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Chapter 1 Transgenic Crop Plants for Resistance to Biotic Stress N. Ferry and A.M.R. Gatehouse 1.1 Introduction We couldn’t feed today’s world with yesterday’s agriculture and we won’t be able to feed tomorrow’s world with today’s. – Lord Robert May, President of the Royal Society, March 2002 The human population is everexpanding; conservative estimates predict that the population will reach ten billion by 2050 (United Nations Population Division), and the ability to provide enough food is becoming increasingly difficult (Chrispeels and Sadava 2003). The planet has a finite quantity of land available to agriculture and the need for increasing global food production has led to increasing exploitation of previously uncultivated land for agriculture; as a result wilderness, wetland, forest and other pristine environments have been, and are being, encroached upon (Ferry and Gatehouse 2009). The minimization of losses to biotic stress caused by agricultural pests would go some way to optimizing the yield on land currently under cultivation. For nearly 50 years, mainstream science has told us that this would be impossible without chemical pesticides (Pimental 1997). The global pesticide market is in excess of $30 billion per year (Levine 2007); despite this, approximately 40% of all crops are lost directly to pest damage (Fig. 1.1). These figures are simplified rough estimates; in reality crop losses to biotic stress are extremely difficult to quantify and vary by crop, year, and region. N. Ferry (*) School of Biology, Institute for Research on Environment and Sustainability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK e-mail: [email protected] C. Kole et al. (eds.), Transgenic Crop Plants, DOI 10.1007/978-3-642-04812-8_1, # Springer-Verlag Berlin Heidelberg 2010 1

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Chapter 1

Transgenic Crop Plants for Resistance

to Biotic Stress

N. Ferry and A.M.R. Gatehouse

1.1 Introduction

We couldn’t feed today’s world with yesterday’s agriculture and we won’t be able to feed

tomorrow’s world with today’s. – Lord Robert May, President of the Royal Society, March

2002

The human population is everexpanding; conservative estimates predict that the

population will reach ten billion by 2050 (United Nations Population Division), and

the ability to provide enough food is becoming increasingly difficult (Chrispeels

and Sadava 2003). The planet has a finite quantity of land available to agriculture

and the need for increasing global food production has led to increasing exploitation

of previously uncultivated land for agriculture; as a result wilderness, wetland,

forest and other pristine environments have been, and are being, encroached upon

(Ferry and Gatehouse 2009). The minimization of losses to biotic stress caused by

agricultural pests would go some way to optimizing the yield on land currently

under cultivation. For nearly 50 years, mainstream science has told us that this

would be impossible without chemical pesticides (Pimental 1997). The global

pesticide market is in excess of $30 billion per year (Levine 2007); despite this,

approximately 40% of all crops are lost directly to pest damage (Fig. 1.1).

These figures are simplified rough estimates; in reality crop losses to biotic stress

are extremely difficult to quantify and vary by crop, year, and region.

N. Ferry (*)

School of Biology, Institute for Research on Environment and Sustainability, Devonshire

Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK

e-mail: [email protected]

C. Kole et al. (eds.), Transgenic Crop Plants,DOI 10.1007/978-3-642-04812-8_1, # Springer-Verlag Berlin Heidelberg 2010

1

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1.2 Biotic Stress due to Insect Pests

1.2.1 Crop Losses due to Insect Pests

[A]nimals annually consume an amount of produce that sets calculation at defiance; and,

indeed, if an approximation could be made to the quantity thus destroyed, the world would

remain skeptical of the result obtained, considering it too marvelous to be received as truth.

– John Curtis, 1860

Arthropods are the most widespread and diverse group of animals, with an esti-

mated 4–6 million species worldwide (Novotny et al. 2002). While only a small

percentage of arthropods are classified as pests, they cause major devastation of

crops, destroying around 14% of the world annual crop production, contributing to

20% of losses of stored grains and causing around US$100 billion of damage each

year (Nicholson 2007).

Herbivorous insects and mites are a major threat to food production for human

consumption. Larval forms of lepidopterans are considered the most destructive

insects, with about 40% of all insecticides directed against heliothine species

(Brooks and Hines 1999). However, many species within the orders Acrina, Cole-

optera, Diptera, Hemiptera, Orthoptera and Thysanoptera are also considered

agricultural pests with significant economic impact (Fig. 1.2).

Insect pests may cause direct damage by feeding on crop plants in the field

or by infesting stored products and so competing with humans for plants as a

food resource. Some cause indirect damage, especially the sap-feeding (sucking)

insects by transmitting viral diseases or secondary microbial infections of crop

plants.

Fig. 1.1 The world agricultural cake

2 N. Ferry and A.M.R. Gatehouse

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1.2.1.1 The Phylloxera Plague

In the late nineteenth century, a phylloxera epidemic destroyed most of the

vineyards for wine grapes in Europe, most notably in France. Grape phylloxera

(Daktulosphaira vitifoliae, family Phylloxeridae) is a pest of commercial grape-

vines worldwide, originally native to eastern North America. These minute, pale

yellow sap-sucking insects feed on the roots of grapevines. In Vitis vinifera, theresulting deformations and secondary fungal infections can damage roots, gradually

cutting off the flow of nutrients and water to the vine. Phylloxera was inadvertentlyintroduced to Europe in the 1860s. The European wine grape V. vinifera was highlysusceptible to the pest and the epidemic devastated most of the European wine-

growing industry. Some estimates hold that between two-thirds and nine-tenths of

all European vineyards were destroyed. Native American grapes Vitis labrusca are

naturally Phylloxera-resistant. The grafting of European grape vines onto resistant

grape rootstock is the preferred method to cope with the pest problem even today

(http://www.calwineries.com). Thus, phylloxera provides a clear example of how a

single insect pest can nearly devastate a whole industry.

Innumerable examples exist of insect pests that are highly injurious to agri-

cultural production. The most notable for their destructive capacity being the

Fig. 1.2 Phylloxera, a sap-sucking pest of grape, almost devastated the European wine industry in

the nineteenth century

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migratory locust (Locusta migratoria), Colorado potato beetle (Leptinotarsadecemlineata), boll weevil (Anthonomus grandis), Japanese beetle (Popillia japon-ica), and aphids, which are among the most destructive pests on earth as vectors of

plant viruses (many species in ten families of the Aphidoidea), and the western corn

rootworm (Diabrotica virgifera virgifera), also called the billion dollar bug becauseof its economic impact in the US alone.

Curiously, one of these pests, the cotton boll weevil, responsible for near-

destruction of the cotton industry in North America, is also ultimately responsible

for subsequent diversification of agriculture in many regions, thus warranting a

monument in the town of Enterprise, Alabama, in profound appreciation of its role

in bringing to an end the state’s dependence on a poverty crop (Fig. 1.3).

The global challenge facing agriculture is to secure large and high-quality crop

yields and to make agricultural production environmentally sustainable. Control of

insect pests would go some way towards achieving this goal.

1.2.2 Insecticides

Insecticides have been, and still are, a highly effective method to control pests

quickly when they threaten to destroy crops. The chemical nature of the

Fig. 1.3 Monument to the

cotton boll weevil Source:Wikimedia Commons

4 N. Ferry and A.M.R. Gatehouse

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insecticides used has evolved over time. In early farming practices, inorganic

chemicals were used for insect control; however, with the advances in synthetic

organic chemistry that followed the two world wars the synthetic insecticides

were born. In the 1940s, the neurotoxic organochlorine, DDT, was the pesticide

of choice, but following its indiscriminate use it was reported to bio-accumu-

late in the food chain where it affected the fertility of higher organisms – such

as birds. Rachel Carson first highlighted this in the book Silent Spring pub-

lished in 1962; while her presumptions have since been proven to be wrong,

the book was nevertheless an important signature event in the birth of the

environmental movement. This pesticide was subsequently replaced by the

comparatively safer organophosphate and carbamate-based pesticides (both

acetylcholinesterase inhibitors) and many of these were replaced in turn by

the even safer pyrethroid-based pesticides (axonic poisons). Synthetic pyre-

throids continue to be used today despite the fact that they are broad-spectrum

pesticides.

The major limiting factor on the insecticide strategy is the occurrence of

resistance in insect populations. In fact, resistance to insecticides has now been

reported in more than 500 species (Nicholson 2007). Furthermore, resistance has

evolved to every major class of chemical. The underlying causes of insecticide

resistance are manyfold. Owing to wide usage and narrow target range, arthropods

have been put under a high degree of selection pressure (Feyereisen 1995). Insecti-

cide resistance may be characterized by:

(a) Metabolic detoxification (upregulation of esterases, glutathione-S-transferases,and monoxygenases)

(b) Decreased target site sensitivity (via mutation of the target receptor)

(c) Sequestration or lowered insecticide availability

In addition, cross-resistance to different classes of chemicals has occurred

because of the fact that many insecticides target a limited number of sites in the

insect nervous system (Raymond-Delpech et al. 2005). The five target sites in

insects comprise: nicotinic acetylcholine receptors (e.g., imidacloprid), voltage-

gated sodium channels (e.g., DDT, pyrethroids), g-aminobutyric acid receptors

(e.g., fipronil), glutamate receptors (e.g., avermectins), and acetylcholinesterase

(AChE) (e.g., organophosphates and carbamates). The world insecticide market is

dominated by compounds that inhibit the enzyme AChE. Together, AChE inhibi-

tors and insecticides acting on the voltage-gated sodium channel, in particular the

pyrethroids, account for approximately 70% of the world market (Nauen et al.

2001).

Unfortunately, as insecticide target sites are conserved between invertebrates

and vertebrates, insecticides have undesirable nontarget effects and unaccept-

able ecological impacts. Insecticides are implicated in the poisoning of nontar-

get insects, other arthropods, marine life, birds, and humans (Fletcher et al.

2000). The poisoning of nontarget organisms has obvious implications for

biodiversity.

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1.2.3 Integrated Pest Management and Organic Agriculture

In parallel to the development of modern insecticides, the specific microbial toxins

produced by the soil-dwelling bacterium Bacillus thuringiensis (Bt) are increas-

ingly being adopted as biopesticides. In fact, microbial sprays of Bt are used in

organic agriculture. This shift is due in part to a demand for increased safety both

for humans and for the environment.

Organic agriculture is a form of agriculture that relies on crop rotation, green

manure, compost, biological pest control, and mechanical cultivation to maintain

soil productivity and control pests, excluding or strictly limiting the use of synthetic

fertilizers and synthetic pesticides, plant growth regulators, and genetically modi-

fied organisms (Directorate General for Agriculture and Rural Development of the

European Commission).

Integrated pest management (IPM) has also been proposed as a sustainable

control system for insects. Several control systems are combined, including the

judicious application of chemicals and biopesticides, use of trap crops, biological

control, rotation, good husbandry and cultural control to manage all the pests of a

particular crop (Gatehouse and Gatehouse 1999). Increasing crop varietal resistance

is critical to both IPM and organic agriculture.

It is unfortunate that ultimately neither organic agriculture nor IPM will be able

to feed the world. In order to feed an increasing world population, more food must

be produced in the future and on either the same amount, or less land (Ferry and

Gatehouse 2009). Neither of these farming methods will be as productive as will be

necessary to meet increased demands (Amman 2009).

1.2.4 Transgenic (Genetically Modified) Crops

Genetically modified (GM) maize and cotton varieties that express insecticidal

proteins derived from B. thuringiensis (Bt) have become an important component

in agriculture worldwide. At present, 20.3 million hectares of land is planted with

insect-protected transgenic Bt cotton and maize (James 2007), with economic

benefits from Bt cotton estimated at US$9.6 billion and maize US$3.6 billion

(James 2007). Significantly, Phipps and Park (2002) showed that on a global

basis GM technology has reduced pesticide use. These authors estimated that

pesticide use was reduced by a total of 22.3 million kg of formulated product in

2000 alone.

1.2.4.1 Bacillus thuringiensis Toxins

B. thuringiensis (Bt) is a soil-dwelling bacterium of major agronomic and scientific

interest. Whilst the subspecies of this bacterium colonize and kill a large variety of

6 N. Ferry and A.M.R. Gatehouse

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host insects, each strain tends to be highly specific. Toxins for insects in the orders

Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), Coleoptera

(beetles and weevils), and Hymenoptera (wasps and bees) (Table 1.1) have been

identified (de Maagd et al. 2001), but interestingly none with activity towards

Homoptera (sap suckers) have, as yet, been identified, although a few with activity

against nematodes have been isolated (Gatehouse et al. 2002). Further, there is little

evidence of effective Bt toxins against many of the major storage insect pests.

Bt toxins (also referred to as d-endotoxins; Cry proteins) exert their pathologicaleffects by forming lytic pores in the cell membrane of the insect gut. On ingestion,

they are solubilized and proteolytically cleaved in the midgut to remove the

C-terminal region, thus generating an “activated” 65–70 kDa toxin. The active

toxin molecule binds to a specific high-affinity receptor in the insect midgut epithelial

cells. Following binding, the pore-forming domain, consisting of a-helices, insertsinto the membrane; this results in cell death by colloid osmotic lysis, followed by

death of the insect (de Maagd et al. 2001). A number of putative receptors in the

insect gut have been identified and include aminopeptidase N proteins (Knight et al.

1994; Sangadala et al. 1994; Gill et al. 1995; Luo et al. 1997), cadherin-like proteins

(Vadlamudi et al. 1995; Nagamatsu et al. 1998; Gahan et al. 2001) and glycolipids

(Denolf 1996).

Transgenic plants expressing Bt toxins were first reported in 1987 (Vaeck et al.

1987) and following this initial study numerous crop species have been transformed

with genes encoding a range of different Cry proteins targeted towards different

pest species. Since bacterial cry genes (genes encoding Bt toxins) are rich in A/T

content compared to plant genes, both the full-length and truncated versions of

these cry genes have had to undergo considerable modification of codon usage and

removal of polyadenylation sites before successful expression in plants (de Maagd

et al. 1999). Crops expressing Bt toxins were first commercialized in the mid-1990s,

with the introduction of Bt potato and cotton. Currently, 20.3 million hectares of

land is planted with Bt cotton and maize (James 2007).

Bt Maize

Lepidopteran pests such as European corn borer Ostrinia nubilalis, fall armyworm

Spodoptera frugiperda, and corn earworm Helicoverpa zea perennially cause leaf

Table 1.1 Insecticidal properties of Bt toxins

Insect order Cry protein

Lepidoptera Cry1A, Cry1B, Cry1C, Cry1E, Cry1F, Cry1I, Cry1J, Cry1K, Cry2A, Cry9A,

Cry9I, Cry15A

Coleoptera Cry1I, Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, Cry8C, Cry14A, Cry23A

Diptera Cry2A, Cry4A, Cry10A, Cry11A, Cry11B, Cry16A, Cry19A, Cry20A, Cry21A

Hymenoptera Cry22A

Nematodes Cry5A, Cry6A, Cry6B, Cry12A, Cry13A, Cry14A

Liver fluke Cry5A

1 Transgenic Crop Plants for Resistance to Biotic Stress 7

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and ear damage to corn. The Bt concept was particularly attractive for maize, since

it made it possible to combat European corn borer larvae hidden inside the stem of

the plant for the first time. Bt maize has now been grown on a large scale for over a

decade, particularly in the US. In 2007, insect-resistant Btmaize was grown on 21%

of the total maize cultivation area, and Bt maize with a combination of insect and

herbicide resistance was grown on a further 28% (James 2007). Various Bt maize

varieties are also authorized in the EU. In 2007, there was notable cultivation of Btmaize primarily in Spain, where it was grown on around 75,000 hectares (Ortego

et al. 2009).

Transgenic corn hybrids expressing the insecticidal protein Cry1Ab from

B. thuringiensis (Bt) var. kurstaki were originally developed to control European

corn borer, and offer the potential for reducing losses by fall armyworm and corn

earworm. Several events of transgenic Bt corn have been developed with different

modes of toxin expression (Ostlie et al. 1997). Amongst the most promising events

were Bt11 expressing the cry1Ab gene from B. thuringiensis subsp. kurstaki(Novartis Seeds) and MON810 expressing a truncated form of the cry1Ab gene

from B. thuringiensis subsp. kurstaki HD-1 (Monsanto Co.). In both events, the

endotoxins are expressed in vegetative and reproductive structures throughout the

season (Armstrong et al. 1995; Williams and Davis 1997). Crops containing either

of these events are collectively referred to as having “YieldGard technology.”

Furthermore, a modified cry9C gene from B. thuringiensis subsp. tolworthi strainBTS02618A is expressed in maize (tradename StarLink – marketed by Aventis

CropScience). StarLink corn has only been approved in the US for livestock

feed use.

In recent years, there has been increasing focus on another maize pest, this

time a Coleopteran (beetle); the western corn rootworm. Western corn root-

worm is one of the most devastating corn rootworm species in North America.

Its larvae are root pests and can destroy significant percentages of corn if left

untreated. In the US, current estimates show that 30 million acres (120,000 km)

of corn (out of 80 million grown) are infested with corn rootworms. The

United States Department of Agriculture (USDA) estimates that corn root-

worms cause US$1 billion in lost revenue each year. To make matters worse,

this pest is extending its geographical range all of the time – including

spreading throughout Europe. Bt maize which is resistant to the western corn

rootworm has been authorized in the US since 2003 and has been grown on a

large scale since. YieldGard Rootworm uses event MON 863 and expresses the

Cry3Bb1 protein from B. thuringiensis (subsp. kumamotoensis) in the plant,

protecting the plant against root feeding from the western and northern corn

rootworm larvae. Products containing both YieldGard Corn Borer (MON810)

and YieldGard Rootworm (MON 863) are marketed under the trade name

YieldGard Plus (http://www.agbios.com). Corn rootworm-resistant maize is

also produced by expression of the cry34Ab1 and cry35Ab1 genes from

B. thuringiensis strain PS149B1 (DOW AgroSciences LLC and Pioneer

Hi-Bred International, Inc.).

8 N. Ferry and A.M.R. Gatehouse

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Bt Cotton

Cotton fibers used in textiles around the world come from the seed hairs of

Gossypium hirsutum. Cotton develops in closed, green capsules known as bolls

that burst open when ripe, revealing the white, fluffy fibers. But cotton is more than

just a fiber for textiles. It is also an important source of raw materials used in animal

feed and for various processed food ingredients, including cottonseed oil, protein-

rich cottonseed meal (mostly used as animal feed) and even leftover fibers can be

used as food additives.

Lepidopteran, particularly heliothine, pests can have an enormously damaging

effect on a cotton crop and controlling these insects in conventional farming

involves treatment with a number of insecticide sprays. In 1996, Bollgard1 cotton

(Monsanto) was the first Bt cotton to be marketed in the US. Bollgard cotton

produces the Cry1Ac toxin from B. thuringiensis (subsp. kurstaki), which has

excellent activity on tobacco budworm and pink bollworm. These two insects are

extremely important as both are difficult and expensive to control with traditional

insecticides and the damage caused by them directly impacts on the harvestable

plant organ, the cotton bolls themselves.

Bollgard II1 was introduced in 2003, representing the next generation of Btcottons. Bollgard II contains Cry1Ac plus a second gene from the Bt bacteria whichencodes the production of Cry 2Ab (also subsp. kurstaki). WideStrike (a Trademark

of DowAgrosciences) was registered for use in 2004, and like Bollgard II, it

expresses two Bt toxins but this time Cry1Ac and Cry1F were used in combination.

Both Bollgard II and WideStrike have better activity on a wider range of caterpillar

pests than the original Bollgard technology. GM Bt cotton has become widespread,

covering a total of 15 million hectares in 2007, or 43% of the world’s cotton. Most

GM cotton is grown in the US and China, but it can also be found in India, South

Africa, Australia, Argentina, Mexico, and Columbia (http://www.agbios.com).

Currently, 20% of the cotton grown commercially in China expresses Cry1Ac in

combination with a plant protease inhibitor, cowpea trypsin inhibitor (CpTI) (He

et al. 2009).

Bt Potato

Potato (Solanum tuberosum L.) is a major world food crop. Potato is exceeded only

by wheat, rice, and maize in terms of world production for human consumption

(Ross 1986). Many commercial potato varieties are highly susceptible to damage by

the Colorado potato beetle. In 1999, 93% of the 1.1 million potato acres grown in

the US were treated with a total of 2.6 million pounds of insecticide (http://www.

usda.gov/). To date, few traditionally bred varieties have been produced with

resistance to this major pest. Unfortunately, many of the pesticides currently used

are broad-spectrum pesticides, killing not only the target pest but most of its natural

enemies as well. The Cry3A d-endotoxin from B. thuringiensis Berliner subsp.

tenebrionis is toxic to coleopterans, particularly chrysomelids (Krieg et al. 1983;

1 Transgenic Crop Plants for Resistance to Biotic Stress 9

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Bauer 1990; MacIntosh et al. 1990). It is insecticidal against the Colorado potato

beetle, L. decemlineata (Ferro and Gerlernter 1989). In 1995, Bt.Cry3A (NewLeafTM)

potato became the first Bt-crop to be commercialized, although they are currently

withdrawn from the US market.

1.2.4.2 Evolution of Resistance in Pest Populations

Perhaps one of the most important issues surrounding the cultivation of Bt cropsrelates to the evolution of target pest resistance, which could limit the life span of the

technology. In the case of Bt toxins, this is a major concern for the organic farming

community, since the potential for insect populations to evolve resistance to Bt willnot only limit the effectiveness of Bt-expressing crops but also Bt-based biopesti-

cides. Bt resistance in insect pests has been reported to develop within 4–5 genera-

tions in the laboratory (Stone et al. 1989). To date, the mechanism of resistance to

Cry toxins in insects has been most commonly ascribed to the loss or inactivation of

specific toxin-binding sites on midgut cell membranes (Ferre and Van Rie 2002).

Other resistance mechanisms that have been proposed include a defect in the toxin

activation by midgut proteases (Oppert et al. 1994; Sayyed et al. 2001), or an

increased repair and/or replacement rate of Cry-damaged midgut cells by stem

cells (Forcada et al. 1999). Studies have also revealed evidence for novel resistance

mechanisms based on active defensive responses (Rahman et al. 2004; Ma et al.

2005). When one considers the ability of insects to evolve resistance to chemical

pesticides (French-Constant 2004), the development of field resistance is inevitable

and in fact was recently reported to have already occurred (Tabashnik and Carriere

2009). Analysis of monitoring data shows that some field populations ofH. zea haveevolved resistance to Cry1Ac, the toxin produced by first-generation Bt cotton(Tabashnik et al. 2008). Nonetheless, resistance of H. zea to Cry1Ac has not causedwidespread crop failures in the field for several reasons (Tabashnik et al. 2008).

First, the documented resistance is spatially limited. Second, from the outset,

insecticide sprays have been used to improve the control of H. zea on Bt cottonbecause Cry1Ac alone is not sufficiently effective to manage this pest. Finally, GM

cotton producing two Bt toxins (Cry2Ab and Cry1Ac) was planted on more than one

million ha in the US in 2006; control of H. zea by Cry2Ab would limit problems

associated with resistance to Cry1Ac (Jackson et al. 2004).

Considerable effort has been devoted to delaying the onset of evolution of

resistance, e.g., the use of refugia has been required/recommended in most regions

growing Bt-crops depending upon the country in question (Tabashnik and Carriere

2009). Gene-stacking and integrated pest management should be combined to

control this problem.

1.2.4.3 Unexpected Benefits

Interestingly, the expression of Bt has resulted in improved crop quality as a

consequence of decreased levels of Fusarium infestation and fumonisin mycotoxin

10 N. Ferry and A.M.R. Gatehouse

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production as a direct result of reduced levels of insect pest damage. This benefit is

particularly important in food crops such as maize (Glaser and Matten 2003).

1.2.4.4 A Global Technology

Of the global total of 12 million biotech farmers in 2007, over 90% were small and

resource-poor farmers from developing countries; the balance of one million were

large farmers from both industrialized countries such as Canada and developing

countries such as Argentina. Of the 11 million small farmers, most were Bt cottonfarmers; 7.1 million in China (Bt cotton), 3.8 million in India (Bt cotton), and the

balance of 100,000 in the Philippines (GMmaize), South Africa (GM cotton, maize

and soybeans often grown by subsistence women farmers) and the other eight

developing countries which grew GM crops in 2007. This modest uptake by

subsistence farmers contributes towards the Millennium Development Goals of

reducing poverty by 50% by 2015 and is a very important development (James

2007).

1.2.5 Other Sources of Insecticidal Molecules

The concept of employing genes encoding Bt toxins to produce insect-resistant

transgenic plants arises from the successful use of Bt-based biopesticides.

A number of other strategies for protecting crops from insect pests actually exploit

endogenous resistance mechanisms (Harborne 1998; Gatehouse 2002a, b). Genes

encoding such defensive proteins are obvious candidates for enhancing crop resis-

tance to insect pests.

1.2.5.1 Enzyme Inhibitors

Interfering with digestion, and thus affecting the nutritional status of the insect, is a

strategy widely employed by plants for defense, and has been extensively investi-

gated as a means of producing insect-resistant crops (Gatehouse 2002a, b). Insect-

digestive proteases tend to fall into four mechanistic classes (serine, cysteine,

aspartic or metallo proteases – depending on the enzyme-active site residue).

Numerous studies since the 1970s have confirmed the insecticidal properties of a

broad range of protease inhibitors from both plant and animal sources (Jouanin et al.

1998; Gatehouse 2002a, b). Proof of concept for exploiting such molecules for crop

protection was first demonstrated with expression of a serine protease inhibitor

from cowpea (CpTI), which was shown to significantly reduce insect growth and

survival (Hilder et al. 1987). These studies were subsequently extended to include a

greater range of target pests (Gatehouse et al. 1994; Graham et al. 1995; Xu et al.

1996), and a broader range of inhibitors and plant species, including economically

1 Transgenic Crop Plants for Resistance to Biotic Stress 11

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important pest species, particularly lepidopterans (Broadway 1997; De Leo et al.

2001).

Since many economically important coleopteran pests predominantly utilize

cysteine proteases for protein digestion, inhibitors for this class of enzyme (cysta-

tins) have also been investigated as a means for controlling pests from this order.

Oryzacystatin, a cysteine protease inhibitor isolated from rice seeds, is effective

towards both coleopteran insects and nematodes when expressed in transgenic

plants (Leple et al. 1995; Urwin et al. 1995; Pannetier et al. 1997). Similarly, the

cysteine/aspartic protease inhibitor equistatin, from sea anemone, is also toxic to

several economically important coleopteran pests, including the Colorado potato

beetle (Outchkourov et al. 2003). More recent studies have included the stacking of

different families of inhibitors to increase the spectrum of activity (Abdeen et al.

2005).

A major limitation, however, to this strategy for the control of insect pests arises

from the ability of some lepidopteran and coleopteran species to respond and adapt

to ingestion of protease inhibitors by either overexpressing native gut proteases, or

producing novel proteases that are insensitive to inhibition (Bown et al. 1997;

Jongsma and Bolter 1997). Thus, detailed knowledge about the enzyme–inhibitor

interactions, both at the molecular and biochemical levels, together with detailed

knowledge on the response of insects to exposure to such proteins is essential to

effectively exploit this strategy. The concept of inhibiting protein digestion as a

means of controlling insect pests has been extended to the inhibition of carbohy-

drate digestion. For example, inhibitors of a-amylase have been expressed in

transgenic plants and shown to confer resistance to bruchid beetles (Shade et al.

1994; Schroeder et al. 1995).

1.2.5.2 Lectins

Lectins, found throughout the plant and animal kingdoms, form a large and diverse

group of proteins identified by a common property of specific binding to carbohy-

drate residues, either as free sugars, or more commonly, as part of oligo- or

polysaccharides. Many physiological roles have been attributed to plant lectins,

including defense against pests and pathogens (Chrispeels and Raikhel 1991;

Peumans and Vandamme 1995).

Although some lectins are toxic to mammals, and are thus not suitable candi-

dates for transfer to crops for enhanced levels of protection, this is by no means

universal. Many lectins are not toxic to mammals, yet are effective against insects

from several different orders (Gatehouse et al. 1995), including homopteran pests

such as hoppers and aphids (Powell et al. 1995; Sauvion et al. 1996; Gatehouse et al.

1997). This finding has generated significant interest, not least since no Bts effectiveagainst this pest order have been identified to date. One such lectin is the snowdrop

lectin (Galanthus nivalis agglutinin; GNA). Both constitutive and phloem-specific

(Rss1 promoter) expression of GNA in rice is an effective means of significantly

reducing survival of rice brown planthopper (Nilaparvata lugens), and green

12 N. Ferry and A.M.R. Gatehouse

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leafhopper (Nephotettix virescens) both serious economic pests of rice (Rao et al.

1998; Foissac et al. 2000; Tinjuangjun et al. 2000). GNA has been expressed in

combination with other genes encoding insecticidal proteins, including the crygenes (Maqbool et al. 2001). Although lectins such as GNA, and ConA are not as

effective against aphids as they are against hoppers, they nonetheless have signifi-

cant effects on aphid fecundity when expressed in potato (Down et al. 1996;

Gatehouse et al. 1997, 1999) and wheat (Stoger et al. 1999).

The precise mode of action of lectins in insects is not fully understood although

binding to gut epithelial cells appears to be a prerequisite for toxicity. In the case of

rice brown planthopper, GNA not only binds to the luminal surface of the midgut

epithelial cells, but also accumulates in the fat bodies, ovarioles and throughout the

haemolymph, suggesting that the lectin is able to cross the midgut epithelial barrier

and pass into the insect’s circulatory system, resulting in a systemic toxic effect

(Powell et al. 1998; Du et al. 2000).

As with protease inhibitors, the levels of protection conferred by the expression

of lectins in transgenic plants are generally not high enough to be considered

commercially viable. However, the absence of genes with proven high insecticidal

activity against homopteran pests may well mean that transgenic crops with partial

resistance may still find acceptance in agriculture, especially if expressed with other

genes that confer partial resistance, or if introduced into partially resistant genetic

backgrounds.

1.2.5.3 A Brief Aside; Plant Parasitic Nematodes

Plant parasitic nematodes include several groups causing severe crop losses. The

most common genera are: Aphelenchoides (foliar nematodes), Meloidogyne (root-knot nematodes), Heterodera, Globodera (cyst nematodes) such as the potato cyst

nematode, Nacobbus, Pratylenchus (lesion nematodes), Ditylenchus, Xiphinema,Longidorus, and Trichodorus. Several phytoparasitic nematode species cause his-

tological damages to roots, including the formation of visible galls (Meloidogyne).Some nematode species transmit plant viruses through their feeding activity on

roots. One of them is Xiphinema index, vector of GFLV (grapevine fanleaf virus),

an important disease of grapes. Bt toxins, lectins (Burrows et al. 1999) and proteaseinhibitors have shown some promise for control, particularly the expression of

cystatins (Cowgill et al. 2002). For a recent review of the topic, the reader is

referred to Fuller et al. (2008).

1.2.5.4 Other Sources of Insecticidal Molecules

Generating insecticidal transgenic crops harboring genes from nonconventional

sources is an extremely active area, with amongst others, foreign genes from plants

(e.g., enzymes inhibitors and novel lectins) and animal sources including insects

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(e.g., biotin-binding proteins, neurohormones, venoms and enzyme inhibitors)

being a major focus (Ferry et al. 2006).

The development of second-generation transgenic plants with greater durable

resistance might result from the expression of multiple insecticidal genes such as

the Vip (vegetative insecticidal proteins) produced by B. thuringiensis during its

vegetative growth. The benefit of such an approach is a broader insect target range

than conventional Bt proteins and the proposed expectation to control current Btresistant pests due to the low levels of homology between the domains of the two

proteins classes (Christou et al. 2006).

With Bt toxins as the classical reference, toxins from other insect pathogens

provide a potential repository of novel insecticidal compounds. Photorhabdus spp.are bacterial symbionts of entomopathogenic nematodes, which are lethal to a wide

range of insects (Chattopadhyay et al. 2004). Photorhabdus toxin expression in

Arabidopsis caused significant insect mortality (see for review Ferry et al. 2006).

Thus, toxins from other insect pathogens are also opening up new routes to pest

control using transgenic-based strategies.

Interesting recent developments include the use of novel proteins from insect

biological control agents and insect hormones to generate transgenic crops. A

teratocyte secretory protein from a hymenopteran endoparasitoid (a parasitic

wasp often used in biocontrol programs) has been expressed in transgenic tobacco

and shown to increase resistance to lepidopteran pests (Maiti et al. 2003). Similar

protection has also been achieved with insect peptide hormones (Tortiglione et al.

2003). Interestingly, they replaced the tomato systemin peptide region of prosyste-

min (a plant signaling molecule) with the insect peptide and showed that this

resulted in the production of biologically active insecticidal peptides.

Reliance on the expression of a single gene product for pest control is a relatively

short-term strategy that parallels the use of exogenously applied chemical pesti-

cides. Thus, pyramiding (stacking) of genes encoding different Bt toxins has beendeveloped as a method for preventing the onset of evolution of pest resistance, and

for conferring greater levels of pest control (Boulter et al. 1990; Maqbool et al.

2001; Zhao et al. 2003). This strategy has now been adopted in commercially

available crops (see above). For example, corn lines have recently been developed

(Moellenbeck et al. 2001; Ostlie 2001) coexpressing two d-endotoxins from Bt forresistance to corn rootworm. Hybrid proteins have also been developed to enhance

and extend the activity of Bt toxins. The use of a single Bt toxin in a crop is limited

in that many insects attack a single crop and toxins generally show very high

specificity towards a single pest species. Therefore, toxins have been engineered

to modify their receptor recognition and pore formation. Each toxin consists of

three domains. Domain I is involved in membrane insertion and pore formation.

Domains II and III are both involved in receptor recognition and binding. Addi-

tionally, a role for domain III in pore function has been found. This approach has

proved successful in both enhancing activity (Karlova et al. 2005) and extending

host range (Singh et al. 2004). Such hybrid/fusion proteins offer an alternative/

complementary strategy to address potential limitations in conventional transgenic

insect pest control.

14 N. Ferry and A.M.R. Gatehouse

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1.2.5.5 Transgenic Plants Expressing Fusion Proteins

The concept of “gene stacking” has recently been extended to the development

and use of fusion proteins. Such proteins not only provide a means of increasing

durability, but also provide a “vehicle” for more effective targeting of insecti-

cidal molecules, including peptides. It thus offers an alternative/complementary

strategy to address potential limitations in conventional transgenic insect pest

control. For example, recognition of binding sites in the insect gut is an important

factor determining the toxicity of Bt. Enhancing toxin-binding capabilities shouldthus extend host range and delay resistance in pest populations. Bt is believed to

bind primarily to aminopeptidase N or cadherin membrane proteins, while the

generation of a fusion protein with the nontoxic B-chain from ricin (RB) was

shown to extend the binding of Bt to include specific glycoproteins. Transgenic

plants expressing the Bt fused RB demonstrated that the addition of the RB-

binding domain provided a wider repertoire of receptor sites within target species

and significantly enhanced the levels of toxicity of Bt. For example, survival of

the armyworm Spodoptera littoralis, a species of insect not sensitive to Bt, wasreduced by ~90% when feeding on transgenic maize expressing the fusion,

compared to plants expressing either Bt Cry1Ac alone, or the RB-binding

domain. Expression of the fusion protein resulted in the insect becoming suscep-

tible to Bt (Mehlo et al. 2005). This strategy has shown great potential beyond

just extending the toxicity of Bt. Zhu-Salzman et al. (2003) have generated fusion

proteins with anchor regions to other insecticidal proteins to the insect gut

epithelium. Using the legume lectin rGSII, they proposed a system to combat

the ability of certain insect species to activate protease inhibitor-insensitive

proteolytic enzymes. The soybean cysteine protease inhibitor soyacystatin N

(scN) was covalently linked to the GlcNAc-specific legume lectin using a natu-

rally occurring linker region from the potato multicystatin. In this instance, the

fusion protein not only has a novel binding ability that is proposed to initiate a

concentration effect by localizing the inhibitor at the anterior of the gut, but the

fused lectin moiety additionally offers a degree of protection to the insecticidal

moiety by blocking the access of scN-insensitive proteases, thereby preventing

proteolytic destruction of the cystatin.

Not only do fusion proteins have potential for use in transgenic crops, but also to

improve the efficacy of biopesticide-based sprays. Neuropeptides potentially offer a

high degree of biological activity, and thus provide an attractive alternative pest

management strategy. There are major drawbacks to their use, particularly as

topical sprays. They are unlikely to be rapidly absorbed through the insect cuticle

to their site of action, and are prone to proteolysis and rapid degradation in the

environment. Should they survive the application process and are then taken up by

the insect, they are then unlikely to survive the conditions of the insect gut or be

delivered to the correct targets within the insect. The discovery that snowdrop lectin

(GNA) remains stable and active within the insect gut after ingestion, and that it is

able to cross the gut epithelium, provided an opportunity for its use as a “carrier

molecule” to deliver other peptides to the circulatory system of target insect

1 Transgenic Crop Plants for Resistance to Biotic Stress 15

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species. This strategy effectively delivered the insect neuropeptide hormone, alla-

tostatin, to the haemolymph of the tomato moth Lacanobia oleracea (Fitches et al.

2002). Subsequent expression of the fusion protein in potato further provided proof

of concept for the efficacy of fusion proteins as a means of delivery. The results

demonstrated significant reduction in mean larval weight when compared to the

controls. GNA can be used to deliver insecticidal peptides isolated from the venom

of the spider Segestria florentina (SFI1) to the haemolymph of L. oleracea (Fitcheset al. 2004). Neither the GNA nor the SFI1 moieties alone were acutely toxic;

however, the SFI1/GNA fusion was insecticidal to first stage larvae, causing 100%

mortality after 6 days. This spider venom neurotoxin is believed to irreversibly

block the presynaptic neuromuscular junctures. Such venom toxins show high

degrees of specificity and thus lend themselves to environmentally benign pest

management strategies.

1.2.6 Manipulation of Plant Endogenous Defenses

Alternative strategies for protecting crops from insect pests, that are not dependent

on the expression of single or stacked genes, seek to exploit the induced endoge-

nous resistance mechanisms exhibited by plants to most insect herbivores. For

further information, the reader is referred to Chap. 10 of this volume. Such induced

defenses are exemplified by the wounding response, first identified as the local and

systemic synthesis of proteinase inhibitors (PIs), which block insect digestion in

response to plant damage (Gatehouse 2002a, b). Many transgenic strategies have

attempted to exploit the potential overexpression of plant PIs to protect crops from

pest damage (Jouanin et al. 1998) but these have relied on the transfer of a single PI

gene, and many insects have been able to adapt to this.

More recent research has shown that induced defenses also involve the plant’s

ability to produce toxic or repellent secondary metabolites as direct defenses,

and volatile molecules, which play an important role in indirect defense (Kessler

and Baldwin 2002). Insect herbivores activate induced defenses both locally and

systemically via signaling pathways involving systemin, jasmonate, oligogalacturo-

nic acid and hydrogen peroxide (Fig. 1.4).

Ecologists have long understood that plants exhibit multi-mechanistic resistance

towards herbivores, but the molecular mechanisms underpinning these complicated

responses have remained elusive (Baldwin et al. 2001). However, recent studies

investigating the plant’s herbivore-induced transcriptome, using microarrays and

differential display technologies, have provided novel insights into plant–insect

interactions. The jasmonic acid cascade plays a central role in transcript accumula-

tion in plants exposed to herbivory (Hermsmeier et al. 2001). A single microarray-

based study revealed that the model plant Arabidopsis undergoes changes in levels

of over 700 mRNAs during the defense response (Schenk et al. 2000). In contrast,

only 100 mRNAs were upregulated by spider mite (Tetranicus urticae) infestationin lima bean (Phaseolus lunatus), although a further 200 mRNAs were upregulated

16 N. Ferry and A.M.R. Gatehouse

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in an indirect response mediated by feeding-induced volatile signal molecules

(Arimura et al. 2000).

Deciphering of the signals regulating herbivore-responsive gene expression will

afford many opportunities to manipulate the response. Signaling molecules such as

salicylic acid, jasmonic acid and ethylene do not activate defenses independently by

linear cascades, but rather establish complex interactions that determine specific

responses. Knowledge of these interactions can be exploited in the rational design

of transgenic plants with increased insect resistance (Rojo et al. 2003; De Vos et al.

2005; Giri et al. 2006).

1.2.6.1 A Special Case: Sap-Feeding Insects

While most herbivorous insects cause extensive damage to plant tissues when

feeding, many insects of the order Homoptera feed from the contents of vascular

tissues by inserting a stylet between the overlying cells, thus limiting cell damage

and minimizing induction of a wounding response. In contrast to wounding, plant

responses following attack by these insects have been shown to be typical of pathogen

attack, with examples of gene-for-gene interactions being known (Walling 2000;

lategenes

(defence)

earlygenes -

signalling

ABA

SA

auxin, SA (-)ethylene, ABA (+)

Herbivory

local peptide hormone release eg.

prosystemin

systemin

receptor binding

linolenic acid

octadecanoid pathway

jasmonic acid

vascular bundle

mesophyllcells

H202

NAPDHoxidase

Insecticidalcompoundse.g. ProteaseInhbitors

plant-plantinteractions(systemicinduction ofPls)

Voltiles e.g.-green leafvolatiles-C10, C15terpenoids-indole

pest deterrent/attraction ofnaturalenemies

methyljasmonate

plant-plantinteractions

polygalactouronidaseoligalacturonic acid

physical forces

ethyleneinsect dervived elicitors

Cel

l wal

l

Fig. 1.4 The generalized plant-wounding response. Generalized overview of the plant wounding

response, and signalling molecules which can modulate it, showing the pathways necessary for

both local and systemic induction of insecticidal proteins. (Adapted from: Ferry et al. 2004)

1 Transgenic Crop Plants for Resistance to Biotic Stress 17

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Moran et al. 2002). However, these pathogen-induced pathways can induce expres-

sion of many of the genes upregulated by wounding because of pathway cross-talk.

Moran and Thompson (2001) demonstrated that phloem feeding by the green

peach aphid (Myzus persicae) on Arabidopsis induced expression of genes asso-

ciated with salicylic acid (SA) responses to pathogens, as well as a gene involved in

the jasmonic acid-mediated response pathway. These results suggest stimulation of

response pathways involved in both pathogen and herbivore responses. Microarray

data has identified genes involved in oxidative stress, calcium-dependent signaling,

pathogenesis-related responses, and signaling as key components of the induced

response (Moran et al. 2002). It may be that transgenic strategies that activate such

signaling cascades could enhance plant resistance to these problematic pests.

1.2.6.2 Indirect Defense (Volatile Production)

The role of plant volatiles in indirect defense has been described as “top-down”

defense (Baldwin et al. 2001). Some volatiles appear to be common to many

different plant species, including C6 aldehydes, alcohols and esters (green leaf

volatiles), C10 and C15 terpenoids, and indole, whereas others are specific to a

particular plant species. Many volatiles are preformed and act in herbivore deter-

rence; furthermore, the wounding response also includes the formation of volatile

compounds. Top-down control of herbivore populations is achieved by attracting

predators and parasitoids to the feeding herbivore, mediated by these volatile

organic compounds (VOCs). For example, genes involved in the biosythesis of

the maize VOC bouquet are upregulated by insect feeding (Frey et al. 2000; Shen

et al. 2000). In addition, herbivore oviposition has been shown to induce VOC

emissions, which attract egg parasitoids (Hilker and Meiners 2002). Herbivore-

induced VOCs can also elicit production of defence-related transcripts in plants

near the individual under attack (Arimura et al. 2000; Dicke et al. 2003). Exposure

to herbivore-induced volatiles in lima bean results in transcription of genes

involved in ethylene biosynthesis (Arimura et al. 2000).

Manipulation of volatile biosynthesis can affect insect resistance. Transgenic

potatoes in which production of hydroperoxide lyase (the enzyme involved in green

leaf volatile biosynthesis) was reduced were found to support improved aphid

performance and fecundity, suggesting toxicity of these volatiles to M. persicae(Vancanneyt et al. 2001). In a review of the topic Degenhardt et al. (2003) discuss

the potential of modifying terpene emission with the aim of making crops more

attractive to herbivore natural enemies.

1.2.6.3 Detoxification and Insect Modulation of the Wounding Response

However, insect pests are able to feed on plants despite their defenses, both

constitutive and inducible. Many insects are able to detoxify potentially toxic

secondary metabolites, using cytochrome P-450 monoxygenases and glutathione-

S-transfereases. These enzymes are induced by exposure to toxic plant secondary

18 N. Ferry and A.M.R. Gatehouse

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compounds, for example, xanthotoxin (a furanocoumarin) induces P-450 expres-

sion in corn earworm (Li et al. 2000). More recently, Li et al. (2002) have shown

that corn earworm uses signaling molecules from its plant host, jasmonate and

salicylate, to activate four of its cytochrome P450 genes, thus making the induction

of detoxifying enzymes rapid and specific. Recent strategies based on RNAi

technology have shown that it is possible to overcome these insect responses

(discussed later in this chapter).

1.2.7 RNAi

Disrupting gene function by the use of RNAi is a well-established technique in

insect genetics based on delivery by injection into insect cells or tissues. The

observation that RNAi could also be effective in reducing gene expression,

measured by mRNA level, when fed to insects (Turner et al. 2006) has led to two

recent articles in which transgenic plants producing double-stranded RNAs

(dsRNAs) which are shown to exhibit partial resistance to insect pests. Transgenic

maize producing dsRNA directed against V-type ATPase of corn rootworm showed

suppression of mRNA in the insect and reduction in feeding damage compared to

controls (Baum et al. 2007). Similarly, transgenic tobacco and Arabidopsis expres-sing dsRNA directed against a detoxification enzyme (Cytochrome P450 gene

CYP6AE14) for the breakdown of gossypol (a defensive metabolite) in cotton

bollworm caused the insect to become more sensitive to gossypol in the diet

(Mao et al. 2007). This approach holds great promise for future development. It

is also proving effective for nematode control (Bakhetia et al. 2005). For a recent

review on RNAi-mediated crop protection against insect pests the reader is referred

to Price and Gatehouse (2008).

Advances in our understanding of induced responses in plants and their regula-

tion, has refocused attention on potential exploitation of endogenous resistance

mechanisms for crop protection. While plant resistance is an integral component of

organic and IPM strategies, it does not afford similar levels of protection as those

provided by the use of “direct” protective methods such as the expression of Bttoxins. The goal of the plant breeder, and now the biotechnologist, is to engineer

durable multi-mechanistic resistance to insect pests in crops, and increased know-

ledge of induced defense mechanisms and their molecular control is likely to play

an important role in realizing this aim.

1.2.8 Environmental Impact of IR Crops

Almost from the beginning of the production of transgenic crops there have been

concerns over their use and introduction into the environment. There is interna-

tional agreement that GM crops should be evaluated for their safety, including their

environmental impact (Dale 2002). During the past 15–20 years, there have been

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extensive research programs of risk assessment, with several areas of major concern

identified.

1.2.8.1 Impact on Nontarget Organisms

Assessing the consequences of pest control on nontarget organisms is an important

precursor to their becoming adopted in agriculture. The expression of transgenes

that confer enhanced levels of resistance to insect pests is of particular significance

since it is aimed at manipulating the biology of organisms in a different trophic

level to that of the plant. Potential risks to beneficial nontarget arthropods exist.

Those groups most at risk include: nontarget Lepidoptera, beneficial insects (polli-

nators, natural enemies) and soil organisms.

Exposure of nontarget Lepidoptera to insecticidal transgene products may occur

through both direct consumption of transgenic plant tissues including via consump-

tion of transgenic pollen; many nontarget Lepidoptera are rare butterflies having

great conservation value. The case of the Monarch butterfly (Danaus plexippus), aconservation flagship species in the US, highlighted the need for ecological impact

research. In a letter to Nature, Losey et al. (1999) claimed that both survival and

consumption rates of Monarch larvae fed milkweed leaves (natural host) dusted

with Bt pollen were significantly reduced, and that this would have profound

implications for the conservation of this species. However, a series of ecologically

based studies rigorously evaluated the impact of pollen from such crops on Mon-

archs and demonstrated that the commercial wide-scale growing of Bt-maize did

not pose a significant risk to the Monarch population (Hellmich et al. 2001;

Gatehouse et al. 2002). In fact, the initial experiments did not quantify the dose

of pollen used, or indeed, if this was a realistic level likely to be encountered in the

field; nevertheless, this work highlighted the importance of studying nontarget

effects. In a separate field studyWraight et al. (2000) showed that Papilio polyxenes(black swallowtail) larvae were unaffected by pollen from Bt expressing maize

event Mon810 at 0.5, 1, 2, 4 and 7 m from the transgenic field edge, highlighting the

need for a case-by-case study of organisms considered to be at risk. In addition to

the potential direct impacts of Bt toxins on susceptible target insects, as in the case

of the Morarch butterfly, some Lepidoptera have been shown to have a reduced

sensitivity to the lepidopteran-specific Bt toxins. For example, S. littoralis can

survive on maize expressing Cry1Ab (Hilbeck et al. 1998) and thus present a

route of exposure to the next trophic level. In the case of Bt Cry3Aa or Cry3Bb

expressing potatoes or maize, some Lepidoptera may represent nontarget secondary

pests, and whilst not directly affected by the transgene product themselves may

again present a route of exposure to the next trophic level, as do other nontarget

herbivores. Organisms such as those belonging to the orders Homoptera, Hemi-

ptera, Thysanoptera, and Tetranychidae are not targeted by Bt toxins expressed in

transgenic plants; however, they do utilize the Bt crop (Groot and Dicke 2002). Thedirect effect that this may have on these insects is dependent on the presence of Btreceptors in the first instance, and it is so far unclear whether such receptors are

20 N. Ferry and A.M.R. Gatehouse

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present in nontarget organisms (de Maagd et al. 2001). In addition, the fate of the

toxin ingested by nontarget herbivores is unclear, since if it retains toxicity then this

may have implications at the next trophic level.

The impacts of insect-resistant transgenic crops at higher trophic levels have also

been considered, where there are concerns over the risks to beneficial arthropod

biodiversity (Schuler et al. 1999; Bell et al. 2001); in particular, predators and

parasitoids, which play an important role in suppressing insect pest populations

both in the field and under specialized cultivation systems (glasshouses). Natural

enemies may ingest transgene products via feeding on herbivorous insects that have

themselves ingested the toxin from the plant; such tritrophic interactions will be

influenced by the susceptibility of the herbivore to the plant protection product. If,

as in the case with Bt toxins, the prey item is susceptible to the toxin, then the

predator will not come into contact with the toxin as the pest will effectively be

controlled, and in target insects the toxin is bound to receptors in the midgut

epithelium that are structurally rearranged and may lose their entomotoxicity (de

Maagd et al. 2001). In nontarget insects (and resistant insects), the toxins do not

bind and may thus retain biological activity. However, the overwhelming weight of

evidence from independent laboratory and field studies show that Bt toxins have alimited ability to affect the next trophic level (reviewed in Sanvido et al. 2007;

Ferry and Gatehouse 2009; Romeis et al. 2008).

Pollinators represent another group of nontarget organisms highlighted as at risk

from Bt toxins in GM crops. The current generation of transgenic crops produce Bttoxin in the pollen as well as in the vegetative tissues. Several studies have been

conducted to determine toxicity of Bt toxins to pollinators (Vandenberg 1990; Sims

1995, 1997; Arpaia 1997; Malone and Pham-Delegue 2001); generally, they all

conclude that neither the adults nor the larvae of bees were affected by Bt toxins. Fora comprehensive review of the impact of transgenic crops on pollinators, the reader

is referred to two recent reviews (Malone et al. 2008; Malone and Burgess 2009).

Finally, nontarget species may come into contact with Bt toxins via the environ-ment. Several studies have shown that Bt toxins released from transgenic plants

bind to soil particles (Palm et al. 1996; Crecchio and Stotzky 1998; Saxena et al.

1999). Soil-dwelling and epigeic insects such as Collembola and Carabidae may

thus be exposed to the toxins. Several studies (Saxena and Stotzky 2001; Ferry et al.

2007) show no differences in mortality or body mass of bacteria, fungi, protozoa,

nematodes and earthworms or carabid beetles exposed to Bt.Exposure to the transgene products, however, does not necessarily imply a

negative impact. Most studies to date have demonstrated that crops transformed

for enhanced pest resistance have no deleterious effects on beneficial insects

(reviewed in Ferry et al. 2003; Romeis et al. 2008).

1.2.9 Conclusions

Ultimately one must consider the impact of transgenic crops and specifically Bttoxins in comparison to other pest control strategies such as conventional crop

1 Transgenic Crop Plants for Resistance to Biotic Stress 21

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protection using insecticides. While pesticides have no doubt brought vast yield

improvements, they have well-documented undesirable nontarget effects (Devine

and Furlong 2007). It is worth remembering that whilst potential risks do exist to

the environment from the cultivation of GM crops, their potential to decrease

reliance on external inputs (less insecticide sprays) and to increase the availability

of genetic resources available to breeders is great (Ferry and Gatehouse 2009).

1.3 Biotic Stress due to Weeds

1.3.1 Crop Losses due to Weeds

Weeds can compete with productive crops or pasture, or convert productive land

into unusable scrub. Weeds are also often poisonous, distasteful, produce burrs, or

thorns that interfere with the use and management of desirable plants by contam-

inating harvests or excluding livestock. Weeds tend to thrive at the expense of the

more refined edible or ornamental crops. They provide competition for space,

nutrients, water and light, although how seriously they will affect a crop depends

on a number of factors. Some crops have greater resistance to competition than

others, for example smaller, slower-growing seedlings are more likely to be over-

whelmed than those that are larger and more vigorous. Weeds also differ in their

competitive abilities, and this can vary according to specific conditions and the time

of year. Tall-growing vigorous weeds such as fat hen (Chenopodium album) canhave the most pronounced effects on adjacent crops. Chickweed (Stellaria media),a low-growing plant, can happily coexist with a tall crop during the summer, but

plants that have overwintered will grow rapidly in early spring and may swamp

crops.

Simply put, weeds are any plant growing in an area where it is not wanted. Of

over 250,000 plant species in the world, only a few hundred are troublesome weeds.

Although no single characteristic clearly defines a weed, two attributes are common

in the worst weeds: competitiveness and persistence. The world’s worst weeds are

shown in Table 1.2 (Chrispeels and Sadava 2003).

1.3.2 Weed Management: Prevention, Control, and Eradication

As weeds are persistent, once they have established in a field they are extremely

difficult to eradicate. One of the most successful attempts in the eradication of a

weed is provided by witchweed (Striga sp.) in the US. Witchweed is a parasitic

plant; its seeds germinate in response to a chemical, strigol, produced by the roots of

the host plant. The germinated seedling attaches to the root through special haus-

toria, as this occurs underground infestation can go unnoticed, consequently serious

22 N. Ferry and A.M.R. Gatehouse

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infestation leading to yield loss occurs before the parasitic plant even appears

through the soil. Once the witchweed comes through the soil it becomes photosyn-

thetically active, but still dependent on its host for water. Such parasitic weeds are

extremely difficult to control and yield losses of approximately 50% can be

expected under drought conditions. In the US, only quarantine of infected areas

reduced witchweed infection from 150,000 hectares to a couple of 1,000 hectares,

but this quarantine lasted for nearly 50 years (Chrispeels and Sadava 2003)!

Table 1.2 World’s worst weeds (from: Chrispeels and Sadava 2003)

Plant family Weed examples Crop

Grass (Poaceae) Bermuda grass (Cynodon dactylon) Wheat

Barnyard grass (Echinochloa crus-galli)Johnson grass (Sorghum halepense)

Sedge (Cyperaceae) Purple nutsedge (Cyperus rotundus) None

Yellow nutsedge (Cyperus esculentus)Smallflower umberella sedge (Cyperus

difformis)Sunflower (Asteraceae) Canada thistle (Cirsium arvense) Sunflower

Common cocklebur (Xanthium strumarium)Common dandelion (Taraxacum officinale)

Buckwheat (Polygonaceae) Wild buckwheat (Polygonum convolvulus) Buckwheat

Curly dock (Rumex crispus)Red sorrel (Rumex acetosella)

Pigweed (Amaranthaceae) Smooth pigweed (Amaranthus hybridus) Grain

amaranth

Spiny amaranth (Amaranthus sinosus)Redroot pigweed (Amaranthus retroflexus)

Mustard (Brassicaceae) Shepherd’s purse (Capsella bursa-pastoris) Canola

Hoary cress (Brassica draba)Wild mustard (Brassica kaber)

Legume (Fabaceae) Black medic (Medicago lupulina) Soybean

Sensitive plant (Mimosa pudica)Kudzu (Pueraria lobata)

Morning glory

(Convolvulaceae)

Field bindweed (Convolvulvus arvensis) Sweet potato

Field dodder (Cuscuta campestris)Swamp morning glory (Ipomoea aquatica)

Spurge (Euphorbiaceae) Leafy spurge (Euphorbia esula) Cassava

Garden spurge (Euphorbia hirta)Spotted spurge (Euphorbia maculata)

Goosefoot (Chenopodiaceae) Common Lamb’s quarters (Chenopodiumalbum)

Sugarbeet

Russian thistle (Salsola iberica)Nettleleaf goosefoot (Chenopodium murale)

Mallow (Malvaceae) Venice mallow (Hibiscus trionum) Cotton

Velvetleaf (Abutilon theophrasti)Arrowleaf sida (Sida rhombifolia)

Nightshade (Solanaceae) Black nightshade (Solanum nigrum) Potato

Jimsonweed (Datura stramonium)Cutleaf groundcherry (Physalis angulata)

1 Transgenic Crop Plants for Resistance to Biotic Stress 23

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For this reason, preventative weed management is critical. This involves keeping

weed seeds and vegetative propagules from getting to the field in the first place and

is mainly achieved through seed cleaning and the purchase of certified clean seed.

Good management practice and the cleaning of farm equipment moved between

different areas are critical in preventative control. Ultimately weed control is

achieved by mechanical, biological and predominately chemical means.

1.3.3 Herbicides

The economic importance of weeds is emphasized by the fact that the herbicides

comprise as large a share of the world agrochemical market as all other pesticides

combined, farmers spend an annual US$10 billion to control weeds in the US alone

(Chrispeels and Sadava 2003). Herbicides have evolved over time, and herbicide

use has seen a move towards more biodegradable chemicals that only need to be

applied at a very low concentration of active ingredient. Thus, the new generation

of herbicides are relatively environmentally benign. Many herbicides exploit the

differences in plant physiology between the crop species and its weeds (usually the

differences between monocots and dicots); they may be systemic or act on contact

(Table 1.3). Major crops such as wheat, rice, and maize; and legumes such as

soybean, common bean, and peanut come from the same plant families as their

weeds, thus creating a control problem.

The mode of action of common herbicides is varied (Naylor 2002). For example,

inhibition of photosynthesis and light-dependent membrane destruction (acting on

photosystems II and I, respectively) are the mode of action of the foliar-acting

nonselective herbicides like atrazine, paraquat and diquat. Hormone herbicides,

such as 2,4-dichlorophenoxyacetic acid (2,4-D) induce abnormal plant growth by

interring with auxin regulation. The sulfonyl ureas, imidazolines and the environ-

mentally benign, but nonselective glyphosate (acting on 5-enolpyruvylshikimate-

3-phosphate synthase) inhibit amino acid synthesis. Others include inhibitors of

lipid synthesis, inhibition of cell division and pigment synthesis. The advantages of

herbicides are clear – they control multiple weed species, control perennial weeds,

cause no injury to the crop plant, and can readily be applied to large areas.

Table 1.3 Herbicide modes of action

Herbicide type

Contact herbicides destroy only that plant tissue in contact with the chemical spray. Generally,

these are the fastest-acting herbicides. They are ineffective on perennial plants that are able to

regrow from roots or tubers

Systemic herbicides are foliar-applied and are translocated through the plant and destroy a greater

amount of the plant tissue. Modern herbicides such as glyphosate are designed to leave no

harmful residue in the soil

Soil-borne herbicides are applied to the soil and are taken up by the roots of the target plant

Pre-emergent herbicides are applied to the soil and prevent germination or early growth of weed

seeds

24 N. Ferry and A.M.R. Gatehouse

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Nonchemical weed control includes: cultural weed control practices such as

intercropping and rotation; mechanical removal of weeds; and biological control,

all of which are labor intensive and unpredictable. Chemical treatment is by far the

most effective method, but it is not without problems.

1.3.3.1 Resistance to Herbicides

A common consequence of repeated use of herbicide application is the evolution of

resistance in weed populations because of strong selection pressure. The first

example of a problematic herbicide resistant weed was reported in the 1970s

(Chrispeels and Sadava 2003) when common groundsel resistant to triazine was

identified in the US. The mechanism of herbicide resistance is usually a change in

the target site with a reduction in herbicide affinity – this may be achieved through a

single point mutation and alteration of a single amino acid. By 2001, over 150 weed

species had resistance to at least one herbicide. Some biotypes of weeds have

resistance to herbicides with different modes of action.

Rigid Ryegrass

When a weed evolves resistance to a particular herbicide, the farmer is forced to use

an alternative control strategy. This often involves the use of another herbicide with

a different mode of action. Subsequent selection pressure may lead to a weed

population acquiring multiple herbicide resistance. In nearly all the cases so far,

resistance to only one or two chemical families has been reported. Rigid ryegrass is

a notable exception. In the most severe cases, biotypes exist that are resistant to nine

chemical families with five different modes of action! Resistance is conferred by

multiple mechanisms; both altered sites of action and the ability to metabolize

particular herbicides, via nonspecific monoxygenases.

1.3.4 Transgenic Crops for Weed Control

The adoption of transgenic herbicide-resistant (HR) crops has made remarkable

changes to global agriculture within the last decade. Currently, an estimated 114.3

million hectares of transgenic crops are planted throughout a variety of agroeco-

systems in 23 developing and industrial countries. Approximately, 90% of the land

area with transgenic crops includes a trait for glyphosate resistance (GR) (Owen

2008).

While there are a number of herbicide-tolerant genetically modified crops that

have been developed for several herbicides with different modes of phytotoxic

action, the primary influence in world agriculture are glyphosate-resistant crops

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(GRCs) (Duke 2005; Duke and Powles 2008). More specifically, of the 23 countries

that grow plant glyphosate-resistant crops, the US, Canada, Argentina and Brazil

are the countries that account for most hectares (Owen 2008). In these countries, the

principle GRCs planted include maize (Zea mays), soybean (Glycine max), cotton(G. hirsutum), and canola (Brassica napus). For further information, the reader is

referred to Chap. 3 of this volume.

1.3.4.1 Round-up1 Ready Crops

Glyphosate (Round-up1) is a highly effective broad-spectrum herbicide that

inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a branch point

enzyme in aromatic amino acid biosynthesis. A naturally occurring EPSP synthase

gene (cp4) was identified from Agrobacterium sp. strain CP4, whose protein

product provided glyphosate tolerance (Fig. 1.5) in plants (Padgette et al. 1995).

COO–

OH

OH

OP

COO–

OH

OP O COO–

CH2

COO–

OH

O COO–

CH2

COO–

OP CH2+

SHKP

H3PO4

shkG

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase)

phosphoenol-pyruvate

(PEP)

H3PO4

shkH

5-enolpyruvylshikimate-3-phosphate (EPSP )

chorismate synthase

chorismate (CHA)

GLYPHOSATE

Fig. 1.5 Glyphosate; mode of action. Glyphosate kills plants by interfering with the synthesis of

the amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme

5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shiki-

mate-3-phosphate (SHKP) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phos-

phate (EPSP). EPSP is subsequently dephosphorylated to chorismate, an essential precursor in

plants for the aromaticamino acids: phenylalanine, tyrosine and tryptophan. These amino acids

are used as building blocks in peptides, and to produce secondary metabolites such as folates,

ubiquinones and naphthoquinone

26 N. Ferry and A.M.R. Gatehouse

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Furthermore, glyphosate detoxification pathways are known in microbes involving

glyphosate oxidoreductase (gox) genes (Jacob et al. 1988). These two genes confer

glyphosate resistance in selected crops.

Crops engineered for resistance to this broad-spectrum herbicide allow control

of multiple weeds without injury to the main crop. The economic benefits to farmers

of glyphosate-resistant crops are estimated at US$17.5 billion, mainly accrued from

reduced sprays and lower inputs of labor. In addition, herbicide-tolerant crops

reduce the amounts of active ingredient required for weed control; they also

promote the usage of no/low till farming and thus lower fuel consumption with

direct benefits to both soil structure and carbon emissions (James 2007).

1.3.4.2 Other Transgenic Herbicide-Tolerant Crops

Traits for resistance to three other classes of herbicides have been developed, but

have not reached the same level of popularity as glyphosate resistance. Resistance

to oxynil herbicides conferred by the BXN nitrilase from Klebsiella pneumoniae(subsp. ozaenae) (Stalker et al. 1988) was the first trait engineered in cotton

(developed by Calgene, Davis, CA [now Monsanto]). Because glyphosate is less

expensive and controls more weed species, interest in using the oxynil herbicides

has waned and 2004 was the final year of BXN1 cotton sales. BXN canola was

commercialized by Rhone-Poulenc Canada (now Bayer Crop Science, Monheim,

Germany) and subsequently discontinued. Phosphinothricin acetyltransferase (PAT

or BAR) detoxifies phosphinothricin- or bialaphos-based herbicides (glufosinate).

The pat gene is native to Streptomyces viridichromogenes and bar is from

S. hygroscopicus where they act in both the biosynthesis and detoxification of the

tripeptide bialaphos (De Block et al. 1987). Like glyphosate, phosphinothricin

herbicides control a broad spectrum of weed species and break down rapidly in

the soil so that the problems with residual activity and environmental impact are

greatly reduced. Bayer CropScience markets this trait as LibertyLink1 in several

species. The pat and bar genes are also popular plant transformation markers in the

research community. Finally, BASF (Ludwigshafen, Germany) markets nontrans-

genic CLEARFIELD1 imidazolinone-resistant canola, wheat, sunflower, corn,

lentils, and rice, while DuPont (Wilmington, DE) markets nontransgenic STS1

soybeans with tolerance to sulfonylurea herbicides. A sulfonylurea-tolerant flax

variety called CDC Triffid, developed by the University of Saskatchewan (Canada),

was grown commercially in Canada in 2000 but is no longer offered. All these crops

contain mutagenized versions of the acetohydroxyacid synthase, also called acet-

olactate synthase (ALS), which are not inhibited by imidazolinone and/or sulfonyl-

urea herbicides (Devine and Preston 2000). Herbicides that inhibit ALS are

considered low or very low use-rate herbicides with a good spectrum of weed

control and are likely to remain an important part of weed resistance management

programs (Castle et al. 2006).

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1.3.5 HT Canola

There are two GM traits for herbicide resistance in canola: glyphosate and glufo-

sinate resistance. Resistance to glyphosate herbicides is conferred by the genes

5-enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4

epsps) (event GT73, Monsanto) that is targeted to the chloroplast, and a glyphosate

oxidoreductasegene from Achromobacter sp. strain LBAA (gox v247) (both

Roundup Ready1). Resistance to phosphinothricin herbicides is conferred by

phosphinothricin-acetyltransferance (pat gene), and marketed as LibertyLink1

by AgrEvo (now Bayer CropScience).

Herbicide-resistant canola (oilseed rape) allows post-emergence application of a

single herbicide with a wide spectrum of activity. For effective control, it has to be

applied before the weeds reach 10 cm height. This extends the potential time period

for spraying (Benbrook 2004). The timing is more flexible and the application of a

single herbicide simplifies weed control (Firbank and Forcella 2000). Since many

herbicides allow post-emergence applications, HT crops do not generally provide a

new option. However, post-emergence spraying with non-GMHT oilseed rape is

confined to a short 3–5 week period after crop emergence (Pallutt and Hommel

1998). Thus, GMHT canola offers greater flexibility (Owen 1999).

With HT oilseed rape, the intention is to reduce the amount of active ingredient

of herbicide used and to rely preferably on one broad-spectrum herbicide only

(http://www.canola-councildemo.org). The intent is also to reduce the number of

spraying rounds, which helps reduce soil compaction and erosion (Madsen et al.

1999). During the first years of cultivating GMHT oilseed rape, most farmers had

reduced active ingredient rates and applications (Champion et al. 2003). GMHT

oilseed rape was usually sprayed only once, with an average active ingredient

amount that was either lower (Champion et al. 2003) or not significantly different

from conventional oilseed rape (Schutte et al. 2004). However, in Canada (where

HT canola is widely cultivated), application of herbicide was seen to actually

increase (Canola Council of Canada 2001). In fact, after years of continued

GMHT oilseed rape cultivation, secondary adverse effects on application rates

were reported. This was due to weeds becoming herbicide tolerant on- and off-

site (Devos et al. 2004; Hayes et al. 2004). HT oilseed rape volunteers occurred in

subsequent rotations (Devos et al. 2004), and multiple tolerances of volunteers to

herbicides were recorded because of seed impurities and seed banks after out-

crossing events between fields (Hall et al. 2000). Outcrossing to weedy relatives

also occurred (Daniels et al. 2005); these weedy relatives, however, have not yet

been proved to be fit enough to persist on cultivated fields (Hails and Morley 2005).

Nevertheless, improved weed suppression with HT oilseed rape in many agro-

nomic respects has been demonstrated (Westwood 1997; Bohan et al. 2005), and

ultimately the technology remains popular with farmers because of its simplicity

and reduced costs (Canola Council of Canada 2001; Graef et al. 2007). Genes have

always moved between the natural and agroecosystem, and to date – despite the

formation of hybrids between HT canola and wild relatives in Canada (Gressel

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2008) – the technology has proven safe and effective (Cerdeira and Duke 2006;

Darmency et al. 2007; James 2007; Gressel 2008).

1.3.6 HT Maize

C. album, Amaranthus retroflexus, Abutilon theophrasti and several annual grass

species cause economic losses in maize, if left uncontrolled. The option to use

glyphosate in these systems is extremely valuable because it is a broad-spectrum

herbicide that provides excellent control of a wide range of weed species. Resis-

tance to glyphosate-herbicides in maize is conferred by the epsps gene in event

GA21 (the GA21 trait for glyphosate-resistant maize relies on a modified maize

epsps gene) (Roundup Ready), developed by DeKalb (now Monsanto) in 1998.

This is now being largely replaced by event NK603, Roundup Ready corn 2, with

two epsps expression cassettes under the transcriptional regulatory control of the

rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced cauliflower mosaic virus

35S (P-e35S) promoters to impart fully constitutive expression. Maize has also

been developed with resistance to phosphinothricin herbicides via transformation

with the pat gene in events T14 and T25, developed by Aventis (now Bayer

CropScience), and marketed as LibertyLink.

Maize is an open-pollinated, wind-facilitated species and gene flow via pollen is

well recognized (Haslberger 2001). Thus, the movement of GM traits is a signifi-

cant consideration in maize production (Luan et al. 2001; Ma et al. 2004). Gener-

ally, the introgression of GR traits in seed maize can be managed successfully

(�1% outcross) by establishing isolation distances of 200 m between fields (Ma

et al. 2004). However, in typical maize production regions of the US, these isolation

distances are not possible and GR trait introgression into non-GR fields is prevalent.

The occurrence of the GR transgene in non-GM maize can have significant

economic consequences, if the grower of the non-GM maize has a contract to

provide a GM-free product. Furthermore, incidences of GM gene introgression in

local landraces of maize in Oaxaca, Mexico have been reported (Quist 2001). The

implications for transgene occurrence reflect concerns for the maintenance of the

genetic resource of the landrace maize. However, the initial report of transgene

introgression was followed by a second report that suggested that no transgenes

existed in these landraces of maize (Ortiz-Garcia et al. 2005). Regardless, given the

adoption of GR maize, the discovery of transgene introgression into landrace maize

is likely in the longer term.

1.3.7 HT Cotton

Cotton is a slow-growing plant, and only a limited selection of herbicides can be

used for weed control. These two factors sometimes make weed control difficult.

Cotton is a semitropical, perennial plant, although it is grown as an annual crop.

1 Transgenic Crop Plants for Resistance to Biotic Stress 29

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Its growth is especially slow in the northern cotton belt in the US, where it is often

planted early into cool soils. Post-emergence broadleaf control is accomplished

with herbicides that can injure cotton but that are applied in a directed spray that

misses most of the cotton plant; thus, weed control in cotton is particularly difficult.

Cotton with resistance to glyphosate herbicides has been developed based on the

expression of the CP4 epsps in eventsMON1445/1698,Monsanto, Roundup Ready1.

Resistance to phosphinothricin herbicides is based on the expression of the bargene in LLCotton25, Bayer CropScience LibertyLink1. Herbicide-tolerant cotton

expanded from around 2% of the cotton acreage in 1996 to 26% in 1998, and

reached 46% in 2000 (James 2001). In 2008, adoption of GM cotton in the US with

either or both herbicide tolerance and Bt reached 86% (James 2007).

There are limited reports that cotton demonstrates introgression at low frequen-

cies (Ellstand et al. 1999). Pollen movement in cotton is dependent on insects.

Cotton is predominantly self-pollinated and natural outcrossing is typically quite

low (Xanthopoulos and Kechagia 2000). Thus, there is a very low probability that

the GR transgene would move into non-GR cotton cultivars via pollen flow. While

gene flow via GR cotton seed can occur, the consequences of this are not thought to

be important.

1.3.8 HT Soybean

Soybean engineered for resistance to glyphosate herbicides has been developed by

Monsanto with the CP4 epsps gene, event GTS-40-3-2 and is marketed as Roundup

Ready1.

Glyphosate-resistant soybeans were one of the earliest transgenic crops brought

to market and they have experienced rapid adoption to the point that over 85% of

US soybeans and 56% of soybeans globally are now glyphosate-resistant (James

2007). Soybeans are an autogamous (self-fertilizing) species with limited opportu-

nity for pollen-directed gene flow (Palmer et al. 2001). Spontaneous gene flow in

cultivated soybeans ranges from 0.02 to 5% depending on distance and is facilitated

by thrips (Thrips tabaci) and honeybees (Apis mellifera). While the movement of

the GR transgene has been observed in soybeans, there are extremely limited

opportunities for this occurrence and pollen-mediated gene flow in GR soybeans

is essentially a nonissue (Abud et al. 2004; Owen 2005). However, gene flow by

seed is highly probable and represents a significant economic problem (Swoboda

2002; Owen 2005).

1.3.9 Other GMHT Crops

In terms of commercial crops, glyphosate-resistant alfalfa had been developed and

was launched in 2006 (James 2007).

30 N. Ferry and A.M.R. Gatehouse

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However, of special interest genetically engineered herbicide-resistant crops are

being tested as a solution to the parasitic plants Orobanche spp. (broomrapes) and

Striga spp. (witchweeds), which parasitize the roots of important crops, heavily

reducing yields. Joel et al. (1997) showed that the use of three target-site resistances

decimatedOrobanche, demonstrating the potential of transgenic herbicide-resistant

crops in the control of parasitic weeds. This is potentially of great importance as

every year parasitic plant damage to crops accounts for an estimated US$7 billion in

yield loss in sub-Saharan Africa, and affects the welfare and livelihood of over 100

million people. For this technology to have a great impact in Africa, crops adapted

to African agroecologies need to be available to farmers.

1.3.10 Changes in Agronomic Practice

The adoption of GM Crops, and specifically glyphosate-resistant crops, has resulted

in significant changes in agronomic practices. Most obviously, these changes

include the increase glyphosate use at the cost of other herbicides and the manner

and frequency in which glyphosate is used. In addition, the amount of tillage that is

conducted for crop production has significantly changed (Young 2006; Service

2007a, b; Foresman and Glasgow 2008). This reduction in tillage has an important

benefit of reducing the use of petroleum-based fuels as well as an implicit gain in

time use efficiency by growers. A significant reduction in pesticide use has been

attributed to the adoption of herbicide-tolerant (HT) crops (Sankula 2006). Further-

more, the benefits ascribed to herbicide-tolerant crops have dramatically changed

the crop cultivars selected by growers and have hastened the development of new

transgenic crops for commercial distribution worldwide (Duke 2005; Dill et al.

2008).

Despite wide scale commercial plantings of GM soybean, canola, cotton and

maize, several herbicide-resistant crops have been developed but have not been

commercially introduced. Notably, sugarbeet (Beta vulgaris) cultivars that are

resistant to glyphosate were deregulated in 1999, but not commercially offered

until 2008 because of concerns about the acceptability of sugar refined from a GM

crop (Duke 2005; Gianessi 2005). The development of GM rice (O. sativa) cultivarsmodified to be resistant to glufosinate herbicide proceeded from 1998 to 2001, but

were withdrawn and commercial development terminated because of concerns

about market acceptance of the GM rice (Gealy and Dilday 1997; Gealy et al.

2007). Similarly, wheat (Triticum aestivum) glyphosate-resistant cultivars were

under development but the program was terminated in 2004 (Dill 2005). While

the GR-based wheat production systems demonstrated excellent opportunities for

improved weed management, concerns about the acceptance of the flour made from

GRwheat cultivars as an export commodity in GM-adverse countries resulted in the

decision to halt further development (Stokstad 2004; Howatt et al. 2006).

In addition to concerns centered on consumer acceptability, several concerns

over potential environmental impact have been raised. These are addressed below.

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1.3.11 Weeds, Gene Flow, Invasiveness, and Biodiversity Impacts

There have been significant concerns regarding the potential of GM crops, particu-

larly glyphosate-resistant crops, to become major agricultural problems (by inva-

sion, volunteerism) or to create “superweeds” by cross-pollination (Ellstrand 2003).

1.3.11.1 Gene Flow

While GM crops do have the potential to cross-pollinate other crops and wild

relatives, there are four basic elements determining the likelihood and conse-

quences of gene flow: first, the distance of pollen movement from the GM crop;

second, the synchrony of flowering between crop and pollen recipient; third, sexual

compatibility between crop and recipient; and fourth, ecology of the recipient

species (Dale et al. 2002). Research has shown that pollination declines sharply

with distance from the pollen source (Lutman 1999) and one could reduce the

chances of GM pollen reaching other crops through the use of isolation or buffer

zones, although pollenmay travel further if plants are insect-pollinated. Ellstrand et al.

(1999) reviewed the sexual compatibility of crops with weeds and feral species. For

example, oilseed rape (canola), barley, wheat and beans can hybridize with weeds in

some countries; however, in the UK, for example, the probability of hybridization

with weeds is considered minimal for wheat, low for oilseed rape and barley,

and high for sugarbeet. Although sugarbeet can readily hybridize, in the case of

herbicide-tolerant varieties of sugarbeet the crop would be harvested before flower-

ing and hence shed no pollen. Indeed, methods have been developed to block

expression in the pollen of transgenic plants, including engineering of the chloro-

plast genome (Heifetz 2000) and transgene mitigation strategies (Gressel 2008).

Transgene Mitigation Strategies

Transgenic crops may interbreed with nearby weeds, increasing their competitive-

ness, and may themselves become a “volunteer” weed in the following crop. The

desired transgene can be coupled in tandem with genes that would render hybrid

offspring or volunteer weeds less able to compete with crops, weeds and wild

species. Genes that prevent seed shatter or secondary dormancy, or that dwarf the

recipient could all be useful for mitigation and may have value to the crop. Many

such genes have been isolated in the past few years (Gressel 1999). Examples

include: apomixis (asexual reproduction) as a fail-safe so that the seed is actually of

vegetative origin and not from sexual pollination (Zemetra et al. 1998); chromo-

some-specific cytogenetic fail-safes (the risk of transgenic traits spreading into

weeds can be reduced by orders of magnitude by using cytogenetic mapping to

locate transgenes and releasing only those transgenic lines in which it is on a genome

incompatible with local weeds (Gressel and Rotteveel 2000)); plastome-specific

cytogenetic fail-safes (it is possible to introduce some traits into the chloroplast or

32 N. Ferry and A.M.R. Gatehouse

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mitochondrial genomes;thus, in species where these genomes are completely

maternally inherited, the transgenes cannot move into other species (Daniell et al.

1998)); seed dormancy (the transgenic abolition of secondary dormancy is neutral

to the crop but deleterious to weeds (Gressel 1999)); seed ripening and shattering

(uniform ripening and anti-shattering genes are harmful to weeds but neutral for

some crops (Schaller et al. 1998)), and dwarfing (dwarfing is disadvantageous for

weeds, because they can no longer compete with the crop for light (Gressel 1999)).

However, these strategies are not as of yet fully developed and in use, with the

exception of plastid transformation.

1.3.11.2 Invasiveness

The potential exists for GM crops to become invasive. There has been a great deal

of concern that such crops could persist in the wild and disperse from their

cultivated habitat. However, recent studies (and experience from the field) have

indicated that the ability of GMHT crops to invade and persist was actually no

better than that of their conventional counterparts (Crawley et al. 2001). Finally,

GM crops persisting in fields after harvest thus becoming a weed in a different crop

may be dealt with in two ways; simple treatment with an appropriate herbicide or

mitigation technologies that prevent the transgene being carried over to the next

generation (Gressel 2008).

1.3.11.3 A Dose of Reality

In order to put these concerns into perspective, one must understand that flow from

the agroecosystem to natural ecosystems has always occurred. Gene flow is a

continuing process and is the source of biological diversity (Thies and Devare

2007). There has always been gene flow from commercial crops to relatives living

in near proximity (Ferry and Gatehouse 2009). In reality, the vast majority of the

major cultivated crops have no wild or weedy relatives outside of their centers of

origin (Gressel 2008); however, some crops are grown in areas where gene flow

may occur and appropriate measures must be taken in these areas.

1.3.12 Coexistence of GM and Non-GM Crops

A pervasive problem that exists with the production of GR crops is their coexis-

tence with non-GM crops (Byrne and Fromherz 2003). The issue of coexistence

includes three possibilities: (1) introgression of the trait via pollen (pollen drift), (2)

containment of plant products during the production year (grain segregation), and

(3) volunteer GRC plants in following years (Owen 2005).

While GR crops and non-GM crops can coexist, growers must go to great lengths

to accomplish segregation (Anonymous 2007). Grain segregation, while difficult to

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maintain, can be accomplished and will effectively minimize the impact of GR

crops on non-GM crops. Given that the mixing of grain is equivalent to loss of

intellectual property (IP), this can be costly to growers; thus, appropriate tactics to

isolate GR crops from non-GM crops must be in place (Owen 2000). Controlling

volunteer GR crops is also relatively easy, depending on the rotational crop, but

does require diligence on the part of the grower (Owen 2005). The introgression of

the HT trait via pollen movement is another possibility and management of this

problem is considerably more difficult, particularly in open-pollinated crops such as

maize (Luan et al. 2001; Palmer et al. 2001; Westgate et al. 2003; Abud et al. 2004).

A number of factors affect the success of maize pollen movement and subsequent

pollination, and generally, the greater the distance between the pollen source and

the donor, the less likely is the introgression of the GR trait (Luan et al. 2001;

Westgate et al. 2003). However, given the tolerance levels established for some GM

traits in non-GM crops, the isolation distances required to mitigate the risks of gene

flow are too large to be realistic (Matus-Cadiz et al. 2004). Other open-pollinated

crops have also undergone a great deal of scrutiny (Charles 2007; Fisher 2007;

Harriman 2007; Weise 2007). It is suggested that the issues of the coexistence of

GRCs with non-GM crops will continue to be a concern as long as there are

economic differences between the crop cultivar types (Hurburgh 2000; Ginder

2001; Hurburgh 2003). Nevertheless, coexistence guidelines and buffer zone (iso-

lation zone) regulations are in existence (Boller et al. 2004).

1.3.13 Stacked Traits

Stacked products are a very important feature and future trend for GM crops, which

meets the multiple needs of farmers and consumers; these are now increasingly

deployed by ten countries – US, Canada, the Philippines, Australia, Mexico, South

Africa, Honduras, Chile, Colombia, and Argentina (James 2007). In fact, 37% of all

GM crops in the US in 2007 were stacked products containing two or three traits

that delivered multiple benefits. Currently, there are a number of GM crops that

have stacked herbicide resistance and Bt events (Owen 2006).

In some events, the pat gene is used as a marker for the Bt – the pat gene also

confers resistance for glufosinate herbicide. Interestingly, the Bt trait is combined

with a GR hybrid, the resultant GM cultivar is resistant to both glyphosate and

glufosinate. Monsanto has announced new transgenic maize cultivars that will

combine several Bt events plus two herbicide resistance events.

The Bt event functions ecologically differently compared to herbicide-resistant

transgenes; the transgene for herbicide resistance can be considered benign to the

weed population and has no impact until the herbicide is applied (Owen 2009). In

contrast, Bt exerts continuous selection pressure on the target insect whether the

insect population is at economic threshold or not. Furthermore, given the potential for

the introgression of transgenes into near-relatives, Bt could potentially improve the

fitness of compatible weed species (Snow and Pedro 1997). For example, the fitness

of canola was increased by the inclusion of Bt (Steward et al. 1997); thus, gene flow

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was kept in check. However, there is no reason to believe that stacked traits will

have any greater impact on nontargets than the crops with single traits, and for Btnon-target impacts have been shown to be minimal (Ferry and Gatehouse 2009).

1.3.14 Conclusions

Herbicide-resistant crops, specifically GR crops, have been globally adopted as the

basis for the production of maize, soybean, cotton and canola (Dill et al. 2008).

Their adoption provides economic benefits to agriculture and has major positive

impacts on the environment; specifically, conservation tillage which can reduce soil

erosion (Duke and Powles 2008; Shipitalo et al. 2008). However, growers must

adopt measures to proactively sustain the technology (Mueller et al. 2005; Johnson

and Gibson 2006; Sammons et al. 2007; Christoffoleti et al. 2008) and to steward

GR crops to avoid the evolution of GR weeds (Duke and Powles 2008; Owen 2009).

While there are tactics that are capable of mitigating some of the other concerns,

including the risks of transgene introgression into near-relative plants (Snow and

Pedro 1997; Gepts and Papa 2003), an improved process to assess the environmen-

tal risk of GR crop technologies and communicating those risks to the lay public

should be in place (Owen 2009).

1.4 Biotic Stress due to Plant Pathogens

1.4.1 Crop Losses due to Plant Pathogens

Plants are challenged constantly by many different potential pathogens (Table 1.4).

There are hundreds of thousands of viral, bacterial, and fungal species in the world

and thousands of these are pathogens that infect plants (Chrispeels and Sadava 2003).

Any one pathogen can severely depress the yield of a given crop. Pathogens may

reduce yield by causing tissue lesions; by reducing leaf, root, or seed growth; or by

clogging up vascular tissue and causing wilt. Even in the absence of obvious symp-

toms, pathogens can still be a major metabolic drain that reduces productivity. They

may also cause pre- or postharvest (stored products) damage to the harvested product.

Table 1.4 Organisms that cause infectious disease in plants

Types of plant pathogens

FungiAscomycetes e.g., Fusarium, VerticilliumBasidiomycetes e.g., Rhizoctonia, Puccinia (rust)

Oomycetes e.g., Phytopthora (blight)

Bacteria e.g., Xanthomonas, PseudomonasPhytoplasmas and Spiroplasmas

Viruses Viroids and virus-like organisms

Nematodes, protozoa, and parasitic plants are also considered plant

pathogens

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1.4.1.1 Current Disease Control Measures

Disease control in commercial crops is both economically important and environ-

mentally controversial. Disease management includes: quarantine, cultural prac-

tices such as modification of the plant environment (e.g., soil management) to

reduce pest numbers, host plant resistance, biological control and chemical control.

Chemical control can be highly effective. The first fungicide was discovered in the

1880s when Bordeaux mixture (a mixture of copper sulfate and lime) was found to

suppress grape downy mildew. Numerous compounds with antifungal or antibac-

terial activity have since been discovered, most often applied as sprays, dusts or

seed coatings. Many older compounds are broad spectrum and toxic with the newer

chemistries acting systemically with a narrower target range. Such chemicals are

expensive and normally reserved for use on high-value fruit and vegetable crops

(Ferry and Gatehouse 2009). They are expensive to manufacture and a great deal of

investment must be put into human and ecological safety testing before a product

can be released (Chrispeels and Sadava 2003). As with other chemical control

strategies, pathogens can evolve resistance to these compounds.

Thus, plant breeders have often relied on genetic disease resistance traits to

manage pathogens of particular crops. In classical plant breeding, this has relied on

crosses between elite crops and wild relatives (that are more genetically diverse) to

introduce new disease resistance traits into the crops. Extensive backcrossing of the

elite line is then required to eliminate the undesirable traits in the wild relative and

thus makes traditional breeding a time-consuming process with a time of about

15 years required before a new resistant variety is available for release to growers.

Nevertheless, such traditional plant breeding has had significant successes. For

example, the development of F1 hybrid crops (derived from crossing two inbred

lines) resulted in vastly improved yields (Chrispeels and Sadava 2003). Despite this

enormous success, the F1 hybrids also serve as a warning as to the dangers of large-

scale monoculture and the need for effective and durable disease resistance. In

1970, the highly successful but genetically uniform F1 maize crops in the US were

left devastated by a disease, southern corn leaf blight (Bipolaris maydis Nisik),which caused damage to 710 million bushels of maize (Ullstrup 1972).

In extreme cases, crop disease can change political as well as agricultural

landscapes!

1.4.1.2 The Irish Potato Famine

The Irish Potato Famine (the Great Hunger) started in 1845, lasted until 1849–1852

and led to the death of approximately one million people through starvation and

disease; a further million are thought to have emigrated as a result of the famine.

Some estimate that the population of Ireland was reduced by 25% (Kinealy 1995).

The cause of the famine was a potato disease commonly known as late blight caused

by the oomycete, Phytophthora infestans. Although blight ravaged potato crops

throughout Europe, the impact and human cost in Ireland was high as a third of the

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population was entirely dependent on the potato for food; it was also exacerbated by

a host of political, social and economic factors. The famine was a watershed in the

history of Ireland. Its effects permanently changed the island’s demographic,

political and cultural landscape. For both the native Irish and the resulting diaspora,

the famine became a rallying point for nationalist movements. The fallout of the

famine continued for decades afterwards and Ireland’s population still has not

recovered to prefamine levels.

1.4.1.3 The Bengal Famine of 1943

The Bengal famine of 1943 occurred in British administered Bengal. It is estimated

that around four million people died from starvation and malnutrition during the

period. During the SecondWorld War, the British had just suffered defeat in nearby

Burma and British authorities feared a subsequent Japanese invasion of India

by way of Bengal, so emergency measures were introduced to stockpile food for

British soldiers and prevent access to supplies by the Japanese in case of an

invasion. However, in the rice-growing season of 1942 weather conditions were

exactly right to encourage an epidemic of the rice disease brown spot following a

cyclone and flooding; brown spot in rice is caused by the fungus Helminthosporiumoryzae. When food shortages became apparent, the Bengal government reacted to

the crisis incompetently refusing to stop the export of food from Bengal to allied

soldiers and failing to provide adequate famine relief in Bengal itself. Winston

Churchill was Prime Minister at the time and while his involvement in the disaster,

and indeed his knowledge of it remains unclear, it has been suggested that in

response to an urgent request by the Secretary of State for India to release food

stocks for India, Churchill responded with a telegram asking if food was so scarce,

“why Gandhi hadn’t died yet” (Mishra 2007). Needless to say, whether this is

completely true or is in fact an unfortunate misquote used out of context, the British

Administration in India declined in popularity and there is little doubt that this

incident fuelled feelings for Indian Independence.

Given the vast array of diseases that threaten crops, and the scale of disaster that

can ensue, it is a wonder that crops be produced at all?

1.4.2 Plant Defense Against Pathogens

Plants have evolved mechanisms to resist pathogen invasion that consist of different

defense layers. Firstly, waxy coatings on epidermal cells provide a physical barrier;

secondly, plants contain large amounts of preformed secondary metabolites that

have antimicrobial activity (constitutive defenses including glucosides, saponins,

alkaloids, antifungal proteins, antifeedants and enzyme inhibitors), these are effec-

tive in many cases – but pathogens have evolved enzymes capable of detoxifying

these compounds. Often induced defenses are the plants’ last line of defense against

pathogens and are sufficient to (partially) ward off invading microorganisms.

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Essentially, plants have two major types of induced disease resistance, basal defense

and resistance (R) gene-mediated defense. All plants have basal defense, this

is a general immune response to pathogens and other environmental stresses.

R-gene-mediated defense is more specific and is only found in certain plant species.

R-gene-mediated defense involves recognition of a specific pathogen effector by a

plant ligand receptor. These pathogen effectors can suppress plant basal defense,

making any plant without the R-gene defense susceptible. The ligand-effector

recognition can result in a dramatic immune response such as cell death. In tomato,

Solanum lycopersicum, theMi gene, a member of a large family of R-genes, mediate

resistance to potato aphids, whiteflies, and root-knot nematodes (Kaloshian and

Walling 2005). Both types of plant defenses (R and basal) involve signaling via

three major plant hormones: salicylic acid, jasmonic acid, and ethylene (ETH). In

some instances, defense responses are induced distal to the site of infection and this

is referred to as systemic acquired resistance (SAR).

At least three nonspecific induced defense pathways are described which are

triggered by these specific signaling molecules:

(a) The salicylic acid (SA)-dependent pathway is induced by necrosis inducing

pathogens and triggers systemic acquired resistance (SAR)

(b) A second pathway is triggered by nonpathogenic rhizobacteria, it is dependent

on jasmonic acid (JA) and ethylene (ETH) and constitutes induced systemic

resistance (ISR)

(c) JA and ETH regulate a third pathway that is effective against a different set of

pathogens and not affected by ISR

Most of the inducible defense-related genes are regulated by these signaling

pathways (Delaney et al. 1994; Sticher et al. 1997; Van Loon 1997; Reymond and

Farmer 1998; Knoester et al. 1998; Ananieva and Ananiev 1999).

Defense gene regulation has been extensively studied and severa1 rapid pro-

cesses characteristic of the hypersensitive response (HR) appear to involve prima-

rily activation of preexisting components rather than changes in gene expression.

One of these rapid processes is the striking release of reactive oxygen species.

1.4.2.1 Oxidative Burst

The oxidative burst, a rapid production of reactive oxygen species (ROS), is a well-

documented early plant response to biotic stress (e.g., Apel and Hirt 2004). ROS

comprise radicals and other nonradical but reactive species derived from oxygen.

Among them, the superoxide anion (O�2 ) and hydrogen peroxide (H2O2) exert

various effects on cells, directly or in cooperation with other molecules. In excess,

ROS pose a threat to important biomolecules and cell membranes. One of the

consequences of ROS activity is oxidative damage of membrane integrity due to

lipid peroxidation processes which may result in the generation of highly cytotoxic

compounds. Glutathione-S-transferases (GSTs), induced upon pathogen attack,

may detoxify lipid peroxides by conjugating them with glutathione (GSH). These

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enzymes can also catalyze the GSH-dependent reduction/inactivation of H2O2,

forming glutathione disulfide (GSSG) and increasing GSH synthesis by feedback

induction (Marrs 1996). On the other hand, numerous studies indicate an essential

role of ROS in plant defense responses to biotic stress. In addition to direct

antimicrobial activity and contribution to the strengthening of barriers against

pathogens, recent reports point to H2O2 and O�2 as signal transduction agents

activating defense pathways and as key mediators in cell death during hypersensi-

tive response (HR) (Grant and Loake 2000). To maintain a balance between

negative and beneficial functions of ROS, their levels are strictly controlled by a

complex and flexible network of antioxidant systems (Mittler et al. 2004). The

major enzymatic ROS-scavenging components of this network are superoxide

dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Superoxide

dismutases dismute O�2 to H2O2, an excess of which may be subsequently detox-

ified by CATs and/or APX. Ascorbate peroxidases, in contrast to CATs localized in

peroxisomes, are present in almost all cellular compartments; they exhibit a high

affinity to H2O2 and are considered to be responsible for the fine modulation of ROS

level (Mittler 2002). Moreover, APX, in cooperation with two main low-molecular

antioxidants, ascorbate (Asc) and glutathione (GSH), and enzymes for their regen-

eration, monodehydroascorbate reductase, dehydroascorbate reductase and gluta-

thione reductase (GR), constitute the ascorbate–glutathione (Asc–GSH) cycle,

believed to be the central part of the antioxidant network (Noctor and Foyer

1998). Ascorbate, present at high concentrations in all cellular compartments and

capable of direct scavenging of O�2 and hydroxyl radicals, is considered to be the

most powerful cell antioxidant (Noctor and Foyer 1998). Ascorbate and glutathione

are the major cellular redox buffers, which together with their oxidized forms,

dehydroascorbate (DHAsc) and glutathione disulfide (GSSG), enable cells to main-

tain a redox balance. Changes in the levels or redox state of ascorbate and

glutathione pools as well as in H2O2 homeostasis, and thereby in cellular/compart-

ment redox state, are considered to be pivotal signaling events influencing gene

expression and modulating the plant defense response (Pastori and Foyer 2002;

Foyer and Noctor 2005).

Numerous genes are induced during the plant defense response and presumably

these function in host plant pathogen defense. Induced defenses include the activa-

tion of phytoalexins (including terpenoids, glycosteroids and alkaloids) and PR

proteins (including chitinase, b-1,3 glucanase and other antimicrobial proteins) and

the production of reactive oxygen species (ROS) leading to a hypersensitive

response (HR).

1.4.3 Utilizing Defense Mechanisms

Most conventional breeding strategies have relied on identifying major resistance

genes (often in wild or ancestral plants) while fewer have concentrated on breeding

for polygenic resistance. New opportunities and strategies to enhance disease

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resistance are afforded by genetic engineering of plants – these may follow the

single gene model, or may involve manipulation of defense-activating mechanisms.

1.4.4 Transgenic Disease Resistance

Many transgenic-based approaches exist for conferring enhanced levels of disease

resistance, as exemplified by Fig. 1.6, which provides a simplified model.

1.4.4.1 Expression of Single Genes

R-Genes

A very effective defense mechanism in plants is gene-for-gene resistance, this

induced resistance mechanism is based on the interaction between plant-derived

NucleusHost cell

Pathogen

Pathogenicity

factors

(1a) Constitutive defense

(3) Pathogen mimicry

(1b) Induceddefense

(2a) Detection ofpathogen

(2b) Defenseregulation

Transgenic Strategies. 1a. Constitutive expression of antimicrobial factors,1b. Pathogen induced expression of one or more genes, 2a. Altering

recognition of the pathogen (e.g. R-genes) and 2b. altering downstreamregulators, 3. Priming recognition of pathogens (genetic vaccination).

Fig. 1.6 Simplified model of transgenic strategies to enhance plant defense

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resistance (R) gene products and an avirulence (avr)-gene product (elicitor) pro-

duced by the pathogen. The interaction is generally very specific and results in the

triggering of strong resistance responses including hypersensitive response (HR)

and localized cell death at the point of infection. There is no common structure

between avirulence gene products, except that most are secreted proteins. Because

there would be no evolutionary advantage to a pathogen keeping a protein that only

serves to be recognized by the plant, it is believed that the products of avr genes,sometimes known as effector proteins, play an important role in virulence in

genetically susceptible hosts. The guard hypothesis model proposes that the R

proteins interact, or guard, a protein known as the guardee which is the target of

the Avr protein. When it detects interference with the guardee protein, it activates

resistance. R-gene specificity (recognizing certain avr gene products) is believed tobe conferred by the leucine-rich repeats (LLRs). LRRs are multiple, serial repeats

of a motif of approximately 24 amino acids in length, with leucines or other

hydrophobic residues at regular intervals. LRRs are involved in protein–protein

interactions, and the greatest variation amongst resistance genes occurs in the LRR

domain. LRR swapping experiments between resistance genes in flax rust resulted

in the specificity of the resistance gene for the avirulence gene changing (Bergelson

et al. 2001). Gene-for-gene resistance thus provides obvious targets to engineer

disease resistance in transgenic plants. However, pathogens can often breakdown

R-gene-mediated resistance if the corresponding avr gene mutates becoming inac-

tivated. Pathogen adaptation is also seen in conventionally bred crops. The use of

R-genes is limited by them conferring resistance to only a single pathogen or race

of pathogen; however, they can provide effective (even broad spectrum) resistance

if transformed by genetic engineering into new species or new genera of plant

(Oldroyd and Staskawicz 1998). For example, Rxo1, an R-gene derived from

maize, a nonhost of the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola,was successfully transformed into rice and shown to confer resistance against this

pathogen (Zhao et al. 2005). Interspecies differences do radically influence R-genefunction making it preferable to use R-genes from related species (Ayliffe and

Lagudah 2004); however, the transgenic approach circumvents the tedious and

time-consuming backcrossing required to introduce a single trait via traditional

crop breeding.

Constitutive Expression of Inducible Antimicrobial Factors

Several examples of transgenic strategies to enhance plant constitutive defenses are

described below. This is an extension of the single gene paradigm that has worked

so well for insect-resistant transgenic plants.

Expression of Chitinases

Chitinases are glycosyl hydrolases catalyzing the degradation of chitin, an insoluble

linear b-1,4-linked polymer of N-acetylglucosamine. They are produced by a wide

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range of organisms from microbes to insects and mammals, including plants

(Kasprzewska 2003). Plants produce chitinases as defense against chitin-containing

fungal pathogens by inhibiting spore germination, germ tube elongation and

degrading hyphal tips (Khan et al. 2008).

Transgenic plants expressing chitinase ChiC genes have already been produced

in several species. In carrot, the tobacco class I ChiC gene has shown resistance to

Botrytis cinerea (Punja and Raharjo 1996). Transgenic tobacco transformed with

bean class I ChiC exhibited enhanced resistance to Rhizoctonia solani (Broglieet al. 1991) and Alternaria alternata (Lan et al. 2000). The rice chitinase gene

(RCC2) exhibited increased resistance to Sphaerotheca humuli in transgenic straw-berry (Asao et al. 1997). Peanut (Arachis hypogae) plants transformed with tobacco

ChiC gene were resistant to a fungal pathogen (Cercospora arachidicola), thecausal organism of leafspot disease (Rohini and Rao 2001). Potato has been

transformed with the Streptomyces griseus ChiC gene and resistance to Alternariasolani (causal agent of early blight) demonstrated (Kahn et al. 2008) and cotransfer

and expression of ChiC, glucanase, and bialaphos resistance (bar) genes in creepingbent grass conferred resistance to the fungal pathogens Sclerotinia homoeocarpaand R. solani (Wang et al. 2003).

Expression of Defensins

Defensins are the best characterized cysteine-rich antimicrobial proteins in plants

(Broekaert et al. 1995). All known members of this family have four disulphide

bridges and are folded in a globular structure that includes three b-strands and an

a-helix (Segura et al. 1998). Inhibition of fungal growth by defensins seems to

occur by permeabilization of the plasma membrane through binding to a putative

receptor (De Samblanx et al. 1997). Genes encoding plant defensins are develop-

mentally regulated, with predominant expression in the outer cell layers, and are

induced in response to pathogen infection and stress (Segura et al. 1998). Enhanced

tolerance to the fungus Alternaria longipes is observed in transgenic tobacco

overexpressing a radish defensin (Rs-AFP2) (Chiang and Hadwinger 1991). In

support of this role, Manners et al. (1998) show that the promoter of the plant

defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal patho-

gens and responds to methyl jasmonate, but not to salicylic acid.

Expression of Oxalate Oxidase and H2O2 -Generating Enzymes

Rapid generation of superoxide and accumulation of H2O2 is a characteristic early

feature of the hypersensitive response following perception of pathogen avirulence

signals. In germinating barley and wheat seeds as well as in barley leaves

challenged with powdery mildew, oxalate oxidase activity has been identified as

a generator of H2O2 (Lane et al. 1993; Zhou et al. 1998). Oxalate oxidase utilizes

oxalic acid and oxygen as substrates producing H2O2 and CO2. For certain necro-

trophic fungi, such as Sclerotinia sclerotiorum, oxalic acid is a major pathoge-

nicity determinant, significantly altering environmental pH (Cessna et al. 2000).

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Pathogen-inducible oxalate oxidase both acts as a generator of H2O2, killing the

invading pathogen, and simultaneously detoxifies the acid, which is phytotoxic at

high concentrations. Sunflower plants expressing a wheat oxalate oxidase accumu-

late enhanced levels of salicylic acid and PR1 even in the absence of pathogen

infection and display improved tolerance to Sclerotinia infection (Hu et al. 2003).

This effect is most likely due to the production of H2O2 from endogenous oxalic

acid. Amine oxidases are a class of enzymes mainly found in the plant apoplast that

act on a variety of amine substrates, including mono-, di- and polyamines and

release the corresponding aldehyde as well as NH3 and H2O2. Amines are present in

the plant apoplast and accumulate in response to environmental stress (Bolwell

et al. 1999). Thus, in plants several systems are available that can produce H2O2

following pathogen attack. The importance of H2O2 in plant defense has clearly

been shown by several groups who have reported increased pathogen resistance in

transgenic plants by introducing either H2O2-generating systems or by inhibiting

H2O2-degrading systems. The expression of a fungal glucose oxidase resulted in

enhanced resistance to P. infestans and Erwinia carotovora in potato (Wu et al.

1995). Similarly, the prevention of H2O2 breakdown resulted in higher levels of

H2O2 and increased disease resistance (Chamnongpol et al. 1998). Whether this

improved pathogen tolerance is due to the direct antimicrobial effect of H2O2, or

due to the fact that the plant defense system is induced by the increased levels of

H2O2, was, in either case, not investigated.

Inducible Expression of Single Antimicrobial Factors

Stilbene Synthase

Production of stilbene, a phytoalexin, is a well-characterized defense reaction in

grape vine. Stilbene plays an important role in resistance to fungal and bacterial

infection in plants. It strongly inhibits the growth of fungi and sprout of spores.

Stilbene synthase gene (Vst1) is responsible for the synthesis of resveratrol (trans-3,405-trihydroxystilbene) when plants are challenged by fungal diseases. Thus,

introduction of stilbene synthase genes into transgenic plants can be used to induce

synthesis of phytoalexins. Vst1 has been transferred into many plants to enhance

fungal resistance including common spring wheat using biolistic transformation,

where plantlets with mild resistance to powdery mildew were identified (Leckband

and Lorz 1998). Other crops transformed to produce stilbene include: tomato

(Thomzik et al. 1997); apple (Szankowski et al. 2003); and Vst1 overexpressed in

its native grape (Fan et al. 2008).

1.4.4.2 Activation of Expression of Multiple Genes

Polygenic Resistance

There are several types of disease resistance in terms of the effects of genes on the

plant and pathogen. However, in terms of the number of genes involved, there are

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two general types of resistance. The first (as described above for R-genes) is calledmajor-gene or single-gene resistance. This type of resistance is well defined and

more easily measured; it is sometimes called qualitative resistance because plants

are either resistant or susceptible, without intermediate levels. The second type,

called polygenic resistance, involves several or many genes. This type of resistance

is harder to define; exactly which genes are involved may be unknown. It usually is

effective against all races of a pathogen. This type is often called quantitative,

because there are intermediate levels ranging from resistant to susceptible. It is also

harder to measure than major-gene or single-gene resistance. Often polygenic

resistance does not give a plant as high a level of resistance as major gene

resistance. Polygenic durable resistance to multiple diseases of a crop would be

highly desirable. In nature, most host plant resistance is based on multiple genes

and a diverse set of resistant factors. This diverse, polygenic resistance system helps

to prevent plant-feeding microorganisms from overcoming the resistance in the host

plant. At present, polygenic resistance may be bred into a crop via quantitative trait

loci (QTL) analysis, and the use of molecular markers in molecular breeding

approaches; promising transgenic strategies (as discussed below) activate poly-

genic resistance via elicitor-induced resistance and activation of multiple genes.

Detection of Pathogens; Expression of Elicitors and Defense Activators

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats

(LRRs), a nucleotide binding site (NBS), and a putative amino-terminal signaling

domain, termed NBS–LRR proteins. The LRRs of a wide variety of proteins from

many organisms serve as protein interaction platforms, and as regulatory modules

of protein activation. Genetically, the LRRs of plant R proteins are determinants of

response specificity, and their action can lead to plant cell death in the form of the

hypersensitive response (HR). It is thought that this halts pathogen growth. In the

absence of specific recognition, a basal defense response also occurs, which is

apparently driven by pathogen-associated molecular patterns (PAMPS), such as

flagellin and lipopolysaccharides (LPS) elicitors of defense responses (Belkhadir

et al. 2004). The basal defense response overlaps significantly with R-protein-

mediated defense, but is temporally slower and of lower amplitude. However,

Takakura et al. 2008 show that expression of a bacterial flagellin gene (N1141) intransgenic rice triggers disease resistance and enhances resistance against blast

(Magnaporthe grisea). Furthermore, a number of transgenic plants expressing

NBS–LRR proteins under the control of the CaMV 35S promoter have been

described (Mindrinos et al. 1994; Ellis et al. 1999; Stokes et al. 2002). For example,

Grant et al. (2003) show that an Arabidopsis mutant, adr1 (activated disease

resistance), contains both elevated levels of SA and ROIs, accumulates a number

of defense-related gene transcripts and exhibits resistance against a number of

microbial pathogens. ADR1 encodes a distinct NBS–LRR protein which possesses

N-terminal kinase subdomains. Furthermore, transient expression of ADR1 is suffi-

cient to engage defense-related gene expression and establish disease resistance

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in the absence of significant seed yield penalty. ADR1 plants showed striking

resistance against both P. parasitica (Noco2) and another biotrophic pathogen,

E. cichoracearum (UED1).

Pathogen Phytosensing

A related strategy for crop defense involves engineered phytosensors indicating the

presence of key plant pathogens to provide an important first line of defense

(Mazarei et al. 2008).

Plant defense mechanisms are highly regulated on the transcriptional level, and

can be induced by chemical elicitors produced by pathogens. These elicitors have

been shown to cause changes in gene expression, which initiate a whole plant

response from a localized encounter with a pathogenic organism (Metraux et al.

2002). This is controlled by signal transduction pathways, inducible promoters and

cis-regulatory elements corresponding to key genes involved in HR, SAR, ISR, and

pathogen specific responses, any of which could be useful in building phytosensors.

Cis-acting elements are conserved among plant species, which enables them to

be used efficiently as synthetic inducible promoters. Employing synthetic promo-

ters with potential inducible elements to engineer plants that can sense the presence

of plant pathogens at the molecular level provides novel technologies for monitor-

ing and increasing resistance to diseases (Gurr and Rushton 2005). Identified

inducible promoters and cis-acting elements could be utilized in plant sentinels,

or “phytosensors,” by fusing these to reporter genes to produce plants with altered

phenotypes in response to the presence of pathogens. Mazarei et al. (2008) have

employed cis-acting elements from promoter regions of pathogen-inducible genes

as well as those responsive to the plant defense signal molecules salicylic acid,

jasmonic acid, and ethylene. Synthetic promoters were constructed by combining

various regulatory elements supplemented with the enhancer elements from the

cauliflower mosaic virus CaMV 35S promoter to increase basal level of GUS

expression. Histochemical and fluorometric GUS expression analyses showed that

both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohor-mone treatments with increased GUS expression when compared to untreated

plants. Pathogen-inducible phytosensor studies analyzed the sensitivity of the

synthetic promoters against virus infection. Transgenic tobacco plants infected

with alfalfa mosaic virus showed an increase in GUS expression when compared

to mock-inoculated control plants. The end goal of such studies is to engineer

transgenic plants for the purpose of early pathogen detection.

1.4.4.3 Regulation of Inducible Defenses

The overexpression of regulatory genes provides another tool to activate plant

defenses in response to pathogen attack. For example, transduction of the SA signal

requires the function of NPR1 (also known as NIM1), a regulatory protein that was

1 Transgenic Crop Plants for Resistance to Biotic Stress 45

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identified in Arabidopsis through genetic screens for SAR-compromized mutants

(Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Mutant npr1 plants

accumulate normal levels of SA after pathogen infection but are impaired in their

ability to express PR genes and to mount a SAR response. The NPR1 gene encodesa protein with a BTB/BOZ domain and an ankyrin-repeat domain (Cao et al. 1997).

Upon induction of SAR, NPR1 is translocated to the nucleus (Kinkema et al. 2000)

where it interacts with members of the TGA/OBF subclass of basic domain/leucine

zipper (bZIP) transcription factors (Zhang et al. 1999; Despres et al. 2000; Zhou

et al. 2000; Subramaniam et al. 2001; Fan and Dong 2002) that are involved in the

SA-dependent activation of PR genes (Lebel et al. 1998; Niggeweg et al. 2000).

Physical interaction between NPR1 and TGA transcription factors has been shown

to be required for the binding activity of these factors to promoter elements that

play a crucial role in the SA-mediated activation of PR genes (Despres et al. 2000;

Fan and Dong 2002).

In separate studies NPR1 overexpression and enhanced resistance are correlated

with either elevated or earlier expression of PR transcripts (Cao et al. 1998). Genes

with high sequence similarity to NPR1 are found in Arabidopsis, tobacco, tomato,

rice and maize (Campbell et al. 2003). Overexpression of NPR1 in rice has been

shown to enhance resistance to bacterial blight (X.o.o) (Chern et al. 2001).

1.4.5 Pathogen Mimicry and Virus Resistance

Numerous reports concern transgenic resistance to plant viruses (Fuchs and

Gonsalves 2007) in which RNA-mediated gene silencing is the predominant strat-

egy (viral RNA is degraded and viral DNA is deactivated by methylation). Most of

these strategies can be categorized as pathogen mimicry. Transgenes constitutively

expressed to provide RNA-mediated virus resistance fall into three main types:

1. Sense or antisense viral sequences

2. Inverted repeats/hairpin RNA of viral sequences, and

3. Sequences of engineered microRNAs targeted against the virus

RNA-mediated resistance against viruses was first reported by Lindbo et al.

(1993). Since the discovery of what is now generally called RNA silencing in

plants, the same specific RNA degradation mechanism has been identified in nearly

all eukaryotes (Baulcombe 2004). In plants, genetic and molecular analyses have

revealed at least three natural pathways for RNA silencing: cytoplasmic short

interfering RNA (siRNA) silencing; endogenous mRNA silencing by microRNA

(miRNA); and the silencing associated with DNA methylation and suppression of

transcription (Baulcombe 2004). All these pathways involve the cleavage of double-

stranded RNA molecules into short 21–26 nuclotide RNAs, known as siRNAs and

miRNAs (Baulcombe 2004). To date, it has primarily been the cytoplasmic siRNA

silencing pathway that has been exploited by genetic engineering to confer resis-

tance to plant viruses.

46 N. Ferry and A.M.R. Gatehouse

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The best documented examples of commercial transgenic resistance involve type

(1) resistance, although the mechanism of resistance was not initially known. In the

1990s, the papaya industry in Hawaii suffered a 50% reduction in production because

of papaya ringspot virus; the solution came through the expression of viral coat protein

in transgenic plants (thought to be immune priming, analogous to vaccination).

1.4.5.1 Papaya Ringspot Virus

Papaya ringspot is a destructive disease (a potyvirus) characterized by yellowing and

stunting of the crown of papaya trees, a mottling of the foliage, damage to younger

leaves, water-soaked streaking of the petioles (stalks), and small darkened rings

on the surface of fruit. Papaya ringspot virus is transmitted from infected papaya

trees to healthy trees by the feeding action of various species of aphids, especially

the green peach aphid and melon aphid. The virus is transmitted in a nonpersistent

manner, meaning that the virus does not multiply within the aphid but is instead

carried on its mouth parts and is transmitted from plant to plant while feeding.

In 1995, American researchers developed a transgenic papaya resistant to the

virus, by expressing a copy of a viral coat protein in the plant (Ling et al. 1991).

It was field-tested in Hawaii where it was shown to be effective against the virus.

The virus-resistant papaya is now widely used by commercial papaya producers in

Hawaii. The presence of small RNA species, presumably siRNA, corresponding to

regions of the viral coat protein gene was later shown to be present in transgenic

lines resistant to PRSV; thus it is posttranscriptional gene silencing involved in the

establishment of resistance (Krubphachaya et al. 2007). Pathogen-derived resis-

tance has also been shown to be effective against maize streak virus (Shepherd et al.

2007), potato leaf roll virus (Vazquez Rovere et al. 2001) and tomato yellow leaf

curl (Yang et al. 2004).

An important drawback of RNA-based approaches to enhanced resistance is the

high level of sequence specificity required for RNA degradation. Viruses contain-

ing >10% nucleotide divergence are insensitive to RNA degradation. Another

drawback is the size of the transgene, commonly >300 bp, required to trigger

efficient RNA silencing. However, Parizotto et al. (2004) has provided experimen-

tal evidence using genetically engineered microRNA (miRNA) to show that a 21

nucleotide sequence complementary to GFP mRNA was sufficient to trigger com-

plete GFP silencing in transgenic plants. In the future, similar miRNA-derived

strategies could provide resistance to a large number of plant viruses. However, the

major technical limitation for technologies based on RNA silencing is that many

important plant crop species are difficult or impossible to transform, precluding the

constitutive expression of constructs directing production of double-stranded RNA.

Moreover, public concerns over the potential ecological impact of virus-resistant

transgenic plants have so far significantly limited their use (Fermin et al. 2004).

For DNA viruses (particularly, Gemini viruses), transgenic resistance involves

both RNA-mediated resistance and mutated viral proteins exerting negative effects

on viral replication (Vanderschuren et al. 2007).

1 Transgenic Crop Plants for Resistance to Biotic Stress 47

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1.4.6 Conclusions

While insect-resistant and herbicide-tolerant transgenic plants are the most widely

grown GM crops, very few strategies using genetically engineered plants for

disease resistance have had a similar impact (Collinge et al. 2008). Given the effort

put into biotechnological strategies for disease resistance over the last two decades,

why have so few crops expressing these traits become commercialized crops?

The development of molecular markers, which are particularly useful in screen-

ing for disease resistance and facilitate conventional breeding, provide a higher

commercial incentive for seed companies because of strong public opposition to

GM crops. Often, transgenic disease resistant plants have only partial resistance,

or resistance to rapid breakdown (single genes), again giving a low economic

incentive to developers. At present, clear field results show that transgenic virus

resistance is effective; however, there are no signs that commercial bacterial- or

fungal-resistant crops will be introduced onto the market at any point soon.

1.5 Transgenic Crops for Resistance to Biotic Stress:

Conclusions

Approximately 10.3 million farmers in 22 countries grew transgenic (genetically

modified) crops in 2006. Yet this technology remains one of the most controversial

agricultural issues of current times. Many consumer and environmental lobby

groups believe that GM crops will bring very little benefit to growers and to the

general public, and that they will have a deleterious effect on the environment. The

human population is currently 6.1 billion and it is predicted to increase to 9–10

billion in the next 50 years (Fig. 1.7). This is at a time when food and fuel are

competing for land (Fig. 1.8) and climate change threatens to compromise current

resources. Population growth, changing diets, higher transport costs and a drive

towards biofuels are forcing food prices up (Fig. 1.9). The UN’s Food and Agricul-

ture Organization (FAO) stated that the food crisis had thrown an additional 75

million people into hunger and poverty in 2007 alone.

2.5bn 4.1bn 6.1bn 8.0bn 9.2bn

World Population Growth (billion people)

1950 1975 2000 2025 2050

Fig. 1.7 World population growth

48 N. Ferry and A.M.R. Gatehouse

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It is, and will continue to be, a priority for agriculture to produce more crops on

less land. The minimization of losses to biotic stress caused by agricultural pests

would go some way to optimizing the yield on land currently under cultivation.

Traditionally, agricultural production has kept pace, even outstripped human

population growth; however, we currently face a set of unique challenges. One of

the greatest dangers to agriculture is its vulnerability to global climate change. The

expected impacts are for more frequent and severe drought and flooding, and

shorter growing seasons. The performance of crops under stress will depend on

their inherent genetic capacity and on the whole agroecosystem in which they are

Biofuel use:47m tonnes

Total:80m tonnes

Demand for Biofuels

Rise in use of course grains 2005-7 (FAO/OECD).

Fig. 1.8 Demand for biofuels

31%

74%

87%

130%

Corn Rice Soya Wheat

Price Rises in a Single Year (Mar 2007-Mar 2008)Source: Bloomberg, FAO/ Jackson Son & Co

Fig. 1.9 Price rises of major food crops (2007–2008)

1 Transgenic Crop Plants for Resistance to Biotic Stress 49

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managed. It is for this reason that any efforts to increase the resilience of agriculture

to climate change must involve the adoption of stress-resistant plants as well as

more prudent management of crops, animals and the natural resources that sustain

their production. Currently, we may be at the limit of the existing genetic resources

available in our major crops (Gressel 2008). Thus, new genetic resources must be

found and only new technologies will enable this.

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