa

sugarbeet

Beta vulgaris subsp. vulgaris

cabbage

Brassica oleracea

cauliflower

Brassica oleracea

turnip

Brassica rapa

tea

Camelia sinensis

103

10
+

papaya

Carica papaya

chestnut

Castanea sativa

chicory

Cichorium intybus

lemons

Citrus limon

tangerine

Citrus reticulata

13.5

oranges

Citrus sinensis

117

coconuts

Cocos nucifera

inhame

Colocasia antiquorum

2.5
64
+
87

melon

Cucumis melo

courgettes

Cucurbita pepo

pumpkin

Cucurbita pepo

carrots

Daucus carota

figs

Ficus carica

strawberry

Fragaria ananassa

cotton

Gossypium hirsutum

sweet potato

Ipomoea batatus

lettuce

Lactuca sativa
Litchi chinensis

410
+

+
450
+

tomatoes

Lycopersicon esculentum

100

apples

Malus x domestica

195

mangoes

Mangifera indica

manioc (tapioca)

Manihot ultissima

bananas

Musa spp.

tobacco

Nicotiana tabacum

rice

Oryza sativa

passion fruit

Passiflora edulis

avocados

Persea americana

dates

Phoenix dactylifera

cherry

Prunus avium

plum

Prunus domestica

peach

Prunus persica

pears

Pyrus communis

*
*

sugarcane

Saccharum officinarum

rye

Secale cereale

eggplant

Solanum melongena

potatoes

Solanum tuberosum

cocoa

Theobroma cacao

wheat

Triticum aestivum

vanilla

Vanilla planifolia and other spp.

wine grapes

Vitis vinifera

maize

Zea mays

+
+
6000

+
8295
+

11.8
85
+
22
10.5

14300

240

125

1600

+
+
+
+
+
+
+
+

beans

+
80

beans, French

9
+

+
+

beans, green
flowers

(*)

+
+

lychees

Canary
Islands

DEFRA Contract CPEC 47

Mascarenes (Ellstrand 2003c). In addition, coffee is not listed as a major crop in

Guyane (Table 2.8) thus the risks of transgene escape via hybridisation during this
trial would be minimal.

2.2.3

Forestry

Forestry production is difficult to quantify, as survey data of forest resources are not
collected according to species. This is also true for forest plantations. However,
recent global forest surveys show that in most regions of the world, relatively few
tree species make up the majority of standing wood volume (FAO 2005). A list of
dominant forest species has been compiled for all the countries of Europe from the
FAO Forest Resources Assessment, 2000 (FAO 2000). These data are consolidated
in Table 2.9 to give a list of species for Europe as a whole. There are 20 species
listed of which the majority are native to Europe. Only six species are introduced to
Europe, namely Eucalyptus globulus, Larix kaempferi, Picea sitchensis, Pinus
contorta var. latifolia, Pinus radiata and Pseudotsuga menziesii, the majority of which
will account for the planted forest area. The largest areas of natural forest occur in
Sweden (26,565 thousand ha), Finland (21,935 thousand ha), France (14,380
thousand ha), Spain (12,466 thousand ha) and Germany (10,740 thousand ha) (FAO
2000). Areas of planted forest are much less in comparison. The largest planted
areas occur in the UK (1928 thousand ha), Spain (1904 thousand ha), Bulgaria (969
thousand ha), France (961 thousand ha) and Portugal (834 thousand ha) (Stace
1975; Simmonds 1976; Fehr and Hadley 1980; Smart and Simmonds 1995;
Ellstrand 2003b).

DEFRA Contract CPEC 47

Table 2.9 Dominant forest species of Europe

Species list includes tree species of both natural woodland and forest plantations.
Europewide Totals
1000ha

EU25 Totals 1000ha

Planted
forest

10011

8403

Natural
forest

154445

131062

Natural woodland and planted species

Silver fir

Abies alba

Sycamore

Acer pseudoplatanus

Alder

Alnus spp.

White / Silver birch

Betula pendula

GM
Field
Trials

Downy birch

Betula pubescens

Birch

Betula spp.

Sweet chestnut

Castanea sativa

Tasmanian blue gum

Eucalyptus globulus

Beech

Fagus sylvatica

Ash

Fraxinus excelsior

European larch

Larix decidua

Japanese/hybrid larch

Larix kaempferi

Norway spruce

Picea abies

Sitka spruce

Picea sitchensis

Lodgepole pine

Pinus contorta var. latifolia

Corsican pine

Pinus nigra subsp. laricio

Maritime pine

Pinus pinaster

Monterey pine

Pinus radiata

Pines

Pinus spp.

Scots pine

Pinus sylvestris

Poplar

Populus spp.

Quaking aspen

Populus tremula

Douglas fir

Pseudotsuga menziesii

Oak

Quercus spp.

Cork oak

Quercus suber

Elm

Ulmus spp.

DEFRA Contract CPEC 47

2.3

Potential gene flow: recipient species and wild relatives of

European crops

All crop species on the GM field trials list (Table 2.2) have been assessed with
regard to the potential for transgene escape to wild relatives by investigating known
hybridisation events with related species (Tutin et al. 1964-1980; Stace 2001;
Ellstrand 2003b; Jrgensen & Wilkinson 2005). In addition, crop species that have
wild relatives native to Europe have been screened to evaluate the potential for
further hybridisation events and the subsequent ecological impact with respect to
endemism and rarity of related taxa. The extent of known escape and naturalisation
of crop species has also been recorded where data are available.
The results identify those crop species with known ability to form spontaneous
hybrids with wild species, those crops that may have the potential to hybridise under
certain conditions, and those which pose no risk of transgene spread to wild relatives
in Europe (Table 2.10).

2.3.1

Crops posing less risk of transgene spread

Of the 172 crops listed in Table 2.10, only 17 appear to pose a low risk of transgene
escape via hybridisation with wild species in Europe. These are kiwi, pineapple,
papaya, watermelon, Tasmanian blue gum, soybean, sweet potato, lily, tomato,
cassava, banana, rice, aubergine, potato, sorghum and maize. Of these 17, only two
are in the top ten crop species of Europe, namely maize and potatoes, although rice,
soybean and tomato are relatively high-ranking crops (30, 32 and 34 respectively).
The remainder of crops posing limited risk are relatively minor, with four being
cultivated only in the Overseas Departments (pineapple, papaya, sweet potato and
banana) (Table 2.10).

DEFRA Contract CPEC 47

Table 2.10 Crops from GM Field Trials with wild relatives and hybridisation potential in Europe.
Rank order of crop species is based on the area under production in 2004 (Table 2.6). Plant family
and the geographical origin of crops are shown.
Rank

Risk

Native to
China

Actinidia deliciosa

Actinidaceae

Velvet bentgrass

Agrostis canina

Gramineae

W Asia, Europe

Bentgrass

Agrostis spp.

Gramineae

N temperate

Creeping bentgrass

Agrostis stolonifera

Gramineae

Macaronesia, N Africa, Temperate Asia, Europe

Onion

Allium cepa

Liliaceae

only cultivated

Leek

Allium porrum

Liliaceae

only cultivated

osd

13,osd

Family

Kiwi

41,
osd
95

Crop

Allegheny servis berry

Amelanchier laevis

Rosaceae

E N America

Pineapple

Ananas comosus

Bromeliaceae

Brazil

Lily

Anthurium andraeanum

Araceae

Tropical America

Arabidopsis

Arabidopsis spp

Brassicaceae

N temperate

Thale Cress

Arabidopsis thaliana

Brassicaceae

N temperate

Peanut

Arachis hypogaea

Leguminosae

South America

Asparagus

Asparagus officinalis

Liliaceae

Europe - N Africa

Belladonna / Deadly
nightshade

Atropa belladonna

Solanaceae

Mediterranean-Eurasia

Oat

Avena sativa

Gramineae

Mediterranean basin

Begonia

Begonia semperflorens

Begoniaceae

SE Brazil, NE Argentina

maritima
Beta vulgaris subsp
vulgaris
Beta vulgaris subsp
vulgaris
Beta vulgaris subsp
vulgaris

Beet

Fodder beet

17,osd

Sugar beet

Chenopdiaceae

Europe, near East, Asia to China

Silver birch

Betula pendula

Betulaceae

Europe

Birch

Betula spp.

Betulaceae

N. hemisphere

Ethiopian mustard

Brassica carinata

Brassicaceae

Europe

Indian mustard

Brassica juncea

Brassicaceae

Europe, Asia

Canola/Oilseed rape

Brassica napus

Brassicaceae

Europe

128

Swede

Brassica napus

Brassicaceae

Europe

Canola

Brassica napus/rapa?

Brassicaceae

Europe

Brown mustard

Brassica nigra

Brassicaceae

Europe, Asia Minor, Iran, Asia and Far East

B. oleracea

Brassica oleracea

Brassicaceae

Europe

Broccoli

Brassica oleracea

Brassicaceae

Europe

57,osd

Cabbage

Brassica oleracea

Brassicaceae

Europe

46,osd

Cauliflower

Brassica oleracea

Brassicaceae

Europe

B. rapa

Brassica rapa

Brassicaceae

Europe

Canola/Turnip rape

Brassica rapa

Brassicaceae

Europe

Chinese cabbage

Brassica rapa

Brassicaceae

Europe

Turnip

Brassica rapa

Brassicaceae

Europe

Brassica

Brassica spp

Brassicaceae

Europe

Chilli

Capsicum annuum

Solanaceae

New World tropics

105,
osd

osd

Sweet pepper

Capsicum annuum

Solanaceae

New World tropics

Papaya

Carica papaya

Caricaceae

Tropical Americas

Safflower

Carthamus tinctorius

Asteraceae

Turkey, Lebanon, Israel to NW India

DEFRA Contract CPEC 47

Table 2.10 continued

Rank

Risk

Crop

Family

Native to

American chestnut

Castanea dentata

Fagaceae

Asia, introduced to N America

Sweet chestnut

Castanea sativa

Fagaceae

Mediterranean-Caucasus

Chrysanthemum

Chrysanthemum spp.

Asteraceae

N Africa, Europe-Asia

Chicory

Cichorium intybus

Asteraceae

Mediterranean

Watermelon

Citrullus lanatus

Cucurbitaceae

Tropical and Southern Africa

Lime

Citrus aurantiifolia

Rutaceae

Southeast Asia

86,osd

Lemon

Citrus limon

Rutaceae

Southeast Asia

51,osd

Sweet orange

Citrus sinensis

Rutaceae

Southeast Asia

Citrange

Citrus sinensis X Poncirus

trifoliata

Rutaceae

Southeast Asia

Citrus

Citrus spp.

Rutaceae

Southeast Asia

Grapefruit

Citrus x paradisi

Rutaceae

Southeast Asia

Coffee

Coffea arabica

Rubiaceae

SW Ethiopia-Sudan

osd,fs

80,osd

121

63,osd

Robusta coffee

Coffea canephora

Rubiaceae

Zaire, Sudan, Uganda, NW Tanzania

Cantaloupe

Cucumis melo

Cucurbitaceae

Subgen Melo Africa-India

Cucurbit

Cucumis melo

93,osd

Courgette

Cucurbita pepo

Cucurbitaceae

Tropical America

87,osd

Cucurbit/Squash

Cucurbita pepo

Cucurbitaceae

Tropical America

Squash

Cucurbita texana

Cucurbitaceae

Tropical America

Bermudagrass

Cynodon dactylon

Chloridoideae

Pan-tropical, extending into warm temperate

Tamarillo/tree tomato

Cyphomandra betacea

Solanaceae

Tropical America

Carrot

Daucus carota

Umbelliferae

Mediterranean region

Orchid

Dendrobium spp

Orchidaceae

Tropical Asia-Australia-Pacific

Carnation

Dianthus caryophyllus

Caryophyllaceae

Mediterranean

Persimmon

Diospyros virginiana

Ebenaceae

SE USA

Murray red gum

Eucalyptus camaldulensis

Myrtaceae

Australia

Tasmanian blue gum

Eucalyptus globulus

Myrtaceae

Australia

Flooded gum

Eucalyptus grandis

Myrtaceae

Australia

Lisianthus

Eustoma grandiflorum

Gentianaceae

N America

Tall fescue

Festuca arundinacea

N temperate Europe, Asia, Americas

Gladiolus

Gladiolus spp

Iridaceae

Africa

Soybean

Glycine max

Leguminosae

C & N Asia

Cotton

Gossypium hirsutum

Malvaceae

Seasonally arid tropics and sub-tropics

38,osd

62,osd

32
28,osd

DEFRA Contract CPEC 47

Table 2.10 continued

Rank

Risk

Crop
Sunflower

Family
Helianthus annuus

Native to

Asteraceae

Temperate N America
Domesticated from wild races found in SW Asia/near
East

Barley

Hordeum vulgare

Gramineae

Sweet potato

Ipomoea batatus

Convolvulaceae

South America

Walnut

Juglans ssp.

Juglandaceae

SE Europe - China

59,osd

Lettuce

Lactuca sativa

Asteraceae

Mediterranean basin

Lentils

Lens culinaris

Leguminosae

Mediterranean basin & SW Asia

Lily

Lilium longiflorum

Liliaceae

Easter I, Bermuda, Japan

Statice

Limonium spp.

Plumbaginaceae

Maritime & arid N hemisphere

Flax

Linum usitatissimum

Linaceae

Eurasia

Sweetgum

Liquidambar styraciflua

Hammamelidaceae

N-C America

Italian ryegrass

Lolium multiflorum

Gramineae

C&S Europe

3
osd

U
47,60

Perennial ryegrass

Lolium perenne

Gramineae

Europe

Narrow-leaved lupin

Lupinus angustifolius

Leguminosae

Mediterranean basin

Tomato

Lycopersicon esculentum

Solanaceae

W S America, C America, subtropics of Old World

Paradise apple

Malus pumila

Rosaceae

Asia Minor, Caucasus, Soviet Central Asia

Apple

Malus x domestica

Rosaceae

Asia Minor, Caucasus, Soviet Central Asia

34,osd

26,osd

Cassava

Manihot esculentum

Euphorbiaceae

S & C America

Alfalfa/Lucerne

Medicago sativa

Leguminosae

SW Asia

Barrel clover

Medicago truncatula

Leguminosae

Europe-Mediterranean-S Africa

Peppermint

Mentha x piperita

Labiatae

Europe

Banana / plantain

Musa spp.

Musaceae

SE Asia and Pacific

Watercress

Nasturtium officinale

Brassicaceae

Europe

Nicotiana

Nicotiana attenuata

Solanaceae

W N America

Tobacco

Nicotiana benthamiana

Solanaceae

Australia

Tobacco

Nicotiana sylvestris

Solanaceae

S America

osd
19

osd

50,osd

30,osd

125,
osd

Tobacco

Nicotiana tabacum

Solanaceae

S & C America

Olive

Olea europea

Oleaceae

Mediterranean basin

Rice

Oryza sativa

Gramineae

Asia

Marigold

Osteospermum ecklonis

Asteraceae

Southern Africa

Poppy - oilseed

Papaver somniferum

Papaveraceae

W Mediterranean - Asia

Guayule

Parthenium argentatum

Asteraceae

Texas-N Mexico

Bahia grass

Paspalum notatum

Paniceae

New World

Scented Pelargonium

Pelargonium
odoratissimum

Geraniaceae

Southern Africa

Pelargonium

Pelargonium spp.

Geraniaceae

Southern Africa

Avocado

Persea americana

Lauraceae

Americas

Petunia

Petunia petunia X
P. hybrida

Solanaceae

(Sub)tropical S America

Petunia

Petunia ssp.

Solanaceae

(Sub)tropical S America

Canary grass

Phalaris canariensis

Gramineae

W Mediterranean

Picea abies

Pinaceae

N & C Europe

Norway spruce

Spruce

Picea ssp.

Pinaceae

Cool N hemisphere

Monterey pine

Pinus radiata

Pinaceae

California

Pine

Pinus spp.

Pinaceae

N temperate

Scots pine

Pinus sylvestris

Pinaceae

Eurasia

Pea

Pisum sativum

Leguminosae

Mediterranean, near East

DEFRA Contract CPEC 47

Table 2.10 continued

Rank

Risk

40,osd

53,osd

Family

Native to

Poa pratensis

Gramineae

Mediterranean-Eurasia

Meadow grass

Poa pratensis X Poa

arachnifera

Gramineae

Common meadow
grass / Kentucky
bluegrass

European aspen

Populus alba X P. tremula

Salicaceae

Grey poplar

Populus alba X P. tremula

Salicaceae

Cottonwood

Populus deltoides

Salicaceae

E N America

Black poplar

Populus nigra

Salicaceae

Europe, W Asia
N temperate

Poplar

Populus spp.

Salicaceae

Poplar/Spruce

Populus spp. / Picea spp.

Salicaceae

Hybrid aspen

Populus tremula X
P. tremuloides

Salicaceae

Sweet cherry

Prunus avium

Rosaceae

Western Asia

Cherry / Plum

Prunus domestica

Rosaceae

Europe

European plum

Prunus domestica

Rosaceae

Europe

Russian wildrye

Psathyrostachys juncea

Gramineae

Eurasia

Pear

Pyrus communis

Rosaceae

Asia Minor, Caucasus, Soviet Central Asia

Wild radish

Raphanus raphanistrum

Brassicaceae

Europe, Asia

Rhododendron

Rhododendron spp.

Ericaceae

Temperate N hemisphere

Rose

Rosa hybrida

Rosaceae

N temperate

96,osd

16,osd

Sorghum

Sorghum bicolor

Gramineae

Semi-arid tropical East Africa

St. Augustine grass

Stenotaphrum secundatum

Gramineae

Warm Americas

African/Cape
marigold

Tagetes erecta

Asteraceae

C America

Torenia

Tourenia fournieri

Scrophulariaceae

Vietnam

Hares foot clover

Trifolium arvense

Leguminosae

Europe

White clover

Trifolium repens

Leguminosae

Europe, Asia, N Africa

Subterranean clover

Trifolium subterraneaum

Leguminosae

W Europe, Asia minor, N Africa

1,osd

Wheat

Triticum aestivum

Gramineae

only cultivated

Wheat, durum

Triticum turgidum ssp.

durum

Gramineae

only cultivated

American elm

Ulmus americana

Ulmaceae

E N America

Blueberry

Vaccinium spp.

Ericaceae

Cranberry

Vaccinium spp.

Ericaceae

Adzuki bean

Vigna angularis

Leguminosae

18,54,
osd

NE Asia

Grape

Vitis berlandieri X V. riparia

Vitaceae

N America

Grape

Vitis berlandieri X
V. rupestris

Vitaceae

N America

Grape - sand grape

Vitis rupestris

Vitaceae

N America

Vitaceae

Wild V. vinifera C Asia, Afghanistan to Black & Caspian

Seas

Grape

Vitis vinifera

DEFRA Contract CPEC 47

Table 2.10 continued

Rank

5,8,osd

Risk

Crop

Family

Native to

Grape

Vitis vinifera X V. berlandieri

Vitaceae

Corn/maize

Zea mays

Gramineae

S&C America

Zoysiagrass

Zoysia matrella/japonica

Gramineae

Tropical Asia

Definitions:
osd
fs
grey text
plain text &
background

2.3.2

cultivated in overseas departments of Europe

European forestry species
not cultivated in Europe
cultivated in Europe with no potential for gene flow
hybrids recorded in Europe

related species native/naturalised in Europe

DEFRA Contract CPEC 47

2.3.3.2

Trifolium

Of the two species of Trifolium for which GM varieties are being developed, more is
known about the hybridisation potential of T. repens than T. subterraneaum.
Nonetheless, both species occur wild throughout most of Europe (Tutin et al. 19641980). T. repens will hybridise with fifteen other species of Trifolium in Europe, of
which five are endemic to certain areas. T. repens itself is a complex of six
subspecies, of which four are endemic (Smartt & Simmonds 1995).
Field trials of Trifolium species have been conducted in Australia, Canada and New
Zealand (Table 2.2). These trials have been testing varieties with resistance to
herbicide, viral disease and stress.
A small number of trials of transgenic Trifolium arvense have been conducted in New
Zealand (Table 2.2). This species occurs across Europe and the UK but it is not
widely cultivated as a forage species (Smart & Simmonds 1995). The hybridisation
potential of this species has therefore not been considered here.
2.3.3.3

Lupin

Hybridisation of cultivated lupins is a recognised problem as species are highly interfertile and sweet varieties cultivated for fodder are known to become bitter due to
hybridisation with sympatric wild types (Tutin et al. 1964-1980). The European flora
contains ten species of lupin, of which six are native annual species, including
Lupinus angustifolius (Table 2.2), and four are introduced American perennials (Tutin
et al. 1964-1980; Stace 1975).
Lupins have been field trialled in Australia (Table 2.2) for traits including altered seed
composition and sulphur levels.
2.3.3.4

Summary of forage and fodder crops

DEFRA Contract CPEC 47

These data show that for all the potential GM forage crops that may be cultivated in
Europe, transgene escape to wild and related wild species is probable, with the
exception of green fodder maize.

2.3.4

Forestry Species

Unlike arable crop species, forestry species are much longer lived and are often
farmed from natural/semi-natural woodland as well as from planted forest. The
species utilised are thus not improved varieties as for conventional crop species. As
such, forestry species are identical to those species growing in the wild and thus,
hybridisation with wild populations will have no genetic barrier and is of primary
concern.
The increasing number of applications for field-testing of GM forest trees in the US is
shown in Figure 2.2. Of the 20 major forest species in Europe (Table 2.9), six have
been included in GM field trials, of which four involve native species present across
Europe. However, details of the exact species transformed are not given in some

Figure 2.2 Field test applications for GM forest trees in the US

(Source: APHIS data from FAO 2004)

applications (i.e. Betula spp.) so an exact number is not possible at this point. The
generic field trials applications name Betula spp., Picea spp., Pinus spp., Populus
spp. and Ulmus spp. all of which have species native in Europe. Development of GM
varieties and hybridisation between species of Betula, Picea, Pinus and Populus are
discussed below.

DEFRA Contract CPEC 47

Commercial, and academic, interest in transgenic tree breeding (Walter and Killerby
2003; Boerjan 2005) is based on efforts to increase tree productivity, improve
manufacturing efficiency and product quality, and introduce disease resistance and
control over reproductive traits (Cooke et al. 2004; Powell et al. 2005). There is also
an emerging interest in the use of GM trees to generate bio-pharma products and for
phytoremediation (Peuke & Rennenberg 2005).
Insect resistance has been engineered into trees species by the insertion of the Bt
insecticidal toxin genes in a wide range of tree species including poplar (McCown et
al. 1991), eucalyptus (Harcourt et al. 2000), larch (Shin et al. 1994), and spruce
(Pea & Sguin 2001). Alternative approaches to confer insect resistance have been
developed; these include the over-expression of proteinase inhibitors that interfere
with digestive processes in herbivorous insects (Lepl et al. 1995; Delledonne et al.
2001).
There are arguments that suggest the genetic modification of native and landscape
trees may be beneficial in a conservational context, since introduced pests and
diseases have devastated populations of many native tree species of the North
Temperate Zone. For example, populations of the English elm (Ulmus procera) have
been decimated by Ophiostoma ulmi, Dutch elm disease. As trees have such an
extended life span, generating disease resistant lines by conventional methods may
well be insufficient. Adams et al. (2002) argue that limited transfer of resistant genes
from the original host species may enable many North Temperate tree species to
retain and resume their ecological niche.
2.3.4.1

Poplar

Of all forest tree species most transgenic research has been conducted on poplar
(Marchadier & Sigaud 2005) (Figure 2.3) with the majority of this research
concentrated in the USA (Figure 2.4) (see also www.fao.org/docrep/008/ae574e
/ae574e00.htm). Populus is a good model species for genetic studies in trees as it
grows rapidly, can be vegetatively propagated, is amenable to tissue culture and to
transformation protocols and it has a relatively small genome size (Cooke et al.
2004). The Populus genus includes species that are native in Europe and are
39

DEFRA Contract CPEC 47

reasonably widespread. In addition, there are species that have been introduced
from North America and Asia. Both the native and the introduced species will

Figure 2.3 Transgenic research on trees according to genus

(Source: Marchadier and Sigaud 2005)

Figure 2.4 Transgenic research on poplar according to country

(Source: Marchadier and Sigaud 2005)

hybridise and these hybrids are sufficiently common to have been given specific
names. Poplar trees, including transgenic specimens, are also capable of vegetative

DEFRA Contract CPEC 47

propagation offering a means of reproduction without pollination (Tutin et al. 19641980; Stace 1975).
The predominant focus of this research has been herbicide tolerance (Figure 2.5).
Herbicides are used in intensive forestry management to reduce competition from
weedy species and thus the development of herbicide-resistant trees would be
advantageous to the industry. Resistance to glyphosate has been engineered into
poplars (Donahue et al. 1994; Meilan et al. 2002) and into larch (Shin et al. 1994),
and resistance to glufosinate ammonium into poplar and eucalyptus (Confalonieri et
al. 2000; Harcourt et al. 2000).

Figure 2.5 Transgenic research on poplar according to gene of interest

(Source: Marchadier and Sigaud 2005)

Improvements to wood quality and a reduction in the environmental impact of the

pulping process may be attained through the genetic modification of the lignin
biosynthesis pathway. This pathway is well documented and has been the subject of
much investigation in the past due to its importance in wood and paper quality. Thus
more progress has been made on manipulating lignin than any other trait affecting
wood quality (Cooke et al. 2004). However, modification of lignin can result in
aberrant tree growth and may have negative effects on fibre characteristics and
indeed, may render the wood more susceptible to infection from fungal pathogens
and disease (Vance et al. 1980; Legrand et al. 2000).

DEFRA Contract CPEC 47

Several strategies have been used in poplar to confer general disease resistance
including expression of oxalate oxidase from wheat (Liang et al. 2001), introduction
of a bacterio-opsin gene from Halobacterium halobium (Mohamed et al. 2001), and
expression of synthetic antimicrobial peptides that cause pathogen mortality (Liang
et al. 2002). Engineering resistance to specific diseases is also being explored. For
example, resistance against the fungal rust Melampsora has been ascribed to a
single dominant gene in Populus spp. (Newcombe et al. 1996) and diminished crown
gall formation by Agrobacterium tumefaciens was achieved by transformation of
Populus spp. with antisense iaaM (Ebinuba et al. 1997).
As a consequence of this emphasis on Populus species in transgenic tree research
(Fladung et al. 2003; 2004; Kumar and Fladung 2004) they represent the only GM
trees that are grown extensively under non-restricted conditions, albeit only in China
to date. Growth of GM poplars in China has been ongoing since 1993 (Tian 1998,
Wang 2004) and is continuing. An insect resistant line of P. nigra expressing Bt
(Poplar-12) is being grown over 200 ha of the northern regions of China including
Hebei, Beijing, Liaoning and Ningxia. Another commercialised transgenic species,
Poplar-741, expressing Bt plus another enzyme, is growing in sporadic plantations
totalling less than 3 ha across nine provinces and municipalities including Hebei,
Beijing, Tianjin and Shandong. Both lines are reported to be female poplars with
altered fertility, and are unable to release pollen (China Daily, 1st April 2005,
http://www.china.org.cn/english/environment/124471.htm).
Recent related studies on inducing improved salinity tolerance have included tests
on the utility of mannitol-1-phosphate dehydrogenase, which catalyzes the
biosynthesis of mannitol from fructose. The gene expressing this enzyme (mtlD) was
cloned from E. coli and transferred to poplar (Populus tomentosa) through
Agrobacterium-mediated transformation (Hu et al. 2005).
Wang (2004) reports that in May 2003, the GM poplar expressing this gene was
officially registered for the protection of breeders right under a Chinese common
name,

Taiqing

No.1

poplar

(PVP

Office

2003;

http://www.cnpvp.net/

anouncement1.htm). In June 2003, the environmental release was officially

approved by the Ministry of Agriculture to establish 100 mu (equal to about 6.6 ha) of
42

DEFRA Contract CPEC 47

plantations in Tianjin City and Shandong Province. In December 2003, the research
project was thoroughly reviewed; the GM poplar was given another Chinese
common name, Zhongtianyang, and assigned a cultivar name as (Populus
xiaozhuanica W.Y. Hsu and Liang cv. Balizhuangyang-zhongtian). This GM line has
now established in coastal areas in Shandong Province, where the soil has been so
seriously saline that there is a limited choice of tree species for planting (Wang
2004). Table 2.11 provides a summary of the status of GM tree research in China.
Table 2.11 Summary of genetic modification in forest trees in China.
R: research; E: environmental release; C: commercial planting. (Source: Wang 2004)
Species

Status

Traits

Betula platyphylla

Insect resistance

Eucalyptus camaldulensis

Eucalyptus urophylla

Lowering lignin content

Resistance to disease
caused by Pseudomonas
solanaceanum

Poplar hybrid 741

(P. alba [P. davidiana + P.
simonii] P. tomentosa)
Populus xiaozhuanica W.Y.
Hsu et Liang cv.
Balizhuangyang-zhongtian
Populus deltoides
Populus deltoides P.
cathayana
Populus deltoides P. simonii
(N-106)

Gene
Spider insecticidal
peptide
C4H

Development
On going

cecropin D

On going
Applied for commercial
plantings in 2001

On going

E, C

Resistance to leaf-eating
insects

Bt Cry1 and API

Salt tolerance

mtlD

Insect resistance
Resistance to leaf-eating
insects
Resistance to leaf-eating
insects
Resistance to leaf-eating
insects
Salt tolerance.
Resistance to disease and
stress

Approved for environment

release in 2003. Granted
breeders right in 2003
On going

mtlD/gutD

On going

AaIT

On going

R
R

Populus nigra

E, C

Populus simonii P. nigra

Populus tomentosa

Bet-A

Applied for commercial

Eucalyptus

One of the introduced timber species that has reached GM field trials is Tasmanian
blue gum (Eucalyptus globulus). There are no native species of Eucalyptus present
in Europe, although many have been introduced as timber species. As yet, there are
no details of hybridisation events between these species in Europe (Tutin et al.
1964-1980; Stace 1975) but spontaneous hybridisation between eucalypt species
has been noted in parts of Australia (Barbour et al. 2003; 2005).
Eucalyptus species have been engineered to confer tolerance to glyphosate
(Llewellyn 2000) and to insect infestation (Harcourt et al. 2000). In addition, field
trials have been carried out in the US of eucalypts with altered lignin biosynthesis
pathways (http://www.isb.vt.edu/cfdocs/) and recently (1 Nov 2005) it was
announced that Nippon Paper Industries (NPI) has started an outdoor cultivation
experiment with salt tolerant eucalypts in collaboration with the University of
Tsukuba. These trees express a choline oxidase gene from Arthrobacter globiformis,
and their salt tolerance has been confirmed in the previous greenhouse experiment

DEFRA Contract CPEC 47

in which the trees were supplied with saltwater of 30% salinity, the same salinity as
seawater. Additional field trial applications for GM eucalypts have been made in
Brazil, Portugal, South Africa, Spain and the UK (Table 2.2).
2.3.4.7

Elm

Although there have been no field trials of genetically modified English Elm (Ulmus
procera) there are two applications and one pending from New York State University
to grow genetically modified American elm (Ulmus americana). The trees have been
engineered to express synthetic magainin and cecropin genes (Figure 2.6; US
Patent 5856127) as a means to improve tolerance to Dutch elm disease. These field
trials began in 2005. Transformation of Ulmus procera, and regeneration of
transformants has been demonstrated by research at the University of Abertay,
Dundee (Gartland et al. 2000; 2001). Should the American trials prove successful,
the development of English elm resistant to Dutch elm disease should also be
possible.

Figure 2.6 Transgenic American elm

(Source: http://www.esf.edu/efb/powell/research.html)

2.3.4.8

Oak

The recent problem of sudden oak death (Phytophthora ramorum) has also
generated renewed interest in protection against this disease. The pathogen itself is
the subject of a genome sequencing project funded by the US Department of Energy
(http://genome.jgi-psf.org/euk_cur1.html), and although there have been no reported
trials of transgenic oak, there are laboratory studies of the cork oak Quercus suber
(lvarez et al. 2004) and the sawtooth oak Q. acutissima (Taniguchi et al. 2003).

DEFRA Contract CPEC 47

2.3.4.9

Summary of forestry species

All timber species for which GM field trial applications have been made have the
potential to hybridise with tree species that occur in the wild, with the exception of
Eucalyptus globulus, the Tasmanian blue gum. However, the cross-compatibility
relationships between the blue gum and the other Eucalyptus species present in
Europe requires further investigation before the risk of transgene spread can be
completely ruled out. In addition, an initiative launched last year in the US to improve
hardwood quality via genetics will undoubtedly increase the number species for
which GM technology has been developed (Merkle & Nairn 2005). A new National
Science Foundation research centre dedicated to improving the quality of valuable
hardwood tree species will be lead by Purdue University. This will advance work in
improving the quality of hardwood tree species native to Indiana and much of the
Midwest, such as black walnut, northern red oak and black cherry, which are highly
prized by the fine furniture and cabinetry industries.

DEFRA Contract CPEC 47

2.3.5

Orchard crops

Small numbers of field trials have been conducted on a variety of fruit trees and
orchard crops that are cultivated in Europe (Table 2.2, 2.6).
2.3.5.1

Olives

Olives are the highest-ranking orchard crops in Europe in terms of area (Table 2.6).
The cultivated olive (Olea europea) is native to the Mediterranean basin and often
occurs with is wild progenitor species O. sylvestris (Tutin et al. 1964-1980). The two
species are fully interfertile and sporadic hybridisation occurs where they are in
proximity (Tutin et al. 1964-1980; Smartt & Simmonds 1995). Cultivated olive trees
also occur as feral populations (Ellstrand 2003c).
To date there have been two field trials of GM olives conducted in Italy by Universit
degli Studi della Tuscia (Table 2.2).
2.3.5.2

Apples

Apples rank 26 in terms of acreage in Europe (Table 2.6) and they are present in
most European states under cultivation and as escapes (Tutin et al. 1964-1980). The
cultivated apple will readily hybridise with most species of Malus (Smartt &
Simmonds 1995) of which there are six present in Europe, including one endemic
species in the Balkan States and Italy (Tutin et al. 1964-1980).
To date, there have been 48 field trial applications for GM apples in Argentina,
Belgium, Germany, The Netherlands, New Zealand, Sweden, UK and USA (Table
2.2).

DEFRA Contract CPEC 47

2.3.5.3

Plums and cherries

Plums and cherries are less widely cultivated in Europe, ranking 40 and 42 in terms
of acreage (Table 2.6). The cultivated plum (Prunus domestica) and the sweet cherry
(P. avium) will cross with many of the other Prunus species occurring in Europe
including wild types, those cultivated for timber and ornament, as well as the other
fruit-bearing Prunus species (peach, almond, damson, greengage) (Stace 1975;
Smartt & Simmonds 1995). In all there are 21 species of Prunus in Europe, including
one species endemic to southern Spain (Tutin et al. 1964-1980).
Despite plums being a lower acreage crops than olives and apples, the development
of GM lines of plums is further developed. To date there have been 10 field trial
applications for GM plums in the US, Italy and Spain (Table 2.2). In addition ARS
currently have a petition pending in the US for the deregulation of a GM line of plum
resistant to plum pox virus (Appendix VI). There have been fewer trials of GM
cherries, with only three applications being made in Italy (Table 2.2).
2.3.5.4

Pear

Pears (Pyrus communis) are native to Asia Minor and the Caucasus and rank 53 as
a European crop (Table 2.6). Most cultivated pears are able to cross with other
Pyrus species and varieties (Smartt & Simmonds 1995) and possibly with Sorbus
aria (whitebeam) (Stace 1975). There are 13 species of Pyrus in Europe, the
majority of which are endemic (Tutin et al. 1964-1980) and includes one of Britains
rarest trees, the Plymouth pear (Pyrus cordata).
Development of GM lines of pear have so far been limited with only six field trial
applications having been made in Sweden and the US (Table 2.2).
2.3.5.5

Walnut

Walnuts (Juglans regia) rank 83 in terms of acreage in Europe (Table 2.6) and are
native to the area extending across the southeastern states of Europe (Greece and
the Balkan Peninsula) to China. There are three species of Juglans present in
50

DEFRA Contract CPEC 47

Europe of which only J. regia is native. The other species have been introduced for
fruit and timber production and have become naturalised (Tutin et al. 1964-1980)
Many Juglans species are interfertile and it is likely that these species will be crosscompatible with each other (Simmonds 1976).
To date there have been 15 field trial applications for lines of GM walnut, all of which
have been conducted in the US (Table 2.2).
2.3.5.6

Citrus

There are nine species of Citrus present in Europe all of which have been introduced
and are cultivated on different scales for their fruit and essential oils (lemon, lime,
sweet orange, Seville orange, mandarin, grapefruit, bergamot, pomelo/ugli fruit and
citron) (Tutin et al. 1964-1980). Of these species, oranges are the most widely
cultivated in terms of area, ranking 51, followed by lemon (86), grapefruit (121) and
lime (133). Citrus species are native to Southeast Asia and are widely interfertile
between species. Hybrids of interspecific crosses are also often fertile and this has
been exploited by breeders to generate a wide variety of citrus fruit forms (Smartt &
Simmonds 1995).
There have been relatively few GM field trial applications for Citrus trees (lime, 1;
lemon, 2; orange, 1; grapefruit, 6) based in the US, Mexico, Italy and Spain (Table
2.2) demonstrating the limited development of GM technology in this group of crops.

2.3.5.7

Avocado

Avocados (Persea americana) have been introduced into Europe from the Americas
and they are cultivated on a relatively small scale in parts of France and Cyprus,
ranking only 125 in terms of area (Table 2.6). The three cultivated races of avocado
constitute subspecies of P. americana and these are all cross-compatible with each
other (Smartt & Simmonds 1995). P. americana does not appear to have escaped
cultivation and does not exist in feral populations. However, there is one species of
51

DEFRA Contract CPEC 47

Persea (P. indica) native to some parts of Europe. This species is native to Madeira
and to the Canary Islands and has been introduced and become naturalised in the
Azores (Tutin et al. 1964-1980). It is as yet unclear whether P. americana and P.
indica are cross-compatible with each other.
There has been very little development of GM lines of avocado with only one field
trial application having been made to date in the US (Table 2.2).
2.3.5.8

Papaya

Papaya is cultivated on a very small scale in Europe, with data suggesting cultivation
only occurs in Madeira (Table 2.7). The species is native to tropical America and as
such there are no related species in the European flora (Tutin et al. 1964-1980). The
development of GM lines of papaya is relatively well developed, however, with one
variety of virus-resistant GM papaya that has been deregulated by APHIS (Appendix
V) and a second variety with an application for deregulation currently pending
(Appendix VI). These two varieties have been developed in the US by Cornell
University, the University of Hawaii and the University of Florida, and represent the
only GM fresh fruit on the market in the world. In addition to field trials in the States,
field trial applications for GM papaya have also been made in Australia, Brazil,
China, Cuba, Japan and Mexico (Table 2.2).
2.3.5.9

Summary of orchard crops

The majority of orchard crops planted in Europe pose some risk of transgene escape
to wild relatives with olives, apples, plums, cherries, pears and walnuts all native to
Europe or the Mediterranean area and all possessing cross-compatibility with related
species. Development of GM lines of these crops is not advancing rapidly, however,
with only small numbers of field trial applications having been made. The most
developed GM variety is a plum pox resistant plum tree developed by ARS in the US
currently with an application for deregulation currently pending.
The remaining fruit trees (Citrus, avocado, papaya) are all introduced to Europe and
are cultivated on very small scales. As these trees have no wild relatives in the
52

DEFRA Contract CPEC 47

European flora, the risk of transgene spread is thus restricted to crop-to-crop

pollination. There are two virus-resistant lines of GM papaya, one which has been
deregulated in the US and one with an application for deregulation pending. These
are the only GM fruit grown commercially in the world. GM lines of Citrus and
avocado are not well developed.

2.3.6

Grasses

As in the case with tree species, grasses cultivated for forage or amenity are often
very similar to those species growing in the wild. Of the 13 species of grass for which
GM field trial applications have been made, ten occur in Europe. Grasses have a
very effective self-incompatibility mechanism that makes the group inherently outcrossing. Many grasses are cross-compatible with species in the same genus, and in
some cases in other genera.
2.3.6.1

Agrostis

GM field trials of two species of Agrostis (A. canina and A. stolonifera), have been
conducted (Table 2.2). These species will hybridise with each other and both occur
across most of Europe (Tutin et al. 1964-1980). In addition, A. stolonifera is known to
cross with at least two other species in the genus. All 25 species within the genus in
Europe are believed to have the potential to hybridise with each other (Tutin et al.
1964-1980).
Only one field trial of GM A. canina has been conducted (Table 2.2). This trial of a
phosphinothricin tolerant cultivar took place in the US by the University of Rhode
Island. A. stolonifera conversely, has been extensively trialled in the US and Canada
(Table 2.2). The transgenic traits expressed most commonly are herbicide tolerance,
but include many others such as tolerance to shade, pests and diseases, drought,
salt, heat and aluminium. Most field trial applications have been made by Monsanto,
Scotts and Rutgers University.

DEFRA Contract CPEC 47

The most commercially advanced GM grass material comprises herbicide tolerant

Agrostis. Scotts and Monsanto have made a joint application to APHIS for the
deregulation of their ASR368 line of glyphosate tolerant A. stolonifera. This
application is currently pending. A. stolonifera is known to hybridise with eight
species of Agrostis and three of Polypogon in the US. In addition, there are a further
20 Agrostis species in the US for which the hybridisation potential with A. stolonifera
is unknown. It is a fast growing perennial grass that can spread vegetatively via
stolons with wind-pollinated flowers and small, wind and water dispersed seed. It
used extensively in golf greens, municipal parks and lawns (APHIS 2004) where
herbicide resistance would be a great advantage to reduce management
requirements. There is growing public concern, however, over the potential
deregulation of this grass due to its ability to hybridise. It is claimed that none of the
potential recipient species for transgene escape via hybridisation with GM A.
stolonifera are a serious weed problem, as they do not appear of the Federal
noxious weed list (APHIS 2004). It is claimed that the transfer of herbicide resistance
from A. stolonifera as a result of transgene escape has the potential to generate a
new set of problem weeds in the US. However, the Weed Science Society of
America does not consider this to be the case as glyphosate is not commonly used
to control Agrostis (Banks et al. 2004).
2.3.6.2

Festuca and Lolium

There are three species of Festuca and Lolium for which GM field trial applications
have been made (Table 2.2). These species are all important forage grasses in the
UK and Europe (Smartt & Simmonds 1995). Festuca arundinacea is wind pollinated
and highly self-incompatible. It is native in Europe and will hybridise with all four of
the other broad-leaved fescue species present in Europe. In addition, F. arundinacea
will also hybridise with Lolium perenne and L. multiflorum, both of which have had
GM field trials (Table 2.2). These two Lolium species are also native and will intercross freely between themselves and with the other three species of Lolium that
occur in Europe (Tutin et al. 1964-1980).
F. arundinacea has been trialled in France and the US for herbicide tolerance,
drought tolerance and disease resistance. Trials were also conducted in France with
54

DEFRA Contract CPEC 47

lines having altered lignin biosynthesis. GM L. perenne lines have been trialled in the
Netherlands to test genes inhibiting flowering; in the US drought and salt tolerant
varieties have been trialled. The Noble Foundation in the US has been trialling L.
perenne and L. multiflorum lines with transgenically reduced pollen allergens.
2.3.6.3

Other grasses

The remaining grass species with GM field trial applications are Canary grass
(Phalaris canariensis), Russian wildrye (Psathyrostachys juncea) and St. Augustine
grass (Stenotaphrum secundatum) (Table 2.2).
There are several species of Phalaris native to Europe and although Phalaris
canariensis is introduced, it is widely naturalised in the Mediterranean regions of
Europe. There has been one field trial of GM P. canariensis in Canada for a cultivar
developed with herbicide tolerance. It has been cultivated as a minor cereal in the
past but is now mainly cultivated for birdseed.
Psathyrostachys juncea does not have any related species in Europe, but occurs in
the eastern border states of Europe in Russia and Belorussia. It is native to the
steppes and deserts of Russia and China and is very tolerant to cold, drought, and to
fire. It is also moderately tolerant of flooding. Individual plants are very long lived and
are planted in the US for soil improvement, stabilisation and rehabilitation. As a
forage grass it is nutritious and digestible (Taylor 2005). GM field trials of P. juncea
remain at the experimental stage with the Noble Foundation trialling lines containing
marker genes.
Stenotaphrum secundatum is introduced to Europe and so there are no related
species present. The species is naturalised in some habitats along the seashores of
the Mediterranean (Tutin et al. 1964-1980; Stace 1975). It is widely used as a lawn
grass in parts of the US and can be used for erosion control. Scotts have conducted
field trials of GM S. secundatum in the US with glyphosate resistance and with
altered growth rates and morphology (Table 2.2).

DEFRA Contract CPEC 47

2.3.6.4

Summary of grasses

Of the eight species of grass that have GM varieties in development, six are native in
Europe and pose a risk of transgene escape. Agrostis stolonifera is one of these
native species and Monsanto and Scotts have a pending application for the
deregulation of glyphosate tolerant A. stolonifera in the US. Of the remaining three
species in field trials, two exist as naturalised, escape populations in some areas of
the Mediterranean.

2.3.7

Ornamentals

Cultivation of ornamentals as cut flowers or as garden plants does not occur as a

major activity in the agricultural statistics as it does not cover any significant area of
land and is a specialised commercial activity. Gene escape from certain cultivated
ornamentals to wild relatives is possible in Europe and has the potential to affect
populations of numerous species that are endemic to regions of Europe.
2.3.7.1

Rose

Rosa is a large genus of over 100 species native to the north temperate regions of
the world. There are 47 species of native roses across Europe, and innumerable
introduced ornamentals (Tutin et al. 1964-1980). Rosa is a very complex genus with
a unique self-incompatibility system in that many of the species have unbalanced
gametes in terms of their chromosome complement. Hybrids are common between
species and can produce morphologically different offspring from the parental
phenotypes (Stace 2001).
One of the most innovative of recent GM ornamental products (Chandler & Lu 2005)
is the development of a genetically modified blue rose announced by a joint venture
between Suntory and CSIRO (http://www.csiro.au/index.asp?type=faq&id=bluerose&
stylesheet=divisionFaq) in April 2005. This product has been produced using a threegene construct consisting of a pansy gene encoding flavonoid 3'5'-hydroxylase, an

DEFRA Contract CPEC 47

RNAi construct to inhibit the endogenous dihydroflavinol reductase (DHR) and a

novel DHR gene from iris (Figure 2.7).

Figure 2.7 Method for production of blue rose

The plant expresses the blue pigment delphinidin, produced by introduction of the gene encoding the
enzyme flavonoid 3'5'-hydroxylase from pansy and down regulation of the endogenous dihydroflavinol
reductase gene. (Source: http://www.florigene.com/news/news.php)

Figure 2.8 Transgenic blue roses (Source: http://www.florigene.com/news/news.php)

This plant and its derivatives (Figure 2.8) may possibly become the first commercial
product using RNAi technology. However, it is likely to be several years before this
product goes through the regulatory process required prior to commercial release.
Hybrid roses have also been transformed to confer disease resistance (Marchant et
al. 1998; Li et al. 2003b). Relatively few field trial applications have been made for
GM roses and these have been in the US and Australia (Table 2.2).

DEFRA Contract CPEC 47

2.3.7.2

Carnation

Forty one GM field trial applications have been made for carnations (Dianthus spp.)
in Australia, Japan, Mexico and The Netherlands (Table 2.2). Florigene, now part of
the Japanese Company Suntory, have been selling GM varieties of Dianthus
caryophyllus with modified flower colour (Fukui et al. 2003), under the names
Moondust, Moonshadow, Moonlite, Moonshade Moonaqua and Moonvista
(Figure 2.9) since 1995 in Australia and 1997 in Europe. During this period, they
have

developed

considerable

patent

portfolio

covering

most

the

anthocyanin/flavonoid pathway genes that control flower colour in petunia and

numerous homologues from a diverse number of species, as well as methods for
transformation of several ornamentals (Table 2.12).

Figure 2.9 Transgenic carnations with modified flower colours (Source: www.florigene.com)

The regulatory evaluation of this material has considered the occurrence of the many
Dianthus species across Europe. This genus is native to the Mediterranean and
there are 115 species present across Europe, of which there are 92 endemic taxa
(either species or subspecies) (Tutin et al. 1964-1980). Most species are cross
compatible but hybridisation is minimal, as the different species tend to be
geographically isolated. Where species are sympatric, hybridisation is seen to occur
(Stace 1975). This geographic isolation may prevent any transgene spread from
population to population, however, the GM varieties of D. caryophyllus used as cut
flowers are largely male sterile and there is no history of pollen dispersal or
hybridisation with other Dianthus species despite many decades of growth
throughout the world.

DEFRA Contract CPEC 47

Table 2.12 Florigene patent portfolio

Roses
US 5792927
Genetically transformed rose plants and methods for their production
US 5480789
WO 9200371
Rose plants and methods for their production and genetic transformation
US 5530182
Methods for production of hybrid rose plantlets from rose somatic embryos
Carnations
AU 703841
Transgenic carnations exhibit prolonged post-harvest life
WO 9635792
WO 9217056
Carnation plants and methods for their transformation and propagation
US 5589613
Chrysanthemums
US 5567599
Method for producing transformed chrysanthemum plants
WO 9203041
General Change in Pigmentation
Genetic sequences encoding flavonoid pathway enzymes for use in the genetic
WO 9732023
manipulation of flower pigments

2.3.7.3

Statice

Another ornamental species native to Europe is statice (Limonium spp.). There are
87 species of Limonium in Europe, of which 79 are endemic taxa (species or
subspecies) (Tutin et al. 1964-1980). Although hybridisation between species is less
common owing to the presence of a self-incompatibility mechanism, hybrids between
species have been recorded in the UK.
Limonium species have been transformed to alter their form to induce dwarfing and
early flowering, and in a more experimental study, to have green fluorescent petals
by the insertion of the green fluorescent protein gene GFP (Mercuri et al. 2001a; b).
Field trial applications have been made in Italy (Table 2.2).
2.3.7.4

Other ornamentals

Most other ornamental species for which GM field trial applications have been made
[chrysanthemum, orchids, gladiolus, pelargonium, petunia, African violet, cape
marigold, Lisianthus (Bradley et al. 2000)] represent less risk, as they are introduced
species with no wild relatives present in the European flora. Some ornamental

DEFRA Contract CPEC 47

species have escaped cultivation, however, and may become locally naturalised
representing some potential for transgene spread.
2.3.7.5

Summary of ornamentals

Carnations are the most developed transgenic ornamental with GM varieties

commercially available in some parts of the world. This species, together with rose
(also under GM development), both have wild relatives present in the European
flora. However, cultivated Dianthus species are unlikely to hybridise with wild
species. Many other ornamental species with GM varieties under development are
not native to Europe and present minimal opportunities for transgene escape.

2.3.8

Summary of European crops with potential for

transgene escape

These crops are all cultivated in Europe, have GM varieties in development

and could hybridise with wild/naturalised relatives in the European flora:

major crops

wheat, barley, oilseed rape, durum wheat,

sunflower, oat, sugar beet, grapes

fodder crops

alfalfa/lucerne, barrel clover, lupin

forestry species

silver birch, sweet chestnut, Norway spruce, Scots

pine, poplar, elm

orchard crops

olive, apple, plum, cherry, pear, walnut

grasses

Agrostis canina, A. stolonifera, Festuca arundinacea,

Lolium perenne, L. multiflorum

ornamentals

rose, statice

DEFRA Contract CPEC 47

3. Review

Containment

Methods

Conventional Crop Species

The incentive for containment of transgenic crops, especially those producing
pharmaceutical compounds (see Section 4), derives principally from the recognition
of the difficulties of maintaining separation of such crops from their conventional
equivalent. The most well known example of this problem is the so-called Starlink
episode in 2000 (http://pewagbiotech.org/resources/issuebriefs/starlink/starlink.pdf),
where material from a non-approved line of Bt corn was found in processed food
products in the US. This was followed in 2002 by identification of corn expressing
trypsin as volunteers in the following years soybean crop. This latter incident led to
the incineration of 63 ha of crops from around the site involved. In addition there are
widespread concerns regarding the introgression of transgenes into native
populations of wild species leading to undesirable ecological change, or into non-GM
crops leading to the loss of non-GM or organic status of a crop.
There are a range of methods and technologies presently being investigated in an
attempt to overcome this problem and to provide reassurance to the regulators and
the public (Ellstrand 2003a). Amongst the approaches there has been extensive
discussion of improved management oversight of the production process (Anon
2005, Wolt et al. 2004) but most activity is now focussed on more active methods to
reduce or prevent the possibility of transgene dispersal (Gepts 2004).
These different alternative approaches, which range from physical isolation to
complex molecular technology, will be discussed below.

DEFRA Contract CPEC 47

3.1

Physical separation

The simple physical separation of the transgenic crop from its non-GM equivalent
has been proposed as the most obvious method of maintaining purity and avoiding
contamination. In its simplest form this includes the use of contained growth cabinets
and greenhouses but much larger scale contained facilities have been developed.
This was first exemplified by a project in which transgenic tobacco expressing a
human recombinant glycoprotein B (gB) of human cytomegalovirus were grown in an
underground copper and zinc mine in Flin Flon, Manitoba, Canada (Tackaberry et
al.

2003).

The

company

involved,

Prairie

Plants

Systems

Inc.,

(http://www.prairieplant.com/home.htm) have operated the facility, 360 metres below

the ground, for 13 years during which time research has been conducted on over
500 plant species including various softwood and hardwood species, culinary herbs,
nutraceuticals, ornamentals and agricultural crops (Figure 3.1). Examples include
species of Nicotiana, Taxus, Maize, Brassica, Populus, Echinacea, and Ocimum.
The company has also been awarded a five-year contract by Health Canada for the
development of comprehensive operations for the cultivation and fabrication of
marijuana in Canada. In 2000, operations were extended to the USA where the first
underground chamber for the U.S. subsidiary, SubTerra LLC, was constructed and
commenced operations in White Pine, Michigan. In the same year, SubTerra LLC
was contracted for the growth, production and harvest of transgenic tobacco seed
used in cancer research.

Figure 3.1 Underground growth facilities of Prairie Plant Systems, Inc

(Source: http://www.prairieplant.com/home.htm)

DEFRA Contract CPEC 47

Since that time, interest in this approach has increased significantly. For example,
the advantages of growing these crops underground are the theme of a submission
from

Numedloc

Inc.

APHIS

2003

(http://www.fda.gov/ohrms/dockets

/dailys/03/Jan03/011603/8004ac94.pdf). In this response to Docket Number 02D0324 (Guidance for Industry - Drugs, Biologics, and Medical Devices Derived from
Bioengineered Plants for Use in Humans and Animals, Draft Guidance) the company
point out the wide availability of underground chambers, principally limestone mines,
across the US. It is stated:
The largest single mine we have examined has 1,600 acres of mineral depleted useable
space. Several states have inventories of developable space. A single mine in a
metropolitan area or serving a specific industry (steel, etc.) may create >40 acres of new
space per year. Martin Marietta, the largest limestone producer in the US has operations
in over 350 locations across the US. They and other companies can actually mine
limestone deposits to specs to create large-scale underground production facilities.
Limestone mines are typically at depths between 50 and 200 feet below the surface, have
temperatures ranging between 50 and 70oF, and do not contain minerals such as Cu or
Pb that represent direct health risks to workers.
Underground limestone mines offer significant advantages in environmental control for
bioengineered crop production relative to the aboveground environment and regarding
exposure of both workers and the public to undefined risks. Unlike greenhouses
(including double envelope designs) and aboveground windowless facilities, underground
mines are unaffected by wind, hail, snow or ice storms, tornados, fire, etc. Mineraldepleted limestone mines offer a secure protected biosecurity environment which lends
itself to production spaces engineered on the clean corridor/dirty corridor concept widely
used in animal research facilities, industrial clean rooms, etc. An underground facility can
use the mass of the rock as a thermal sink for heat produced by electric lighting and
avoids environmental heat gain from sunlight and the environment.

This approach has since been developed by Controlled Pharming Ventures

(http://www.controlledpharming.com/) a company founded by Doug Ausenbaugh of
Purdue University. Their production facility is located in a 50 acre depleted limestone
quarry in Marengo, Crawford County, Indiana. In a transcript of a UDSA meeting with
various

stakeholders

held

February

2004

(http://www.aphis.usda.gov/

DEFRA Contract CPEC 47

brs/stakeholder/Controlled_Pharming_Ventures.pdf) it is stated by Ausenbaugh that

for IP reasons their first crop would be corn, followed by alfalfa, tomatoes, and leafy
vegetables.

press

article

(http://www.impactlab.com/modules.

php?name=News&file=article&sid=5767) John Turner, director of policy coordination

in the USDAs Biotechnology Regulatory Services branch, is quoted as saying We
probably wouldnt have regulatory authority inside a contained facility such as a
mine. Similarly, as part of their own web site advertising it is stated:
The contained nature of our production system removes the USDA/BRS field testing
permit requirement, which can be cumbersome, and
The contained nature of our production system provides an exemption to the
USDA's permitting requirements for field tests, dramatically reducing the time to
market.

3.1.1
The

physical

Summary of physical separation

containment

crops

includes

growth

chambers

and

greenhouses and large-scale underground facilities. The contained growth of

transgenic plants under such conditions is commercially attractive as it
requires considerably less regulation, as there is no intended release into the
environment.

DEFRA Contract CPEC 47

3.2

Biological Containment

Several publications include some discussion of the range of methods under

development to reduce or eliminate the transfer of transgenes from genetically
modified plants. Thorough summaries are provided in several publications (Lu 2003;
Gleba et al. 2004; Davison 2005; Gressel & Al-Ahmad 2005b), and the relevance of
these approaches in the context of invasive ornamentals is discussed by Li et al.
(2004).
The term molecular containment includes natural mechanisms that are the result of
the biology of the plant, and any mechanism that is imposed by a recombinant DNA
technology.

3.2.1

Natural Genetic Containment

The biology of certain plant species provides opportunities through careful

management to eliminate the potential for pollen development. This would apply
mostly to molecular pharming activities that use of the vegetative parts of the plant to
produce pharmaceutical products and there is no need for pollination and seed
development. For example, materials produced in the vegetative parts of alfalfa or
tobacco can be harvested before flowering. (See section 4 of this report for a review
of molecular pharming technologies). An alternative to the standard genetic
engineering method of inserting genes into chromosomes is to insert genes into
chloroplasts.

3.2.2

Plastid Transformation

3.2.2.1

numbers in leaves, but not in roots. Thus, its application has some limitations and it
68

DEFRA Contract CPEC 47

is not certain how effective the insertion of transgenes into chloroplast DNA would be
for phytoremediation purposes (Davison 2005).
3.2.2.3

Commercial applications

Tobacco has been genetically modified using plastid transformation technology to

confer insect resistance, herbicide tolerance, disease resistance and heavy metal
tolerance in addition to the expression of a wide range of vaccine antigens and
pharmaceutical proteins (see section 4). Recent permits for field trials of chloroplast
transformed tobacco plants in the United States have been issued by APHIS (Animal
and Plant Health Inspection Service, USDA) for the following material:
Phenylalanine ammonia lyase: University of Kentucky (in collaboration with Icon
Genetics, Munich) issued 7June 2005
Gene construct not detailed: Chlorogen, Inc. issued 19 May 2005
Human serum albumin: Chlorogen, Inc. issued 25 May 2004 and 15 May 2003
Insulin like growth factor: Chlorogen, Inc. issued 25 May 2004
Interferon: Chlorogen, Inc. issued 25 May 2004
Mullerian inhibiting substance: Chlorogen, Inc. issued 25 May 2004

In addition, an application was made to APHIS in 2004 by Chlorogen, Inc to field trial
tobacco expressing cholera toxin B, and protective antigens from Bacillus anthracis
and Yersinia pestis and later withdrawn.
Chlorogen, Inc (http://www.chlorogen.com/) is a commercial concern based upon the
chloroplast transformation technology developed by Henry Daniell (Daniell et al.
2005) and is one of the foremost companies specializing in this technology (Maliga
2004). The company has a considerable intellectual property portfolio (Table 3.2).In
November 2004, Chlorogen signed a joint development and supply agreement with
Sigma-Aldrich Fine Chemicals, which is expected to produce the first commercial
products from chloroplast transformation technology. Sigma-Aldrich will fund an
undisclosed portion of Chlorogens efforts to produce four specific proteins in
tobacco plants. The proteins will be sold to the reagent and cell culture markets and
have pre-identified applications as active pharmaceutical ingredients.

DEFRA Contract CPEC 47

Table 3.2 Chlorogen, Inc patent portfolio

Inventors
H. Daniell,
B.A. McFadden
H. Daniell,
B.A. McFadden
H. Daniell,
K. Wycoff
H. Daniell

Date
2004

Title
Chloroplast genetic engineering

Table 3.3 Icon Genetics patent portfolio.

Patent number
WO 05054478
WO 05049839
WO 04108934
WO 04101797
WO 04067748
WO 04067749
WO 04046359
WO 04046360
WO 04046361
WO 04015115
WO 03102197
WO 03020927
WO 03020928
WO 03020938
WO 03012035

Date
16 June 2005
2 June 2005
16 Dec 2004
25 Nov 2004
12 Aug 2004
12 Aug 2004
3 June 2004
3 June 2004
3 June 2004
19 Feb 2004
21 March 2003
13 March 2003
13 March 2003
13 March2003
13 Feb 2003

WO 03004658
WO 03001900

16 Jan 2003
9 Jan 2003

WO 02101006
WO 02101060
WO 02096192
WO 02097080
WO 02088369

19 Dec 2002
19 Dec 2002
5 Dec 2002
5 Dec 2002
7 Nov 2002

WO 02083867
WO 02079481

24 Oct 2002
10 Oct 2002

WO 02077246
WO 02068927
WO 02068664
WO 02057466
WO 02055651
WO 0246440
WO 0229068
WO 0170939
WO 0111020
WO 0070019
WO 9855636
WO 9854342

3 Oct 2002
6 Sept 2002
6 Sept 2002
25 July 2002
18 July 2002
13 June 2002
11 April 2002
27 Sept 2001
15 Feb 2001
23 Nov 2000
10 Dec 1998
3 Dec 1998

3.2.2.5

Title
Controlling Gene Expression in Plastids
RNA-Virus-Derived Plant Expression System
Safe Production of a Product of Interest in Hybrid Seed
Process of Producing a Plastid-Targeted Protein in Plant Cells
Plant Transformation with in vivo Assembly of a Trait
Plant Transformation with in vivo Assembly of a Sequence of Interest
Method of Controlling Processes in Plants or Plant Cells
Method of Controlling Cellular Processes in Plants
Method of Controlling a Cellular Process in a Multi-Cellular Organism
Plastid Transformation Using Modular Vectors
Transgenic Plants with Controlled Distribution of a Trait to Progeny
Creation of Artificial Internal Ribosome Entry Site (IRES) Elements
Identification of Eukaryotic Internal Ribosome Entry Site (IRES) Elements
Method of Protein Production in Plants
Commercial Use of Arabidopsis for Production of Human and Animal
Therapeutic and Diagnostic Proteins
Gene Expression in Plastids Based on Replicating Vectors
Process of Controlled Shuffling of Chromosome Fragments for Plant
Breeding
Production of Proteins in Plants
Processes and Vectors for Producing Transgenic Plants
Process of Producing Environmentally Safe Transgenic Organisms
Amplification Vectors Based on Trans-Splicing
Processes and Vectors for Amplification or Expression of Nucleic Acid
Sequences of Interest in Plants
IRES Enabled Gene Trapping in Plants
Method of Encoding Information in Nucleic Acids of a Genetically
Engineered Organism
Site-targeted Transformation Using Amplification Vectors
Using Viruses to Detect or Purify Proteins
Recombinant Viral Switches for the Control of Gene Expression in Plants
Processes and Vectors for Plastid Transformation of Higher Plants
Processes and Vectors for Plastid Transformation
Processes and Vector Systems for Producing Transgenic Plants
Vector Systems for Plants
Methods for Transforming Plant Plastids and Making Transplastomic Plants
Method of Making Plant Artificial Chromosomes
Process of Rapid Variety-Independent Plant Transformation
Recombinant Construct for Enhancement of Gene Expression in Plants
Methods for Coexpression of more than one Gene Using at least one
Internal Ribosome Entry Site (IRES)

Other species

Although tobacco remains the species most amenable to chloroplast transformation,

recent successes have been achieved with Petunia (Zubko et al. 2004; US Patent
Application 040199937), cotton (Kumar et al. 2004b), carrot (Kumar et al. 2004a)
(Figure 3.5), tomato (Ruf et al. 2001; US Patent Application 040237131), soybean

DEFRA Contract CPEC 47

(Dufourmantel 2004), lettuce (Lelivelt 2005), Brassica (Nugent et al. 2006; US Patent
6891086) and another crucifer species, Lesquerella fendleri (Skarjinskaia et al. 2003;
US Patent Application 030200568), but stable integration of transgenes into the
chloroplast of rice (Khan and Maliga 1999) and oilseed rape (Hou et al. 2003) have
so far been unsuccessful.
Food crops with developed plastid transformations include tomato, carrot and
soybean and these are all important crops in European agriculture (Table 2.6).
Carrots are native to the Mediterranean and cultivated carrots will hybridise with their
wild relatives, of which some in Europe are endemic (Tutin et al. 1964-1980). Carrots
are biennial, however, and under normal circumstances should not flower under
cultivation as the root develops during the first years growth and the inflorescence in
the second year. In addition, carrots are thought to undergo strict maternal
inheritance of the chloroplast (Vivek et al. 1999). Transformation of carrots via the
plastid genome has been used to infer high levels of salt tolerance but this material
has not yet reached field trials.
Tomato and soybean represent a low-risk for transgene escape to wild relatives in
Europe as there are no related species in the European flora (Table 2.10), although
crop-to-crop geneflow remains a possible escape for transgenes for all crop species.
The development of plastid transformed tomato and soybean cultivars remain in the
experimental stage as successful plastid transformation of these species has only
been developed recently (Grevich & Daniell 2005; Koya & Daniell 2005). Similarly for
cotton, chloroplast transformation technologies have been developed as alternative
to nuclear transformation as a means of reducing the public concern against GM
cotton (Grevich & Daniell 2005). Cotton is an important crop in Europe ranking 28
(Table 2.6), but it also represents a low risk of transgene escape to wild relatives in
the European flora (Table 2.10).

DEFRA Contract CPEC 47

3.2.3

Genetically Engineered Gene Containment

3.2.3.1

Conditional Lethality

Transgenic plants may be engineered to contain a conditionally lethal gene that is

capable of converting a biologically inert toxin precursor into a toxic compound. As
the gene is only expressed in transgenic plants, spraying fields with the precursor
only kills the transgenic plants and non-transgenic plants are unaffected. Such
chemical compounds based on the herbicides glyphosate and glufosinate have been
developed by Monsanto and Hoechst respectively.
Kriete et al. (1996) isolated a gene from E. coli that converts non-toxic N-acetyl-Lphosphinothricin (N-ac-Pt) into cytotoxic L-phosphinothricin (Pt, glufosinate).
Tobacco plants transformed with the gene argE died following application of N-ac-Pt
whereas untransformed plants showed no reaction. This gene has also been
employed to induce male sterility by a process in which the argE gene is fused to a
DNA fragment known to function exclusively in the tapetum of the pollen grain. The
release of glufosinate in the developing anthers following N-ac-Pt application
resulted in male-sterile plants. In the absence of N-ac-Pt application plants showed
normal pollen development and full fertility (Kriete et al. 1996).
Other genes have been isolated that can be used to the same effect. For example,
the pehA gene also works on the basis of known herbicide chemistry and converts
the non-toxic phosphonate of glyphosate into glyphosate (US Patent 5254801)
leading to induced tissue damage and death of the plants containing the transgene
converter. Similarly, the iaaH gene converts non-toxic levels of naphthalene
acetamide into toxic levels of the auxin naphthalene acetic acid (Klee et al. 1987).
Other such systems have been developed for negative plant selection and inducible
male sterility (Kobayashi et al. 1995; OKeefe et al. 1994; Perera et al. 1993; patents
WO 9204454, WO 03003816).
Examination of the EU, USDA (APHIS) and OECD databases of GM field trials
(http://biotech.jrc.it/deliberate/gmo.asp;

http://www.isb.vt.edu/cfdocs/fieldtests1.cfm;

DEFRA Contract CPEC 47

http://webdomino1.oecd.org/ehs/biotrack.nsf) does not identify any applications for

deliberate release of plants transformed with genes used for conditional lethality.
Complete information of transgene constructs is not available for all field trial
applications, however, owing to business confidentiality.
3.2.3.2

Inducible Promoters

Transgenes can be activated by an artificial stimulus such as a chemical spray. In

the absence of the stimulus, the transgene would not express the trait and there will
be no conferred advantage to wild or weedy relatives receiving the transgene
through hybridisation.
Many different types of promoter elements have been described and those that
result in high levels of expression or limit expression to certain organs such as
leaves or seeds are valuable for molecular farming purposes. Promoter elements
can also be inducible and thus associated genes are only activated under certain
conditions. Induction may be dependent on natural or physiological parameters such
as heat, cold, day length, stage of development or may be dependent on the
presence of a certain chemical or biochemical. Promoters that are specifically
induced by applied chemicals can be used to drive expression of genes only when
these chemicals are applied to the plants, thereby restricting the expression of the
transgene to limited periods. For example, a crop may be sprayed to induce
herbicide resistance prior to herbicide application. Several gene regulation systems
have been developed that may be used to confer inducible herbicide resistance in
plants. These include those induced or repressed by the antibiotic tetracycline, the
Triple-Op promoter (Gatz and Quail 1988) and the Top10 promoter (Weinmann et
al. 1994); the GAL4-UAS promoter induced by the steroid glucocorticoid (Aoyama
and Chua 1997) and the GRH or similar promoter induced by the insect
prohormone ecdysone (Martinez et al. 1999; Unger et al. 2002). There are also
systems based on induction via copper (Mett et al. 1993; Granger & Cyr 2001),
acetaldehyde (Junker et al. 2003) and the steroid dexamethazone (Tang et al. 2004).
The most advanced of these technologies is the ethanol-induced alcA promoter
(Caddick et al. 1998, Sweetman et al. 2002) developed by Syngenta to induce
resistance to glyphosate (Patent WO 9706269). No records of field trials of crops
74

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a containment strategy and there are a substantial number of patent applications that
describe various GURT concepts and elements (Appendix III). Two main types of
GURT mechanisms have been developed to date: V-GURTs, that produce sterile
seeds; and T-GURTs that only express their introduced trait if treated with a specific
chemical inducer. The biological (Visser et al. 2001), economic (Eaton & van
Tongeren 2002; Eaton et al. 2002) and social (Gar 2002; Lence & Hayes 2005)
aspects of this subject have been reviewed in detail elsewhere and only a summary
will be provided here.
The first type (V-GURT) restrict the use of the entire variety through interference with
the reproduction process, and within this type, three subtypes of V-GURT can be
distinguished (Visser et al. 2001):

In the first, the seeds are fertile, but through the application of a chemical a
dormant 'lethal' gene can be activated. This gene inhibits full seed
development. As a consequence, seeds are fit for consumption but infertile.

In a second, a lethal gene is expressed in the seed, resulting in sterility.

Breeders can apply a chemical compound that activates another gene to
safeguard the fertility of the seed and the reproduction of the variety, but they
will stop doing so before selling the seed.

DEFRA Contract CPEC 47

The third, and complementary, strategy concerns vegetatively reproducing

crops, e.g. root and tuber crops and ornamentals. The described method
involves prevention of growth during storage, which may be in the interest of
breeders, growers and/or consumers. Normally, a gene that blocks growth is
expressed, but growth can be restored by activation of a second gene.
Regulation of hormone metabolism and function lies at the basis of this
strategy.

In contrast, in the T-GURT concept, one or more genes conferring a single trait are
switched on or off at will through chemical inducers. The seed itself remains viable.
It is important to realize that, although current patent applications apply to plants,
GURTs can be built into any organism, including farm animals and fish.
Sometimes known as Terminator Technology, seed sterility V-GURT systems have
been much criticised in the media and elsewhere as they have been portrayed as a
vehicle for large multi-national seed companies to suppress the freedom of farmers
on the basis that it prevents them from saving and re-sowing seeds.
The original GURT patent (US Patent 5723765), entitled Control of Plant Gene
Expression, was granted in 1998 to the USDA and Delta & Pine Land, a cotton
breeding company (http://www.deltaandpine.com/). The patent was based on
research conducted under a Cooperative Research and Development Agreement
(CRADA) signed in 1993 between Delta & Pine Land Company and the Agricultural
Research Service, a subunit of the USDA. The European version of this patent (EP
775212) was granted on 5 October 2005. The seed lethality system proposed
provides a method to protect against transfer of novel traits to other crops and plant
species as it causes seed death at a late stage in seed germination
(http://www.biotech-info.net/howto.html;
http://filebox.vt.edu/cals/cses/chagedor/terminator.html).
There are three components to the specific seed-lethality mechanism described in
this patent:

DEFRA Contract CPEC 47

1. A bacterial tetracycline-responsive Tet repressor gene (from Tn10) under the

control of a constitutive promoter. The repressor is inactivated in the presence
of tetracycline.
2. A phage P1 site-specific recombinase gene (cre) under the control of a
promoter that is subject to repression by the Tet repressor.
3. A gene coding for a cyto-toxic protein (a ribosomal inhibitor protein from
Saponaria officinalis) expressed from a seed-specific plant promoter (late
embryogenesis abundant, LEA), active only at a very late stage of seed
maturation. The expression of this toxin gene is prevented by a DNA spacer
sequence blocking its transcription. This latter sequence is bounded by the
sites of action (loxP) of the site-specific recombinase Cre.
The construct is triggered by tetracycline, which acts as a specific stimulus. In the
absence of the stimulus, the presence of the DNA spacer region in the gene coding
for the cyto-toxic protein prevents its expression and allows seed germination to
occur as normal. The application of the stimulus triggers the Tet repressor to activate
the site-specific recombinase gene cre, which excises the DNA spacer region by
acting on the loxP sites at either side. The result is a complete, uninterrupted gene
encoding the cytotoxic protein, which is expressed at the specified late stage of seed
maturation resulting in specific destruction of seed tissues.
A diagrammatic representation of such a method is shown below in Figures 3.2, 3.3
and 3.4 (adapted from http://cls.casa.colostate.edu/TransgenicCrops/terminator
diagram.pdf).

DEFRA Contract CPEC 47

Figure 3.2 Terminator technology consists of three genes

Gene I is a repressor gene that produces a repressor protein that interacts with a binding site near
Gene II. Gene II is a recombinase gene that is controlled by a promoter. Between the gene and the
promoter is a binding site for the repressor from Gene I. The recombinase gene produces a
recombinase protein that is an enzyme and snips out pieces of DNA. Gene III produces a toxin that is
lethal to embryos. The gene is controlled by a late promoter, which is active only during the late stage
of seed development when the embryo is developing. Between the late promoter and the toxin gene
is a piece of DNA called a blocker, which interferes with the ability of the promoter to turn on the toxin
gene. INDUCER: The inducer is a chemical applied to the seed by the seed company that will initiate
the terminator gene interactions.

Figure 3.3. Terminator technology: production of viable seeds

If the seed company does not want to initiate the terminator genes, it will not apply the inducer. This
allows the repressor protein to bind to the binding site on Gene II, preventing the production of
recombinase. In the absence of recombinase, the blocker on Gene III is not snipped out, and the toxin
is not produced. This allows the seed company to raise enough seed to sell to farmers.

DEFRA Contract CPEC 47

Figure 3.4 Terminator technology: inhibition of seed viability

Before the seed company sells the seed to the farmers, it applies the inducer. The inducer blocks the
binding site on Gene II preventing the repressor protein to bind. Gene II then produces recombinase
which snips out the blocker on Gene III. With the blocker removed, the late promoter is able to turn on
production of the toxin gene late in the season.

Similar strategies within the same transgene construct have been proposed, using
alternative stimuli, such as temperature or osmotic shock to regulate the mechanism,
and by using abnormal levels of plant hormones as the cytotoxic element leading to
cell destruction (Daniell 2002).
In a related study, Schernthaner et al. (2003) propose a repressible seed lethal
system whereby viable seeds are produced by the GM crop plant, when crossed (or
selfed) by another GM crop plant in the same field, but non-viable seed are produced
when the crop plant is crossed (in either direction) with non-GM plants. This method
also uses seed lethal genes with a repressor element, where the seed lethal (SL)
element is tightly linked to the desired novel trait. The repressor element (R) is
crossed into the seed during hybrid seed production; thus the planted seed contains
both the SL and R element. The presence of the repressor silences the seed lethality
gene and thus the seed generated by the crop is fully viable. Therefore, the seed
that result from the selfing of the crop are able to germinate.

DEFRA Contract CPEC 47

Should out-crossing or in-crossing occur between the SL/R crop plant and a plant
lacking the repressor element R, the two are separated. In the absence of the R
element, the seed lethality gene is activated in the seed embryo and thus any seed
containing the novel trait will not germinate (Schernthaner et al. 2003).
Separation of the SL and R elements does enable the escape of the repressor
element into other crops or wild populations. Contamination of conventional crops
with a transgene of any kind presents issues with regard to their non-GM status. The
escape of the repressor element into wild or weedy populations offers the possibility
for escape of the functioning SL transgene should a wild plant carrying the repressor
element be grown in proximity to a crop carrying the same construct in subsequent
years. In addition, seed escape during harvest would enable the GM crop to become
feral as any spilt seed will be viable and will express the novel trait.
Gressel and Al-Ahmad (2005a) claim that this particular system is technically
impractical as the seed lethal trait and the repressor element must be simultaneously
inserted at the same locus on homologous chromosomes in the hybrid seed and
site-specific insertion of transgenes has not yet been achieved in higher plants. In
addition, the hemizygous seed lethal parent would not be able to produce viable
seeds, and of the seed produced only 25% would contain both the seed lethality and
repressor element, with the remaining 75% containing only one element (Gressel &
Al-Ahmad 2005a). Such a system, therefore, requires additional development in
order to become a reliable containment methodology.
All relevant national and international agencies have considered the potential impact
of the various GURT technologies. For example, In 2000, the United Nations
Convention on Biologicial Diversity (CBD) (http://www.biodiv.org/programmes/
areas/agro/gurts.asp) recommended that "in the current absence of reliable data on
genetic use restriction technologies, without which there is an inadequate basis on
which to assess their potential risks, and in accordance with the precautionary
approach, products incorporating such technologies should not be approved by
Parties for field testing until appropriate scientific data can justify such testing, and
for commercial use until appropriate, authorized and strictly controlled scientific
assessments with regard to, inter alia, their ecological and socio-economic impacts
85

DEFRA Contract CPEC 47

and any adverse effects for biological diversity, food security and human health have
been carried out in a transparent manner and the conditions for their safe and
beneficial use validated".
Apart from Monsanto and Delta & Pine Land, other companies having patents or
applications relating to GURT technology include Syngenta, DuPont, and BASF. As
regards the present state of GURTs, the strength of public opposition has led to seed
companies withdrawing such systems from commercial development. Analysis of the
relevant databases suggests that there are no confirmed field trials of these
technologies (if defined to mean production of sterile seed), though presumably
research programmes may be continuing in glass houses etc. However, it should be
noted that there have been field tests of various chemically switchable promoters
(Section 3.2.3.2), which may be one component of a GURT system. Although the
various mechanisms are considered too complex and unreliable by some critics,
others suggest the systems may represent a useful research tool and more robust
methods are being developed (Budd 2005). Since such technology could be adopted
in one form or another as a gene containment strategy it could be particularly useful
in phytoremediation and in bio-pharming (see Section 4 below) where replanting
saved seeds is not a priority, in situations where the seeds are not intended for
human and animal consumption, and where environmental dissemination is to be
avoided. In a recent review of various genetic containment methods (Lee & Natesan
2006) the authors conclude Because they are generally lethal in hybrids, GURTs
can have an important role in controlling gene flow and introgression. If particular
caution is required, multiple strategies can be used to manage the concerns
surrounding transgenes with the greatest potential for environmental or human
health impacts.
The present Defra position on GURTs is available at:http://www.defra.gov.uk/environment/gm/eu/gurts-0602.htm
3.2.3.5

Apomixis

The use of apomixis (i.e. the production of fruit or seed without the need for
fertilisation and pollination) is a potentially important tool for the containment of
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transgenes. In addition, as seed is produced without pollination the maternal

genotype becomes fixed, thus enabling the fixation of uniform and superior
generations (Daniell 2002; Anon 2004). However, the genetic regulation of apomixis
is complex and is most likely polygenic. As such, it is likely that a complex gene
construct would be required to induce genetically engineered apomictic crop
production (Gleba et al. 2004). The transfer of apomixis to crop species via
conventional methods of crossing and backcrossing over a forty-year period has so
far failed (Perotti et al. 2004) and current understanding of apomixis is not at a point
where it can be engineered.
Some recent advances have been made however, as Spielman et al. (2003) suggest
that part of the difficulty in transferring apomixis to other species is due to the nature
of endosperm development. In seeds produced from sexual reproduction, the
endosperm is derived from two maternal cells and one paternal cell. In apomicts, the
endosperm is entirely maternal. Spielman et al. (2003) suggest that modification of
DNA methylation could alter the imprinting system that determines endosperm
development and induce endosperm development in the absence of a male gamete.
Apomixis is considered by some to have a limited application in terms of containment
of GM (Gleba et al. 2004). In particular, there are several concerns regarding the
leakiness of apomictic systems. Absolute apomicts are rare in nature and many
apomicts retain a low to moderate level of sexual reproduction, and in some cases
moderate to high levels of pollen fertility are common. Some apomicts require
pollination as a stimulus to seed formation although gamete fusion does not take
place (Anon 2004). If viable pollen containing engineered apomixis genes is
produced by the crop then the transgenes are not contained. The escape of
apomictic genes into wild populations of plant species not naturally carrying such
genes could lead to considerable damage to the natural population structure. Due to
the physiological cost of sex by the production of flowers, apomicts will always
replace out-crossing organisms when all else is equal (Anon 2004). Thus,
transgenes could spread quickly throughout a population leading to the apomicts
becoming invasive and the natural population becoming extinct by swamping (Anon
2004).

DEFRA Contract CPEC 47

3.2.3.6

Cleistogamy

Cleistogamous plants have flowers that do not open. Self-pollination and fertilisation
takes place within the closed bud and there is no release of pollen or exposure of the
gynoecium to out-crossed pollen. According to Lu (2003), transgene containment by
the use of cleistogamy appears to be innovative for some crop species, but it is not
practical for all crop species. Many crops such as maize, common buckwheat,
cassava, and most cucurbits are allogamous, and it is extremely difficult to create
cleistogamous plants for these crops. Even in the case of autogamous crops, the
manipulation of cleistogamy may alter floral structures and natural flowering habit of
some species. Such alteration might affect yield of those crops whose grains/seeds
are the part harvested.
As with apomixis, no wild plant species produce entirely cleistogamous flowers and
there will be a need to ensure leakiness does not develop. For example, in rice lines
that exhibit cleistogamy, gene flow occurs between feral and cultivated forms
(Daniell 2002). Despite the potential benefits, cleistogamy is not being developed as
a potential containment strategy (Anon 2004) and identification of genes that could
be used to engineer cleistogamy remains remote (Daniell 2002).
3.2.3.7

Transgene Mitigation (TM)

This concept has been championed by Jonathan Gressel who has published
extensively in the last few years (Al-Ahmad & Gressel 2005, 2006; Al-Ahmad et al.,
2005, 2006; Gressel 1999; Gressel & Al-Ahmad 2004, 2005a). It is also the subject
of a patent application (US 040172678).
It is argued that most transgene containment mechanisms are unidirectional, as they
will only prevent gene flow in one direction and as such are not absolute (Gressel
and Al-Ahmad 2005a). As these mechanisms are leaky there is a need to add
additional genes to mitigate against the spread of transgenic hybrids in wild
populations by expressing traits that render hybrids unable to compete against wild
plants, but that will not reduce the performance of the crop plants in the field. Thus,
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DEFRA Contract CPEC 47

the transgene-of-interest conferring the desired trait could be part of a tandem

construct containing a second gene that is beneficial, or neutral, under agricultural
conditions, but disadvantageous in the wild. Examples include genes that prevent
seed-shatter or secondary dormancy or that induce dwarfism. Proof of principle was
obtained using a cassette consisting of a semi-dominant gibberellic-acid-insensitive
gene that causes dwarfing, and a model desired-trait for herbicide resistance.
Greenhouse experiments with tobacco and oilseed rape showed that plants
containing the cassette were unable to compete with normal plants when sown in
close spacing in the absence of herbicide treatment to mimic competition in the wild.
In the absence of competition, then both the tobacco and oilseed rape
plantsproduced a good crop with reduced stem height, yet with increased leaf and
flower production (Al-Ahmad et al. 2004, Al-Ahmad & Gressel 2006). For phytoremediation purposes, this technique is inapplicable in its present form, since
competition with wild plants may be a desired trait.
According to Lu (2003), for TM to be successful, the inserted gene cassettes must
comprise tightly linked tandem constructs of the TM trait and the desired trait of
interest such that their separation during meiosis is extremely rare. However, there
are some concerns with TM as a method of transgene containment. First, this
technology does not address the problem of transgene escape from a GM crop to
non-GM crop varieties. In addition, transgene escape is allowed to occur to weedy or
wild species through gene flow, even though any resulting hybrids are engineered
not to persist in the population. The destiny and long-term consequences of the
mitigation genes in crop and weedy/wild populations are as yet unpredictable as the
traits used to compromise fitness such as seed dormancy, seed shattering, and
delayed seed ripening, are usually controlled by dominant alleles. Second, the
organization of tandem constructs with tightly linked genes will require considerable
efforts of multigene engineering. Envisaging future attempts to transfer multiple
genes with different functions into one crop variety in later generations of transgenic
biotechnology make the constructs particularly difficult and as such, TM technology
is still at the developmental stage.

DEFRA Contract CPEC 47

3.2.3.8

Recoverable Block of Function

A system of transgene containment has been developed to include a two-factor

blocking sequence and a recovering sequence within the same transgene
construct as the transgene of interest. This recoverable block of function system
developed by Kuvshinov (2001a,b; 2004; US Patent Applications 050039229,
040107457, 040025200) inseparably links the blocking sequence to the transgene of
interest by inserting it into an artificial intron within the transgene sequence. The
expression of the blocking sequence may be used to confer cell death or to prevent
sexual reproduction. The recovering sequence is induced by an external stimulus
such as chemical application or heat shock and expression results in the expression
of the normal phenotype (Gleba et al. 2004).
This system has been demonstrated in tobacco where the ribonuclease barnase was
used as the blocking sequence and is expressed from a sulphydryl endopeptidase
promoter, active at the time of seedpod development, thus preventing seed
germination. The antidote to barnase is the expression of the barstar gene, which is
placed under the control of a heat-shock promoter. Prolonged heating of the
developing seeds to 40C in a greenhouse results in barstar production and removal
of barnase expression, thus permitting correct seed development and germination.
Any seed formed from hybridisation between wild relatives and the GM crop will be
unable to germinate due to the expression of the blocking construct as they will not
encounter the prolonged high temperatures necessary for removal of the barnase
block, and so would fail to germinate (Kuvshinov et al. 2001a).
This system has been tested experimentally, but there is no record of this system
having been trialled on a field scale.
3.2.3.9

Inteins

Protein splicing elements, known as inteins are fragments of a protein sequence that
are spliced out or excised during the post-translational process to form a new
mature protein (Zeidler et al 2004; Perler 2005; Xu & Evans 2005). The action of
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inteins makes them natural protein engineering elements, which can be harnessed in
a number of ways, such as the synthesis and purification of proteins.
Inteins can be used to reconstitute a functional protein from fragments of an inactive
protein. This function can be exploited in order potentially to limit transgene spread
by reducing the possibility that a transgene expressing an active protein can escape
into wild populations or non-GM crops (Chen et al. 2001; Sun et al. 2001; Chin et al.
2003). The transgene is divided into two sections, each of which expresses an
inactive, truncated protein. These gene fragments can be inserted into different
locations in the plant cell, such as the nuclear and plastid genomes. Expression of
the genes occurs separately within the nuclear and plastid genomes, a localisation
signal from the plastid is able to target the gene product in the nucleus via the
cytoplasm, and the nuclear expressed gene product is trans-located to the
chloroplast. Within the chloroplast, the two products are trans-spliced via intein
mediation to generate a full-length transgene product (Evans et al. 2005).
By separating the two gene fragments between the nuclear and chloroplast genome,
pollen cells from such a plant will encode only a truncated, inactive transgene
product. Furthermore, should some plastid DNA escape it also encodes only a
portion of the inserted protein. Additional security could be developed for some crops
by the insertion of the nuclear transgene fragment into a genome that is not
compatible with weedy relatives. For example, crops such as oilseed rape and wheat
have multiple genomes as they are derived from hybrids of different wild relatives.
Specifically, wheat is comprised of three genomes designated A, B and D (Hedge &
Waines 2004), whereas its wild relative, jointed goatgrass (Aegilops cylindrica) has
only two, C and D. The location of the nuclear encoded fragment on the A or B
genome of wheat would therefore significantly reduce the risk of transgene spread to
wild species, but would not prevent crop-to-crop movement.
Intein mediated trans-splicing of proteins has been demonstrated in Arabidopsis
(Yang et al. 2003) and in tobacco (Chin et al. 2003) using marker-based expression
systems and herbicide resistance genes. Proof of concept of the use of inteins in
transgene containment (Evans et al. 2005; Khan et al. 2005a; Zhao et al. 2004) and
molecular pharming (Morassutti et al. 2002) has been achieved in a small number of
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experimental studies (Chen et al. 2001; Sun et al. 2001; Chin et al. 2003; Yang et al.
2003). There is also a series of patents relating to this methodology (US Patent
Applications 040172688, 040096938, 030167533).
Despite reported successes of this methodology, the trans-splicing of the proteins
within the plant cell requires optimal temperature and pH conditions in the laboratory
to ensure the proteins fold into the correct structure, making the process still
experimental and not yet sufficiently robust to be performed in the field. To date, no
field trial application has been made for plants with the inserted intein SspDnaE.
Regulatory issues regarding this technology must still consider that the partial
construct of engineered, non-native DNA may still enter the genepool of wild
species and non-GM crops. The genetic engineering of crop species with the transsplicing technique may offer an additional level of protection for transgene
containment but is unlikely to prevent transgene escape in isolation (Evans et al.
2005).
3.2.3.10

Auxotrophy

Auxotrophy is a method adopted in microbial research as a means of containing

fungal, bacterial, viral strains and cell cultures within the laboratory environment and
of preventing sample contamination. The organism is first transformed to become
dependent upon a compound that is made exogenously available, and as a
consequence it is then unable to survive or develop normally in the absence of that
compound, and untransformed organisms cannot survive in the presence of the
compound.
This has been demonstrated experimentally in plants with manipulation of the
methionine synthesis pathway in Nicotiana plumbaginifolia and Arabidopsis (Kim &
Leustek 2000; Frankard et al. 2002). Interruption of the methionine biosynthesis
pathway by the antisense expression of the CGS gene in Arabidopsis resulted in
severe growth stunting, morphological abnormalities and an inability to flower. These
traits were all reversed by the exogenous supply of methionine metabolites (Kim &
Leustek 2000).
92

Selection of a new organism

may set a project back in
time and increase costs
Cultivation, harvesting and
processing may not be
established for new
organisms

Expression of bio-pharming
products in non-conventional
crop species under
development (see section 4)

Does not prevent escape via

wild-to-crop pollination
Not all plants have 100%
maternal inheritance
Many desirable traits cannot
be produced by proteins in
the chloroplast

Conditional lethality

Control of lethality may be

timed to prevent flowering or
the development of
reproductive organs

Incomplete expression may

occur if the application of the
chemical inducer is
insufficient or fails to
penetrate plant tissues

Successful technique in
tobacco
Demonstrated in potato,
tomato, petunia, cotton,
carrot, soybean
Field trials of tobacco in the
US expressing
pharmaceutical proteins
Not yet demonstrated in the
field

Inducible promoters

Gene only activated when

necessary

Confines the trait but not the

transgene
Not applicable to traits
required throughout the life of
the plant
Incomplete expression may
occur if the application of the
chemical inducer is
insufficient or fails to
penetrate plant tissues

Natural genetic containment

Plastid transformation

Not yet demonstrated in

transgenic crops

DEFRA Contract CPEC 47

Table 3.4 continued

Technique
Engineered male sterility

Advantages
Prevents out-crossing to wild
and to non-GM crop plants
Well developed in a wide
range of crop plants

Disadvantages
Seed crops require additional
pollen source
Potential for volunteer seed
dispersal
Male sterility often leaky
Recombination of
chromosomes during meiosis
could separate transgene
from sterility system

The terminator concept and

seed lethality

Prevents transgene escape

via out-crossing and
volunteer seed dispersal

Apomixis

Prevents transgene escape

via out-crossing and
volunteer seed dispersal

Cleistogamy

Prevents transgene escape

via out-crossing and
volunteer seed dispersal

Transgene mitigation

Prevents introgression of
transgenes into wild or
weedy populations
Addresses crossing of GM
crop in both directions

Recoverable block of
function

Transgene and containment

system are inseparable
during chromosome
recombination
Can be engineered to control
pollen or seed sterility

Recombination of
chromosomes during meiosis
could separate transgene
from sterility system
Not suitable for crops where
seed is saved
Negative public perception
Genes controlling apomixis
not yet identified but likely to
involve a complex gene
construct
Likely to be leaky
GM apomicts likely to be
invasive
Does not prevent seed
dispersal
Genes controlling floral
development not yet
identified
Likely to be leaky
Seed escape may still lead to
volunteers
Not suitable for all crops
Does not prevent seed
dispersal
Does not prevent gene flow
from GM crops to non-GM
crops
May be harmful to natural
populations of wild relatives
Allows formation of
transgenic F1 hybrids
Depends on weed
competition to succeed
Recombination of
chromosomes during meiosis
could separate transgene
from mitigation system
Incomplete expression may
occur if the application of the
chemical inducer is
insufficient or fails to
penetrate plant tissues

Status
Barnase based male sterility
demonstrated in tobacco,
rice, maize, alfalfa, oilseed
rape, tomato, wheat, citrus
and birch
121 field trials in the US of
maize, oilseed rape and
Brassica oleracea crops with
barnase based male sterility
19 field trials in Europe of
crops with barnase based
male sterility
There are 2 lines or oilseed
rape, 2 of maize and 1 of
chicory commercially
available with barnase based
male sterility
Male sterile oilseed rape is
widely grown in Canada and
is pending release in the EU
under directive 2001/18.
terminator technologies
withdrawn from commercial
development and not been
demonstrated in the field

Genes controlling apomixis

not yet identified
Not yet demonstrated in
transgenic crops

Very little development as a

containment strategy
Genes controlling
cleistogamy not yet identified
Not yet demonstrated in
transgenic crops

Demonstrated experimentally
in tobacco and oilseed rape
Requires further
development

Demonstrated experimentally
in tobacco

DEFRA Contract CPEC 47

Table 3.4 continued

Technique
Inteins

Advantages
Prevents escape of a
complete, functioning
transgene

Auxotrophy

Prevents survival of
transgenic plant outside
controlled environment

Transgene excision

May be engineered to
produce non-transgenic
seed/fruit from transgenic
plants

Disadvantages
Allows escape of non-native
but non-functioning DNA
May contaminate non-GM
crops
Function of the recombinase
requires optimal conditions
that may not be obtainable in
the field
Does not prevent formation
of F1 hybrids
Recombination of
chromosomes during meiosis
could separate transgene
from mitigation system
High costs on a large scale
Requires complete
expression of the
recombinase
Recombinase excision site
will remain as non-native
DNA
Deletion products may
remain intact within the cell
and may be passed on to the
next generation
Not applicable to traits
expressed in seeds

Status
Demonstrated experimentally
in tobacco and Arabidopsis

Not well developed as a

containment mechanism
Demonstrated experimentally
in Nicotiana plumbaginifolia
and Arabidopsis

Requires further
development

DEFRA Contract CPEC 47

3.3

3.3.2

Containment options

The induction of transgenic sterility would appear to address most concerns with GM
trees. Not only would this eliminate the concerns of gene flow via pollen (Williams &
Davis 2005), such a strategy would prevent production of pollen allergens. In
addition, it has the potential to lessen the slowing of growth associated with
maturation and therefore to increase productivity (Campbell et al. 2003; Meilan et al.
2004).
Genes controlling flowering appear to be conserved among angiosperms and even
extend to some gymnosperms (Campbell et al. 2003) and transgenic sterility can be
induced in a number of ways (Ellis et al. 2001; Anon 2004; Meilan et al. 2004). Floral
tissues can be prevented from developing fully via cell ablation. Cytotoxic elements
linked to floral specific promoters can use used to induce the early death of cells e.g.
the ribonuclease barnase. Dominant negative mutations can be engineered to
encode proteins that suppress the activity of the native proteins expressed during
floral organ development. Post-transcriptional gene silencing can be induced by RNA

101

DEFRA Contract CPEC 47

interference (RNAi). This latter technique uses the introduction of a transgene that
contains an inverted repeat region of a sequence corresponding to part of a
transcribed region of the endogenous gene targeted for silencing. The repeat region
interferes with the protein coding process and floral organs will not develop fully (Ellis
et al. 2001; Meilan et al. 2004).
Stability of sterility traits is again an issue as some tree rotations last more than 40
years (Anon 2004) and occasional reversions should be expected (Meilan et al.
2004; van Frankenhuyzen & Beardmore 2004). Field trialling of sterility traits requires
taking trees through to maturity, and this will necessitate additional containment
measures such as biological buffer zones and physical isolation which will also
increase the economic costs of the trial (Campbell et al. 2003).
Complete sterility may not be required in all cases and the absence of flowers and
fruit from forest plantations will further reduce any biodiversity in the area. In some
instances, it may be beneficial to wildlife for forest trees to produce intact flowers and
fruit (Meilan et al. 2004).
Several other options for containment have potential but require further
development. Triploid poplars have been used in the past and have a high level of
innate sterility. Methods of tissue specific expression may offer alternative
containment strategies as leaf and vasculature specific promoters have been
identified in poplar and spruce (Gray-Mitsumune et al. 1999; No et al. 2000) but
tissue specific expression has not yet been demonstrated (Anon 2004).
Methods of transgene containment for GM trees require further development and
trialling. In particular, monitoring of field trials over long periods remains an issue and
the need for large areas further increases development costs (Anon 2004). The long
time lag between research, development, trialling and demonstrated commercial
potential is a barrier to the continuation of GM development in forest trees (van
Frankenhuyzen & Beardmore 2004). Indeed the benefits of insect resistance and
herbicide tolerance may not provide sufficient return on the investment to make
commercial release viable (Valenzuela & Strauss 2005). Levels of industrial interest
in the development of GM forest species are low at present, as researchers in some
102

DEFRA Contract CPEC 47

parts of the world turn away from GM technology due to concerns over public opinion
and the costs of development and trialling. However, some workers express
concerns over the difficulties in the development of GM trees and the missed
opportunities for their use in phytoremediation (Peuke & Rennenberg 2005) and
disease resistance and in reducing the economic pressure on native forests
(Valenzuela & Strauss 2005).
Research into the commercial development of lines of GM forest and fruit trees
remains active and field trials are still taking place. At present 280 applications have
been made to trial GM forestry trees and 83 to trial GM fruit trees (Table 2.2). In the
United States, Cornell University have successfully developed a GM line of ringspot
virus resistant papaya that has been deregulated by APHIS for commercial planting
(Appendix V). In addition, The University of Florida have developed another line of
GM papaya resistant to ringspot virus, and ARS have developed a GM variety of
plum resistant to plum pox both of which have applications for deregulation pending
under APHIS (Appendix VI).

103

DEFRA Contract CPEC 47

3.3.3

Summary of containment issues relating to trees

Gene flow from GM trees is a concern because:

Out-breeding, wind borne pollen capable of travelling long distances

Long-lived perennial

Forestry species are undomesticated genotypes

Cultivated close to cross-compatible wild relatives

Exotic species often escaped cultivated and feral populations exist

Dominant species in their ecological niche

Interact with many organisms

Containment options:

Male sterility

Ploidy levels

Tissue specific expression

Problems:

Long term transgene stability over lifetime of organism

Length of trial often restricted to juvenile phase of growth to prevent

flowering
o

Trialling long term stability restricted

Trialling of sterility restricted

104

DEFRA Contract CPEC 47

3.4

Containment issues relating specifically to grasses

Improvement of grasses via genetic modification offers a variety of benefits from

improved digestibility for forage grasses to increased manageability of turf grasses.
The large turf and amenity grass market has focussed development of transgenic
grasses towards the turf grass species, widely used in landscaping and golf courses.
Beneficial traits such as herbicide and disease resistance will maintain species
stands and reduce chemical input required to manage infection and infestation by
other species. Improved drought and stress tolerance in grasses will reduce the
need for irrigation and increase the use potential of marginal land. In addition, the
induction of insect and pest resistance traits will reduce the need for pesticide
applications. Horticultural qualities can be engineered to change pigmentation or
confer a stay-green appearance (Luo et al. 2004). Indeed, Monsanto are developing
a GM turf grass that will require less mowing (Anon 2004).

3.4.1

Concerns of GM grasses

Containment of GM grasses presents several challenges. Grasses are perennial,

inherently out-crossing, capable of hybridisation with related, weedy species, and
able to propagate themselves vegetatively. The perenniality of grasses means that
any spontaneous hybrids occurring between species will persist and flower from
season

season,

increasing

opportunities

for

further

backcrossing

and

introgression. Most grass species are self-incompatible and out-crossed by wind

borne pollen. Grass pollen is known to travel great distances and workers have
taken the opportunity to assess transgene movement during GM field trials of
grasses in the US. Wang et al. (2004b) examined pollen mediated transgene flow in
Festuca arundinacea and found transgenic traits in populations of grasses up to
150m from the crop, but no further than 200m. Watrud et al. (2004) analysed
geneflow from a transgenic crop of Agrostis stolonifera and found that most gene
escape occurred within 2 Km in the direction of the prevailing winds. Some pollen
was found to travel much further with the maximum gene flow distances measured
being 21 Km from the field site (Watrud et al. 2004). Gene flow and introgression

105

DEFRA Contract CPEC 47

between non-transgenic Sorghum bicolor (cultivated sorghum) and S. halepense

(Johnson grass) has been assessed using a range of crop specific markers and
found that gene flow occurs between sympatric populations, with up to 32% of
individuals in a population containing DNA of sorghum origin (Morrell et al. 2005). In
addition, populations of Johnson grass with no recent exposure to sorghum were
found to contain sorghum alleles; this suggests that sorghum pollen had travelled
long distances and that the introduced alleles had become stabilised in the
population (Morrell et al. 2005).
Grasses can also reproduce by vegetative means via rhizomes and stolons and
these in turn be moved around by agricultural machinery. In addition, seed can be
dispersed by birds and mammals feeding and foraging in the grass (Anon 2004).

3.4.2

Containment options

Most of the containment strategies discussed in Section 3 of this report may be

applied to grasses. Development has focussed on male sterility as pollen mediated
gene flow over long distances causes most concern. Male sterility via cell ablation
has been demonstrated in several grass species including Agrostis stolonifera (Luo
et al. 2004; US Patent Application 050235379). Interruption of the pollen
development pathway will also reduce the amount of pollen in the air and be of
benefit to allergy sufferers.
To date there have been just over 200 field trial applications for GM grasses. The
vast majority of these have been for Agrostis stolonifera in the US and Canada.
Indeed there is currently a petition pending in the US for the deregulation of
glyphosate tolerant A. stolonifera from Monsanto and Scotts (Appendix VI).

106

DEFRA Contract CPEC 47

3.4.3

Summary of containment issues relating to

grasses

Gene flow from GM grasses is a concern because:

out-breeding, wind borne pollen is capable of travelling long distances

grasses are perennial

cultivated species are undomesticated genotypes

will hybridise with wild species

will hybridise with close relatives, which may be weedy species

some species have wind borne seed

some species are capable of vegetative propagation

107

DEFRA Contract CPEC 47

4. Review of Transgenic Methodologies for

the Bio-Pharming of Pharmaceutical
Products
The adoption of plants as generators for industrial and pharmaceutical compounds is
a rapidly expanding area of development. The production of plant based
pharmaceuticals (pharming) offers many benefits compared to conventional
mammalian cell culture techniques (Webster 2004). Plant based platforms are less
costly with increased flexibility in terms of scale and storage and can return high
yields (Table 4.1). The benefits are such that the World Health Organisation are now
developing plant based production of antibodies for diseases such as rabies in
collaboration with the biotech industry (WHO 2004), and the potential commercial
market is estimated to grow at a compound annual growth rate of 104.3% over the
period 2005-2011 (Figure 4.1) (Webster 2004).
Plant based pharmaceuticals present a different set of problems for the regulators
(Howard & Donnelly 2004; Stewart & Knight 2005). The contamination of crops
destined for consumption by humans or livestock by genes that express potentially

Figure 4.1 Forecast for the total North American market for plant biopharmed products for
pharmaceutical use (Source: Webster 2004)

108

DEFRA Contract CPEC 47

Table 4.1 Comparison of the features of recombinant protein production for the various
bioreactor systems currently available (modified from Fischer et al. 1999b and Walker et al. 2005)
a
In terms of large-scale fermentors and associated equipment; bIn relation to contamination of human
therapeutic proteins with viral sequences, oncogenes or endotoxins
Production System
Features

Bacteria

Yeast

Mammalian
cell culture

Transgenic
animals

Transgenic
plants

Transgenic
microalgae

Production time
Production cost
Scale up cost
Storage cost
Storage method
Production scale
Propagation
Distribution
Delivery vehicle
Gene size
Glycosylation
Multimeric
protein assembly
Protein yield

Short
Medium
Higha
Low
-20oC
Limited
Easy
Feasible
No
Unknown
Absent
No

Medium
Medium
Higha
Low
-20oC
Limited
Easy
Feasible
No
Unknown
Incorrect
No

Long
High
Higha
High
Liquid N2
Limited
Difficult
Difficult
No
Limited
Correct
No

Long
High
Higha
High
Liquid N2
Limited
Feasible
Difficult
Yes
Limited
Correct
Yes

Long
Low
Low
Low
Room temp
Worldwide
Easy
Easy
Possible
Not limited
Correct
Yes

Short
Very low
Very low
Low
Room temp
Worldwide
Very easy
Easy
Possible
Not limited
Correct
Yes

Medium

High

Unknown

Riskb
Safety
Ethical concerns

Yes
Low
Low

Unknown
Unknown
Medium

Mediumhigh
Yes
Medium
Medium

Yes
High
High

Unknown
High
Medium

harmful compounds is a cause for real concern, as has already been demonstrated
by the Starlink episode. A number of novel approaches are being investigated and
adopted in order to reduce this risk. Physical containment strategies discussed in
Section 3 of this report also apply to bio-pharming crops. In addition, various
biological methods are available to reduce or eliminate gene transfer via pollen or
any other route, with the choice of species being the most obvious consideration.
Bio-pharming systems have been developed for many existing crop species since
there are established harvesting and processing structures in place and a high level
of knowledge on the biology and genetic modification of many crop species.
Following concerns regarding the extra levels of containment required for biopharma crops (Andow 2004), some groups have chosen to use non-crop plants as
the basis of their bio-pharming technology as these will be easier to separate from
existing agricultural and food processing systems and so will be less likely to enter
the food chain.

109

DEFRA Contract CPEC 47

4.1

Conventional crop species

(NEPA

document,

http://www.aphis.usda.gov/brs/ph_permits.html).
Other recent experimental studies on rice include the production of the glucagon-like
peptide that has potential in diabetes therapy (Sugita et al. 2005, Yasuda et al.
2005), a mammalian transglutaminase (Capell et al. 2004) and expression of cedar
pollen induced T-cell epitopes (Takagi et al. 2005).
The containment strategies for GM barley are similar to those required for a
transgenic rice crop. Barley is 99% self-pollinating and is rarely visited by insects. A
fallow zone of 50 feet (15 m) is required around the crop and an isolation distance of
0.25 miles (0.4 Km) from the nearest barley crop. As the transgene is expressed in
the seed, the remaining stubble is considered to contain insignificant amounts of the
transgene product and may be ploughed under. The field must also be burnt when
the conditions allow. Seed dormancy is not observed in barley and there are no
sexually compatible wild relatives in the area of the Ventria facility. Dedicated
handling equipment is again required and processing must take place on site (NEPA
document, http://www.aphis.usda.gov/brs/ph_permits.html).

113

DEFRA Contract CPEC 47

All plants expressing pharmaceutical products in the US are regulated and monitored
by APHIS and as such an application for deliberate field trial must be made and the
trial must be carried out in accordance with the specifications set out when the
licence is issued. There are no plans to deregulate crops expressing pharmaceutical
products in the US (Ma et al. 2005a).

4.1.2

Pharmed products close to the market

There are numerous bio-pharmed products that are currently close to the market.
These include the antibody CaroRx, collagen from tobacco, human gastric lipase,
and vaccines induced in edible crops (Horn et al. 2004). A summary of the status of
some of these programmes is provided below (Table 4.2) (Ma et al. 2005a).
It should be noted that the early emphasis on the idea of using fruit or vegetables
directly as edible vaccines has now been superseded by realisation of the difficulty
of controlling the dosage and the problems associated with separating such GM
products from the non-GM food chain. The present terminology is orally active
thermostable vaccines and the future programmes in this area are likely to involve a
processed rather than a fresh product.
Table 4.2 Plant-derived pharmaceutical proteins that are closest to commercialization for the
treatment of human diseases (From Ma et al. 2005a)
Crop
Transgenic
Arabidopsis
Transgenic lettuce

Class
Dietary

Product
Human intrinsic factor

Vaccine

Hepatitis B virus
surface antigen

Transgenic maize

Dietary

Lactoferrin

Transgenic maize

Gastric lipase

Transgenic potato
Transgenic potato

Therapeutic
enzyme
Vaccine
Vaccine

Transgenic potato

Vaccine

Transgenic tobacco
Transgenic tobacco
Viral vectors in
Nicotiana
benthamiana
Viral vectors in
spinach

Antibody
Vaccine
Antibody

Vaccine

Disease
Vitamin B12
deficiency
Hepatitis B

E. coli heat labile toxin

Hepatitis B virus
surface antigen
Norwalk virus capsid
protein
CaroRx
E. coli heat labile toxin
Various single chain Fv
antibody fragments
Rabies glycoprotein

Company/Organisation
Cobento Biotech AS

Status
Phase I

Thomas Jefferson
University/Polish Academy of
Sciences
Meristem Therapeutics

Phase I

Meristem Therapeutics

Phase II

Tacket et al. 1998

Richter et al. 2000

Phase II
Phase I

Norwalk virus
infection
Dental caries
Diarrhoea
Non-Hodgkins
lymphoma

Tacket et al. 2000

Phase I

Planet Biotechnology Inc

Prodigene Inc
Large Scale Biology Corp

Phase II
Phase I
Phase I

Rabies

Yusibov et al. 2002

Phase I

Gastrointestinal
infections
Cystic fibrosis,
Pancreatitis
Diarrhoea
Hepatitis B

114

Phase I

DEFRA Contract CPEC 47

4.1.3

Tobacco

Due to the long history of genetic modification in tobacco, this was the most obvious
first target species for pharming application and indeed, more transgenes have
been expressed in tobacco than all other species combined (Grevich & Daniell
2005). Tobacco is a very efficient production system, with one plant producing a
million seeds and one acre producing in excess of 40 metric tonnes of leaves per
year (Cramer et al. 1999). As a bioreactor species, tobacco is significantly less costly
than standard E. coli production systems (Kusnadi et al. 1997) and as it is neither a
food nor a feed crop it is unlikely to contaminate the food chain (Grevich & Daniell
2005). In addition, there is an established process for harvesting leaf material and
this can be done before flowering takes place. Several companies are using tobacco
for pharmaceutical production.
One example of this is the recent announcement (12 July 2005) by Large Scale
Biology Corporation (LSBC) and Planet Biotechnology of increased production of
Planets lead product, CaroRxTM, a plant-made antibody to control dental caries.
CaroRxTM is the worlds first recombinant plant-made antibody (plantibody) and has
been shown in clinical studies to prevent the adhesion to the tooth surface of the
decay causing bacteria Streptococcus mutans (Figure 4.2).

Figure 4.2 Working hypothesis for mode of action of CaroRxTM

(Source: Planet Biotechnology)

115

DEFRA Contract CPEC 47

Many species of bacteria inhabit the mouth and teeth (Figure 4.2) but most of these
are harmless. Antiseptic treatment will kill and remove most of these bacteria
including S. mutans from between the teeth. The removal of bacterial leaves an
ecological niche previously occupied by S. mutans and this will be recolonised
under normal circumstances. With CaroRx treatment recolonisation of S. mutans
is blocked by preventing the adhesion of the bacteria to the surface of the teeth.
Colonization of other oral bacteria occurs unimpeded and after a sufficient period the
niche once occupied by S. mutans is occupied by some other organism, or is altered
in some other way to exclude S. mutans.
Planets tobacco plants expressing the proprietary CaroRxTM protected SIgA are
being grown in Kentucky and extracted by Large Scale Biology Corporation (LSBC)
(www.lsbc.com) at its Owensboro manufacturing facility in Kentucky. It was stated in
a press release on 12th July 2005 (http://www.lsbc.com/pdfs/Planet_LSBC_7_12_
2005.pdf) that CaroRx is currently approved for sale as a medical device in the
European Union, although no further details of this approval are known and this
statement is no longer (13th December 2005) on their web site. LSBC is a
biotechnology

company

developing

and

bio-manufacturing

bio-therapeutics,

vaccines and industrial proteins for important life science industry markets. They
have an extensive patent portfolio in areas relevant to this technology (Table 4.3),
and have been financially aided in 2005 by the commercial arm of the University of
Kentucky. LSBC use transiently expressed viral vectors to induce recombinant
protein production in non-transformed Nicotiana (see section 4.2).
Planet Biotechnology Inc. (www.planetbiotechnology.com) is a privately held,
clinical-stage company whose mission is to discover, develop and commercialise
new monoclonal-antibody-based therapeutic and preventative products to meet
significantly underserved medical needs.
Icon Genetics (http://www.icongenetics.com/) are also using tobacco in a similar
project announced in August 2005. In a joint experiment in collaboration with the
Kentucky Tobacco Research and Development Center (KTRDC), University of

116

DEFRA Contract CPEC 47

Table 4.3 The Large Scale Biology Corporation patent portfolio

Title
Plant Viral Vectors Having Heterologous Subgenomic
Promoters for Systemic Expression of Foreign Genes
Recombinant Plant Viral Nucleic Acids
Viral Amplification of Recombinant Messenger RNA in
Transgenic Plants
Recombinant Plant Viral Nucleic Acids
Recombinant Plant Viral Nucleic Acids
Viral Amplification of Recombinant Messenger RNA in
Transgenic Plants
The Cytoplasmic Inhibition of Gene Expression by Viral RNA
Viral Amplification of Recombinant Messenger RNA in
Transgenic Plants
Production of Peptides in Plants as Viral Coat Protein Fusions
Viral Amplification of Recombinant Messenger RNA in
Transgenic Plants
The Cytoplasmic Inhibition of Gene Expression by Viral RNA
Viral Expression Vectors
Production of Peptides in Plants as Viral Coat Protein Fusions
Cytoplasmic Inhibition of Gene Expression in Transfected
Plants by Tobraviral Vector
Cytoplasmic Inhibition of Gene Expression and Expression of
a Foreign Protein in a Monocot Plant by a Plant Viral Vector
Production of a Parvovirus Vaccine in Plants as Viral Coat
Protein Fusions
Method for Conferring Herbicide, Pest or Disease Resistance
in Plant Hosts
Method of Compiling a Functional Gene Profile in a Plant by
Transfecting a Nucleic Acid Sequence of a Donor Plant into a
Different Host Plant in an Anti-Sense Orientation
Process for Isolating and Purifying Viruses, Soluble Proteins
and Peptides From Plant Sources
Process for Isolating and Purifying Viruses, Soluble Proteins
and Peptides From Plant Sources
Method for Recovering Proteins From the Interstitial Fluid of
Plant Tissues
Method for Recovering Proteins from the Interstitial Fluid of
Plant Tissues
Method for Recovering Proteins from the Interstitial Fluid of
Plant Tissues

US Patent
5316931

Issue Date
31 May 1994

Technology
GENEWARE, Plant
GENEWARE, Plant
GENEWARE, Plant

5866785
5889190
5889191

31 December 1996
22 September
1998
2 February 1999
30 March 1999
30 March 1999

5922602
5965794

13 July 1999
21 October 1999

GENEWARE, Plant
GENEWARE, Plant

5977438
6462255

2 November 1999
8 October 2002

GENEWARE, Plant
GENEWARE, Plant

6479291
6656726
6660500
6700040

12 November 2002
2 December 2003
9 December 2003
2 March 2004

GENEWARE, Plant
GENEWARE, Plant
GENEWARE, Plant
GENEWARE, Plant

6800748

5 October 2004

GENEWARE, Plant

6730306

4 May 2004

Vaccines

6303848

16 October 2001

Genomics

6426185

30 July 2002

Genomics

6033895

7 March 2000

Biomanufacturing

6037456

14 March 2000

Biomanufacturing

6284875

4 September 2001

Biomanufacturing

6441147

27 August 2002

Biomanufacturing

6617435

9 September 2003

Biomanufacturing

5589367
5811653

GENEWARE, Plant
GENEWARE, Plant
GENEWARE, Plant

Kentucky, Lexington, Icon will field trial transgenic plants with genetically engineered
chloroplasts containing a phenylalanine ammonia lyase (PAL) gene from Arabidopsis
thaliana. A release permit has been obtained from APHIS, the United States
Department of Agricultures' Animal and Plant Health Inspection Service. According
to Zaitlin et al. (2004) KTRDC believe that an F1 hybrid strategy can prevent
transgene escape. For production of industrial or pharmaceutical proteins, the
maternal (tobacco) cultivar is transformed and homozygous transformants with highlevel expression are selected and crossed with other Nicotiana species. It is the
resulting sterile, morphologically distinct, F1 hybrids that are grown in the field
(Figure 4.3).

117

DEFRA Contract CPEC 47

Figure 4.3 Nicotiana crop-producing materials for medical and other applications
The crop is close-grown and machine-harvested, with up to five harvests per season. Tobacco plants
are harvested at night to ensure lowest possible temperatures of the plants for transfer to the
bioprocessing facility. (Source: http://www.uky.edu/KTRDC/TOB%20Tech9%2005.pdf).

Overexpression of the PAL gene in tobacco chloroplasts is aimed at both

pharmaceutical and industrial applications. The purified enzyme can serve as a drug
for the treatment of the inherited disease phenylketonuria (PKU) and also serves as
a biocatalyst for industrial biochemical synthesis. In addition, strong expression of
PAL leads to accumulation of metabolites from the phenylpropane pathway, which
Table 4.4 US field trials of GM tobacco expressing pharmaceutical products
CBI: confidential business information. (Source: APHIS http://www.aphis.usda.gov/brs/pharmaceutical
.html)
Permit
99-041-02r
99-041-01r
00-070-01r
00-070-02r
01-074-01r
01-074-02r
02-080-01r
03-063-01r
04-044-01r

Date Issued
8 March 1999
8 March 1999
19 April 2000
19 April 2000
11 May 2001
11 May 2001
7 May 2002
15 May 2003
28 April 2004

Company
CropTech
CropTech
CropTech
CropTech
CropTech
CropTech
CropTech
Chlorogen Inc
Planet Biotechnology

04-114-04r

25 May 2004

Chlorogen Inc

04-355-01r
05-061-01r

19 May 2005
7 June 2005

05-053-01r

8 June 2005

Chlorogen Inc
University of
Kentucky
Planet Biotechnology

05-087-01r

7 July 2005

Planet Biotechnology

118

Modification
CBI
CBI
CBI from Man
CBI from Man
CBI
CBI
CBI from Man
Serum albumin from Man
Antibody from Mouse
Antibody from Oryctolagus cuniculus
Albumin from Man; Insulin-like growth factor
from Man; Interferon from Man; Mullerian
inhibiting substance from Man
CBI from Man; CBI from Cow
Phenylalanine
ammonia
lyase
from
Arabidopsis thaliana
Common cold antibody from CBI; Tooth
decay antibody from Mouse; Tooth decay
antibody from Oryctolagus cuniculus
Tooth decay antibody from Mouse

DEFRA Contract CPEC 47

are also of commercial value. It is claimed that chloroplast-located transgenes

generally do not spread into the environment via pollen flow. Moreover, the
genetically engineered plants do not contain antibiotic resistance genes, since they
were created using ICON's proprietary resistance marker removal technology.
A modified version of the tobacco production system is that reported by Menassa et
al. (2001) who describe the use of a hybrid male-sterile low-alkaloid tobacco (MSLA)
for the production of human interleukin-10.
The majority of these products have been produced in field trials in the US. Since
1992 there have been 16 field trial applications for tobacco expressing
pharmaceutical and industrial proteins in the US. Fourteen of these have been
granted to CropTech, Planet Biotechnology, Chlorogen Inc, and the University of
Kentucky (Table 4.4). Further details of these field trials are listed in Appendix IV.
Table 4.5 European field trials of tobacco expressing pharmaceutical products
(Source: http://gmoinfo.jrc.it/gmp_browse_geninf.asp)
Permit
B/FR/98/05/07
B/FR/97/05/17
B/FR/99/02/13
B.FR/95/02/18CON
B/FR/96/01/10CON
B/FR/96/01/11CON
B/FR/97/05/15
B/FR/97/05/16
B/FR/95/02/17CON
B/FR/96/01/12CON
B/ES/97/32
B/ES/97/33
B/ES/97/24
B/IT/01/02

Country
France
France
France
France

Company
Seita Institut du Tabac
Biocem SA Limagrain Group
Meristem Therapeutics
Biocem SA Limagrain Group

Modification
Synthesis of antibodies
Synthesis of collagen
Synthesis of collagen
Synthesis of dog gastric lipase

France

Biocem SA Limagrain Group

DEFRA Contract CPEC 47

APHIS demands that field trials of transgenic tobacco expressing pharmaceutical

products are topped to prevent flowering. Non-transgenic crops less than one mile
from the field site are also prevented from flowering.
There have also been a number of trials in Europe of transgenic tobacco expressing
pharmaceutical products (Table 4.5) all of which took place between 1995 and 2001
and no trials have been conducted more recently. The containment strategies in
place during these trials are no longer available through the European Commissions
Joint Research Centre website as the trials are not recent.
There are numerous other studies on transgenic tobacco (Elliott et al. 1996; Alli et al.
2002) expressing a wide range of pharmaceutical products. These are not yet at a
commercial stage of development and summarised below:
Anthrax antigen: Watson et al. 2004; Aziz et al. 2002
Antibodies: Almquist et al 2004; Bakker et al. 2001; Bruyns et al. 1996; Cabanes-Macheteau
et al. 1999; Frigerio et al. 2000; Galeffi et al. 2005; Kathuria et al. 2002; Ko et al.
2005; Le Gall et al. 1998; Magee et al. 2004; Makvandi-Nejad et al. 2005; Nuttall et
al. 2002; Rodriguez et al. 2005; Schillberg et al. 1999; Stevens et al. 2000; Vaquero
et al. 2002; Weintraub et al. 2005; Wu et al. 1997
Antigens: Daniell et al. 2001; Ehsani et al. 2003; Elbers et al. 2001; Ramirez
et al. 2003; Rukavtsova et al. 2003.
Birch pollen allergen Bet v 1: Krebitz et al. 2000
Bovine growth hormone (bGH): Oh et al. 2003
Cholera toxin B subunit: Wang et al. 2001;
Colorectal cancer antigen GA733-2: Verch et al. 2004
Desmodus rotundus salivary plasminogen activator alpha1: Schiermeyer et al. 2005
Escherichia coli enteropathogenic pilin subunit A: da Silva et al. 2002
Escherichia coli heat-labile enterotoxin (LTB), non-toxic B subunit: Kang et al. 2003
Hantavirus serotype Puumala, Nucleocapsid protein: Khattak et al. 2004
Helicobacter pylori strain ZJC02, UreB antigen: Gu et al. 2005
Honeybee royal jelly MRJP1: Judova et al. 2004
Human alpha-lactalbumin: Takase et al. 1998
Human acetylcholine esterase-R: Geyer et al. 2005
Human CD 14: Blais & Altosaar 2005
Human cytomegalovirus glycoprotein B: Tackaberry et al. 1999, 2003; Wright et al. 2001
Human epidermal growth factor: Wirth et al. 2004

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Human erythropoietin: Cheon et al. 2004

Human granulocyte-macrophage colony stimulating factor: Sardana et al. 2002
Human growth hormone: Leite et al. 2000
Human hepatitis B virus core antigen: Tsuda et al. 1998; Valdes et al. 2003; Yano &
Takeoshi 2004
Human interferon: Leelavathi & Reddy 2003
Human immunodeficiency virus, diagnostic reagent: Schunmann et al. 2002
Human immunodeficiency virus type I (HIV-1) p24 capsid protein: Zhang et al. 2002;
Obregon et al. 2006
Human insulin-like growth factor precursor prohormone IGF-1B: Panahi et al. 2003,
2004
Human islet autoantigen glutamic acid decarboxylase: Porceddu et al. 1999; Avesani et
al. 2003
Human lactoferrin: Salmon et al. 1998; Samyn-Petit et al. 2003
Human papillomavirus type 16 virus-like proteins: Biemelt et al. 2003; Liu et al. 2005,
Varsani et al. 2003
Human placental -glucosidase: Reggi et al. 2005
Human somatotropin: Staub et al. 2000
Hydroxylated human homotrimeric collagen I: Ruggiero et al. 2000; Merle et al. 2002;
Osorio 2004
Immunogenic Puumala virus nucleocapsid protein: Kehm et al. 2001
Interleukin-18: Zhang et al. 2003
Interleukin-4: Ma et al. 2005b
K88 fimbriae, major subunit: Huang et al. 2003b; Joensuu et al. 2004
Measles virus, haemagglutinin protein: Huang et al. 2001, Webster et al. 2005
Plasmodium falciparum C-terminal region of merozoite surface protein (PfMSP1(19)), a
potential malaria vaccine candidate: Ghosh et al. 2002
Preproricin: Sehnke & Ferl 1999
Porcine epidemic diarrhoea virus, protein: Bae et al. 2003; Kang et al. 2004, 2005b
Porcine parvovirus, Capsid protein VP2: Rymerson et al. 2003
Porcine relaxin: Buswell et al. 2005
Rabies virus, surface glycoprotein: Ashraf et al. 2005
Recombinant dog gastric lipase: Gruber et al. 2001
Rinderpest virus, haemagglutinin protein: Khandelwal et al. 2003
Rotavirus VP6: Birch-Machin et al. 2004
SARS-coronavirus (CoV) spike protein: Pogrebnyak et al. 2005
Single-chain human interleukin-12: Gutierrez-Ortega et al. 2004
Staphylococcus aureus, staphylokinase: Dobrowolska et al. 2004
Toxoplasma gondii, surface antigen: Clemente et al. 2005
Tetanus toxin Fragment C: Tregoning et al. 2003, 2005

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Thrombomodulin: Schinkel et al. 2005

Xylanase: Leelavathi et al. 2003

4.1.4

Alfalfa

Alfalfa (or lucerne) is a perennial plant that does not die after harvesting but grows
back to maturity in five weeks, without flowering, sexual crossing or producing seeds.
The new leaves generated after harvesting are identical, perfectly natural clones of
the ones they replace, thus alfalfa is a highly efficient protein production system. In
addition, vegetative propagation allows for ultra-fast multiplication by stem-cutting,
producing identical clones. Alfalfa can be harvested at least ten times a year for ten
years or more, providing 10g of recombinant protein for every square metre of
greenhouse space.
The most advanced production system using this crop is that developed by
Medicago in Canada (http://www2.medicago.com/en/). Alfalfa plants are grown in hitech greenhouses under their proprietary system named ProficiaTM. This system is
claimed to offer high volume potential of plant-based technology together with the
safety and control of confined environment production.
Medicago has currently four products under early stage development. Preclinical
trials are planned to start in 2005 for two products: monoclonal antibodies and
plasmatic proteins. A recently announced collaboration (2 June 2005) with
InterveXion

Therapeutics

(http://www.intervexion.com/)

will

use

Medicagos

technology for the production of monoclonal antibodies therapeutics designed to

treat drug abuse. InterveXion has received a $3 million grant to conduct clinical trials
for the first antibody treatment for addiction to the drug known as phencyclidine, or
PCP. It is claimed that that working closely with FDA and NIDA will allow an
accelerated clinical program to get the drug approved in three to four years.
Earlier this year (31 January 2005) Medicago announced a research collaboration
with Bayer CropScience to assess the feasibility of using the Proficia system for
the production of an undisclosed human therapeutic protein.

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Table 4.6 US field trials of GM alfalfa expressing pharmaceutical products

(Source: APHIS http://www.aphis.usda.gov/brs/pharmaceutical.html)
Permit
94-362-02r

Date Issued
12 April 1995

Company
University of
Wisconsin

97-052-02r

23 April 1997

University of
Wisconsin/Madison

93-088-02r

4 June 1993

University of
Wisconsin

92-183-01r

10 October 1992

Noble Foundation

Modification
Amylase from Bacillus licheniformis;
Lignin peroxidase from Phanerochaete
chrysosporium
Cellulase; Inositol hexaphosphate
phosphohydrolyase from Aspergillus
niger; Lignin peroxidase
Alpha-amylase from Bacillus
licheniformis; Lignin peroxidase from
Phanerochaete chrysosporium
Cholera toxin B from Vibrio cholera

Since 1992 there have been four field trials of transgenic alfalfa in the US carried out
by the University of Wisconsin and the Noble Foundation (Table 4.6). Further details
of these trials are listed in Appendix IV.
The Medicago crop of alfalfa was grown under physical containment. For field
release of GM alfalfa APHIS requires an isolation distance of 900 feet to the nearest
alfalfa crop. Most commercial crops of alfalfa are harvested every 20-40 days to
prevent seed formation.
To date there have been no field trials in Europe of alfalfa expressing pharmaceutical
proteins.
Other projects using a transgenic alfalfa system (DAoust et al. 2004a, 2004b;
Vzina et al. 2002) include:
Antibodies: Bardor et al. 2003; Busse et al. 2002; Khoudi et al. 1999
Bovine rotavirus, VP4 protein: Wigdorovitz et al. 2004
Classical Swine Fever Virus, E2 glycoprotein: Legocki et al. 2005
Foot and mouth disease virus, structural protein VP1: Wigdorovitz et al. 1999
Foot and mouth disease virus, immunogenic epitope: Dus Santos et al. 2002; Dus Santos
& Wigdorovitz 2005
Human group A rotavirus, VP6 protein: Dong et al. 2005
Mannheimia haemolytica, leukotoxin gene from: Ziauddin et al. 2004
Root exudation of recombinant protein: Tesfaye et al. 2005

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Promising results from M. truncatula have also been reported (Abranches et al.
2005).

4.1.5

Sugarcane

Another species adopted for use in bio-pharming techniques is sugar cane as it is a

non-flowering crop. Wang et al. (2005b) developed 200 independent sugarcane lines
producing the human cytokine granulocyte macrophage colony-stimulating factor
(GM-CSF). Analysis of recombinant protein accumulation and activity levels of GMCSF protein ranged from undetectable to 0.02% of total soluble protein. Human bone
marrow cells (TF-1), which require GM-CSF for cell division, proliferated when
growth media was supplemented with transgenic sugarcane extracts. Comparison to
purified commercially produced GM-CSF indicated the sugarcane-produced protein
had essentially identical activity levels. The field trial was conducted in Hawaii and
the trial plants did not flower, thus no pollen or seed was produced from the crop. In
addition, the test plants were dried, burnt and buried to prevent the transgenic
sugarcane entering the environment or food supply. It was suggested therefore that
sugarcane might provide a highly secure system for biofactory production of
pharmaceutical proteins. As a food crop, regulations need to be in place to ensure
the containment of transgenes from pharma-sugarcane.
In a related publication (http://tlo.tamu.edu/tlo/documents/INDR/1436.pdf) Texas
A&M University are offering to produce any recombinant protein in sugar cane. They
claim that sugarcane is an ideal bio-pharming crop for the following reasons:

Sugarcane displays the highest biomass per acre of any annual crop.

Plant materials are not part of the food chain since they are seldom consumed directly by
humans, unlike corn or rice.

Sugarcane economics can be leveraged by a variety of valuable by-products, e.g., the

biogases following crushing can be burned to generate power and the sucrose can be used
for the production of ethanol.

Does not cross-pollinate since it can be grown in geographic areas where the plant does not
flower.

Protein expression demands relatively little post-transcriptional modification.

Due to sugarcanes high-water content, protein purification is relatively simple.

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Plants harbour no animal pathogens.

Texas A&M University System have already (2003) signed a license agreement with
proCANE LLC, a subsidiary of ECOR Corp. of Sedona, Arizona, to produce
pharmaceutical-grade proteins in sugarcane plants.
Since 1992 there has been only one field trial of transgenic sugarcane expressing
pharmaceutical products in the US. This was the Hawaii Agricultural Research
Centre permit 01-306-01r issued on 11 January 2002. There have been no field trials
in Europe.

4.1.6 Lettuce
Lettuce has also been adopted for pharmaceutical production, as it is another leafy
species for which harvesting can occur prior to flowering. The following products
have been isolated from transgenic lettuce:
Antibodies: Negrouk et al. 2005
Human growth factor: Zuo et al. 2001
Vaccine: Kapusta et al. 1999, 2001; Kawashima et al. 2001; Koprowski 2002; Legocki et al.
2005

Lettuce has advantages in that it is self-pollinating and can be commercially

produced in large quantity under well-defined conditions with controlled hydroponic
growth in animal pathogen free green houses (Figure 4.4). In terms of commercial
development, Altor acquired intellectual property rights to the transgenic plant
expression technology from Sunol Molecular Corporation (Figure 4.5) in 2005 and
has a collaborative agreement with Dow Chemical Company on therapeutic antibody
production in transgenic plants (http://www.sunolmolecular.com/).
As transgenic lettuce expressing pharmaceutical products can be grown in physical
containment there have been no applications for field releases of pharma-lettuce in
the US or in Europe.

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Figure 4.4 Transgenic lettuce

(Source: www.sunolmolecular.com)

Recent relevant progress with this crop includes the first report of plastid
transformation (Lelivelt et al. 2005), and high-level transient expression using an A.
tumefaciens method (Joh et al. 2005; Negrouk et al. 2005) (see Section 4.2).

Figure 4.5 Lettuce transformation

(http://www.burnet.edu.au/freestyler/gui/files/plantmvposter.pdf)

4.1.7 Potato
Potato was one of the first species used for pharmaceutical production. In 1998 Axis
Genetics, a private UK company, commissioned American Ag-Tec International to
grow potatoes containing hepatitis B vaccine for clinical trials. Since then potato has
been used to produce a wide range of pharmaceutical products:

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Antibody: Artsaenko et al. 1998; Conrad U and Fiedler U 1998; De Wilde et al. 2002;
Ehsani et al. 1997; Hendy et al. 1999
Antigen: Arakawa et al. 1999; Dogan et al. 2000; Joung et al. 2004; Kong et al. 2001;
Lauterslager et al. 2001; Ma & Jevnikar 1999; Maloney et al. 2005; Shulga et al.
2004; Thanavala et al. 2005; Yu & Langridge 2001
Avian coronavirus infectious bronchitis virus: Wu et al. 2003b; Zhou et al. 2003, 2004
Bovine group A rotavirus, VP6 protein: Matsumura et al. 2002; Wu et al. 2003a
Cholera toxin: Choi et al. 2005
Escherichia coli enterotoxin (recLT-B), non-toxic B subunit: Lauterslager et al. 2001
Foot and mouth disease virus structural protein VP1: Carrillo et al. 2001
Hepatitis B surface antigen: Thanavala et al. 2005
HIV diagnostic reagent: Schunmann et al. 2002
HIV-1 Tat transduction domain rotavirus enterotoxin fusion protein: Kim & Langridge
2004
Human interferon: Ohya et al. 2001, 2005
Human interleukin-2: Park & Cheong 2002
Human interleukin-4: Ma et al. 2005b
Human papillomavirus particles: Biemelt et al. 2003; Warzecha et al. 2003
Human serum albumin: Farran et al. 2002
Human -amyloid peptide: Kim et al. 2003
Norwalk virus capsid protein: Tacket et al. 2000
Porcine epidemic diarrhoea virus, neutralizing epitope: Kim et al. 2005
Puumala virus nucleocapsid protein: Kehm et al. 2001
Rabbit hemorrhagic disease virus, structural protein VP60: Castanon et al. 1999,
2002
Sea anemone equistatin: Outchkourov et al. 2003
Swine-transmissible gastroenteritis coronavirus, spike protein: Gomez et al. 2000

The most commercially advanced of these projects are probably those of the two
companies Quantum Tubers Corporation of Wisconsin (www.quantumtubers.com)
and Planton (Kiel, Germany). This latter company specializes in the production of
human anti-microbial peptides and claims an extraction rate of approximately 10 15mg of these compounds from one Kg of transgenic potato tubers (Figure 4.6). It is
stated The proof of concept for the drug has been established, and pre-clinical trials
with the product are ongoing. Meanwhile Planton has succeeded in establishing a
co-operation with a big industrial partner to market its products.

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Figure 4.6 Potato plants in vitro

(Source: http://www.biotech-online.com/artimg/a20051934236820.PDF)

No evidence can be found of field trials of GM potatoes expressing pharmaceutical

proteins. It must therefore be assumed that potatoes generated to express
recombinant products are being produced in physical containment in greenhouses or
controlled environment growth rooms.
There have been two field trials of transgenic potatoes in Germany expressing
industrial compounds. These were both conducted by the Institute of Plant Genetics
and Crop Plant Research. The first trial took place between 2002-4 of potatoes
expressing non-plant carbohydrates but no further details of this trial can be found at
present (notification number B/DE/02/138; http://biotech.jrc.it/deliberate/DE.asp). The
second trial took place between 2003-4 of potatoes expressing non-plant spider silk
proteins (notification number B/DE/02/146; http://biotech.jrc.it/deliberate/DE.asp).
According to the notification report the risk of transgene escape was considered to
be nil as no sexually compatible wild species are present in Europe and no other
potato crop was raised near the release site (http://gmoinfo.jrc.it/gmp_browse_geninf
.asp).

4.1.8 Other species

Other crop species used for pharmaceutical production include:
Arabidopsis: Downing et al. 2006; Gil et al. 2006; Wu et al. 2004a,b
Banana: Kumar et al. 2005
Carrot: Alli et al. 2002, Bouche et al. 2003, 2005; Marquet-Blouin et al. 2003; Porceddu et al.

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1999; Sciutto et al. 2002

Lotus corniculatus: Kato et al. 2005
Lupin: Kapusta et al. 1999, Smart et al. 2003
Papaya: Sciutto et al. 2002
Pea: Perrin et al. 2000; Saalbach et al. 2001
Pigeon pea: Satyavathi et al. 2003
Spinach: Karasev et al. 2005, Sussman, 2003; Yusibov et al. 2002
Sunflower: Sciutto et al. 2002
Sweet potato: Berberich et al. 2005
Tomato: Fletcher et al. 2004; Huang et al. 2005; Jani et al. 2002; Kwon et al. 2003a; Ma et al.
2003; McGarvey et al. 1995; Mor et al. 2001; Pogrebnyak et al. 2005; Ruf at el. 2001;
Sandhu et al. 2000; Shchelkunov et al. 2004
White clover: Lee et al. 2001, 2003

Of these plant species, there are only two that have been taken forward into field
trials. There has been one field trial of tomato expressing a classified pharmaceutical
protein in the US. This trial was carried out by Prodigene and the release permit was
issued in 1998. In the Netherlands, Van der Have carried out a field trial of sunflower
in 1995 expressing a variety of traits including albumin. This sunflower line was also
modified to have male sterility.
The crop most recently suggested as a production system is flax. The newly
established company Agragen announced plans in 2005 to develop transgenic
plants expressing therapeutic proteins to be grown in North Dakota. Selection of flax
as a production system is considered by Agragen to be ideal as it is self-pollinating
and pollen travel a very short distance. In addition, flax pollen is short lived, reducing
the risk of pollen mediated transgene escape. As flax is currently cultivated in North
Dakota, a skills base exists in relation to farming and processing, however there are
local concerns regarding the biological safety of the conventional flax crop.
The remainder of these expression systems are in the developmental stage at
present.

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4.1.9

Identity preservation

The use of food crops as production systems for recombinant proteins has been
quite extensive, and although alternative crop species are undergoing continuous
development, there still remains considerable focus on the conventional species. For
this reason there are several suggestions about ways that phenotypic markers may
be introduced into the crop to enable instant recognition of conventional crops
expressing recombinant proteins and the use of inducible promoters to control
expression of the product (Ma et al. 2005a). For example, alteration of fruit colour or
fluorescent marker proteins such as the green fluorescent protein of DsRed could be
added into the transgene cassette and be used not only to generate a visually
distinct pharma crop, but also as a means of monitoring transgene escape without
the need for molecular lab work (Ma et al. 2005a).

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4.1.10

Summary of conventional crop species production

platforms

Several products generated in GM plants are commercially available:

products generated in maize (Prodigene)

Avidin

-glucuronidase

Trypsin

products generated in rice (Ventria)

lactoferrin

Numerous products are in clinical trials and are close to market release:

products generated in tobacco

collagen

antibodies against tooth decay

antibodies against Non-Hodgkins lymphoma (transient expression)

products generated in maize

human gastric lipase

therapeutic enzymes

dietary supplements

products generated in other crop species

vaccines against Hepatitis B and Norwalk virus in potato

vaccines against rabies in spinach (transient expression)

dietary supplements in Arabidopsis

Tobacco is widely used as a production platform. It is an efficient production

system with an established knowledge base in genetics, manipulation,
cultivation and production. It has been adopted as a production platform by
Planet Biotechnology Inc, The Large Scale Biology Corporation, Icon Genetics
and Chlorogen, Inc. Field trials have been conducted in the US and Europe.
Alfalfa and lettuce have also been adopted as production platforms. The
leaves can be harvested repeatedly and flowering can be controlled. There

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have been few field releases as both can easily be grown in physical
containment.
A wide range of products have been expressed and produced in potato tubers.
These are also mostly grown in physical containment.
Several other crops have also been adopted as production platforms but
remain mostly experimental:

Arabidopsis; banana; carrot; Lotus; lupin; papaya; pea; pigeon pea;

spinach; sugarcane; sunflower; sweet potato; tomato; white clover

Development of morphological markers to distinguish pharma-expressing

crops is suggested to aid identification and traceability without molecular
techniques.

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4.2

Transient expression

Expression of recombinant proteins in plants can be achieved by the development of

stable, transgenic plant lines (as discussed above), or via the transient expression of
a gene inserted into a non-GM plant by a vector. Transient gene expression enables
the production of GM products from non-GM plants as it is based on the expression
of traits in specific tissues (e.g. leaves) or from short-lived messenger RNA. This
method, therefore, does not involve the stable insertion of heritable traits into
genomic DNA of the host organism.
Transient expression is a useful research tool that offers greater flexibility, lower
costs and rapid results compared to the development of a line of stably transformed
plants. It can produce relatively large amounts of recombinant proteins rapidly over a
number of days. The nature of the system means that there are some limitations of
scale and as such, transient expression methodologies are not always comparable
with the type of large-scale production that can be achieved with fields of transgenic
plants. It is often been used to verify that gene products are structurally and
functionally stable before progression onto large-scale transgenic production
(Fischer et al. 1999b; Twyman et al. 2003). Recent improvements in the technology
have enabled production to be scaled up to some extent and have improved yield
such that some commercial production is being developed.
There are several approaches to deliver genes into the cells of whole plants. These
include biolistic delivery, infiltration with recombinant Agrobacterium, and infection
with modified viral vectors (Grill et al. 2005).
Biolistic delivery inserts segments of DNA into plant cells via particle bombardment.
It is a very targeted approach and requires that the DNA segments reach and are
encoded into the nucleus for the genes to be expressed. Genes will only reach a
small number of cells and as such this method is not suitable for the expression of
large amounts of recombinant protein but may be used for limited analysis of protein
stability (Christou 1996; Fischer et al. 1999b).

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Infiltration with recombinant Agrobacterium (agroinfiltration) can be used to target

many more cells and can be used to induce expression in leaves that have been
removed from the plant. The genes of interest are cloned into vectors and inserted
into strains of Agrobacterium. The bacterial cells are then inserted into the leaf
tissues via vacuum infiltration. The Agrobacterium vector inserts the new genes
directly into the nucleus of the cells via bacterial proteins in the same way as callus
cells are transformed for the generation of stably transformed plants (Kapila et al.
1996). This system produces rapid results and sufficient quantities of expressed
product for characterisation and assessment of protein stability (Fischer et al.
1999b).
Gene delivery to the whole plant can also be achieved via inoculation with
recombinant viral vectors (Caizares et al. 2005, 2006). The gene of interest is
inserted into the genome of a plant virus and this is then used to infect the plant.
Once inoculated, the virus will systematically spread throughout the plant, replicating
in the cytoplasm and will infect most of the cells. Many transcripts of the gene are
therefore formed within the plant resulting in high levels of expression of the protein
(Fischer et al. 1999b).
High-throughput viral expression systems have been developed with Nicotiana
benthamiana using the Tobacco Mosaic Virus (TMV) vector (Escobar et al. 2003).
The efficiency of the system is such that the most time consuming step is the
engineering of the viral vector itself. Marillonnet et al. (2004) have devised a
procedure to increase the efficiency of this stage by using Agrobacterium to deliver
DNA segments of the viral vector and gene of interest into the plant. The various
components then re-assemble once inside the plant removing the need for vector
engineering in vitro. High yield of recombinant product were then obtained within 314 days (Marillonnet et al. 2004), and the method has been recently adopted for the
high-level expression (I mg/gm fresh weight) of human growth hormone (Gils et al.
2005).

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Table 4.7 Large Scale Biology Corporation products generated via viral transient expression
*Follow on biologicals are established products under patent protection that has or will soon expire.
Product type
Therapeutics

Product
Alpha-galactosidase A
Lysosomal acid lipase

Follow on
biologicals*

Vaccines

Aprotinin

Alpha interferon 2a & 2b; G-CSF;

Hepatitis B vaccine antigens;
cytokinins; growth factors; enzymes;
enzyme inhibitors
Non-Hodgkins lymphoma
personalised cancer vaccines
Papillomavirus vaccines

Parvovirus

Description
Replacement therapy for the
metabolic disorder Fabry disease
Reduces fatty acid deposits in
vasculature, used to treat
atherosclerotic plaques (heart
disease)
Natural protease inhibitor that
reduces blood loss during
cardiovascular bypass surgery
Various

Customised patient specific

therapeutic cancer vaccines
Therapeutic vaccines against various
forms of animal and human
papillomavirus
Vaccine against infection of
companion and agricultural animals

Development
Preclinical trials
Preclinical trials

Product introduced 2004;

distributed through Sigma
Various stages of
development

Early stage clinical trials

Preclinical trials

Initial clinical trials

The Large Scale Biology Company (LSBC) in the US has developed viral vectors for
the production of recombinant proteins in Nicotiana plants and uses this system for
the commercial scale production of pharmaceutical proteins (Marillonnet et al. 2004).
LSBC has several vaccines and therapeutics in preclinical and initial clinical trials, all
produced through viral transient expression (Table 4.7).
LSBC are developing personalised cancer vaccines for non-Hodgkins lymphoma
patients using sequences obtained from tumour hybridoma or human tumour biopsy
cells. The sequences are cloned into Tobacco Mosaic Virus vectors and these are
used to inoculate N. benthamiana plants, which then secrete the scFv vaccines
(McCormick et al. 2003). The rapid turn around of the viral vector system enables
patient specific therapeutic vaccines to be produced in 6-10 weeks as opposed to
several months by conventional means. These vaccines have completed early stage
clinical trials (http://www.lsbc.com/thera.html).
LSBC have demonstrated that their TMV vectors are not pollen or seed transmitted
and do not pose any containment issues. In addition, LSBC have based their system
around Nicotiana benthamiana which is more susceptible to TMV than the
commercial cultivars of N. tabacum that have been bred to confer inherent resistant
to TMV. All of LSBCs pharmaceutical and veterinary products are manufactured in
controlled greenhouses and growth rooms, although external field trials have been

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carried out. Growing the plants indoors offers more environmentally stable
conditions, resulting in higher yields and more consistent recovery of recombinant
products.
It has been demonstrated that recombinant TMV viruses are not likely to persist in
populations of tobacco should they escape confinement. Rabindran and Dawson
(2001) examined the progression of transgenic TMV vectors in N. benthamiana
plants and found that as the virus spread, the transgene was excised and any
hybrids that formed between the released and wild-type viruses were poor
competitors against the wild type TMV.
Transient expression has also been demonstrated as commercially viable in other
leafy crops. Negrouk et al. (2005) optimized an agro-infiltration procedure to produce
as much as 20-80 mg of functional antibody per kilogram of fresh lettuce leaf tissue
in less than a week. This procedure has been used to produce a humanized IgG1
kappa anti-tissue factor antibody (hOAT) as well as other antibodies. The results
demonstrate that transient expression of recombinant antibodies in lettuce is very
efficient, inexpensive and readily scalable. Thus, it is a useful tool for small-scale
production of functionally active antibodies and other pharmaceutically important
proteins. In addition, it can be used to facilitate humanization or optimisation of
antibodies or to produce quantities of antibody (in the range of 100 mg) sufficient for
in vivo studies. Commercialisation of the process has taken place through Altor
Bioscience (http://www.altorbioscience.com/) who recently (31 July 2005) announced
that it had received a Phase I Small Business Innovation Research (SBIR) grant
from the National Cancer Institute (NCI) to support development of its proprietary
processes for making therapeutic antibodies in transgenic lettuce. In the NCIsupported project, Altor will extend the published project (Negrouk et al. 2005) and
conduct preclinical efficacy testing in tumour models using antibodies already
produced at high levels in stable transgenic lettuce. A related study on lettuce has
recently been reported by Joh et al. (2005)

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Further examples of transient expression in plants are:

Rabies vaccines in spinach and tobacco using viral vectors: Yusibov et al. 2002
Human respiratory syncytial virus G-protein in tobacco using alfalfa mosaic virus:
Belanger et al. 2000

Despite having addressed the regulatory hurdles and public opposition that
accompanies stable, genetic transformation with plants, there remain containment
concerns relating to transient expression technologies. Although the plants are
untransformed, the vectors used to induce expression in both the Agrobacterium and
viral based systems are still genetically modified organisms and will require
regulation should deliberate release be requested. The use of genetically modified
plant viruses will require some containment strategies to be in place to prevent the
long-term persistence of a viral strain encoding a pharmaceutical product.

4.2.1

Summary of transient expression

Transient expression enables the production of GM products from non-GM

plants. Traits may be introduced into specific tissues of whole plants via:

Biolistics

Agrobacterium

Viruses

Generates rapid results and is less costly than the development of stable
transgenic lines but with some limitation of scale.
The Large Scale Biology Corporation use only transient expression using
Nicotiana benthamiana and tobacco mosaic virus vectors in physically
contained, greenhouse conditions.
Containment of the GM vector is the major regulatory issue with this
technology.

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4.3

Alternative non-crop species

The adoption of species not conventionally cultivated for food or feed as production
platforms for industrial and pharmaceutical proteins offers additional containment
reassurances as these products have a minimal chance of entering the food chain.
In addition, such species will require the development of specific handling and
processing facilities and, depending on the choice of organism, may be grown in
physically contained conditions.

4.3.1

Flowering plants

4.3.1.1

Safflower

Safflower (Carthamus tinctorius) has been adopted as a production system for biopharming by Syngenta and SemBioSys Genetics Inc (www.sembiosys.com).
Safflower is an old crop previously cultivated for dyes and food colouring before
cheaper aniline based dyes came on to the market. It is a highly productive oilseed
crop and thus only relatively low acreages are required. It is also self-pollinating and
SemBioSys claim that virtually no pollen is transported by the wind and that no
weedy relatives are found in the Americas.
SemBioSys have developed and patented a recombinant protein production system
for safflower, whereby the transgene product is linked to an oleosin fusion protein
and is captured on oil bodies in the seed. The harvested seed are milled in an
aqueous buffer and centrifuged to separate the oil and aqueous fractions. The oil
bodies are then easily removed and the transgene product purified and processed
for its downstream application. This StratosomeTM Biologics System enables the
recovery of 1Kg recombinant protein from 1 tonne of seed.
SemBioSys have carried out four field trials in the US and 17 in Canada of safflower
expressing pharmaceutical proteins or aquaculture feed additives. They have also
produced a safflower crop in Mexico and Chile (as stated on their website,
www.sembiosys.com). Their main pharmaceutical products are insulin (Nykiforuk et
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al. 2006) and apolipoprotein Al (a cardiovascular therapy for heart attacks and
angina) both of which are in the research phase and planned to enter clinical trials in
2007-8. In addition, SemBioSys are developing an immunostimulatory protein feed
additive for shrimp that improves their ability to survive viral infection. This product,
ImmunoSphereTM has been successfully tested in tank trials and is due to enter pond
trials in 2006. SemBioSys is also developing animal vaccines using their
StratosomeTM Biologics System with Dow AgroSciences.
4.3.1.2

Lemna

The monocot family Lemnaceae is composed of small, free-floating aquatic plants,

the most common of which is duckweed or Lemna. Lemna is distributed worldwide
and has biomass doubling times of 48-72h under controlled, axenic conditions.
Reproduction is typically vegetative with culture mass increasing exponentially until
crowding sets in. Lemna, however, can also flower and produce seeds.
The most commercially advanced species is Lemna minor (Gasdaska et al. 2003), a
fresh water plant used as feed and food, mostly in Asian countries. It is also used in
North America for wastewater management.
The rapid reproductive rate and aquatic nature of these plants makes them ideal for
bio-pharming in physically contained water-based reactors. A transformation method
was developed for L. minor several years ago (Yamamoto et al. 2001). Two
independent companies Biolex (US) (www.biolex.com) and LemnaGene (Lyon,
France) (http://www.lemnagene.com/) exploited this technology but recently (28 July
2005) the US company acquired its French rival and now has a dominant position.
The system developed by Biolex has been named the LEX System (Table 4.8)
and this has been developed to produce hard-to-make proteins (such as peptides
and cytokines) and monoclonal antibodies. Biolex state that:
all the human therapeutic proteins Biolex has attempted (including a peptide,
multimeric proteins, alfa interferon and other cytokines, and monoclonal antibodies)
have been expressed successfully. Each of the proteins characterised to date has

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been demonstrated to have the predicted composition, desired physical structure and
correct biological activity.
Table 4.8 The LEX SystemTM of bio-pharming
LEX SystemTM : the ideal system for therapeutic proteins
Genetic stability without pollen or seed
Clonal
Doubles biomass every 36 hours
Fast
Optimizes scale-up & downstream processing
High expression levels
Simplifies downstream processing
Secretes target protein
Conforms with GMP guidelines
Contained and controlled
Low operating and capital costs
Inexpensive
Proprietary multi-ton production format
Scalable

Compared to conventional mammalian manufacturing plants a LEX facility presents

and eight-fold reduction in start up costs. It is stated that a LEX facility can be built
for $50 million in three years, while a mammalian facility can cost as much as $400
million and may take up to five years due to the control systems needed to prevent
contamination.
Biolex holds several patents relating to the transformation of Lemna and has
continued to develop the LEX SystemTM over the past five years. The broad patent
portfolio relates to the method of transformation, the selection, recovery and growth
of the transgenic plants; the composition of expression and enhanced expression
through to the development of automated high throughput systems (Table 4.9).
Table 4.9 Patent portfolio of the Biolex LEX SystemTM
Patent Number
US 6040498
WO 9907210

Date
31 March 2000

Title
Genetically engineered
duckweed

WO 0210414

7 Feb 2002

Expression of biologically
active polypeptides in
duckweed

WO 02097029

Dec 2002

US 030033630

Dec 2002

US 6680200

20 Jan 2004

Plant and method for high

throughput screening
The use of duckweed in
high throughput screening
LED array for illuminating
cell well plants and
automated rack system for
handling the same

140

Claims made of the invention

methods of: transformation and selection;
transgenic plant regeneration; growth and
recovery; use of multiple genes and vectors;
transformed tissue and plants
methods and compositions for: expression,
recovery; enhanced expression levels;
directed secretion.
automation of the LEX System;
commercial line creation technology
automation of the LEX System;
commercial line creation technology
automation of the LEX System;
commercial line creation technology

DEFRA Contract CPEC 47

In addition, on May 6, 2004 Biolex acquired Epicyte Pharmaceutical and as such has
achieved a dominant intellectual property position via Epicytes Plantibodies patent
portfolio. In September 1997, The Scripps Research Institute granted to Epicyte an
exclusive, worldwide license to the entire patent estate covering the expression of
human and other antibodies in plants. In July 2002, US patent 6417429 was issued,
extending the Plantibodies patent protection to all species of transgenic plants that
produce an antibody and methods of making the transgenic plants. In addition, the
Plantibodies portfolio includes issued patent claims to plant-made antibody
compositions and their methods of use (Table 4.10).
Table 4.10 List of patents issued to PlantibodiesTM
Patent number
US 5202422
US 5639947
US 5959177
US 6417429
JP 3238700

Invention
Composition claims
Ig/Transgenic plants
Secretory antibodies
Ig/Transgenic plants
Ig/Transgenic plants

Pending Plantibodies patents include additional claims to transgenic plants

expressing other multimeric proteins, immunization strategies, pharmaceutical
compositions and plant-based manufacturing methods. The Plantibodies portfolio
also has several continuation applications on file. These applications include claims
to compositions, transgenic plants, plant cells and methods of production for:
Single immunoglobulin molecules including single chain antibodies;
Heteromultimeric proteins not normally produced in plants;
Immunoglobulin molecules including fragments Fab, Fab, Fv;
Methods of passive immunization using plant made antibodies.
Based on the issuance of the July 2002 patent, Biolex believe that the Plantibodies
patent portfolio covers all aspects of producing any antibody in any plant. The most
developed of Biolex products is Locteron, a novel controlled-release form of a
hepatitis C treatment that includes the alpha interferon BLX-883. LocteronTM entered
Phase 1 clinical trials on 24 healthy volunteers, in February 2005. On 26 October
2005 Biolex announced the successful completion of the trial of this product which is

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the first clinical-stage therapeutic candidate produced in Biolex's GMP facilities using
its LEX System(TM). In the trial, BLX-883 was safely administered at a clinically
relevant dose and demonstrated bioactivity consistent with Intron A, a currently
marketed alfa interferon used as a comparator. Alfa interferon is used in the
treatment of hepatitis C, hepatitis B, and multiple cancers and its worldwide sales
currently exceed $3 billion. The study was conducted under an IND submitted to the
United States FDA and a CTA filed with the United Kingdom MHRA. Participants
were administered one of three doses of BLX-883. The 12 participants who received
the clinically relevant dose of 3 million international units also received the same
dose of Intron A to allow for a direct comparison of the two agents. No safety
concerns arose during the study, and the side effects attributable to BLX-883 and
Intron A were comparable. Bioactivity of the two agents was also compared, and the
elevation of specific immune system markers, such as neopterin, measured after
administration of BLX-883 was consistent to the elevation measured after
administration of Intron A.
More recently, 27th October 2005, it was announced that Biolex and Kringle Pharma
had formed a collaboration for the production of a novel protein targeting cancer. The
protein, NK4, is an elastase-generated fragment of hepatocyte growth factor (HGF)
containing four kringle domains, initially discovered as a competitive antagonist of
HGF. Research suggests that it also possesses the anti-angiogenic property relevant
to cancer and neovascularisation disorders and thus has potential as a drug
therapeutic. Preclinical testing of NK4 has shown inhibition of both metastasis and
angiogenesis in multiple animal tumour models. Efficient production of NK4 using the
LEX System will accelerate its rapid clinical development and commercialisation.
As with algae, the rapid reproductive rate that makes Lemna an attractive production
system also raises concerns regarding escape. Physical containment will prevent
hybridisation with wild species and enables absolute control over the whole process.
However, safeguards must be in place to prevent escape of the organism via
wastewater. Any release into the water system of transgenic Lemna expressing
products into the growth medium would be of great concern due to the nature of the
organism and would be very difficult to eradicate without further contamination of the
water.

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4.3.1.3

Spirodela

Spirodela is also a member of the Lemnaceae and as one of the less reduced forms
of duckweed is commonly known as giant duckweed, although the plants are still
comparatively small (Figure 4.8). Spirodela also has a worldwide distribution and the
same biomass doubling capacity as Lemna (48-72h). Unlike Lemna, Spirodela rarely
produces any flower or seed. It is cultivated in some places in the US on dairy farm
wastewater. It has a high protein content (50% dry weight), contains a good
balance of essential amino acids, and may be used as an alternative for alfalfa in
animal feed.
Like Lemna, the rapid reproduction capacity of Spirodela offers an efficient
production system for bio-pharming and as it only reproduces vegetatively, there is
no diversion of resources towards the flowering process. Spirodela oligorrhiza has
been transformed by the Weizmann Institute who have developed stable
transformed plants with expression of transgene products ranging from 0.005% to
about 3% of total protein. As Spirodela is edible, the dried plant material can be used
in the final biotech product without the need for a protein extraction and purification
stage.
Production takes place in totally enclosed growth facilities (Figure 4.7) thus adopting
a physical containment strategy. As with Lemna, the greatest risk of escape would
appear to be via wastewater from the production system. Treatment of wastewater
prior to discharge into the water system would need to be adequate to remove all
organisms to prevent accidental release.

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Figure 4.7 Growth of Spirodela oligorrhiza in culture.

(Source: http://www.lemnagene.com/communication/poster.pdf).

4.3.2

Algae

Algae are a division of plants that inhabit a range of aquatic habitats from marine and
fresh water, to damp terrestrial areas. Some algae are single celled, others are more
structured, but none possess roots, stems or leaves. Algae reproduce by spores and
many species are capable of sexual and non-sexual reproduction i.e. vegetative
propagation. The potential of using transgenic algae as a source of novel products,
including a range of pharmaceutical proteins, has been reviewed recently (El-Sheekh
2005; Franklin & Mayfield 2004, 2005; Walker et al., 2005). The ease with which
algae can be transformed, their short-life cycle from generation to generation, and
the fact that algal cultivation requires some degree of physical containment make
algae an attractive alternative for the bio-pharming of pharmaceuticals and industrial
products. The species involved may be conveniently divided into the single cell forms
and the multicellular species.
4.3.2.1

Single celled species

The single-celled alga Chlamydomonas reinhardtii has long been used as a model
species since it has a small genome (now sequenced) and is readily transformed

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(Mayfield et al. 2003; Fuhrmann 2004; Leon-Banares et al. 2004; Mayfield & Franklin
2005). In addition, the commercial exploitation of other species of non-transgenic
algae as a nutritional supplement is relatively well developed (Table 4.8).
The use of transgenic algal systems as bioreactors for industrial and pharmaceutical
compounds is also developing (Table 4.11) although it has not yet reached the
market place. Single-celled algae offer many benefits compared to higher plants as a
bio-pharming system as they have relatively fast growth rates, can be grown in
physically contained conditions under high densities, and with the right conditions of
light and aeration, can be grown in megalitre volumes (Walker et al. 2005). In
addition, the simple structure of micro-algae compared to higher plants makes the
protein purification process simpler and therefore lest costly. The reduced costs
compared to higher plants have led to an increase in commercial activity in this
sector and the formation of several new biotech companies (Walker et al. 2005)
(Table 4.11)
Table 4.11 Companies currently selling microalgal-based products or developing microalgae
as bioreactors for protein production
Company
Cyanotech
(www.cyanotech.com)

Microalgae
Spirulina pacifica

Martek Biosciences Corp

(www.martekbio.com)
Mera Pharmaceuticals
(www.aquasearch.com)
Earthrise Nutritionals
(www.earthrise.com)

Crypthecodinium
cohnii
Haematococcus
pluvialis
Spirulina sp

PharmaMar
(www.pharmamar.com)
Nikken Sohonsha Corp.
(www.chlostanin.co.jp)

Various

Products
Spirulina extracts as nutritional supplements,
immunological diagnostics, aquaculture
feed/pigments and food colouring
Nutritional fatty acids
Natural astaxanthin as a nutraceutical

Chlorella spp.

Nutritional supplement to inhibit replication

and infectivity of viruses including HIV, CMV,
HSV and influenza
Anticancer drugs derived from marine
microorganisms
Dietary supplements: polysaccharide

Dunaliella spp.
Dunaliella bardowil
Dunaliella salina
Undisclosed

N,-1.3 glucan and -carotene

-carotene powder
Mixed carotenoids
Polyunsaturated fatty acids

Nature Beta Technologies

Cognis (www.cognis.com)
Subitec GmbH
(www.subitec.com)
Solazyme
(www.solazyme.com)
Phycotransgenics
(www.phycotransgenics.com)

Undisclosed

No products yet brought to market

Chlamydomonas
reinhardtii

Algal Biotechnology
(www.algalbiotechnology.com)

Chlamydomonas
reinhardtii

Transgenic microalgae for animal health/feed,

bioremediation, environmental monitoring and
biopesticides
Developing recombinant protein technology

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July

2000,

Martek

Biosciences

(http://www.martekbio.com/home.asp)

Columbia, Maryland were granted US Patent 6027900 (Methods and Tools for
Transformation of Eukaryotic Algae). This patent outlines a process to express nonalgal genes in algal cells in order to generate key pharmaceutical and industrial
compounds within algal bioreactors. The company has an extensive interest in algal
derived food supplements including docosahexaenoic acid, a baby food ingredient
claimed to aid mental development.
The necessary production facilities for microalgal growth are well established as
shown in (Figure 4.8-4.9).
The most topical example of exploiting transgenic algae is the application to grow
large

quantities

transgenic

Chlamydomonas

Hawaii

(http://www.i-

sis.org.uk/PICGA.php). The details of this specific case are as follows. On 1

November 2004, the Hawaii Department of Agriculture received an application from
Mera Pharmaceuticals (http://www.aquasearch.com/) (in collaboration with Rincon
Pharmaceuticals, http://www.rinconpharma.com/Docs/Mera-Rincon%20PR%200505
13.pdf), for a permit to begin large-scale production of a genetically modified alga, C.
reinhardtii, producing a human immunoglobulin-A protein active against a variant of
the herpes simplex virus. On 17 April 2005, Mera Pharmaceuticals revised their
application to include seven additional GM strains expressing a range of human
antibodies, interleukins and nerve growth factors.
The details of the 8 strains involved are:
1. Strain Hsv8, producing a full-length human immunoglobulin-A against a
variant of the herpes simplex virus;
2. Strain aFceR 1r-1, producing a protein targeting the Fc portion of the IgE
molecule, thereby limiting the interaction between circulating IgE molecules
and receptors on mast cells, which is turn limits the release of histamines and
reduces inflammation;
3. Strain aTNFr-1, producing IgG1 anti-tumour necrosis factor antibody;
4. Strain a TNr-1, producing IgG1 anti-microbial antibody;
5. Strain aCRr-1, producing IgG1d anti-cell proliferation antibody;
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Figure 4.8 Microalgae Haematococcus pluvialis as seen under the microscope

(Source: http://www.biopro.de/en/stern/magazin/01402/)

4.3.2.2

Multicellular species

Multicellular algae include filamentous and parenchymatous or thalloid forms such as

seaweed. At present, there is no commercial production of transgenic seaweed but
there is increasing interest in its potential to produce fuels, polysaccharides, fish
feed, and pharmaceutical products and for use in bioremediation (Anon 2004).
Genetic transformation systems have been developed for several seaweed species
including the red seaweeds Porphyra, Gracilaria, Grateloupia, Kappaphycus and
Ceramium, the green seaweed Ulva, and the most commonly cultivated seaweed,
the brown seaweed Laminaria japonica (kelp) (Qin et al. 1999, 2005). The majority of
this development work has been carried out in China and a Chinese Patent covering
the transformation of kelp (96120235 Method of producing transgenic kelp. 23 Oct
1996) has been issued (Qin Song et al.).
Expression of pharmaceutical products has been proven in kelp by the insertion of a
heterologous gene encoding hepatitis B surface antigen (HBsAg) via particle
bombardment. Results of quantitative ELISA showed that HBsAg in this material was
0.529 g/mg soluble proteins on average and the highest value was 2.497 g/mg,
implying that recombinant HBsAg had the natural epitope (Jiang et al. 2002).
In the United States, the Northeastern University has filed a patent application (WO
0062601) relating to transformation of multicellular marine algae via Agrobacterium.
The application describes transformation of the red algae Porphyra, widely used as a
staple food in Japan known as nori, for which worldwide production is estimated at
$1.5 billion dollars annually.
A method of confinement described as biological design has been used to contain
invasive species of Porphyra cultivated in Maine in the US. A non-transformed, nonnative species P. umbilicalis was introduced and cultivated commercially on floating
drafts in coastal waters inhabited by a closely related native species. Fears that the
introduced invasive species would hybridise with and out-compete the native species
were allayed by extensive fieldwork demonstrating that the introduced species was
not invasive outside its native range. The data revealed that the introduced species
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Summary of non-crop species production

platforms

Safflower has been adopted as a production platform for novel proteins by

some companies:

productive oilseed crop

no wind borne pollen

no wild relatives in the Americas

Pharmaceutical and industrial products can be generated in physically

contained, water based bioreactors of simple plants:

Algae

Moss

Lemna and Spirodela (duckweeds)

Genetic containment strategies have not been developed for these plants.

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4.4

Other protein expression techniques

The use of plant cells or specific parts of plants in bio-pharming enables strict
physical containment to be employed and removes the need to express the
transgene in the reproductive and/or vegetative parts of the plant. Therefore, the risk
of transgene escape or entry into the food chain is minimised.

4.4.1

Suspension and callus cell cultures

Cell lines of several species have been used for pharmaceutical production. (Xiao et
al. 2003). For example, tobacco cell lines have been used for the production of:
Heavy chain immunoglobulin G: Shin et al. 2003a
Hepatitis B surface antigen: Kumar et al. 2003, Smith et al. 2002; Sojikul et al. 2003
Human erythropoietin: Matsumoto et al. 1995
Human granulocyte-macrophage colony-stimulating factor (GM-CSF): Bodeutsch et al.
2001; James et al. 2000; Kim et al. 2004; Kwon et al. 2003b; Lee et al. 2004
Human interleukin-2 and interleukin-4: Magnuson et al. 1998
Human lactoferrin: Choi et al. 2003
Human milk protein sCD14: Girard et al. 2004
Human tissue transglutaminase: Sorrentino et al. 2005
Mouse monoclonal antibody heavy chain gamma: Magnuson et al. 1996
Soluble mouse single-chain antibody, scFv: Xu et al. 2002
TMV-specific full-size murine IgG-2b/kappa-antibody: Fischer et al. 1999a

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF)

(Shin et al. 2003b, Hong et al. 2005) and human (1)-antitrypsin (Trexler et al. 2003;
2005) have also been produced in rice cell suspension cultures. Most recently, a
novel system was reported for the production of somatic embryos of Siberian
ginseng (Eleutherococcus senticosus), expressing Escherichia coli enterotoxin B
subunit (Kang et al. 2005a). This involved the use of 130 litre air-lift bioreactors. A
much larger industrial scale production using 10 ton reactors is already in place for
the production of ginseng (CBN Biotech, Korea) and such a system could
presumably be adopted for the production of higher value GM compounds.

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The adoption of cell culture technology on a commercial scale progressed in

February

2004

(http://news.dow.com/prodbus/2004/20040217a.htm)

with

the

announcement from The Dow Chemical Company and NOBEX Corporation

(http://www.nobexcorp.com/index.html), a drug development company specializing in
drug delivery, that they will collaborate on plant-based production of NLC-001, a
proprietary peptide currently in preclinical development by NOBEX as a potential
appetite suppressant to treat obesity.
This project broadens the scope of Dow Plant Biopharmaceuticals beyond their
current work in plant-based pharmaceutical production, which has focused primarily
on antibodies. Under the agreement, the initial expression of the peptide will take
place in plant cell suspension to allow for fast evaluation of the ability of plant tissues
to secrete the peptide. If successful, production may be moved to whole plants for
rapid scale-up.
More recently (7 June 2004) The Dow Chemical Company acquired a license to
certain patents and patent applications from Epicyte Pharmaceutical, Inc. The
license is related to the expression of antibodies and other immunoglobulin-derived
molecules in plants.

4.4.2

Root culture, hairy roots and root exudates

The transformation of tobacco plants to secrete products in their roots is described in

Borisjuk et al. (1999), Drake et al. (2003), and Tesfaye et al. (2005). This approach
engineers the plants to produce recombinant proteins and secrete these products
(Vitale & Pedrazzini 2005) from their roots into medium.
This technology was first used by Phytomedics, in Dayton, New Jersey, which later
absorbed Photosynthetic Harvest Inc. (Willingboro, New Jersey). On August 3 2004
they annonced a joint venture with with ARC Ventures Inc who specialise in the
production of hydroponically raised vegetables and herbs, to form Phytomedicinal
Greenhouses LLC (PMG). The aim of PMG was to develop industrial scale
production platforms of hydroponically maintained transgenic plants to produce a

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range of recombinant products from pharmaceuticals to food supplements. PMG

developed their recombinant protein secretion technology, REPOSTTM as a biomanufacturing process based on the secretion of target protein from hydroponically
cultivated roots.
The process can be used to produce relatively pure recombinant proteins that are
shown to retain their biological activity and are accumulated in higher amounts than
in root tissue. In addition, the exudates have the potential to be used to influence soil
microbes and pathogens that may infect the plant via the rhizosphere, modify soil
nutrients near the plant roots and also may be used in the neutralisation of
environmental pollutants (Borisjuk et al. 1999; Drake et al. 2003; Tesfaye et al.
2005).
In a linked study, Gaume et al. (2003) infected transgenic tobacco plants with
Agrobacterium rhizogenes to induce hairy root disease (Figure 4.15). The expansion
in root surface area that resulted from the proliferation of root growth led to an
increase in the secretion of the transgenically expressed product (Gaume et al.
2003).
If single cells are infected with Agrobacterium rhizogenes, the induced root growth is
genetically stable and of clonal origin. If the cell of origin is transformed to produce a
recombinant protein, the product will be secreted from the root mass into the
supporting media. For this reason, production systems can be developed and can be
used immediately, without going through several generations of self-crossing and
selection, and without the use of antibiotic or herbicide resistance markers.
Furthermore, recombinant proteins and peptides can be engineered to be secreted
and can be continuously extracted from the medium. If it is not possible to target the
proteins for secretion, conventional extraction from hairy roots is made easier due to
the absence of waxes and pigment molecules that are present in leaves.
This procedure has allowed the production of a full-length murine IgG monoclonal
antibody from root culture. Similar procedures have been adopted elsewhere
(Gaume et al. 2003; Komarnytsky et al. 2004; Zhang et al. 2005) using root tissue on
transgenic tobacco plants engineered to secrete a model recombinant protein and

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human secreted alkaline phosphatase. This technology is reviewed by MedinaBolivar and Cramer (2004) and the problems of the stability of antibodies secreted
from roots has been discussed recently (Doran 2005).
The most commercially advanced hairy root culture system is ROOTec
(http://www.rootec.com/english/technology.html) a company that has developed a
bioreactor technology for several species using hairy root cultures generated
infecting tissues with the soil bacterium Agrobacterium rhizogenes (Figure 4.12)
(http://www.bio-pro.de/en/region/rhein/magazin/01440/index.html). This can be used
for many applications including the manufacture of bioactive compounds that are
naturally produced by the roots, for seeking new compounds (biotransformations),
and for the production of recombinant proteins in molecular farming. A new
company, ROOTec bioactives GmbH, has recently been started in Basel,
Switzerland.

Figure 4.12 Fermentors for the production of hairy roots

(Source: www.rootec.com)

4.4.3

Guttation fluid

Guttation is the loss of water through the leaves of plants. It has long been known
that some proteins are naturally secreted into the guttation fluid. The rate of guttation

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can be increased under controlled conditions of high water absorption, high root
pressure and a reduced rate of transpiration.
As a production system, guttation fluid can be collected throughout a plant's life, thus
providing a continuous and non-destructive system for recombinant protein
production. This has the potential of increasing the efficiency of recombinant protein
production technology by increasing yield, abolishing extraction, and simplifying its
downstream processing.
Komarnytsky et al. (2000) used endoplasmic reticulum signal peptides fused to the
recombinant protein sequences to generate transgenic tobacco cv. Wisconsin plants
that secrete three heterologous proteins. These proteins were all of different genetic
backgrounds and included bacterial xylanase, green fluorescent protein of jellyfish
(Aequorea victoria), and human placental alkaline phosphatase. Secretion occurs
through the leaf intercellular space into tobacco guttation fluid. Production rates of
1.1 g/g of leaf dry weight per day were achieved for alkaline phosphatase with this
protein comprising almost 3% of total soluble protein in the guttation fluid
(Komarnytsky et al. 2000).
There appear to be no further published studies on the use of guttation fluid as a
means of recombinant protein production, but this and the above cell and root culture
technologies provide a continuous and non-destructive method of protein production
in a physically contained and controlled environment. The development of guttation
fluid as a means of commercial production requires further development at this point
(Komarnytsky et al. 2000).

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4.4.4 Summary of other techniques for protein expression

Cell culture expression can be carried out in physically contained conditions.
Product can be collected in solution without the need to harvest the plant:

plant suspension and callus cell cultures

used to express a wide range of products

hairy root cultures and root exudation

secretion of recombinant product from roots of GM plants or root

cultures derived from somatic cells into hydroponic media

guttation fluid
o

secretion of recombinant product from the leaves of GM plants

These technologies remain in the developmental stage.

160

products are mainly diagnostic and laboratory reagents with a relatively limited
market. These have been raised in crops in the US under strict regulation by APHIS
using large isolation distances, constant monitoring programmes, and the safe
disposal of all plant tissues following harvest. Despite predicted growth in the biopharming sector, there are no plans to deregulate any crops in the US expressing
recombinant proteins and all crops will continue to be grown under licence from
APHIS.
In contrast to the GM plant derived products already commercially available, the
second wave of plant-derived products include a range of human vaccines and
antibodies. These pharmaceutical proteins are being developed to prevent and treat
a wide range of human conditions (tooth decay, cystic fibrosis, diarrhoea,
gastrointestinal infection, hepatitis B, Non-Hodgkins lymphoma, Norwalk virus,
pancreatitis, rabies, and vitamin B12 deficiency) and are currently in Phase I and
Phase II clinical trials.
Production platforms are being developed for an even wider range of products
including insulin (Farinas et al. 2005b; Nykiforuk et al. 2005), human and animal
vaccines (Dus Santos & Wigdorovitz 2005; Lamphear et al. 2005; Streatfield 2005)
and dietary supplements (Drakakaki et al. 2005; Haas et al. 2005).
As the plant itself is not the desired end product, production platforms are more
flexible and containment issues may be more easily addressed. Tobacco has so far
been most widely adopted due to the presence of extensive scientific knowledge in
this crop. However, alternative non-crop species are being adopted to prevent
contamination of conventional crops with transgenes expressing pharmaceutical
products. This includes the use of algae, moss and duckweed species in contained
water-based facilities or the use of greenhouse based crops that do not require
flowering (lettuce, alfalfa, potato). Indeed, whole plants may not be required and
products can be expressed in cell or organ culture.
Considerable success has been achieved with the use of transient expression as a
commercially adopted production platform by the Large Scale Biology Corporation
using Nicotiana benthamiana and viral vectors. LSBC have several products in preclinical and clinical trials and have utilised the rapid turnaround time of transient

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expression to generate personalised cancer vaccines for non-Hodgkins lymphoma

patients over a much shorter timescale than by conventional means. [Note added in
proof: LSBC ceased trading on 23 December, 2005.]
The scale of transient expression technology currently allows crops to be raised in
contained growth facilities. However, movement of this technology to the field will
require regulation of the GM vector and not the plant production platform.
Issues for the future
The containment of GM technology requires continued development, assessment
and regulation to ensure all avenues of transgene escape have been considered and
addressed. As no single containment strategy can at present guarantee transgene
containment, it is likely that a number of strategies will be adopted to reduce escape
potential to a minimum. The adoption of any containment strategy will require
constant assessment and monitoring to ensure that any breakdown in the
mechanism is picked up and acted upon.
As GM technology continues to develop and innovate, regulatory oversight must
continue to move forwards. The adoption of techniques and technologies by industry
that require less regulation must be assessed for their environmental impact and
risk of transgene escape. The use of contained facilities such as greenhouses,
polytunnels and underground mines requires the development and use of standard
protocols to ensure that outputs from these facilities, such as biological waste and
waste water, contain no living transgenic organism or propagule and pose no
environmental risk. The use of lower plants such as algae and mosses in contained
facilties should be investigated in the light of their potential for development as
pharmaceutical production systems. The factors that make them an attractive
production system (fast replication, ease of cultivation) also increase their potential
to cause damage if they are accidentally released into the environment. Equally the
use of transgenic viral vectors to induce recombinant protein production in non-GM
plants in transient expression systems requires assessment. The escape of GM
plant viruses from containment has the potential to damage crops and wild plants
and may lead to unwanted ecological change.
The next generation of GM crop currently under field trials confer resistance to
abiotic stresses such as drought and salt. These crops will allow the cultivation of
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DEFRA Contract CPEC 47

areas currently unsuitable for crop production and will offer great potential improving
the agricultural output of marginal lands, but also present a new set of issues for
containment. For example, areas of land where soils are dry or have a high salt
content are inhabited by native non-GM plants and have developed resistance to
these conditions and form part of a specific ecosystem. In the event that crop plants
or transgenes escape that are also resistant to such conditions, the native plants are
likely to be poor competitors in comparison and as such, feral crop plants are likely
to become invasive. The potential impact of crop plants resistant to abiotic stresses
therefore requires assessment and containment strategies introduced to prevent
unwanted ecological change from their introduction.
Seed escape during the sowing of crops and at harvest can lead to the
establishment of feral populations of crop plants. Should these crops be GM, seed
escape cannot be tolerated. A review of farming practice may identify issues that can
be improved upon to reduce and prevent seed escape. Feral populations of non-GM
crop plants may be less of a concern, but if they are persistent these plants offer
gene flow routes for transgenes from GM plants via cross-pollination. This may occur
as crop-feral pollination whereby the feral plant develops seed conferring the GM
trait, or as feral-crop pollination whereby the crop is pollinated by the feral plant,
which may lead to a breakdown in the containment strategy of the GM crop
dependent on pollination from another GM crop plant.
The extent to which seed mediated gene flow is an issue for European crops is not
well quantified. An assessment of the relative importance of seed mediated and
pollen mediated gene flow would allow containment stategies and farming practices
to be tailored to improve the containment of GM varieties. It is likely that this will be
different crop to crop and will need to be studied on a case by case basis with due
consideration given to the composition of the local flora in areas of cultivation to
identify wild plants that may also offer potential for gene flow.
The development of a landscape scale system of testing and monitoring of crops in
the field may also be required in the future to ensure that any breakdown in
biological containment mechanisms are operating efficiently over a large area. All
transgenes, whether for herbicide tolerance, protein expression or containment
require extensive field trialling to ensure their long-term stability and function. For
example, gene silencing is known to occur via changes in the natural methylation
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DEFRA Contract CPEC 47

patterns of plants, especially for genes that have a high physiological cost. Should
transgenes be turned off in such a manner it is important to be able to detect this at
the field-trial stage so that appropriate action be taken to ensure containment.
The rapid developments in the use of GM plants and transient expression in the
production of pharmaceutical and industrial proteins offers great potential for the
industry and viable alternative to mammalian-based production systems. The
development of new technologies will require regulatory procedures to be developed
and applied to ensure that these technologies can be developed in a safe manner to
their full potential. It is therefore important that all aspects of transgene escape and
environmental impact are addressed whilst these technologies are under
development. In the light of this review, a list of recommendations for the
development and implementation of containment of GM is given.

Overall Summary of Conclusions and Recommendations

In the short term, a review of the regulatory oversight of plants grown in physical
containment, such as glasshouses, polytunnels and underground facilites is required
to ensure that best practice is adopted to reduce the risk of transgene escape. This
should include an assessment of the use of lower plants such as moss and algae
expressing pharmaceutical proteins and contained transient expression systems,
where viral vectors are used to induce transgene expression in non-GM plants. The
main areas of concern in these production systems being the escape of transgenic
algae or plant viruses.
In the longer term, scientific investigations are required to develop a landscape scale
system for the testing and monitoring of genetic containment systems. This could
include investigations into the effects of gene silencing via methylation and how this
may affect the long term stability of transgenes expressing the desired products and
of transgenes used to control gene escape. Issues of seed spillage during sowing
and harvest should also be investigated with regard to the formation and persistence
of feral crop populations. Improvement of farming practice to reduce seed spillage
will reduce the formation of feral populations and lessen the opportunities for
transgene escape via seed. Persistent feral populations and compatible wild
populations allow an escape route for transgenes via pollen mediated gene flow

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either by crop-wild or wild-crop pollination. The impact of crop-wild/feral pollination

on male sterile crops also requires investigation. An assessment of the relative
importance of seed versus pollen-mediated gene flow will aid the targeting of
containment strategies to have the greatest impact.
In addition, an environmental impact assessment is required to assess the new
generation of GM crops resistant to abiotic stress. Should they escape, these crops
have the potential to become invasive and out-compete plants in delicate
ecosystems, leading to unwanted environmental change.

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Recommendations
1. Assess the thoroughness of the regulatory oversight of GM plants
grown in physically contained conditions, including glasshouses,
polytunnels and underground mines (section 3.1)
2. Conduct a detailed environmental risk assessment of the use of
algae and other lower plants as production platforms for high value
and pharmaceutical products (section 4.3)
3. Assess the environmental impact of the next generation of GM crops
with conferred resistance to abiotic stress factors (section 1.1)
4. Commission analysis of the application of transient transformation
systems, including those using viral vector systems for the
production of high value and pharmaceutical products (section 4.2)
5. Investigate the persistence of feral populations of crop plants and
maternal based introgression into populations of wild relatives and
fields of crops plants from persistent feral populations (sections 1.2,
2.3, 3.2, 3.3)
6. Develop a robust technique for the assessment of genetic and
environmental factors affecting gene transfer via plastids in pollen
(section 3.2.2)
7. Investigate the relative importance of seed versus pollen mediated
gene exchange between crop wild relatives (sections 1.2, 2.3, 3.2)
8. Develop a robust system for testing the efficacy and stability of
genetic containment mechanisms on a landscape scale (section 5)
9. Assess the effect and frequency of pollination of male sterile crop
plants by cross-compatible wild relatives (section 3.2.3.3)
10. Conduct

assessment

the

stability

genetic

based

containment mechanisms and the effect of natural methylation based

silencing on their function during introgression (sections 3.2, 5)

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US 050019930. Staub JM, Ye G, Broyles DL. (2005). Method for the transformation of plant
cell plastids. Monsanto. United States. 27 January 2005.
US 050039229. Kuvshinov V, Koivu K, Kanerva A, Anissimov A. (2005). Double recoverable
block of function. UniCrop Ltd. United States. 17 February 2005.
US 050042752. Crete P, Klahre G, Meins F. (2005). Methods of inducing gene expression.
United States. 24 February 2005.
US 050044596. Smith AG. (2005). Methods to confer enhanced floral properties to plants.
United States. 24 February 2005.

227

Organism

Status

Issued/
Effective

Gene(s)

Phenotype(s)/Product
Industrial enzyme

Acreage

Alfalfa

Issued

04/12/95

Amylase from Bacillus licheniformis; B-glucuronidase from E. coli; Lignin peroxidase from
Phanerochaete chrysosporium

Alfalfa

Issued

04/23/97

Cellulase; Inositol hexaphosphate phosphohydrolyase from Aspergillus niger; Lignin peroxidase

Industrial enzyme

Alfalfa

Issued

06/04/93

Alpha-amylase from Bacillus lichenformis; Lignin peroxidase from Phanerochaete chrysosporium

Industrial enzyme

Alfalfa

Issued

10/10/92

Cholera toxin B from Vibrio cholera

Pharmaceutical proteins

04/16/04

Lysozyme from Man; Phosphinothricin acetyl transferase*

Novel protein

Pharmaceutical proteins

3
3

Barley

Acknowledged

Barley

Issued

03/22/01

Amylase from Barley; Antithrombin from Man; Antitrypsin from Man; Green fluorescent protein*;
Lactoferrin from Man; Lysozyme from Man; Phosphinothricin acetyl transferase*; Serum albumin from
Man

Barley

Issued

04/18/00

CBI

Novel protein

06/01/04

Hygromycin phosphotransferase; Lactoferrin Man; Phosphinothricin acetyl transferase

Value added protein for human

consumption

CBI; Phosphinothricin acetyl transferase*

Novel protein

Barley

Issued

Barley

Issued

10/10/00

Barley

Withdrawn

07/10/04

Issued

03/30/04

04-009-01r

ARS

Brassica
juncea

01-012-05n

ProdiGene

Corn

01-012-06n

ProdiGene

Corn

01-229-05n

ProdiGene

Corn

01-032-01n

ProdiGene

Corn

01-045-06n

ProdiGene

Corn

94-279-06n

Agracetus

Corn

00-228-03n

ProdiGene

Corn

95-026-08n

Pioneer

Corn

95-026-06n

Pioneer

Corn

95-026-05n

Pioneer

Corn

95-026-04n

Pioneer

Corn

95-026-03n

Pioneer

Corn

Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged

0.05

Pharmaceutical proteins

0.01

ATP sulfurylase from Arab. thaliana; Cystathionine synthase from Arab. thaliana; Glutamylcysteine
synthetase from E. coli; Hygromycin phosphotransferase*;Methionine methyltransferase from Arab.
thaliana; NptII* SMT sulfurylase from Astragalus bisulcatus; Selenocystine lyase from Mouse;
Sodium/hydrogen ion exchanger from Arab. thaliana

Salt tolerance; Industrial

enzyme

0.3

03/12/01

CBI; Phosphinothricin acetyl transferase*

Novel protein

02/08/01

Phosphinothricin acetyl transferase*

Novel protein

09/20/01

Antibody

10/03/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.3

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

Institution

00-224-03n

ProdiGene

95-256-02n

Agracetus

Corn

00-228-05n

ProdiGene

Corn

00-228-06n

ProdiGene

Corn

00-221-03n

ProdiGene

Corn

95-093-18n

Agracetus

Corn

95-093-19n

Agracetus

Corn

00-228-04n

ProdiGene

Corn

02-081-10n

ProdiGene

Corn

01-045-07n

ProdiGene

Corn

01-193-05n

ProdiGene

Corn

01-194-01n

ProdiGene

Corn

02-113-09n

ProdiGene

Corn

01-194-02n

ProdiGene

Corn

02-081-13n

ProdiGene

Corn

02-081-11n

ProdiGene

Corn

02-081-08n

ProdiGene

Corn

01-194-03n

ProdiGene

Corn

01-324-01n

ProdiGene

Corn

02-081-12n

ProdiGene

Corn

95-335-02n

Pioneer

Corn

95-340-01n

Agracetus

Corn

01-045-08n

ProdiGene

Corn

01-045-09n

ProdiGene

Corn

01-045-10n

ProdiGene

Corn

01-190-02n

ProdiGene

Corn

01-045-12n

ProdiGene

Corn

Organism
Corn

Status
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged

Issued/
Effective

Gene(s)

Phenotype(s)/Product

09/12/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

10/18/95

CBI; Hygromycin phosphotransferase* from E. coli

Antibody

10/03/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.06

09/12/00

CBI; Phosphinothricin acetyl transferase*

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.36

03/16/01

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.54
0.06

03/16/01

CBI; Phosphinothricin acetyl transferase*

Novel protein

08/09/01

Laccase from Turkey tails; Phosphinothricin acetyl transferase*

Industrial enzyme

03/16/01

CBI; Phosphinothricin acetyl transferase*

Novel protein

244

0.12

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

Institution

Organism

01-150-01n

U of Hawaii

Corn

01-045-11n

ProdiGene

Corn

99-055-10n

ProdiGene

Corn

99-055-09n

ProdiGene

Corn

00-060-06n

ProdiGene

Corn

99-055-12n

ProdiGene

Corn

98-356-05n

ProdiGene

Corn

99-055-13n

ProdiGene

Corn

98-273-11n

ProdiGene

Corn

98-273-10n

ProdiGene

Corn

99-278-01n

ProdiGene

Corn

95-284-08n

Pioneer

Corn

99-271-05n

ProdiGene

Corn

99-340-09n

ProdiGene

Corn

99-340-08n

ProdiGene

Corn

99-302-13n

ProdiGene

Corn

99-294-04n

Pioneer

Corn

99-294-03n

Pioneer

Corn

99-274-01n

ProdiGene

Corn

99-055-11n

ProdiGene

Corn

99-271-04n

ProdiGene

Corn

99-271-03n

ProdiGene

Corn

99-271-02n

ProdiGene

Corn

00-049-13n

ProdiGene

Corn

99-145-03n

ProdiGene

Corn

99-106-11n

Iowa State U

Corn

00-049-14n

ProdiGene

Corn

Issued/
Effective

Novel protein

01/25/99

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.1

04/01/99

Novel protein

10/25/99

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.28

01/04/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

12.2

01/04/00

CBI; Phosphinothricin acetyl transferase*

Antibody

0.13

11/23/99

CBI; Phosphinothricin acetyl transferase*

Novel protein
Visual marker;
Phosphinothricin tolerant;
Novel protein
Visual marker;
Phosphinothricin tolerant;
Novel protein

Acreage
1

11/08/99

CBI; CBI from Corn; Phosphinothricin acetyl transferase* from Strep. hygroscopicus

11/15/99

CBI; CBI from Corn; Phosphinothricin acetyl transferase* from Strep. hygroscopicus

11/01/99

ProdiGene

Corn

99-055-14n

ProdiGene

Corn

96-317-03n

Agracetus

Corn

00-109-07n

ProdiGene

Corn

00-122-05n

ProdiGene

Corn

97-098-07n

ProdiGene

Corn

96-094-08n

Agracetus

Corn

96-094-07n

Agracetus

Corn

96-079-15n

Pioneer

Corn

96-079-14n

Pioneer

Corn

96-079-08n

Pioneer

Corn

00-192-08n

ProdiGene

Corn

00-067-11n

ProdiGene

Corn

98-085-42n

ProdiGene

Corn

98-068-12n

Monsanto

Corn

Organism

98-068-11n

Monsanto

Corn

96-317-02n

Agracetus

Corn

00-067-10n

ProdiGene

Corn

97-148-03n

Agracetus

Corn

00-067-09n

ProdiGene

Corn

97-113-03n

Agracetus

Corn

97-164-02n

Agracetus

Corn

97-253-03n

ProdiGene

Corn

02-081-09n
00-060-04n
97-106-20n
01-257-01r
98-274-02r
98-274-01r
99-343-01r
99-034-03r

ProdiGene
ProdiGene
Limagrain
Monsanto
Monsanto
Monsanto
ProdiGene
ProdiGene

Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn

Status
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Acknowledged
Denied
Denied
Denied
Issued
Issued
Issued
Issued
Issued

Issued/
Effective

Gene(s)

Phenotype(s)/Product

03/24/99

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.6

04/01/99

CBI; Phosphinothricin acetyl transferase*

Novel protein

0.05

12/06/96

CBI; Hygromycin phosphotransferase*

Antibody

05/30/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

06/13/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

Novel protein

0.1

04/27/98

CBI; Phosphinothricin acetyl transferase*

Novel protein

04/10/98

CBI

Antibody

04/17/98

CBI

Antibody

204

12/06/96

CBI; Hygromycin phosphotransferase*

Antibody

04/18/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

06/09/97

CBI; Hygromycin phosphotransferase*

Antibody

04/18/00

CBI; Phosphinothricin acetyl transferase*

Novel protein

4.12

05/15/97

CBI; Hygromycin phosphotransferase*

Antibody

5.25

07/01/97

CBI; Hygromycin phosphotransferase*

Antibody

10/09/97

CBI; Phosphinothricin acetyl transferase*

03/22/02
03/07/00
04/18/97
01/11/02
02/03/99
02/04/99
02/22/00
03/03/99

CBI
CBI
CBI
CBI; Phosphinothricin acetyl transferase*

246

Novel protein
Novel protein
Antibody
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins

Acreage

21.2
5

0.2
0.16
20.8
10
1

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

Institution

99-034-01r
99-034-02r
00-363-01r
00-363-02r

ProdiGene
ProdiGene
Monsanto
Monsanto

00-021-04r

00-021-03r

02-023-01r

01-022-03r

Pioneer

Organism
Corn
Corn
Corn
Corn

Corn

Status
Issued
Issued
Issued
Issued

Issued

Issued/
Effective
03/03/99
03/04/99
03/21/01
03/21/01

Gene(s)

Phenotype(s)/Product

CBI; Phosphinothricin acetyl transferase* from Strep. viridochromogenes

CBI; Phosphinothricin acetyl transferase*
C transcriptional activator
C transcriptional activator

Novel protein
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins

03/30/00

Amino polyol amine oxidase; Aspartokinase from E. coli; B-glucuronidase*; CBI from Rapeseed; CBI
from Soybean; CBI from Potato; CryIA(b) from Btk; DNA adenine methylase; from E. coli;
Dihydrodipicolinate synthase from Corynebacterium glutamicum; Esterase; Glycogenin from Corn;
Glycogenin antisense from Corn; Luciferase*; Lysine ketoglutarate reductasefrom Corn; NptII*;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep.
Viridochromogenes; Saccharopine dehydrogenase from Corn; Starch branching enzyme II from Corn;
Starch synthase from Corn; Storage protein from Corn

Altered maturing; Yield

increased; Ear mold resistant;
Smut resistant; Cyanamide
tolerant; Phosphinothricin
tolerant; Coleopteran resistant;
Lepidopteran resistant; Novel
protein; Animal feed quality;
Fumonisin degradation; Grain
processing improved

03/31/00

Amino polyol amine oxidase from Exophiala spinifera; Aspartokinase from E. coli; B-glucuronidase*;
CBI; CBI from Potato; CBI from Soybean; CBI from Rapeseed; CBI from Pentataclethra macrebola;
CryIA(b) from Btk; DNA adenine methylase from E. coli; Dihydrodipicolinate synthase from
Corynebacterium glutamicum; Esterase from Exophiala spinifera; Glycogenin from Corn; Glycogenin
antisense from Corn; Luciferase*; Lysine ketoglutarate reductase from Corn; NptII*; Phosphinothricin
acetyl transferase*; Phosphinothricin acetyl transferase ; Strep. viridochromogenes; Saccharopine
dehydrogenase from Corn; Starch branching enzyme II from Corn; Starch synthase from Corn;
Storage protein from Corn

Altered maturing; Yield

increased; Ear mold resistant;
Smut resistant; Cyanamide
tolerance; Phosphinothricin
tolerant; Coleopteran resistant;
Lepidopteran resistant; Novel
protein; Animal feed quality;
Fumonisin degradation; Grain
processing improved

04/10/02

Aspartokinase from E. coli; CBI; Citrate lyase from Corn; Cystathionine synthase from Corn;
Dehydroascorbate reductase from Corn; Dihydrodipicolinate synthase from Corn; Dihydrodipicolinate
synthetase from Corynebacterium glutamicum; Lysine ketoglutarate reductase from Corn;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep. hygroscopicus;
Saccharopine dehydrogenase*; Starch synthase from Corn; Storage protein from Corn

Altered maturing; Fertility;

Increased stalk strength; Ear
mold resistant;
Phosphinothricin tolerant;
Coleopteran resistant;
Lepidopteran resistant; Visual
marker; Increased
transformation frequency;
Industrial enzyme; Novel
protein; Animal feed quality;
Fumonisin degradation

04/13/01

Adenine methylase from E. coli; Amino polyol amine oxidase from Exophiala spinifera; Bglucuronidase*; CBI*; CBI frin Arabidopsis thaliana; CBI; CBI from Corn; CBI from Soybean;
Dihydrodipicolinate synthase from Corynebacterium glutamicum; Esterase; Esterase from Exophiala
spinifera; Flavin amine oxidase from Exophila spinifera; Lysine ketoglutarate reductase from Corn;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep.
virodochromogenes; Phosphinothricin acetyl transferase from Strep. hygroscopicus; Saccharopine
dehydrogenase from Corn; Storage protein from Corn

Altered maturing; Fertility;

Fumonisin degradation;
Increased stalk strength; Yield
increased; Ear mold resistant;
Phosphinothricin tolerant;
Lepidopteran resistant; Novel
protein; Animal feed quality;
Grain processing improved

247

Acreage

32
33.4

508

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

Institution

Organism

Status

Issued/
Effective

Gene(s)

Phenotype(s)/Product

Yield increased; Ear mold

resistant; Phosphinothricin
tolerant; Coleopteran resistant;
Lepidopteran resistant; Novel
protein; Visual marker; Animal
feed quality; Grain processing
improved

Acreage

01-022-04r

Pioneer

Corn

Issued

04/13/01

Adenine methylase from E. coli; Aspartokinase from Corn; CBI*; CBI; CBI from Soybean; CBI from
Bt; CBI from Corn; CBI from E. coli; CBI - from Potato; CBI from Pentaclethra macrobola; CBI from
Arabidopsis thaliana; Cry1F from Bt azawai; Dihydrodipicolinate synthase from Corn;
Dihydrodipicolinate synthase from Corynebacterium glutamicum; Esterase from ATTC 55552;
Esterase from Exophila spinifera; Glycogenin from Corn; Lysine ketoglutarate reductase from Corn;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep.
viridochromogenes; Phosphinothricin acetyl transferase from Strep. hygroscopicus; Saccharopine
dehydrogenase from Corn; Saccharopine dehydrogenase from Yarrowia lipolytica; Starch branching
enzyme II antisense from corn; Starch synthase from corn; Storage protein from Corn

99-050-01r
01-016-01r
01-016-02r
01-016-03r

Agracetus
ProdiGene
ProdiGene
ProdiGene

Corn
Corn
Corn
Corn

Issued
Issued
Issued
Issued

04/14/99
04/17/01
04/19/01
04/19/01

CBI
CBI; NptII*
CBI; NptII*
CBI; NptII*

Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins

24
10
0.7
0.25

Amino polyol amine oxidase from Exophila spinifera; B-glucuronidase*; CBI from Arab. thaliana; CBI
from Rye; CBI from Rice; CBI from Pea; CBI from Corn; CBI; CBI from Barley; Dihydrodipicolinate
synthase from Corynebacterium glutamicum; Hygromycin phosphotransferase*; Luciferase*; Lysine
ketoglutarate reductase from Corn; NptII*; Phosphinothricin acetyl transferase from Strep.
hygroscopicus; Storage protein from Corn

Altered maturing; Fertility;

Increased stalk strength; Yield
increased; Ear mold resistant;
Phosphinothricin tolerant;
Increased transformation
frequency; Novel protein;
Animal feed quality; Fumonisin
degradation; Grain processing
improved

3200

04/20/04

Amino polyol amine oxidase from Exophila spinifera; B-glucuronidase*; C1 regulatory gene from
Corn; CBI from Arab. thaliana; CBI from Corn;; CBI from Pea; CBI from Rice; CBI from Rye; CBI; CBI
from Barley; Dihydrodipicolinate synthase from Corynebacterium glutamicum; Hygromycin
phosphotransferase*; Luciferase*; Lysine ketoglutarate reductase from Corn; NptII*; Phosphinothricin
acetyl transferase from Strep. hygroscopicus; Storage protein from Corn

Altered maturing; Fertility;

Increased stalk strength; Yield
increased; Ear mold resistant;
Phosphinothricin tolerant;
Increased transformation
frequency; Novel protein
produced; Animal feed quality
improved; Fumonisin
degradation; Grain processing
improved

7475

33.2

04-020-04r

Pioneer

Corn

Issued

04/20/04

04-020-03r

Pioneer

01-057-01r

Horan Bros.
Agri. Enterprises

Corn

Issued

04/25/01

CBI; NptII*

Pharmaceutical proteins

00-067-02r

ProdiGene

Corn

Issued

04/26/00

CBI

Pharmaceutical proteins

04-040-01r

ProdiGene

Corn

Issued

04/28/04

Aprotinin from Cow; Brazzein from Pentadiplandra brazzeana; CBI; CBI from Man; Epitope from CBI;
Epitope from TGEV; Glycoprotein gp120 from Human immunodeficiency virus; Phosphinothricin
acetyl transferase*; Trypsin from Cow

Novel protein

00-067-01r

ProdiGene

Corn

Issued

05/01/00

CBI; Phosphinothricin acetyl transferase*

Pharmaceutical proteins

53.5

Pharmaceutical proteins

0.25

01-023-03r

ProdiGene

Corn

Issued

05/08/01

Aprotinin from Bos taurus; CBI; Enterotoxin subunit B from E. coli; NptII*; Phosphinothricin acetyl
transferase*; Surface antigen from Hepatitis virus B; Surface antigen from TGEV; gp120 (glycoprotein
120) - from Simian immunodeficiency virus

01-023-04r

ProdiGene

Corn

Issued

05/08/01

CBI; NptII*

248

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

Institution

Organism

Status

Issued/
Effective

Gene(s)

Phenotype(s)/Product
Altered plant development;
Altered stalk attributes; Fertility;
Maturity; Reduced plant
growth; Yield increased; Yield
stability; Glyphosate tolerant;
Phosphinothricin tolerant;
Coleopteran resistant;
Lepidopteran resistant;
Selectable marker; Visual
marker; Increased
transformation frequency;
Novel protein; Amino acid
composition; Starch content;
Digestibility improved; Fatty
acid levels; Feed properties;
Fiber quality; Fumonisin
degradation; Fumonisin
mycotoxin levels; Grain
processing improved; Lipid
profile; Nutritional quality; Oil
profile; Phytate reduced;
Protein lysine level increased;
Protein quality; Starch level
increased; Starch metabolism

Acreage

05-024-03r

Pioneer

Corn

Issued

05/12/05

Acetolactate synthase* from Soybean; Adenine methylase from E. coli; B-glucuronidase* from E. coli;
C1 regulatory gene* from Corn; CBI from Secale cereale; CBI from Corn; CBI from Rice; CBI; CBI*;
CBI* from Corn; CBI from Arabidopsis thaliana; CBI from Barley; Dihydrodipicolinate synthase from
Corynebacterium glutamicum; Hygromycin phosphotransferase* from E. coli; Luciferase* from
Photinus pyralis; Lysine ketoglutarate reductase from Corn; Phosphinothricin acetyl transferase from
Strep. viridochromogenes; Phosphinothricin acetyltransferase from Strep. hygroscopicus; Storage
protein from Barley

02-108-01r

Meristem
Therapeutics

Corn

Issued

05/21/02

Antibody (common cold) from Mouse; Antibody (tooth decay) from Mouse; NptII*

Pharmaceutical proteins
Altered plant developmen;
Stalk attributes; Fertility;
Maturity; Reduced plant
growth; Yield enhancement;
Yield stability; Glyphosate
tolerant; Phosphinothricin
tolerant; Coleopteran resistant;
Lepidopteran resistant;
Selectable marker; Visual
marker; Increased
transformation frequency;
Novel protein; Amino acid
composition; Starch content;
Digestibility improved; Fatty
acid levels; Feed properties;
Fiber quality; Fumonisin
degradation; Grain processing
improved; Lipid profile; Lysine
level increased; Nutritional
quality; Oil profile; Phytate
reduced; Protein quality; Starch
levels; Starch metabolism

2800

10285

05-024-04r

Pioneer

Corn

Issued

05/25/05

Acetolactate synthase*; Adenine methylase from E. coli; Amino polyol amine oxidase from Exophila
spinifera; B-glucuronidase*; C1 regulatory gene*; CBI from Arab. thaliana; CBI*; CBI from Barley; CBI
from Corn; CBI; CBI from Rice; CBI from Soybean; Dihydrodipicolinate synthase from
Corynebacterium glutamicum; Hemagglutinin epitope*; Hygromycin phosphotransferase*;
Luciferase*; Lysine ketoglutarate reductase from Corn; Phosphinothricin acetyltransferase from
Strep. viridochromogenes; Phosphinothricin acetyltransferase*; Phosphinothricin acetyltransferase
from Strep. hygroscopicus

00-073-01r
04-131-01r

ProdiGene
Iowa State U

Corn
Corn

Issued
Issued

05/26/00
06/01/04

CBI*; Phosphinothricin acetyl transferase

Enterotoxin subunit B from E. coli; Phosphinothricin acetyl transferase*

Pharmaceutical proteins
Pharmaceutical proteins

2
0.25

02-141-01r

Meristem
Therapeutics

Corn

Issued

06/05/02

CBI*; CBI from Man; CBI

Pharmaceutical proteins

249

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Status

Issued/
Effective

Gene(s)

Phenotype(s)/Product

Corn

Issued

06/05/03

CBI*; CBI

Pharmaceutical proteins

Dow
Iowa State U
Limagrain
Limagrain
Limagrain
Limagrain
Iowa State U
ProdiGene
ProdiGene
ProdiGene
ProdiGene
ProdiGene
Garst
Dow
ProdiGene
Monsanto
Monsanto
Monsanto

Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn
Corn

Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued
Issued

06/07/02
06/10/05
06/10/98
06/12/98
06/12/98
06/12/98
06/14/01
07/12/01
07/14/00
08/23/00
10/05/01
10/13/00
10/14/03
10/23/01
11/03/00
11/03/00
11/03/00
11/03/00

CBI
LT-B heat labile enterotoxin from E. coli; Phosphinothricin acetyl transferase*
Phosphinothricin acetyl transferase*; Serum albumin from Man
Phosphinothricin acetyl transferase; Procollagen* from Man
G glycoprotein
Alpha-hemoglobin from Man; Beta-hemoglobin from Man; Phosphinothricin acetyl transferase*
Enterotoxin subunit B from E. coli; NptII*
CBI; NptII*
CBI; Phosphinothricin acetyl transferase*
CBI*; CBI; Phosphinothricin acetyl transferase*
Enterotoxin subunit B from E. coli; Phosphinothricin acetyl transferase*

Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Novel protein
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins

01-187-01r

ProdiGene

Corn

Issued

11/13/01

Aprotinin from Pig; CBI; Phosphinothricin acetyl transferase*; Surface antigen from Hepatitis virus B;
Surface antigen from TGEV; gp120 (glycoprotein 120) from SIV

Pharmaceutical proteins

99-279-02r
99-279-01r
99-307-01r
98-257-01r
00-207-01r
99-307-02n
98-068-13n

ProdiGene
ProdiGene
ProdiGene
ProdiGene
Monsanto
ProdiGene
Monsanto

Corn
Corn
Corn
Corn
Corn
Corn
Corn

Issued
Issued
Issued
Issued
Issued
Withdrawn
Withdrawn

11/18/99
11/18/99
11/18/99
11/25/98
12/01/00
11/23/99
05/05/98

CBI; Phosphinothricin acetyl transferase*

CBI; Phosphinothricin acetyl transferase*
CBI; Phosphinothricin acetyl transferase*
CBI; Phosphinothricin acetyl transferase*
CBI*; CBI
CBI; Phosphinothricin acetyl transferase*
CBI*; CBI

Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Novel protein
Antibody

04-121-02r

ProdiGene

Corn

Withdrawn

07/02/04

Brazzein from Pentadiplandra brazzeana; CBI; CBI from Man; Glycoprotein gp120 from Human
immunodeficiency virus; Phosphinothricin acetyl transferase*

Value added protein

04-121-01r
04-114-02r
04-065-01r

ProdiGene
ProdiGene
Iowa State U

Corn
Corn
Corn

Withdrawn
Withdrawn
Withdrawn

08/25/04
08/25/04
09/07/04

Aprotinin from Cow; Phosphinothricin acetyl transferase*

Phosphinothricin acetyl transferase*; Trypsin from Cow
Enterotoxin subunit B from E. coli; Phosphinothricin acetyl transferase*

Value added protein

Value added protein
Pharmaceutical proteins

97-273-11n

Monsanto

Rapeseed

Denied

10/02/97

Polymer

97-266-06n

Monsanto

Rapeseed

Denied

09/24/97

Polymer

96-061-02r

Monsanto

Rapeseed

Issued

04/16/96

CBI*; CBI

Polymer

93-048-02r

Cargill

Rapeseed

Issued

05/04/93

CBI; NptII*

Industrial enzyme

Permit

Institution

03-086-01r

Meristem
Therapeutics

02-071-01r
05-069-01r
98-117-03r
98-117-02r
98-117-04r
98-117-01r
01-122-01r
01-114-01r
00-122-01r
00-195-01r
01-190-01r
00-203-04r
03-143-01r
01-212-01r
00-221-01r
00-203-01r
00-203-02r
00-203-03r

Organism

Acetolactate synthase*; CBI from Man; CBI from Mouse

CBI from Human; Phosphinothricin acetyl transferase*
CBI; Phosphinothricin acetyl transferase*
CBI*; CBI
CBI*; CBI
CBI*; CBI

250

Acreage

0.1
0.5
0.2
0.5
0.25
0.2
1
0.5
0.1
36
7.9
15
15
15
0.4
0.2

15
20
12

0.25

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Status

Issued/
Effective

Gene(s)

Phenotype(s)/Product

Rapeseed

Issued

09/17/96

CBI; Phosphinothricin acetyl transferase* from Strep. Hygroscopicus

Pharmaceutical proteins

Monsanto

Rapeseed

Withdrawn

10/20/97

CBI; CBI*

Polymer

Applied
Phytologics

Rice

Issued

Antitrypsin from Human; CBI from Human; Dirgent protein from Forsythia intermedia; Hygromycin
phosphotransferase*; Laccase from Forsythia intermedia; Lactoferrin from Human; Lysozyme from
Human; Pinoresinol reductase from Forsythia intermedia; Pinoresinol-lariciresinol reductase from
Forsythia intermedia; Secoisolariciresinol dehydrogenase from Forsythia intermedia

Novel protein

Rice

Issued

02/25/98

CBI; Hygromycin phosphotransferase*

Pharmaceutical proteins

02/25/98

Antithrombin from Man; Antitrypsin from Man; Hygromycin phosphotransferase* from E. coli; Serum
albumin from Man

Pharmaceutical proteins

Aminoglycoside 3'- adenylyltransferase from Man; Antithrombin from Man; Hygromycin

phosphotransferase*; Serum albumin from Man

Pharmaceutical proteins

Permit

Institution

96-215-01r

Pioneer

97-283-01n

01-206-01r

97-363-01r
98-008-01r
96-355-01r
05-073-01r
02-361-01r

Applied
Phytologics
Applied
Phytologics
Applied
Phytologics
Ventria
Bioscience
Ventria
Bioscience

Organism

Rice

Issued

Acreage

Rice

Issued

03/31/97

Rice

Issued

04/06/05

Lactoferrin from Man; Lysozyme from Man; Serum albumin from Man

Value added protein

Value added protein for human

consumption

00-069-01r

Applied
Phytologics

CBI; Hygromycin phosphotransferase; NptII

Pharmaceutical proteins; Novel

protein

05-117-01r

Ventria
Bioscience

Lactoferrin from Man

Value added protein for human

consumption

Lysozyme from Man

Value added protein for human

consumption

05-117-02r
00-217-01r
99-272-01r
99-272-02r
04-309-01r

Ventria
Bioscience
Applied
Phytologics
Applied
Phytologics
Applied
Phytologics
Ventria
Bioscience

Rice
Rice

Issued
Issued

05/15/00
06/28/05

Rice

Issued

06/28/05

Rice

Issued

10/06/00

Rice

Issued

10/28/99

CBI; Hygromycin phosphotransferase*

Pharmaceutical proteins

Rice

Issued

10/28/99

CBI; Hygromycin phosphotransferase*

Pharmaceutical proteins

Rice

Withdrawn

06/20/05

Lysozyme from Man

Value added protein for human

consumption

100

Rice

Withdrawn

06/20/05

Lactoferrin from Man

Value added protein for human

consumption

100

06/20/05

Lactoferrin from Man; Lysozyme from Man; Serum albumin from Man

Value added protein for human

consumption

4.5

05/28/02

CBI; Phosphinothricin acetyl transferase*

Novel protein

06/03/03

CBI from Cow; Cystatin proteinase from Arab. Thaliana; Growth hormone from Carp; Oleosin from
Arab. Thaliana; Phosphinothricin acetyl transferase*

Industrial enzymes;
Pharmaceutical proteins

05/20/96

CBI; B-glucuronidase* from E. coli

Antibody

0.1

04-302-01r

Ventria
Bioscience

05-004-01r

Ventria
Bioscience

Rice

02-099-13n

Emlay and
Associates

Safflower

03-071-01r

Emlay and
Associates

Safflower

96-113-05n

Agracetus

Soybean

Withdrawn
Acknowledged
Issued
Acknowledged

Novel protein

251

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

Institution

94-257-05n

Agracetus

Soybean

95-093-17n

Agracetus

Soybean

94-279-05n

Agracetus

Soybean

95-074-05n

Agracetus

Soybean

95-074-06n

Agracetus

Soybean

95-093-16n

Agracetus

Soybean

95-101-04n

Agracetus

Soybean

95-268-05n

Agracetus

Soybean

96-086-04n

Monsanto

Soybean

94-136-01n

Agracetus

Soybean

96-113-04n

Agracetus

Soybean

95-019-04n

Agracetus

Soybean

96-172-02n

Agracetus

Soybean

96-198-01n

Agracetus

Soybean

96-198-03n

Agracetus

Soybean

96-277-06n

Agracetus

Soybean

99-074-02n

Monsanto

Soybean

96-113-02n

Agracetus

Soybean

93-321-01n

Agracetus

Soybean

00-021-02r

Pioneer

Organism

Soybean

Issued

Issued/
Effective

Gene(s)

Phenotype(s)/Product

10/04/94

CBI; NptII*

Antibody

Acreage
5

05/05/95

CBI; B-glucuronidase* from E. coli

Antibody

11/02/94

CBI; B-glucuronidase*

Antibody

0.5

04/17/95

CBI; B-glucuronidase* from E. coli

Antibody

0.1

04/17/95

CBI; B-glucuronidase* from E. coli

Antibody

0.1

05/05/95

CBI; B-glucuronidase* from E. coli

Antibody

05/12/95

CBI; B-glucuronidase* from E. coli

Antibody

10/26/95

CBI; B-glucuronidase* from E. coli

Antibody

04/09/96

CBI; CBI*

Polymer

05/24/94

CBI; B-glucuronidase*

Industrial enzyme

0.5

05/01/96

CBI; B-glucuronidase* from E. coli

Antibody

0.3

02/09/95

CBI; B-glucuronidase*

Industrial enzyme

0.1

08/01/96

CBI; B-glucuronidase* from E. coli

Antibody

07/22/96

CBI; B-glucuronidase* from E. coli

Industrial enzyme

0.1

07/22/96

CBI; B-glucuronidase* from E. coli

Antibody; Novel protein

0.1

10/16/96

CBI; B-glucuronidase* from E. coli

Industrial enzyme

0.2

04/19/99

CBI; CBI*

Industrial enzyme

05/20/96

CBI; B-glucuronidase* from E. coli

Industrial enzyme

0.3

12/13/93

CBI; B-glucuronidase* from E. coli

Industrial enzyme

0.1

03/30/00

Acyl-ACP thioesterase from Rapeseed; Acyl-ACP thioesterase from Soybean; Aspartokinase from E.
coli; Aspartokinase II-homoserine dehydrogenase from E. coli; B-glucuronidase*; CBI*; CBI from
Corn;CBI from Soybean; CBI; Conglycinin from Soybean; Cystathionine beta-lyase from Soybean,
Cystathionine synthase from Corn; Cystathionine synthase from Soybean; Delta-9 desaturase from
Soybean; Dihydrodipicolinate synthase from Corynebacterium glutamicum; Dihydrodipicolinate
synthase from Soybean; Dihydrodipicolinate synthase from E. coli; Galactinol synthase from
Soybean; Glycinin from Soybean; Hygromycin phosphotransferase*; Isoflavone synthase from
Soybean; Luciferase*; Lysine ketoglutarate reductase from Soybean; NptII*; Omega 3 desaturase
from Soybean; Omega 6 desaturase from Soybean; Omega 6 desaturase antisense from Soybean;
Phosphinothricin acetyl transferase*; Saccharopine dehydrogenase from Soybean; Saccharopine
dehydrogenase from Yarrowia lypolytica; Storage protein from Corn; Storage protein from Synthetic;
UDP-glucose 4'epimerase from Soybean

Altered maturing; Yield

increased; Ear mold resistant;
Cyanamide tolerant;
Phosphinothricin tolerant;
Coleopteran resistant;
Lepidopteran resistant; Novel
protein; Animal feed quality;
Fumonisin degradation; Grain
processing improved

252

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

02-023-05r

04-020-01r

03-022-03r

04-020-02r

Institution

Pioneer

Organism

Soybean

Status

Issued

Issued/
Effective

04/10/02

04/13/04

04/15/03

04/15/04

Gene(s)

Phenotype(s)/Product

Acetolactate synthase* from Arab. thaliana; Acetolactate synthase from Arab. thaliana; Acyl-ACP
thioesterase from Rapeseed; Acyl-ACP thioesterase from Soybean; Aspartokinase II-homoserine
dehydrogenase from E. coli; B-glucuronidase* from E. coli; B-glucuronidase from Tobacco; CBI from
Alfalfa; CBI from Arab. thaliana; CBI from Soybean; CBI; Conglycinin from Soybean; Cystathionine
synthase from Corn; Cystathionine synthase from Soybean; Delta-9 desaturase from Soybean;
Dihydrodipicolinate synthetase from Corynebacterium glutamicum; Galactinol synthase from
Soybean; Hygromycin phosphotransferase* from E. coli; Hygromycin phosphotransferase from E coli;
Lysine ketoglutarate reductase from Soybean; NptII* from E. coli; NptII from Tobacco; NptII from E.
coli; Omega 3 desaturase from Soybean; Omega 6 desaturase from Soybean; Omega 6 desaturase
antisense from Soybean; Phosphinothricin acetyl transferase* from Strep. viridochromogenes;
Phosphinothricin acetyl transferase from Strep. viridochromogenes; Saccharopine dehydrogenase;
Seed storage protein from Synthetic; Storage protein from Corn; Thioesterase from Rapeseed;
Thioesterase from Soybean; UDP-glucose 4'epimerase from Soybean
Acetolactate synthase*; Acyl-ACP thioesterase from Rapeseed; Acyl-ACP thioesterase from
Soybean; Aspartokinase from E. coli; Aspartokinase II-homoserine dehydrogenase from E. coli; Bglucuronidase*; CBI*; CBI from Agro. tumefaciens; CBI from Arab. thaliana; CBI from Vernonia
galamensis; CBI from Thraustochytrium aureum; CBI from Stigmatella aurantiaca; CBI from Soybean;
CBI from Schizochytrium aggregatum; CBI from Saprolegnia diclina; CBI from Rice; CBI from
Parthenium argentatum; CBI from Mortierella alpina; CBI from Isochrysis galabana; CBI from
Euphorbia lagascae; CBI from Corn; CBI; CBI from Barley; CBI from Bacillus subtilus; CBI from
Bacillus sp.; CBI from Bacillus amyloliquefaciens; CBI from Alfalfa; Conglycinin from Soybean;
Cystathionine beta-lyase from Soybean; Cystathionine synthase from Soybean; Delta-12 desaturase
from Soybean; Dihydrodipicolinate synthase from Corynebacterium glutamicum; Dihydrodipicolinate
synthase from Soybean; Galactinol synthase from Soybean; Glycinin from Soybean; Hygromycin
phosphotransferase*; Luciferase*; Lysine ketoglutarate reductase from Soybean; NptII*; Omega 3
desaturase from Soybean; Omega 6 desaturase from Soybean; Omega 6 desaturase antisense from
Soybean; Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep.
hygroscopicus; Saccharopine dehydrogenase from Yarrowia lipolytica; Storage protein from Corn;
UDP-glucose 4'epimerase from Soybean
10 kDa protein from Corn; Acetolactate synthase*; Acyl-ACP thioesterase from Rapeseed; Acyl-ACP
thioesterase from Soybean; Aspartokinase II-homoserine dehydrogenase from E. coli; Bglucuronidase*; CBI*; CBI from Arab. thaliana; CBI from Alfalfa; CBI from Bacillus amyloliquefaciens;
CBI from Corn; CBI from Isochrysis galabana; CBI from Parthenium argentatum; CBI from Wheat;
CBI from Vernonia galamensis; CBI from Thraustochytrium aureum; CBI from Sunflower; CBI from
Soybean; CBI from Schizochytrium aggregatum; CBI from Saprolegnia diclina; CBI from Rice; CBI
from Mortierella alpina; CBI from Euphorbia lagascae; CBI; Conglycinin from Soybean; Cystathionine
beta-lyase from Soybean; Cystathionine synthase from Corn; Cystathionine synthase from Soybean;
Delta-9 desaturase from Soybean; Dihydrodipicolinate synthase from Corynebacterium glutamicum;
Dihydrodipicolinate synthase from Soybean; Dihydrodipicolinate synthase from E. coli; Galactinol
synthase from Soybean; Hygromycin phosphotransferase*; Lysine ketoglutarate reductase from
Soybean; NptII*; Omega 3 desaturase from Soybean; Omega 6 desaturase antisense from Soybean;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep. hygroscopicus;
Storage protein from Corn; UDP-glucose 4'epimerase from Soybean
Acetolactate synthase*; Acyl-ACP thioesterase from Rapeseed; Acyl-ACP thioesterase from
Soybean; Aspartokinase from E. coli; Aspartokinase II-homoserine dehydrogenase from E. coli; Bglucuronidase*; CBI*; CBI from Alfalfa; CBI from Bacillus sp.; CBI from Barley; CBI from Bacillus
amyloliquefaciens; CBI from Arab. thaliana; CBI from Corn; CBI from Isochrysis galabana;CBI from
Vernonia galamensis; CBI from Thraustochytrium aureum; CBI from Stigmatella aurantiaca; CBI from
Soybean; CBI from Schizochytrium aggregatum; CBI from Saprolegnia diclina; CBI from Rice; CBI
from Parthenium argentatum; CBI from Mortierella alpina; CBI from Euphorbia lagascae; Conglycinin
from Soybean; Cystathionine beta-lyase from Soybean; Cystathionine synthase from Soybean; Delta12 saturase from Corynebacterium glutamicum; Dihydrodipicolinate synthase from Soybean;
Galactinol synthase from Soybean; Glycinin from Soybean; Hygromycin phosphotransferase*;
Luciferase*; Lysine ketoglutarate reductase from Soybean; NptII*; Omega 3 desaturase from
Soybean; Omega 6 desaturase from Soybean; Omega 6 desaturase antisense from Soybean;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep. hygroscopicus;
Saccharopine dehydrogenase from Yarrowia lipolytica; Storage protein from Corn; UDP-glucose
4'epimerase from Soybean

253

Acreage

Fungal susceptibility;
Sclerotinia resistant; Increased
transformation frequency;
Novel protein; Animal feed
quality; Yield increased

182

Longer stems; Shorter stems;

Yield increased; Sclerotinia
resistant; Glyphosate tolerant;
Phosphinothricin tolerant;
Fungal susceptibility; Novel
protein; Transformation
frequency increased; Animal
feed quality

410

Sclerotinia resistant;
Glyphosate tolerant;
Phosphinothricin tolerant;
Fungal susceptibility; Increased
transformation frequency;
Novel protein produced; Animal
feed quality; Yield increased;
Fusarium resistant

520

Longer stems; Shorter stems;

Yield increased; Sclerotinia
resistant; Glyphosate tolerant;
Phosphinothricin tolerant;
Fungal susceptibility; Novel
protein; Transformation
frequency increased; Animal
feed quality

100

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Permit

03-022-04r

05-024-01r

Institution

Pioneer

Organism

Soybean

Status

Issued

Issued/
Effective

Gene(s)

Phenotype(s)/Product

05/01/03

10 kDa protein from Corn; Acetolactate synthase*; Acyl-ACP thioesterase from Rapeseed; Acyl-ACP
thioesterase from Soybean; Aspartokinase II-homoserine dehydrogenase from E. coli; Bglucuronidase*; CBI*; CBI from Alfalfa; CBI - from Bacillus amyloliquefaciens; CBI from Corn; CBI
from Isochrysis galabana; CBIfrom Vernonia galamensis; CBI from Thraustochytrium aureum; CBI
from Sunflower; CBI from Soybean; CBI from Schizochytrium aggregatum; CBI from Saprolegnia
diclina; CBI from Rice; CBI from Parthenium argentatum; CBI from Mortierella alpina; CBI from
Euphorbia lagascae; CBI; CBI from Arab. thaliana; Conglycinin from Wheat; Cystathionine beta-lyase
from Soybean; Cystathionine synthase from Corn; Cystathionine synthase from Soybean; Delta-9
desaturase from Soybean; Dihydrodipicolinate synthase from Corynebacterium glutamicum;
Dihydrodipicolinate synthase from Soybean; Dihydrodipicolinate synthase from E. coli; Galactinol
synthase from Soybean; Hygromycin phosphotransferase*; Lysine ketoglutarate reductase from
Soybean; NptII*; Omega 3 desaturase from Soybean; Omega 6 desaturase antisense from Soybean;
Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase from Strep. hygroscopicus;
Storage protein from Corn; UDP-glucose 4'epimerase from Soybean

Yield increased; Sclerotinia

resistant; Glyphosate tolerant;
Phosphinothricin tolerant;
Fungal susceptibility; Increased
transformation frequency;
Novel protein; Animal feed
quality; Nutritional quality
improved; Oil quality

05/12/05

Acetolactate synthase*; Acyl-ACP thioesterase from Soybean; Acyl-ACP thioesterase from

Rapeseed; Aspartokinase from E. coli; Aspartokinase II-homoserine dehydrogenase from E. coli; Bglucuronidase - Donor: E. coli; B-glucuronidase*; C1 regulatory gene from Corn; CBI from Alfalfa; CBI
from Schizochytrium aggregatum; CBI from Parthenium argentatum; CBI from Euphorbia lagascae;
CBI; CBI; CBI from Arab. thaliana; CBI - from Barley; CBI; CBI from Corn; CBI from Mortierella
alpina; CBI from Rice; CBI from Soybean; CBI from Bacillus amyloliquefaciens; CBI from Vernonia
galamensis; CBI from Sunflower; Conglycinin from Soybean; Cystathionine beta lyase from Soybean;
Cystathionine synthase from Soybean; Cystathionine synthase from Corn; Delta-12 saturase from
Soybean; Delta-9 desaturase from Soybean; Dihydrodipicolinate synthase from Soybean;
Dihydrodipicolinate synthase from Corynebacterium glutamicum; Dihydrodipicolinate synthase from
E. coli; Galactinol synthase from Soybean; Glycinin from Soybean; Hygromycin
phosphotransferase*; Luciferase from Jellyfish; Lysine ketoglutarate reductase from Soybean;
Lysine- and Methionine-rich protein from Synthetic; Omega 3 desaturase from Soybean; Omega 6
desaturase from Soybean; Phosphinothricin acetyl transferase*; Phosphinothricin acetyl transferase
from Strep. hygroscopicus; Saccharopine dehydrogenase from Yarrowia lipolytica

Stem attributes; Yield

increased; Yield stability;
Fungal susceptibility; Mold
resistance; Sclerotinia
resistant; Phosphinothricin
tolerant; Aphid resistant; Visual
marker; Increased
transformation frequency;
Novel protein; Amino acid
composition; Carbohydrate
metabolism; Cell wall; Fatty
acid levels; Feed properties;
Flavinoid levels; Flavinoid level
decreased; Food quality; Lipid
profile; Lysine level increased;
Nutritional quality; Oil quality

915

Stem attributes; Yield

increased; Yield stability;
Fungal susceptibility; Mold
resistance; Sclerotinia
resistant; Phosphinothricin
tolerant; Aphid resistant; Visual
marker; Increased
transformation frequency;
Novel protein; Amino acid
composition; Carbohydrate
metabolism; Cell wall; Fatty
acid levels; Feed properties;
Flavinoid levels; Flavinoid level
decreased; Lipid profile; Lysine
level increased; Nutritional
quality; Oil quality; Protein
quality

125

Acreage

05-024-02r

Pioneer

Soybean

Issued

05/25/05

ACP acyl ACP thioesterase from Rapeseed; Acetolactate synthase*; Acyl-ACP thioesterase from
Soybean; Aspartokinase from E. coli; Aspartokinase II-homoserine dehydrogenase from E. coli; Bglucuronidase from E. coli; B-glucuronidase*; C1 regulatory gene from Corn; CBI from Euphorbia
lagascae; CBI; CBI from Bacillus amyloliquefaciens; CBI*; CBI from Mortierella alpina; CBI from
Parthenium argentatum; CBI from Rice; CBI from Schizochytrium aggregatum; CBI from Soybean;
CBI from Sunflower; CBI from Vernonia galamensis; CBI from Alfalfa; CBI from Arab. thaliana; CBI
from Barley; CBI from Corn; Conglycinin from Soybean; Cystathionine beta lyase from Soybean;
Cystathionine synthase from Soybean; Cystathionine synthase from Corn; Delta-12 saturase from
Soybean; Delta-9 desaturase from Soybean; Dihydrodipicolinate synthase from Soybean;
Dihydrodipicolinate synthase from Corynebacterium glutamicum; Dihydrodipicolinate synthase from
E. coli; Galactinol synthase from Soybean; Glycinin from Soybean; Hygromycin phosphotransferase*;
Luciferase from Photinus pyralis; Lysine ketoglutarate reductase from Soybean; Lysine- and
Methionine-rich protein from Synthetic; Omega 3 desaturase from Soybean; Omega 6 desaturase
from Soybean; Phosphinothricin acetyl transferase from Arab. thaliana; Saccharopine dehydrogenase
from Yarrowia lipolytica; Storage protein from Corn; UDP-glucose 4'epimerase from Soybean

99-147-02n

Monsanto

Soybean

Withdrawn

08/25/99

CBI; CBI*

Industrial enzyme

01-306-01r

Hawaii
Agriculture
Research Ce

Issued

01/11/02

NptII*; CBI from Man

Pharmaceutical proteins

0.5

Acknowledged
Issued

07/18/96

CBI

Novel protein

03/08/99

CBI; NptII*

Pharmaceutical proteins

0.4

Sugarcane

96-184-04n

Biosource

Tobacco

99-041-02r

CropTech

Tobacco

254

DEFRA Contract CPEC 47

Field trial applications of GM crops expressing pharmaceutical and industrial products in the US continued
Institution

99-041-01r
00-070-01r
00-070-02r

CropTech
CropTech
CropTech

04-044-01r

Planet
Biotechnology

Tobacco

Issued

04/28/04

Antibody from Mouse; Antibody from Oryctolagus cuniculus

Pharmaceutical proteins

02-080-01r
01-074-01r
01-074-02r
03-063-01r
04-355-01r

CropTech
CropTech
CropTech
Chlorogen, Inc.
Chlorogen, Inc.

Tobacco
Tobacco
Tobacco
Tobacco
Tobacco

Issued
Issued
Issued
Issued
Issued

05/07/02
05/11/01
05/11/01
05/15/03
05/19/05

CBI from Man; NptII*

CBI
CBI
Amino glycoside adenyl transferase*; Serum albumin from Man
Amino glycoside adenyl transferase*; CBI from Man; CBI from Cow

Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins

0.5

0.1
12.5

04-114-01r

Chlorogen, Inc.

Tobacco

Issued

05/25/04

Albumin from Man; Aminoglycoside 3'- adenylyltransferase*; Insulin-like growth factor from Man;
Interferon from Man; Mullerian inhibiting substance from Man

Pharmaceutical proteins

05-061-01r

U of Kentucky

Tobacco

Issued

06/07/05

NptII*; Phenylalanine ammonia lyase from Arab. thaliana

Pharmaceutical proteins

0.25

05-053-01r

Planet
Biotechnology

Tobacco

Issued

06/08/05

Antibody (common cold) from CBI; Antibody (tooth decay) from Mouse; Antibody (tooth decay) from
Oryctolagus cuniculus; NptII*

Pharmaceutical proteins

05-087-01r

Planet
Biotechnology

Tobacco

Issued

07/07/05

Antibody (tooth decay) from Mouse

Pharmaceutical proteins

04-077-01r

Chlorogen, Inc.

Tobacco

Withdrawn

07/27/04

Aminoglycoside acetyltransferase*; Cholera toxin B from Vibrio cholera; Insulin-like growth factor
from Man; Interferon from Man; Mullerian inhibiting substance from Man; Protective antigen from
Bacillus anthracis; Protective antigen from Yersinia pestis; Serum albumin from Man

Pharmaceutical proteins

97-301-01r

ProdiGene
Applied
Phytologics
Applied
Phytologics

Tomato

Issued

01/29/98

NptII*

Pharmaceutical proteins

Wheat

Issued

05/04/00

CBI; Hygromycin phosphotransferase*

Novel protein

Wheat

Issued

10/10/00

CBI; Hygromycin phosphotransferase*

Novel protein

00-059-01r
00-228-01r

Organism
Tobacco
Tobacco
Tobacco

Status

Issued/
Effective
03/08/99
04/19/00
04/19/00

Permit

Issued
Issued
Issued

Gene(s)

Phenotype(s)/Product

CBI; NptII*
CBI from Man; NptII*
CBI from Man; NptII*

Pharmaceutical proteins
Pharmaceutical proteins
Pharmaceutical proteins

255

Acreage
0.4

DEFRA Contract CPEC 47

Appendix V
Deregulated GM crops in the US

Applicant Documents
Extension
of Petition
Number ***

APHIS Documents

Institution

Regulated
article

Transgenic Phenotype

Transformation Event or Line

92-196-01p

Calgene

Tomato

Fruit ripening altered

FLAVR SAVR

92-204-01p

Upjohn

Squash

WMV2 & ZYMV resistant

ZW-20

92-204-01p_ea

92-204-01p_fr

92-204-01p_com

93-196-01p

Calgene

Cotton

Bromoxynil tolerant

BXN

93-196-01p

93-196-01p_fr

93-196-01p_com

93-258-01p

Monsanto

Soybean

Glyphosate tolerant

40-3-2

93-258-01p

93-258-01p_com

Petition

94-090-01p

Preliminary EA
****
92-196-01p_ea

FR Notice
92-196-01p_fr

Risk Asses.

Final EA &
Determination
92-196-01p_com

Calgene

Rapeseed

9 additional FLAVRSAVR lines

94-230-01p

94-230-01p_com

Potato

Coleopteran resistant

BT6, BT10, BT12, BT16, BT17, BT18, BT23

94-257-01p_ea

94-257-01p_fr

94-257-01p_com

94-290-01p

Monsanto
Zeneca &
Petoseed

Tomato

Fruit polygalacturonase level decreased

B, Da, F

94-290-01p

94-290-01p_com

94-308-01p

Monsanto

Cotton

Lepidopteran resistant

531, 757, 1076

94-308-01p

94-308-01p_com

94-319-01p

Ciba Seeds

Corn

Lepidopteran resistant

Event 176

94-319-01p

94-319-01p_com

94-357-01p

AgrEvo

Corn

Phosphinothricin tolerant

T14, T25

94-357-01p

94-357-01p_com

95-030-01p

95-030-01p_com

94-227-01p

92-196-01p

94-228-01p
94-230-01p

92-196-01p

94-257-01p

95-030-01p

Calgene

Tomato

Fruit ripening altered

20 additional FLAVRSAVR lines

95-045-01p

Monsanto

Cotton

Glyphosate tolerant

1445, 1698

95-045-01p_com

95-053-01p

Monsanto

Tomato

Fruit ripening altered

8338

95-053-01p_com

95-093-01p

Monsanto

Corn

Lepidopteran resistant

MON 80100

95-093-01p_com

95-145-01p

DeKalb

Corn

Phosphinothricin tolerant

B16

95-145-01p_com

Calgene

Tomato

Fruit ripening altered

2 additional FLAVRSAVR lines

95-179-01p_com

Northrup King
Plant Genetic
Systems

Corn

European Corn Borer resistant

Bt11

95-195-01p_com

95-228-01p

Corn

Male sterile

MS3

95-228-01p_com

95-256-01p

Du Pont

Cotton

Sulfonylurea tolerant

19-51a

95-256-01p_com

95-324-01p

Agritope

Tomato

Fruit ripening altered

35 1 N

95-324-01p_com

95-338-01p

Monsanto

Potato

CPB resistant

SBT02-5 & -7, ATBT04-6 &-27, -30, -31, -36

95-338-01p_com

95-352-01p

Asgrow

Squash

CMV, ZYMV, WMV2 resistant

CZW-3

95-352-01p_com

95-179-01p
95-195-01p

92-196-01p

256

DEFRA Contract CPEC 47

Deregulated GM crops in the US continued

Applicant Documents

APHIS Documents

Petition

Extension
of Petition
Number ***

Institution

Regulated
article

Transgenic Phenotype

Transformation Event or Line

96-017-01p

95-093-01p

Monsanto

Corn

European Corn Borer resistant

MON809 & MON810

96-017-01p_com

Cornell U

Papaya

PRSV resistant

55-1, 63-1

96-051-01p_com

96-051-01p
96-068-01p
96-248-01p

92-196-01p

Preliminary EA
****

FR Notice

Risk Asses.

Final EA &
Determination

AgrEvo

Soybean

Phosphinothricin tolerant

W62, W98, A2704- 12, A2704-21, A5547-35

96-068-01p_com

Calgene

Tomato

Fruit ripening altered

1 additional FLAVRSAVR line

96-248-01p_com
96-291-01p_com

96-291-01p

DeKalb

Corn

European Corn Borer resistant

DBT418

96-317-01p

Monsanto

Corn

Glyphosate tolerant & ECB resistant

MON802

96-317-01p_com

97-008-01p

Du Pont

Soybean

G94-1, G94-19, G-168

97-008-01p_com

97-013-01p

Calgene

Cotton

Oil profile altered

Bromoxynil tolerant & Lepidopteran
resistant

Events 31807 & 31808

97-013-01p_com

97-099-01p

Monsanto

Glyphosate tolerant

GA21

97-099-01p_com

97-148-01p

Bejo

Corn
Cichorium
intybus

Male sterile

RM3-3, RM3-4, RM3-6

97-148-01p_com

97-204-01p

Monsanto

Potato

CPB & PLRV resistant

RBMT21-129 & RBMT21-350

97-204-01p_com

97-205-01p

AgrEvo

Rapeseed

Phosphinothricin tolerant

T45

97-205-01p_com

97-265-01p

AgrEvo

Corn

Phosphinothricin tolerant & Lep. resistant

CBH-351

97-265-01p_com

97-287-01p

Monsanto

Tomato

Lepidopteran resistant

5345

97-287-01p_com

97-336-01p

AgrEvo

Beet

Phosphinothricin tolerant

T-120-7

97-336-01p_com

97-339-01p

Monsanto

Potato

CPB & PVY resistant

RBMT15-101, SEMT15-02, SEMT15-15

97-339-01p_com

97-342-01p

Pioneer

Corn

Male sterile & Phosphinothricin tolerant

676, 678, 680

97-342-01p_com

Soybean

Phosphinothricin tolerant

A5547-127

98-014-01p_com

98-173-01p

AgrEvo
Novartis Seeds &
Monsanto

Beet

Glyphosate tolerant

GTSB77

98-173-01p_com

98-216-01p

Monsanto

Rapeseed

Glyphosate tolerant

RT73

98-216-01p_com

98-238-01p

AgrEvo

Soybean

98-238-01p_com

AgrEvo

Rapeseed

Phosphinothricin tolerant
Phosphinothricin tolerant & Pollination
control

GU262

98-278-01p

MS8 & RF3

98-278-01p_com

98-329-01p

AgrEvo

Rice

Phosphinothricin tolerant

LLRICE06, LLRICE62

98-329-01p_com

98-335-01p

U. of
Saskatchewan

Flax

Tolerant to soil residues of sulfonyl urea

herbicide

98-014-01p

96-068-01p

CDC Triffid

98-335-01p_com

98-349-01p

95-228-01p

AgrEvo

Corn

Phosphinothricin tolerant and Male sterile

MS6

98-349-01p_com

99-173-01p

97-204-01p

Monsanto

Potato

PLRV & CPB resistant

RBMT22-82

99-173-01p_com

257

DEFRA Contract CPEC 47

Deregulated GM crops in the US continued

Applicant Documents

Petition

Extension
of Petition
Number ***

00-011-01p

97-099-01p

APHIS Documents

Institution

Regulated
article

Preliminary EA
****

FR Notice

Risk Asses.

Final EA &
Determination

Transgenic Phenotype

Transformation Event or Line

NK603
Line 1507

00-136-01p_com

Cotton Event 15985

00-342-01p_com

Corn

00-136-01p

Monsanto
Mycogen c/o
Dow & Pioneer

Corn

Glyphosate tolerant
Lepidopteran resistant phosphinothricin
tolerant

00-011-01p_com

00-342-01p

Monsanto

Cotton

Lepidopteran resistant

01-121-01p

Vector

Tobacco

Reduced nicotine

Vector 21-41

01-121-01p_com

01-137-01p

Monsanto

Corn

MON 863

01-137-01p_com

MS1 & RF1/RF2

01-206-01p_com

01-206-01p

98-278-01p

Aventis

Rapeseed

Corn Rootworm Resistant

Phosphinothricin tolerant & pollination
control

01-206-02p

97-205-01p

Aventis

Rapeseed

Phosphinothricin tolerant

Topas 19/2

01-206-02p_com

01-324-01p

98-216-01p

Monsanto

Rapeseed

Glyphosate tolerant

RT200

01-324-01p_com

02-042-01p

Aventis

Cotton

Phosphinothericin tolerant

LLCotton25

02-042-01p_com

03-036-01p

Mycogen/Dow

Cotton

Lepidopteran Resistance

281-24-236

03-036-01p_pea

03-036-01p_fr_pc_pet

03-036-01p_com

03-036-02p

Mycogen/Dow

Cotton

Lepidopteran Resistance

3006-210-23

03-036-02p_pea

03-036-02p_fr_pc_pet

03-036-02p_com

03-155-01p

Syngenta

Cotton

COT 102

03-155-01p_pea

03-155-01p_fr_pc_pet

03-155-01p_com

Dow

Corn

Lepidoteran Resistant
Lepidopteran Resistant, Glufosinate
Tolerant

TO-6275

03-181-01p_pea

03-181-01p_fr_pc_pet

03-181-01p_com

03-181-01p

00-136-01p

03-323-01p

Monsanto

Sugar Beet

Glyphosate Tolerant

H7-1

03-323-01p_pea

03-323-01p_fr_pc_pet

03-323-01p_com

03-353-01p

Dow

Corn

Corn Rootworm Resistant

59122

03-353-01p_pea

03-353-01p_fr_pc_pet

03-353-01p_com

03-353-01p

Monsanto
Monsanto &
Forage Genetics

Cotton

Glyphosate Tolerant

MON 88913

04-086-01p_pea

04-086-01p_fr_pc_pet

04-086-01p_com

Alfalfa

Glyphosate Tolerant

J101, J163

04-110-01p_pea

04-110-01p_fr_pc_pet

04-110-01p_com

04-110-01p

258

DEFRA Contract CPEC 47

Appendix VI
GM crops with petition pending for deregulation in the US

Applicant Documents
Extension
of
Petition
Number
Petition
***
03-10401p
04-12501p
04-22901p
04-26401p
04-33701p
04-36201p
05-28001p

APHIS Documents
Final EA &
Institution

Regulated
article

Monsanto & Scotts

Creeping
bentgrass

Glyphosate Tolerant

ASR368

Monsanto

Corn

Corn Rootworn Resistant

88017

Monsanto

Corn

High Lysine

LY038

Transgenic Phenotype

Transformation Event or Line

ARS

Plum

Plum Pox Resistant

University of Florida

Papaya

Papaya Ringspot Virus Resistant

X17-2

Syngenta

Corn

Corn Rootworm Protected

MIR604

Syngenta

Corn

Thermostable alpha-amylase

3272

259

Preliminary
EA ****

FR Notice

04-12501p_pea
04-22901p_pea

03-10401p_fr_pc_pet
04-12501p_fr_pc_pet
04-22901p_fr_pc_pet

Risk
Asses.
0310401p_ra

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