Genetically ModifiedPlantsNigel G Halford, Plant Biology and Crop Science Department, Rothamsted Research,

Harpenden, UK

Based in part on the previous version of this eLS article ‘GeneticallyModified Plants’ (2007) by Paul Christou and Teresa Capell.

The genetic modification of plants is now an established

tool for plant breeders in many parts of the world, with the

area of land used for genetically modified (GM) crop culti-

vation rising to 170 million hectares by 2012. This article

puts genetic modification of plants into the context of sci-

entific plant breeding, and describes the techniques that

are used to transform plants and that define the term

geneticallymodified. The designof transgenes is described,

as is the use of selectable and visible marker genes. The use

of GM crops in commercial agriculture is covered in detail,

including GM crops that may be developed for commercial

use in the near future. The barriers to the continued devel-

opment of crop biotechnology are considered, notably the

cost, the associated issue of regulatory compliance and

the problem of consumer acceptance. The consequences of

science losing the GM crop debate are discussed.

Introduction

It is 19 years since the launch of the first commercialgenetically modified (GM) crop variety and 17 years sincethe first large-scale cultivation ofGMcrops. Since then, theuse of GM crops has risen steadily around the world, withapproximately 170 million hectares being cultivated in 28countries in 2012 (James, 2012). The number of traits thathave been introduced by genetic modification remainslimited and there is still no established market for GM

varieties of rice, wheat and/or potato. Nevertheless, someGM crop traits have been extremely successful and vari-eties carrying them are popular with farmers wherever theyhave been made available. These traits and the reasons fortheir success will be described in this article, as will some ofthe traits that have failed in the marketplace. The articlewill also look forward to the GM traits that could belaunched in the next few years.The success of GM crops has occurred in the face of the

most stringent regulatory systems ever imposed on plantbreeding and a section of the article is devoted to that topic.The regulatory system in the European Union is particu-larly draconian and disproportionate to the relative riskof genetic modification compared with other methods inplant breeding. Scientists and biotechnology companieshave also had to endure a long-running public debate onthe issue that soon outgrew the scientific world and becamepolitical. In Europe, the scientific side in that debate lost,and the implications of that defeat will also be discussed.

Context: Plant Breeding

Farming began in Mesopotamia approximately 10 000years ago, and by 4000 BC breadmaking wheat was beingcultivated throughout that region and Ancient Egypt,while rice had been domesticated in China and potato inPeru. Farmers have been improving crops ever since, partlythrough the selection of the best seed from one year to sowthe next, and partly through finding and exploiting natu-rally-occurring mutants. Modern scientific plant breeding,however, did not arise until the rediscovery of Mendel’s1866 work, ‘Versuche uber Pflanzen-Hybride’, in 1900.See also: Crop Plants: Evolution; History of ScientificAgriculture: Crop PlantsMuch of plant breeding still involves the crossing of

different varieties to exploit heterosis and combine traits.

Introductory article

Article Contents

. Introduction

. Context: Plant Breeding

. Definitions and Methods

. Gene Design for Transformation

. Selectable Marker Genes

. Post-GM Technologies

. Advantages of Genetic Modification

. Commercial Use of GM Crops

. Traits in Commercial Use

. Traits that may be Commercialised in the Near Future

. Barriers to the Development of Crop Biotechnology

. Concluding Remarks

. Acknowledgements

Online posting date: 15th April 2014

eLS subject area: Plant Science

How to cite:Halford, Nigel G (April 2014) Genetically Modified Plants. In: eLS.

John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0003362.pub2

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Genes can also be introgressed into breeding programmesfromwild relatives and even different species, and chemicaland radiation mutagenesis have been used in plant breed-ing since the 1950s. Almost all of the crop varieties used indeveloped countries today are the products of scientificplant breeding.While scientific plant breeding was hitting its stride in

the second half of the last century, there were also rapidadvances being made in molecular biology that eventuallyled to the possibility of transferring genes between speciesand modifying genes artificially in a laboratory beforethey were used. This would open the way to the geneticmodification of plants. In 1977, Chilton et al. described thenatural genetic modification of host plant cells by Agro-bacterium tumefaciens (Chilton et al., 1977) and only6 years after that, in 1983, Hall et al. reported the pro-duction of GM sunflower plants containing a gene fromcommon bean (Murai et al., 1983). Another 11 years on, in1994, a US company, Calgene, launched the first com-mercial GM variety, a tomato variety called ‘Flavr Savr’that had been genetically modified to slow down theripening process. Genetic modification had become a newtool in the plant breeders’ tool box.

Definitions and Methods

Any new crop variety must be genetically different fromvarieties already on themarket. However, new varieties are

not usually described asGM: that termhas come to be usedspecifically to describe a plant or variety that contains agene or genes that have been introduced artificially. Suchplants are also described as being transgenic, having beentransformed, or as genetically engineered.Genetic modification is therefore a term that is defined

by the techniques that are used to produce GM plants. Inthat respect, it is somewhat unsatisfactory and the inevi-table ambiguity of the term and the legislation that is basedon it makes the regulatory situation more complicated.There are several methods currently available to plant

biotechnologists to which the term genetic modificationis applied. The most widely used involves a common soilbacterium, A. tumefaciens, a bacterium that naturallyinfects wounded plant tissue and inserts a short section ofits own deoxyribonucleic acid (DNA), called the transferDNA or T-DNA, into the genome of a plant cell (Chiltonet al., 1977). The cell begins to grow and divide to form atumour-like growth of undifferentiated cells that in natureforms a structure called a crown gall, which on trees can beas large as a football (Figure 1a). It also begins to make andsecrete unusual substances called opines, including nopa-line and octopine, on which the bacteria feed. Undiffer-entiated cells arising from A. tumefaciens infection can becultured in the lab and a clump of these undifferentiatedcells in culture is called a callus.Biotechnologists take advantage of A. tumefaciens’

ability to insert its T-DNA into the genome of a plant cellby replacing the genes in the T-DNA with genes that they

(a)

(c)

(b)

(d)

Figure 1 Production of GM wheat plants using A. tumefaciens. (a) A crown gall caused by the natural infection of a tree by A. tumefaciens, Ashridge Forest,

UK, 2013. (b) Clusters of potentially transgenic cells (callus) derived from infection of wheat embryos by A. tumefaciens. (c) Callus cultures induced to form

shoots by application of a plant hormone. (d) Shoots forming roots to make complete plantlets. Thanks to Caroline Sparks and Rothamsted Research Visual

Communication Unit for (b–d).

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wish to introduce into the plant. Bacteria carrying themodified T-DNA are allowed to infect leaf pieces, stemsegments, protoplasts, or in the case of wheat and othercereals, immature embryos (Figure 1b). The resulting calliare treated with a plant hormone to induce them to formshoots (Figure 1c). Transfer to a medium lacking the hor-mone allows the shoots to form roots, resulting in completeplantlets (Figure 1d). Methods have also been developedthat do not require tissue culture, such as floral dip, inwhich plants at the early stages of flowering are placed ina suspension ofA. tumefaciens in a vacuum jar, a vacuum isapplied to remove air surrounding the plant tissue andallow the bacteria to come into contact with the plant cells,and the plants are grown to seed. Typically, approximately1% of the seeds are GM (Bechtold et al., 1993; Clough andBent, 1998).As well as sometimes being used for A. tumefaciens-

mediated transformation, protoplasts can also be inducedto take up DNA directly, either by treatment with poly-ethylene glycol or by electroporation. This process is calleddirect or DNA-mediated gene transfer and in a smallproportion of the protoplasts, the introduced DNA willintegrate into the host DNA and the protoplast will bestably transformed. The protoplast can then be induced toform callus, from which a GM plant can be regenerated.Electroporation can be applied to intact cells in tissuepieces or in suspension, as well as to protoplasts, but thishas only been shown to work efficiently in a few species.Another direct gene transfer method to have been

developed is silicon carbide fibre vortexing. Plant cells aresuspended in a medium containing DNA and microscopicsilicon carbide fibres. The suspension is vortexed and thefibres penetrate the plant cell walls, allowing the DNA toenter. Finally, there is particle bombardment, in whichplant cells are bombarded with tiny particles coated withDNA (Christou, 1993). Particle bombardment has beenparticularly successful in the production of GM cereals.Genes can also be introduced into plant cells transiently;

in otherwords, a gene is introduced into the cell andmaybeactive, but does not insert into the genome. One way ofdoing this involves the use of plant virus-based vectors. Agene of interest is added to a plant viral genome and theGM virus is allowed to infect a host plant. Once inside thehost cell, the novel gene is expressed along with the otherviral genes. The technique has the advantage that viralgenomes multiply rapidly within infected cells, potentiallyleading to very high levels of expression of the transgene,and the infection process is relatively simple. The high levelof protein that can be derived has led to lots of interest inthe technique for expressing vaccines and other high-valueproteins in plant cells (Nicholson et al., 2006).

Modified viruses have also been used to induce thesilencing of target genes in a plant cell in a technique knownas virus-induced gene silencing (VIGS) (Lindbo et al.,1993). VIGS involves the production of modified plantviruses carrying nucleotide sequences corresponding tothe host gene to be silenced. Infection leads to synthesisof viral double-stranded ribonucleic acid (RNA), as an

intermediate step in the normal viral replication process.This activates the plant’s antiviralRNAsilencing pathway,resulting in degradation of the messenger RNA from thetarget gene.Another variant on the basic technique of genetic mod-

ification is to introduce a gene into the genome of achloroplast instead of the nuclear genome. Plants in whichthis has been achieved are called transplastomic plants(Maliga, 2004). One advantage of this approach is thateach cell of the green parts of the plant contains thousandsof chloroplasts, meaning that there is the potential for veryhigh levels of expression of the transgene (de Cosa et al.,2001). Another is that the chloroplast genome is onlyinheritedmaternally, so pollen froma transplastomic plantdoes not contain the transgene; this may be useful if itis particularly important to prevent gene flow from thetransgenic plant. See also: Plant Transformation

Gene Design for Transformation

A foreign gene is unlikely to function in its new host unlessit is modified first because its promoter will not be recog-nised by the transcriptional machinery in the host cell. Thiscan be overcome by cutting and pasting to remove thegene’s own promoter and replace it with one from agene from the host plant or from a gene that is active inmultiple hosts. The resulting gene, comprising elementsfrom more than one source, is called a chimaeric geneconstruct. The second important consequence is that bio-technologists can use different promoters or short DNAsequences from within promoters to control when andwhere in the host plant the chimaeric gene that is beingconstructed will be active. The types of promoter avail-able vary between different host plants, but may includeconstitutive, tissue-specific/developmentally-regulated andinducible.The most widely used constitutive promoter is the Cau-

liflower mosaic virus 35 S (CaMV35S) promoter (Odellet al., 1985). This promoter has been used particularlysuccessfully in the genetic modification of dicotyledonousplants. It also works in monocotyledonous plants but haslargely been replaced for cereal work with promoters thatare derived from cereal genes. These include a promoter foramaize gene, ubi, encoding ubiquitin, and a rice gene, act1,encoding actin. See also: Transcriptional Regulation inPlantsTissue-specific/developmentally-regulated gene pro-

moters are active only in certain parts of the plant, andoften only at specific developmental stages.One example ofa widely-used tissue-specific promoter from wheat is theglu-D1x promoter (Lamacchia et al., 2001). This promotercomes from a gene that encodes a wheat seed storageprotein that is highly expressed specifically in the endo-sperm. Figure2 shows sections ofwheat seeds that have beentransformed with a chimaeric gene comprising the codingregion of a bacterial gene called uidA that encodes anenzyme called b-glucuronidase, under the control of the

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glu-D1x promoter. This enzyme, sometimes called Gus,converts a colourless substrate called X-Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronide) into a blue product.uidA is one of a number of so-called reporter genes,more ofwhich are discussed in the section ‘Selectable MarkerGenes’.The third type of promoter that a biotechnologist can

use is described as inducible. Promoters of this type are notactive until they are inducedby something such as attackbya pathogen, grazing or application of a chemical.As well as being designed to be expressed in a certain

tissue at a certain time, the genemay bemodified to changethe nature of the protein that it encodes by introducingmutations into the coding region, or designednot tomake anovel protein in the host plant but to reduce the expressionof one that is already there. This is called gene silencing andcan be achieved by the ‘antisense’ technique, using a genewith all or part of the gene of interest spliced in reverseorientation downstream of a promoter. Alternatively thereis the cosuppression technique, in which additional copiesof all or part of a gene in the correct orientation are used tobring about gene silencing. This, of course, is the techniqueused for expressing a novel protein in a GM plant, butcosuppression is sometimes the outcome when the nativeand introduced genes are very similar.

There may be more than one silencing mechanisminvolved in both of these techniques, but the major oneappears to operate post-transcriptionally, causing degra-dation of the RNAmolecule before it can act as a templatefor protein synthesis. Post-transcriptional gene silencinginvolves the production of small, antisense RNAs. Morerecently, this system has been exploitedmore directly usingRNA interference (RNAi), in which a plant is geneticallymodified to synthesise a double-stranded RNA moleculederived from the target gene (reviewed by Baulcombe,2004). See also: Small RNAs in PlantsGene silencing is a powerful technique in plant genetic

research but has also found commercial application to slowdown the ripening process in GM tomato and other fruitsand to modify oil content.

Selectable Marker Genes

The regeneration of GM plants is carried out in the pre-sence of a selective agent, tolerance of which is imparted bythe introduced gene or, more often, an accompanyingselectable marker gene. The most widely used selectablemarker genes make the GMplant resistant to an antibioticor able to tolerate a herbicide that would normally kill it.

(a)

(b)

Development →→ Endosperm

(c)

Endosperm

Embryo

Aleurone

Figure 2 Tissue-specificity of the glu-D1x gene promoter from wheat shown using the uidA gene encoding b-glucuronidase (Gus) as a reporter gene. (a)

Longitudinal and transverse sections of seeds from early to mid-development, showing the gene becoming active in the endosperm (white flour tissue) but

not the embryo as the seed develops; (b) transverse section of a mid-development seed showing expression of the reporter gene in the endosperm and (c)

higher magnification section showing expression of the reporter gene in the cells of the endosperm but not the surrounding aleurone (Lamacchia et al.,

2001). Thanks to Caroline Sparks (Rothamsted Research) for (b).

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Examples of antibiotic resistance marker genes includethe nptII gene, which encodes an enzyme called neomycinphosphotransferase that detoxifies aminoglycosidic anti-biotics such as kanamycin, geneticin or paromycin, andhpt, hph or aph-IV, which encode hygromycin phospho-transferase, an enzyme that detoxifies hygromycin. Theuse of these genes in plant biotechnology has caused con-troversy and biotech companies developing GM crops forcultivation have switched to alternatives, although anti-biotic resistancemarker genes continue tobeusedwidely forplant genetic research. Themost widely-used alternatives toantibiotic resistance marker genes are those that imparttolerance to a herbicide. The third type of marker gene thatis used gives a visible signal. The uidA/gus gene discussedabove in the section on gene design is one of these (Figure 3a).Alternatives include genes encoding luciferase, green fluor-escent protein (GFP) and red fluorescent protein (DsRed).The luciferase system requires two genes, luxA and luxB,from a bacterium, Vibrio harveyi. The GFP gene comesfrom an intensely luminescent jellyfish, Aequorea victoria,and encodes a protein that emits green luminescence whenexcited with blue light (Figure 3b and 3c). The DsRed genecomes from coral (Discosoma spp.) and works in a similarway to GFP but emits red light instead of green (Figure 3d).

Post-GM Technologies

Genetic modification of plants is now almost three decadesold and there are a number of new techniques that may be

regarded as advances on GM, or post-GM technologies.Several of them come under the umbrella term of genomeediting in that they generate targeted mutations. Thesetechnologies use zinc-finger nucleases, meganucleases,transcription activator-like effector nucleases or speciallydesigned oligonucleotides. Another exploits a bacterialdefence system based on clustered regularly-interspacedshort palindromic repeats and the Cas9 nuclease. No cur-rent commercial crop varieties have been produced usingthem, and they are not covered in detail here. However,they are likely to be used in crop biotechnology in thefuture, so it is important to be aware of them.

Advantages of Genetic Modification

Genetic modification of plants has been available as a toolto plant breeders for three decades. By no means is itsweeping away every other technique in plant breeding; farfrom it. Indeed, during the time that genetic modificationhas been developing, there have been huge advances inother genetic techniques and resources that have improvedthe efficiency and scope of conventional plant breeding,not least the availability of complete genome sequencesfor many crop species. Nevertheless, genetic modificationenables plant breeders to do some things that would notbe possible by other methods, and in some cases is not onlythe fastest and most efficient way of achieving a target inplant breeding but also the safest. The advantages are listedin Box 1.

(a)

(c)

(b)

(d)

Figure 3 Visual marker/reporter genes in transgenic wheat embryos. (a) b-glucuronidase (Gus); (b) GFP expressed transiently after particle bombardment;

(c) GFP targeted to the nucleus and (d) DsRed. Thanks to Caroline Sparks, Rothamsted Research, for (a), (c) and (d).

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Commercial Use of GM Crops

Data on the global use of GM crops has been compiled forseveral years byClive Jamesof the International Service forthe Acquisition of Agri-Biotech Applications (ISAAA;www.isaaa.org). According to the ISAAA, the worldwide

area of land plantedwithGMcrops in 2012was 170millionhectares (James, 2012; Table 1). Most (81%) of the globalsoybean production was GM, whereas 81% of cotton,35% of maize and 30% of oilseed rape production wasGM. Other crops with some GM varieties being growncommercially included papaya, squash, tomato, alfalfa,tobacco, sweet pepper, poplar, potato and sugar beet. GMcrops were grown in 28 countries, with the USA, Brazil,Argentina, India and Canada each planting more than 10million hectares of GM crops, and China, Paraguay,Pakistan, South Africa, Uruguay and Bolivia all plantingmore than a million hectares. Herbicide tolerance andinsect resistance were the dominant traits. Varieties car-rying quality (output) traits are beginning to emerge butthese are still grown on relatively small areas comparedwith those carrying input traits (traits that affect the hus-bandry and management of the crop). GM crops withinput traits have sometimes been called first generationGM crops, whereas those with quality traits have beencalled second generation.

Box 1 Genetic modification enables some processes that are

not possible with other methods in plant breeding

. It allows genes to be introduced into a crop plant from any

source.

. It is relatively precise because single genes can be

transferred.

. Genes and their products can be tested extensively in iso-

lation before use to ensure their safety.

. Genes can be ‘cut and pasted’ in the lab to change when and

where in a plant they are active, and to change the properties

of the proteins they produce.

Table 1 Global GM crop cultivation 2012

Country GMcrop area (ha) Crops

USA 69 500 000 Maize, soybean, cotton, oilseed rape, sugar beet, alfalfa, papaya and squash

Brazil 36 600 000 Soybean, maize and cotton

Argentina 23 900 000 Soybean, maize and cotton

India 10 800 000 Cotton

Canada 10 400 000 Oilseed rape, maize, soybean and sugar beet

China 4 000 000 Cotton, papaya, poplar, tomato, sweet pepper, oilseed rape, maize and

soybean

Paraguay 3 400 000 Soybean, maize and cotton

South Africa 2 900 000 Maize, soybean and cotton

Pakistan 2 800 000 Cotton

Uruguay 1 400 000 Soybean and maize

Bolivia 1 000 000 Soybean

Philippines 800 000 Maize

Australia 700 000 Cotton and oilseed rape

Burkina Faso 300 000 Cotton

Myanmar (Burma) 300 000 Cotton

Mexico 200 000 Cotton and soybean

Spain 100 000 Maize

Chile 5100 Maize, soybean and oilseed rape

Colombia 5100 Cotton

Costa Rica 5100 Cotton and soybean

Czech Republic 5100 Maize

Egypt 5100 Maize

Cuba 5100 Maize

Honduras 5100 Maize

Portugal 5100 Maize

Romania 5100 Maize

Slovakia 5100 Maize

Sudan 5100 Cotton

Total 170 000 000 Alfalfa, cotton, maize, oilseed rape, papaya, poplar, soybean, squash, sugar

beet, sweet pepper and tomato

Source: James (2012) and personal communication (China data).

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Traits in Commercial Use

Fruit shelf-life

The first GM variety of any kind to be marketed was the‘Flavr Savr’ variety of tomato, which was developed byCalgene, subsequently acquired by Monsanto, and mar-keted in theUS in 1994. ‘Flavr Savr’ had reduced activity ofpolygalaturonase, one of the enzymes that break downpectin, as a result of antisense inhibition (Sheehy et al.,1988). ‘Flavr Savr’ was not a success and was soon with-drawn.However, Zeneca (now Syngenta), in collaborationwith the University of Nottingham, had developed verysimilar technology (Smith et al., 1988) and applied it toprocessing tomatoes. Tomato paste made from thesetomatoes went on the market in many countries andproved popular due to its lower cost and thicker con-sistency compared with its non-GM counterpart. Twomillion cans of it were sold in the UK between 1996 and1999, but the product was withdrawn when the GM cropissue became increasingly controversial in Europe and isnow not on sale anywhere. However, this and other stra-tegies to prolong fruit shelf life have continued to bedeveloped and slow-ripening GM varieties of tomato andpapaya are being grown commercially in China. See also:Ethylene

Herbicide tolerance

Herbicide tolerance is the most successful and widely usedGM crop trait. The strategy is to genetically modify a cropplant to tolerate a broad range herbicide; that is, a herbi-cide that kills any plant not carrying the tolerance gene.Most herbicides are selective in the types of plant that theykill and a farmer has to use a combination of herbicidesthat are tolerated by the crop but kill the problem weeds.Use of a single broad-range herbicide instead may beeasier, safer and cheaper. The first GM herbicide-tolerant(GM-HT) variety to be introduced was a ‘Roundup-Ready’ soybean, which was produced by Monsanto andhas been marketed since 1996. Varieties carrying this traitnow dominate global soybean production.‘Roundup’ is Monsanto’s trade name for glyphosate, a

broad range herbicide that was firstmarketed in 1974 (longbefore GM crops were introduced). Its target is 5-enol-pyruvoylshikimate 3-phosphate synthase (EPSPS), theenzyme that catalyses the formation of 5-enolpyruvoyl-shikimate 3-phosphate from phosphoenolpyruvate andshikimate 3-phosphate. This reaction is required for thesynthesis of many aromatic plant metabolites, includingthe amino acids phenylalanine, tyrosine and tryptophan.Genetic modification of plants to tolerate glyphosate is

achieved by introducing an epsps gene fromA. tumefaciens(Padgette et al., 1995). The bacterial EPSPS is not affectedby glyphosate, so plants carrying the transgene continue tohave a functional enzyme even when their own EPSPS isinhibited by the herbicide.

Glyphosate tolerance has now been engineered intocotton, oilseed rape, maize, alfalfa, and sugar and fodderbeet. See also: Shikimate Pathway and Aromatic AminoAcid BiosynthesisThe other GM-HT trait on the market at present is

gluphosinate tolerance, which is marketed by Bayer andhas been used in oilseed rape, maize, soybean, sugar beet,fodder beet, cotton and rice. The gene that imparts toler-ance to gluphosinate comes from the bacterium Strepto-myces hygroscopicus and encodes phosphinothrycineacetyl transferase, which detoxifies the herbicide (de Blocket al., 1987).

GM-HT crop varieties have been adopted enthusiasti-cally by farmers wherever they have been authorised foruse. There may be many different reasons for their popu-larity, but the major benefits are given in Box 2. See also:Environmental Impact ofGeneticallyModifiedOrganisms(GMOs)

Insect resistance

The engineering of insect resistance in GM crop plants isbased on the cry genes of a soil bacterium, Bacillus thur-ingiensis, and GM varieties carrying the trait are oftenreferred to as Bt varieties. Bacillus thuringiensis (Bt) itselfhas been used as a pesticide for several decades, in the formof powders, granules or aqueous and oil-based liquids. Thecry genes produce proteins that interfere with insect gutfunction. Different strains of the bacterium have differentcry genes, and each type encodes a protein that is effectiveagainst a different type of insect: CryI proteins, for exam-ple, are effective against the larvae of butterflies andmoths,whereas CryIII proteins are effective against beetles(reviewed bydeMaagd et al., 1999). The cry1 gene has beenused successfully in cotton, sugar beet, rice, soybean andmaize, while the cryIII gene was used in Monsanto’sNewLeaf potato variety in the 1990 s. NewLeaf was resis-tant to the Colorado beetle, but was withdrawn due to alack of enthusiasm for the new variety from fast-foodchains and the development of new, broad-range insecti-cides that controlled not only the Colorado beetle but alsoother pests.One concern over the use of Bt crops is the possibility of

insects developing resistance to what is a fairly mild pesti-cide. For this reason, farmers using Bt crops have to plant

Box 2 Reasons for the popularity of GM herbicide-tolerant

(GM-HT) crop varieties

. Simpler and more flexible weed control.

. Reduced herbicide costs.

. Easier crop rotationbecause the systemuses a herbicide that

is degraded rapidly in the soil.

. The ability to switch to a conservation tillage system,

reducing soil erosion and nitrate leaching.

. Peace of mind because weed problems late in the season can

be dealt with if necessary.

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‘refuges’ of a non-GM variety so that insects that havedeveloped resistance to the Bt toxin are not at a selectiveadvantage. More recently, Bt seed has sometimes beensupplied to farmers ready-mixed with non-GM seed: a‘refuge in a bag’.A more subtle way of deterring insects from feeding on

crops was used in a field trial of GMwheat at RothamstedResearch,UK, in 2011–2012 and 2012–2013 (Figure 4). Thewheat plants were engineered to produce a volatile che-mical called (E)-b-farnesene (EBF), a sesquiterpene (Bealeet al., 2006). EBF is used by aphids as an alarm signal andcauses other aphids to stop feeding and move away. It alsoacts as a repellent to colonising aphids and increasesforaging by predators and aphid parasitoids. The mod-ification involved two genes, both synthetic, one encodingan E-b farnesene synthase, the other a farnesyl pyropho-sphate synthase.

Disease resistance

Virus resistance has been engineered into GM plants byexploiting the phenomenon of cross protection, in whichinfection by a mild strain of a virus induces resistance tosubsequent infection by amore virulent strain. Geneticallymodifying a plant to make a viral coat protein invokes asimilar response. This technology has been used success-fully to engineer papaya to be resistant to Papaya ringspotvirus and a virus-resistant GM variety has been grown inHawaii since 1998 (Ferreira et al., 2002). Virus resistancecan also be achieved by using gene silencing techniques toblock the activity of viral genes. This was used by Mon-santo in the 1990s to engineer resistance to Potato leaf rollvirus into potato by blocking expression of the viral repli-case gene (Lawson et al., 2001). A variety containing this

trait and the Bt insect-resistance trait described above wasmarketed under the trade nameNewLeaf Plus but, like theNewLeaf variety, was not successful. However, virus-resistant papaya, tomato and sweet pepper are being growncommercially for cultivation in China, and Brazil is plan-ning to release a GM common bean (known as pinto beanin Brazil) that is resistant to Bean golden mosaic virus(Bonfim et al., 2007). This bean variety was developed bythe BrazilianAgricultural ResearchCorporation to benefitfarmers for whom the bean is an important subsistencecrop. See also: Viruses and Plant DiseaseGM potato lines engineered to be resistant to the

oomycete Phytophthora infestans, which causes late blightdisease, have been produced by BASF using a gene calledrb from a wild potato species, Solanum bulbocastanum(Song et al., 2003). Two potentially blight-resistant GMpotato lines have also been developed at the John InnesCentre in Norwich, UK: one containing the rpi-vnt1.1gene from Solanum venturii (Foster et al., 2009), theother the rpi-moc1 gene from Solanum mochiquense(Smilde et al., 2005). See also: Resistance Genes (RGenes)in PlantsThis is an example of geneticmodification being not only

the most expedient but also the safest way of achieving thedesired outcome. This is because wild potato species arediploid, while cultivated varieties are tetraploid, makingthe crossing ofwild and cultivated potato species extremelydifficult. Furthermore, potatoes naturally contain toxicsubstances called glycoalkaloids, including solanine andchacocine. Bringing a gene from a wild species into a cul-tivated potato breeding programme by genetic modifica-tion ensures that the gene is not accompanied by unwantedgenetic baggage, such as the genes required for the pro-duction of these toxic chemicals.

GM wheat plot Pollen barrier (non-GM wheat)Separator (barley) Security fencing

Figure 4 Field trial of GM wheat, Rothamsted Research, UK, 2012.

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Modified oil content

Plant oils contain a variety of fatty acids with differentchain lengths and degrees of saturation. Well-knownexamples include lauric acid (12 carbon atoms, no doublebonds, usually written as 12:0) and palmitic acid (16:0),which are found in coconut and palmkernel oil, and stearicacid (18:0), from cocoa butter. Common unsaturated fattyacids include oleic acid (18:1), which contains a doublebond at position 9 with respect to the omega end of themolecule (omega-9 or n-9), and is the major constituent ofolive and oilseed rape oil. Polyunsaturated fatty acidsinclude linoleic acid (LA) (18:2, omega-6), which is foundin safflower, sunflower and maize oil, and makes upapproximately 20% of oilseed rape oil. g-Linolenic acid(GLA) (18:3, omega-6) is identical to LA except that it hasan additional double bond at n-12; it is found in starflower(borage) and evening primrose oil. a-Linolenic acid (18:3,omega-3) is similar to GLA but the double bonds aresituated at different positions, with the first at n-3 withrespect to the methyl end, making it an omega-3 fatty acid.See also: Plant Storage LipidsPolyunsaturated fatty acids are prone to oxidation

during cooking and high-temperature processing, givingrise to lipid peroxides that give a dark colouration, may betoxic, and can break down to form various products thatcause a rancid, ‘off’ flavour and odour. Traditionally, foodprocessors have avoided polyunsaturated fatty acid oxi-dation by chemical hydrogenation of the double bonds,converting the fatty acids to monounsaturates and satu-rates. Saturation of the fatty acids in plant oils also soli-difies them, making them suitable for the production ofmargarines. The problem with the process is that it cancause the formation of trans fatty acids, in which somedouble bonds remain unsaturated but with the twohydrogen atoms on opposite sides of the carbon atomsinstead of on the same side, as they are in the naturally-occurring cis form. Although technically unsaturated,these trans fatty acids cannot legally be designated aspolyunsaturates in food in the USA and Europe becausethey have a similar effect on cholesterol levels as saturatedfats.Soybean oil normally contains a lot of LA,making its oil

prone to trans fatty acid formation. To make a more heat-stable oil that did not require hydrogenation before pro-cessing, PBI, a subsidiary of DuPont, produced a GMvariety, Plenish, in which the activity of a gene encoding anenzyme called delta-12 desaturase was reduced. The delta-12 desaturase converts oleic acid to LA, and the Plenishvariety therefore accumulates high levels of oleic acid(Kinney, 1997). Hydrogenation, with its risk of trans fattyacid formation, is therefore not required. Monsanto alsohas a high oleic acid variety, Vistive, on the market; in thisvariety the high oleic acid trait was developed by muta-genesis, not GM, although the variety also carries a GMtrait for glyphosate tolerance.Genetic modification has also been used to change oil-

seed rape oil: Calgene developed aGMoilseed rape variety

containing a gene from the Californian Bay plant thatencodes an enzyme that causes premature chain termina-tion of growing fatty acid chains, leading to the accumu-lation of high levels (40%) of lauric acid (12:0) (Voelkeret al., 1992). Lauric acid is used in the manufacture ofdetergents and shampoos, and the aim was to enable oil-seed rape oil to compete in that market. The variety wasintroduced in 1995 but was not successful.Other GM plants with more ambitious changes to their

oil profile are in development. The longest fatty acidsmadeby higher plants have an 18-carbon chain, but considerablylonger fatty acids are present in fish oils, including theomega-3 long-chain polyunsaturated fatty acids (Omega-3LC-PUFAs) such as eicosapentaenoic acid (EPA) (20:5)and docosahexaenoic acid (DHA) (22:6). EPA and DHAare in fact made by marine algae, not fish, but they accu-mulate through themarine food chain. Thedevelopment ofGM plants producing EPA and DHA has become animportant target in order to provide a sustainable source ofthese nutritionally-important fatty acids. Several biotechcompanies claim to be close to launching commercial high-EPA/DHA varieties of soybean and oilseed rape, and UKscientists have successfully engineered the metabolic pro-cesses in the seed of false flax (Camelina sativa) to produceup to 12% EPA and 14% DHA, levels very close to thosefound in fish oil (Ruiz-Lopez et al., 2013).

Modified corn for bioethanol and animalfeed

In theUSA, bioethanol production frommaize starch nowaccounts for 42% of the annual maize harvest. Syngentahas produced a GM maize variety that it claims gives abetter yield of ethanol (Johnson et al., 2006). It contains agene, amy797E, encoding a highly thermostable a-amylasefrom a bacterium, Thermococcales spp. This variety wasderegulated (given the go-ahead to proceed to commercialdevelopment) by the US authorities in early 2011.An important aspect of bioethanol production is the

coproduction of a high-protein animal feed. Cereal grainusually has to be supplemented in animal feed because itcontains insufficient amounts of the essential amino acidlysine. Lysine concentration in cereal grains is controlledthrough the feed-back inhibition of a key enzyme inits synthesis, dihydrodipicolinate synthase (DHDPS), andRenessen, a joint venture between Cargill and Monsanto,engineered a maize variety to accumulate more lysine byintroducing a gene from a bacterium, Corynebacteriumglutamicum, encoding a lysine-insensitive DHDPS (Huanget al., 2005). The variety is called Mavera and is currentlybeing grown entirely for US domestic bioethanol/animalfeed production. It also contains a triple stack of inputtraits (resistance to corn rootworm and the European cornborer and tolerance of glyphosate), an indication of wheremaize biotechnology in theUS is heading, with triple inputtraits and a value-adding quality trait as the cherry on top.Nothing like it is likely to be available to European farmersin the foreseeable future.

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Drought tolerance

Drought tolerance is a complex trait controlled by manygenes and involving multiple plant physiological and cel-lular processes. All of the major biotech companies claimtobedeveloping drought-tolerant varieties, butMonsanto,in collaboration with BASF, was the first to the marketwith the release of two drought-tolerant maize varieties in2012. In a typical Monsanto strategy they used a bacterialgene, cspb from Bacillus subtilis, encoding a RNA cha-perone (Castiglioni et al., 2008). RNA chaperones stabiliseRNA molecules under stress conditions. See also: PlantResponse to Water-deficit Stress

Biopharming

Biopharming is a broad term applied to the use of GMplants toproduce pharmaceuticals, vaccines, antibodies andenzymes, in fact any high-value product produced on asmall scale and not intended to enter the food chain. Thecompany ProdiGene, based in Texas, has produced anedible vaccine for the prevention of Transmissible gastro-enteritis virus (TGEV) infection in pigs (Lamphear et al.,2004), and a monoclonal antibody, Guys 13, has been pro-duced in tobacco (Ma et al., 1994). This antibody binds tothe surface protein of Streptococcus mutans, the bacteriumthat causes tooth decay. The technology is now licensed toPlanet Biotechnology Inc. and is apparently undergoingclinical trials under the product name CaroRxTM.An example of the production of a pharmaceutical drug

is insulin, the synthesis of which in GM safflower wasannounced by Sembiosys, Canada, in 2007. Similar tech-nology is being used to make enzymes for industrial uses,such as trypsin, an animal protease that has a variety ofapplications in research and the food industry. ProdiGeneengineered maize to produce bovine trypsin and is alreadymarketing the enzyme under the trade name TrypZean(Woodward et al., 2003). See also: Molecular Farming inPlantsAn important aspect of biopharming is that the GM

crops being used for this purpose should not be allowed toenter the food chain, or to cross with crops that are beinggrown for food.

Traits that may be Commercialised inthe Near Future

Golden rice and other crops with elevatedlevels of b-carotene

Children in developing countries often become vitaminA-deficient because their families rely on subsistence cropssuch as rice and cassava that contain little vitamin A ornone at all.Onepotential solution to this problemwouldbeto distribute GM varieties of these crops that containvitamin A or its precursor, b-carotene, which can be con-verted into vitamin A in the body, and a GM line of rice

containing b-carotene was developed in the late 1990s(Ye et al., 2000). The GM rice producing b-carotene wascrossedwith another line engineered withmultiple genes toimprove iron availability. The high b-carotene/high avail-able iron hybrid was called Golden Rice (Potrykus, 2003).By 2001, Golden Rice was already being crossed into

local varieties by centres such as The Rice Research Insti-tute in Manila, The Philippines. However, its release tofarmers has been held up due to concern about exportmarkets in Europe and Japan, where resistance to GMcrops has been strongest, and the influence of westernpressure groups who have continued to oppose its use.Meanwhile, similar technology is being used in cassava andbanana, both of which are staples in Africa.

Food safety: acrylamide

Acrylamide (C3H5NO) is a familiar industrial and labora-tory chemical. It has been classified as a Group 2A, ‘prob-ably carcinogenic tohumans’, chemical by the InternationalAgency for Research on Cancer. It came as quite a shockfor the food industry, therefore, when it was discovered incommon foods in 2002. The affected foods include coffeeand potato- and cereal-based products such as crisps,French fries, bread, cakes, biscuits, breakfast cereals, crisp-breads, maize snacks and more (Halford et al., 2012).

The precursors of acrylamide are reducing sugars suchas glucose, fructose and maltose, and free asparagine, andreducing the levels of these precursors in crops wouldenable acrylamide levels in foods to be reduced withoutcostly changes to processing methods. Genes encodingasparagine synthetase are potential targets and reductionof expression of these genes in potato by a team fromSymplot Plant Sciences, Idaho, USA, using RNA inter-ference, has already been shown to be effective in reducingacrylamide formation (Rommens et al., 2008). Symplotapplied for deregulation of a low acrylamide GM potatovariety in 2013.

Bioremediation

Another target for biotechnology is to engineer plants totolerate and possibly remove salt or heavy metals, such aslead, cadmium and copper. One way of engineering plantsto cope with the presence of salt and heavy metals is toincrease their ability to sequester undesirable ions in thevacuole and there is an example of this being donesuccessfully for salt. Salt is transferred into the vacuoleagainst a concentration gradient by molecular pumpscalled Na+/H+ antiporters. The over-expression of one ofthese, NHX1, in transgenic tomato has been shown toresult in plants that are able to grow and set fruit whenexposed to 200mMNaCl (Apse et al., 1999).What ismore,the salt accumulated in the leaves but the fruits were edible,suggesting that plants like these could be used to producefood and detoxify soils at the same time. The technologyhas since been applied to oilseed rape, rice and maize, but

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there has yet to be any commercial development of thesecrops. See also: HeavyMetal Adaptation; Plant Salt Stress

Barriers to the Development of CropBiotechnology

Some GM crop varieties have been astonishingly success-ful, but the list of traits that have reached commercialisa-tion in crop biotechnology remains small. There are alsosome notable absentees from the list of crop species forwhich a biotech market has been established, includingwheat, rice and potato. The number of countries that haveallowed farmers to use GM crops is still only 28 (Table 1)and the area of GM crops planted in some of those coun-tries remains small. The only significant cultivation of GMcrops inEurope is that ofGMmaize in Spain, but even thatamounts to less than 100 000 ha. Glyphosate-tolerantsoybean was grown in Romania up to 2006, but this dis-appeared entirely in 2007 because Romania joined theEuropean Union. There are several reasons why thissituation has arisen.

Cost

The cost of developing a commercial GM variety from thefirst identification of a gene associatedwith a trait to the useof that gene in a variety that is available for farmers to growhas been estimated at US$100 million. That figure is waybeyond the means of most plant breeding companies andpublic sector establishments, and represents a significantinvestment for even a large company to risk. Much of thecost associated with bringing a GM crop variety to marketresults not from the science involved but in negotiating theregulatory processes that have been put in place across theworld.

Regulation

The broad consensus amongst plant scientists is that thereis no reason to expect GM varieties necessarily to be morerisky to human health or the environment than varietiesproduced by, for example, wide crossing or the completelyrandom techniques of radiation and chemicalmutagenesis.Most plant scientists would also argue that new varietiesshould be judged on the traits that they carry, not howthose traits were introduced. However, that is not the casewith GM in many parts of the world. The region where theregulation of GM crop varieties is the most convoluted isthe European Union, and Europe’s regulatory systemstymies the development of crop biotechnology across theglobe because the EU is the world’s most lucrative marketfor agricultural commodities. Under its directive, GMFood and Feed Regulation (EC) No. 1829/2003, permis-sion for cultivation, food and feed use of a GM cropvariety, or for food and feed use alone, has to be granted bythe EuropeanCommission, but the EuropeanCommissionfailed to approve a single application between 1998 and

2004. Even when an insect-resistant and gluphosinate-tolerant sweetcorn from Syngenta was approved in 2004 itwas only for food and feed use, not for cultivation, a trendthat has continued since. Currently there are still only twoGM varieties approved for cultivation in Europe: Mon810, aBtmaize variety grown in Spain and to a lesser extentin Romania, Slovakia, the Czech Republic and Portugal,and the Amflora potato.The fate of the Amflora potato illustrates the mess that

Europe has got itself into over crop biotechnology.Amflora was produced by BASF and contained a differenttype of starch to conventional potatoes. Starch is used inthe manufacture of paper, adhesives, gypsum wall boardsand textile yarns, amongstmany other things. It ismade upof chains of glucose units, but it comprises two compo-nents, amylose, consisting of long, unbranched chains, andamylopectin, consisting of branched chains. Amylose andamylopectin have different characteristics and have to beseparated or modified chemically before use in someindustrial processes. In Amflora, the starch is composedalmost entirely of amylopectin as a result of reducedactivity of a granule-bound starch synthase (Visser et al.,1991). Amflora was developed for the European marketand was mired in the EU’s regulatory processes for over adecade. It was finally approved for cultivation in 2010 andBASF grew Amflora in Germany and Sweden in 2011 toproduce seed potatoes. However, the company announcedin 2012 that it was withdrawing from plant biotechnologyin Europe altogether and concentrating on markets else-where, partly because of its experience with Amflora.The development of GM varieties for cultivation in

Europe has now all but been abandoned by biotech com-panies. Instead, companies are focusing on obtaining per-mission forGMcrop products to be imported for food andfeed use so that farmers elsewhere in the world can bereassured that the European market is open to their pro-ducts. Even small-scale field trials of GM lines are extre-mely difficult to run inEurope, and in theUK, for example,there have only been three since 2002, compared with tensof thousands in the USA over the same period. The pictureof the GM wheat trial at Rothamsted Research in 2012(Figure 4) illustrates some of the problems. The trial had tobe protected by fencing and a 24-hour security presenceto prevent vandalism, and the small plots ofGMwheat hadto be surrounded with a separator crop of barley and apollen barrier of non-GM wheat to satisfy the regulators.This is for a species that is almost entirely self-pollinating.

Labelling

Labelling legislation covering GM crop use in food wasintroduced by the EU in 1997 and extended in 2004through directives on the regulation of GM food and feed(1829/2003) and the traceability and labelling of geneticallymodified organisms (1830/2003). In their post-2004 form,the law requires that any food containing material fromGM crops must be labelled, unless the GM material ispresent through accidental mixing and does not exceed

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0.9% of the total. The regulations cover food and animalfeed, but not meat, dairy or other products from GM-fedanimals, or cheeses, yoghurts and other foods that containenzymes such as chymosin produced in GM microorgan-isms (70% of cheese sold in the UK, for example, containschymosin from GM microbes).The fact that meat and dairy products are not covered is

significant because the European animal feed industry usesapproximately 20 million tonnes of imported soybeanevery year, most of it GM. Indeed, European animal feedproduction is heavily dependent on importedGM soybeanand maize.

Acceptance

There has been consumer hostility to GM crop products inEurope since 1999 whenmember states began to ratify andimpose EU labelling regulations. Consumers were parti-cularly angry thatGM foods had been sold unlabelled, andwere wary of a new technology affecting the food chain.Consumer fears were whipped up by pressure groups andthe media (Box 3), and retailers scrambled to portraythemselves as ‘GM-free’. Hostility to GM crop productshas declined; indeed the proportion of respondents to aFood Standards Agency survey who, unprompted, listed

GM food as a concern fell from 18% in 2004 to only 4% in2011 (Food Standards Agency, 2012). Nevertheless, itremains an extremely difficult issue for the food supplychain in Europe and there is currently almost no GM cropmaterial being used for human food consumption in Eur-ope. Some of the headlines that have appeared on GMseem almost comical, but the slogans were designed bypeople who knew what they were doing, and it is worthremembering that the proponents of these scare storieswonthe debate and the science argument lost, with severeconsequences for European biotechnology, plant breedingand farming (Box 4), and for the relationship between sci-ence and the larger community. See also: GeneticallyModified Food: Ethical Issues

Concluding Remarks

Geneticmodification of cropplants is not a panacea but it isa powerful tool that allows plant breeders to improve cropplants in ways that may be impossible by other methods inplant breeding. GM crops have been used safely in agri-culture for almost two decades and while some GM vari-eties have failed to gain a foothold in the market, othershave been adopted with great enthusiasm by farmerswherever they have been made available. The technology isnowwell established, particularly in theAmericas andAsia.

Acknowledgements

Nigel G. Halford is supported via the 20:20 Wheat Pro-gramme at Rothamsted Research by the Biotechnologyand Biological Sciences Research Council (BBSRC) of theUnited Kingdom.

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Further Reading

Chrispeels MJ and Sadava DE (2002) Plants, Genes, and Crop

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Chichester, UK: Wiley.

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of Genetically Modified Crops. Wallingford, UK: CABI.

Halford N (ed.) (2006) Plant Biotechnology: Current and Future

Applications of Genetically Modified Crops. Chichester, UK:

Wiley.

HalfordNG (2012)GeneticallyModifiedCrops, 2nd edn. London,

UK: Imperial College Press.

Slater A, Scott N and Fowler M (2003) Plant Biotechnology: The

Genetic Manipulation of Plants. Oxford, UK: Oxford Uni-

versity Press.

Somers DJ, Langridge P and Gustafson P (eds) (2009) Plant

Genomics: Methods and Protocols. Methods in Molecular Biol-

ogy 513. New York: Humana Press, c/o Springer Science and

Business Media.

Stahl U, Donalies UEB and Nevoight E (2008) Food Biotechnol-

ogy. Advances in Biochemical Engineering/Biotechnology 111.

Berlin, Heidelberg, Germany: Springer-Verlag.

Thompson JA (2002)Genes forAfrica:GeneticallyModifiedCrops

in the Developing World. Landsdowne, South Africa: UCT

Press.

Thompson JA (2007) Seeds for the Future: The Impact of

GeneticallyModified Crops on the Environment. Ithaca: Cornell

University Press.

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