A transgenic plant is defined as one that has had foreign genetic material purposefully introduced and stably incorporated into the plant genome through means other than those that naturally occur in the environment. In other words, transgenic plants are those which carry additional stably integrated and expressed foreign gene(s), usually transferred from unrelated organisms.
The major components for the development of transgenic plants are:
(i) Development of reliable tissue culture and regeneration systems,
(ii) Proportion of gene constructs and transformation with suitable vectors,
(iii) Efficient techniques of transformation for introduction of genes into the crop plants,
(iv) Recovery and multiplication of transgenic plants,
(v) Molecular and genetic characterization of transgenic plants for stable and efficient gene expression,
(vi) Transfer of genes into elite cultivars by conventional breeding methods, and
(vii) Evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental hazard.
Before taking up any attempt to produce transgenic plants to counter insect attack, the following requirements and priorities need to be identified:
(i) The factors for resistance should be controlled by single genes,
(ii) Standardization of methods for transfer of such genes can easily be accomplished.
(iii) Expression of transferred gene should occur in the desired tissues at the appropriate time.
(iv) The transgenic plant should be safe for consumption,
(v) Inheritance of the gene in the successive generations should be very stable, and
(vi) There should be no penalty for yield in terms of other quantitative characters.
Remarkable achievements have been made in the production, characterization and field evaluation of transgenic plants. Both Agrobacterium-mediated gene transfer and direct DNA transfer methods have been used to produce transgenic plants with new genetic properties.
Genes conferring resistance to insects have been inserted into crop plants such as maize, rice, wheat, sorghum, sugarcane, cotton, potato, tobacco, broccoli, cabbage, chickpea, pigeonpea, cowpea, groundnut, tomato, brinjal and soybean.
Development of insect-resistant transgenic crop cultivars has focused on two district approaches:
(i) Integration of bacterial genes encoding for production of toxic proteins, especially from Bacillus thuringiensis (Bt), and
(ii) Integration of plant genes encoding for production of enzyme inhibitors and sugar-binding lectins.
Both the approaches were pioneered in the mid-1980s and thus have developed in parallel. However, the first approach, based in particular on integration of 5-endotoxin genes derived from various subspecies of Bt, has undoubtedly received more attention and thus enjoyed greater progress. To date, all commercially available insect- resistant transgenic cultivars express semi-active Bt toxins, whereas cultivars expressing insecticidal plant proteins are not currently available outside research institutions.
A. Bt Endotoxins:
Bacillus thuringiensis Berliner (Bt) is a gram positive entomocidal spore-forming bacterium. Bt synthesizes an insecticidal crystal protein (Cry) which when ingested by insect larvae is solubilized in the alkaline conditions of the midgut and processed by midgut proteases to produce a protease resistance polypeptide toxic to the insect.
Bt endotoxins are attractive candidates for insect-resistant crop development using transgenic technology because- (i) they have an established safety record, (ii) they act rapidly and are completely biodegradable and proven safe to humans and non-target organisms and the environment, and (iii) the endotoxins are the products of single genes and are highly effective against the larvae of Lepidoptera, a major group of destructive insect pests.
Therefore, the genes encoding the δ-endotoxins were cloned in the early 1980s and expression of modified toxin genes in transgenic tobacco and tomato provided the first examples of genetically engineered insect resistance in plants. Since then, Bt genes have been introduced into and expressed in a wide range of crop species.
Cotton:
Considerable progress has been made in developing cotton cultivars with Bt genes for resistance. The first generation Bt cotton varieties were developed by Monsanto and their seed partners to express the CryIAc protein. Worldwide, several different Cry1Ac events are grown commercially. In the USA and some other countries, Bollgard® event MON531 was used by seed company breeding programmes to develop the first commercial Bt cotton varieties.
In Australia and other countries, Ingard® event MON757 was used. The tobacco budworm, Heliothis virescens (Fabricius) and pink bollworm, Pectinophora gossypiella (Saunders) are very well controlled by Bollgard® cotton whereas the cotton bollworm, Helicoverpa zea (Boddie) is controlled satisfactorily except during the bloom stage when feeding on reproductive parts of flowers which tend to have a lower concentration of Cry protein than other plant parts.
The introduction of Bt cotton in 1996 and 1997 was very timely since P. gossypiella and H. virescens in the USA and H. armigera in China and Australia had become resistant to many of the conventional insecticides and Bt cotton offered a reasonable solution to the problem.
Cotton cultivar Coker 312, transformed with the cry1A(c) gene (having 0.1% toxin protein), has shown high levels of resistance to cabbage looper, Trichoplusia ni (Hubner), tobacco caterpillar, Spodoptera exigua (Hubner) and cotton bolloworms, H. zea and H. virescens. In transgenic cotton, bollworm damage was reduced to 2.3 per cent in flowers and 1.1 per cent in bolls compared to 23 per cent damage in flowers and 12 per cent damage in the commercial cultivar, Coker 312.
The second-generation Bt crops, developed with stacked cry genes, have been shown to improve the level of control and broaden the spectrum of caterpillar pests controlled. China has developed the new events, GK (expressing fused Cry1Ab and Cry1Ac proteins) and SGK (expressing the fused Cry1Ac-Cry1Ab and stacked CpTi proteins).
Monsanto has created Bollgard® II by inserting a synthetic gene from B. thuringiensis, cry2Ab, expressing Cry2Ab protein, into the Bollgard® cotton variety, DP50B, already expressing Cry1Ac through event MON531, creating the new stacked gene event 15985. Bollgard® II is much more effective than Bollgard® at controlling all the caterpillar pests of cotton worldwide, especially H. armigera, S. exigua, S. frugiperda and H. zea.
Laboratory and field studies have shown that Bollgard® II increased mortality of H. zea from 84.2 to 92.2 per cent, S. frugiperda from 16.1 to 100 per cent, S. exigua from 50.1 to 94.9 per cent and T. ni from 1.2 to 97.4 per cent. This high level of pest control is due, in part, to the very high concentration of Cry2Ab protein in Bollgard® II.
Maize:
The first generation Bt proteins engineered into maize were Cry1Ab (from B. thuringiensis) and Cry9c (from B. thuringiensis subsp. tolworthi) and these were highly effective against European corn borer, Ostrinia nubilalis (Hubner). The dominant Cry1Ab events are Monsanto’s YieldGard® maize, event MON810; Syngenta’s BT11 sold under the NK-brand as YieldGard®, and Syngenta’s event 176, known as Knockout®.
The level of expression of Bt proteins tends to be lower in fresh leaves of 176 than MON8IO and BT11. Recently, Cry1F, has been introduced in maize (Dow’s Herculex I®) and maize with a cry3Bb gene or a binary toxin genetic system for control of the maize rootworm (Diabrotica) complex is being developed.
Maize plants transformed with Bt genes have also been found to be effective against the spotted stem borer, Chilo partellus (Swinhoe) and the maize stalk borer, Busseola fusca Fuller in Southern Africa. Maize plants with crylAb gene are also resistant to the sugarcane borers, Diatraea grandiosella Dyar and D. saccharalis (Fabricius).
Transgenic tropical maize inbred lines with crylAb or crylAc genes with resistance to corn earworm, H. zea; fall armyworm, S. frugiperda; Southwestern corn borer, Diabrotica undecimpunctata howardi Barber and Western maize rootworm, D. virgifera virgifera LeConte have also been developed.
More recently, SmartStax maize has been developed by combining eight genes, six for insect resistance and two for herbicide tolerance. SmartStax has been created by crossing four transgene varieties (MON89034 × 1507 × MON88017 × 59122), rather than using genetic transformation of a single maize strain. SmartStax is the first transgenic crop with as many as eight traits; the current transgenic crops in the market have only up to three traits each.
The eight genes in SmartStax maize include three for lepidopteran (moth) resistance (cry 1A. 105, cry2Ab, cry 1 Fa 2), three for coleopteran (corn rootworm) (cry3Bb1, cry35Ab1, cry34Ab1) and two for herbicide tolerance, i.e. Pat (glufosinate tolerance) and CP4 epsps (glyphosate tolerance). SmartStax has proved effective against eight primary pests, viz., six lepidopterans, i.e. H. zea, 0. nubilalis, S. frugiperda, D. grandiosella, A. ipsilon, and Loxagrotis albicosta (Smith), and two coleopterans, i.e. D. virgifera virgifera and D. barberi Smith & Lawrence (www(dot)i-sisorg(dot)uk).
Rice:
Various Chinese universities and research institutes, in cooperation with the International Rice Research Institute (IRRI) and universities in many countries have transformed rice by inserting the cry1Ab gene alone and a fused cry1Ab/cry1Ac into conventional rice varieties and hybrids. Initial field and laboratory tests of these Bt lines against lepidopteran pests have shown good season long control.
Rice plants having 0.05 per cent toxin of the total soluble leaf protein have shown high levels of resistance to the striped stem borer, Chilo suppressalis (Walker) and rice leaf folder, Cnaphalocrocis medinalis (Guenee). Field testing of transgenic rice lines showed high protection against Scirpophaga incertulas (Walker) and C. medinalis.
The percentage of plants with whiteheads was significantly lower on the Bt Shanyou 63 (11%) as compared to control Shanyou 63 (44%) plants. Similarly, transgenic plants showed no damage by C. medinalis as compared to 47.9 per cent damaged plants in non-transgenic rice.
Scented varieties of rice (Basmati 370 and M7) have been transformed with ciyll(a) and are resistant to S. incertulas and C. medinalis. Selected Bt-lines of IR64 and Pusa Basmati 1, having Bt-titres of 0.1 per cent (of total soluble protein) showed 100 per cent mortality of S. incertulas larvae within 4 days of infection in cut-stems as well as at the vegetative stage in whole plant assays.
Potato:
A modified cry3A gene has been expressed in potato plants with resistance to Colorado potato beetle, Leptinotarsa decemlineata (Say). Transgenic potato plants containing the cry1A (b) gene (Bt 884) and a truncated gene cry1A (b) 6 resulted in less damage to the leaves by the potato tuber moth, Phthorimaea operculella (Zeller). However, the size of the leaf tunnels increased over time in plants containing only the Bt 884 gene, while there was no increase in tunnel length in those containing cry1A (b) 6.
The latter also resulted in 100 per cent mortality of the insects in tubers stored up to six months. Several other Bt potato lines showing complete mortality of larvae of P. operculella have also been developed. However, the marketing of Bt potatoes was stopped in 2001 due to opposition from several food producers not to use Bt potatoes in their products.
Vegetables:
Transgenic tomato was one of the first examples of genetically modified plants with resistance to insects. Tomato plants expressing cry1A (b) and cry1A (c) genes are highly effective against Helicoverpa armigera (Hubner). Transgenic broccoli containing cry1A(c) is resistant to Trichoplusia ni (Hubner) and Pieris rapae (Linnaeus).
Synthetic cry1A(c) gene introduced into broccoli provides protection not only from susceptible Plutella xylostella (Linnaeus) larvae, but also from those selected for moderate levels of resistance to Cry1A(c). Synthetic cry1A (b) gene inserted into broccoli cultivar Pusa Broccoli KTS-1 and cry1A (b) in cabbage have shown resistance to P. xylostella.
Transgenic cauliflower plants transformed with synthetic cry9A (a) have also shown high levels of activity against P. xylostella. Transgenic brinjal plants expressing cry1A (c) gene have also shown insecticidal activity against the fruit borer, Leucinodes orbonalis Guenee.
The Genetic Engineering Approval Committee (GEAC) of the Government of India had approved four BT brinjals, viz., MHB-4Bt, MHB-9Bt, MHB-80Bt and MHB-99Bt, for environmental release in 2009. However, the Government of India withheld the commercial release of Bt brinjal in 2010 till more scientific data on biosafety aspects are generated.
B. Plant-Derived Genes:
Largely, plant transformation involving plant genes has focused on:
(1) Protease inhibitors, which disrupt amino acid metabolism.
(2) α-amylase inhibitors, which target carbohydrate metabolism.
(3) Lectins, which cause agglutination and cell aggregation.
(4) Enzymes such as chitinase, which target insect exoskeleton.
The varied modes of action and levels of specificity of these gene products increase the potential target-pest range of transgenic cultivars and allow the possibility of combining (pyramiding) genes that are active at various target sites within a pest insect or against various pests. However, levels of protection provided by genes of plant origin are typically lower than those provided by genes expressing Bt toxins.
Frequently, effects on target insects are sublethal, including reductions in feeding, weight gain, developmental rates and fecundity. Even then, transgenic cultivars with sublethal or chronic effects on target pests may be more attractive components of IPM strategies than cultivars with acute toxic effects because they are more likely to be compatible or act synergistically with other biopesticide strategies.
Thus far, a number of economically important crop plants, such as oilseeds, potato, rice, sugarcane, tobacco, among others, have been genetically transformed to express various genes of plant origin. In many of the cases, significant effects of the plant genes were evident on target-pest mortality rates and/or developmental and reproductive parameters.
1. Protease Inhibitors:
The presence of antimetabolic proteins, which interfere with the processes of digestion in insects, is a strategy for defence that plants have used extensively. Proteins can occur constitutively in tissues that are particularly vulnerable to attack, such as seeds, or mechanical wounding in tissues attacked by chewing insects can induce them.
Analysis of the effects of dietary protease inhibitors has shown that these are detrimental to the growth and development of insects from a variety of genera including Helicoverpa, Spodoptera, Diabrotica and Tribolium. Plants can now be transformed with protease inhibitor genes with strong promoters to express the inhibitor proteins in relatively high levels at specific times.
Several classes of protease inhibitors corresponding to different types of insect gut proteases have been characterized. Many insects, particularly members of Lepidoptera, depend on serine proteases (trypsin, chymotrypsin and elastase like endoproteases) as their primary protein digestive enzymes and genes encoding members of various serine protease inhibitor families have been cloned and introduced into transgenic plants. Insects also produce their own serine protease inhibitors for the regulation of their digestive proteases.
It has been suggested that these could be turned against the insects by expressing them in transgenic plants. Other pests rely on cystein proteases (thiol proteases) as their primary digestive proteases, which act on papain and cathepsin. These have been targeted with cysteine protease inhibitors, which have been shown to exert chronic effects on important pests such as corn root worm, Diabrotica spp., against which there are no effective Bts.
Serine Protease Inhibitors:
The first gene of plant origin to be used in transgenic crop protection was that isolated from cowpea encoding a double-headed trypsin inhibitor (CpTi) and transferred in tobacco. A simple construct was prepared in which a full length coding sequence derived from a cDNA clone was placed under the control of the constitutively expressed cauliflower mosaic virus (CaMV) 35S promoter.
Transformants were screened for CpTi expression, which showed that many of the resulting plants expressed CpTi at levels greater than 0.1 per cent of total soluble protein. Subsequent experience has shown that this is generally the case for expression of genes of plant origin encoding defensive proteins in transgenic plants, in contrast to the very low level of expression observed for unmodified toxin genes of bacterial origin.
CpTi is a small polypeptide of about 80 amino acids; homologous sequences are encoded by a moderately repetitive gene family in the cowpea genome. This protein is considered to be particularly suitable candidate for transfer to other species through genetic engineering because- (i) it is an effective antimetabolite against a wide range of field and storage pests belonging to Lepidoptera, Coleoptera and Orthoptera, (ii) has no deleterious effects on mammals, and (iii) also has a small polypeptide of about 80 amino acids.
Biossays against H. virescens caterpillars showed that transgenic expressing CpTi at the highest levels (about 1% of total soluble protein) caused increased mortality, reduced growth and reduced plant damage. The antimetabolic effects of CpTi expressed in transgenic tobacco have also been observed with other lepidopteran pests including H. zea, S. littoralis and M. sexta.
Subsequent trials carried out in California showed that expression of CpTi in tobacco afforded significant protection against H. zea in the field. Following on from the study using tobacco as a model system, the gene encoding CpTi has been expressed in a range of different crops. (Table 11.4).
For example, constitutive expression of CpTi in rice conferred significantly enhanced levels of resistance towards two species of rice stem borer, viz. Chilo suppressalis (Walker) and Sesamia inferens (Walker) in the field. Furthermore, the trials with CpTi transgenic strawberry plants suggested that these plants were highly resistant to the vine weevil, Otiorhynchus sulcatus (Fabricius).
Cysteine Protease Inhibitors:
Cysteine proteases are used by plants for protein mobilisation and by animals for intracellular lysozomal protein digestion, and protein inhibitors of cysteine proteases (cystatins) are widely distributed throughout all living organisms to regulate these endogenous proteases, even if they are usually present in small amount.
The genes encoding cysteine protease inhibitors have been suggested for use in transgenic plants for the control of coleopteran pests. Although there have been several studies carried out demonstrating in vitro inhibition of insect digestive proteases by cysteine protease inhibitors, with a few examples of their deleterious effects against insects when incorporated into artificial diets, as yet there are few published reports describing their insecticidal effects in plants.
A gene encoding a rice cystein proteinase inhibitor oryzacystatin, has been expressed constitutively in transgenic poplar trees, conferring resistance to the coleopteran pest, Chrysomela tremulae. Corn cystatin (CC) has been introduced into protoplasts of rice and cystatin activity of the transgenic rice plants was assayed against a crude midgut proteinase fraction from Sitophilus zeamais Motschulsky. The results showed that 50 per cent of the midgut protease activity in S. zeamais was inhibited by 2 µg and completely inhibited by 5 µg of transgenic seed protein fraction, whereas untransformed rice seeds had no significant effect.
2. α-Amylase Inhibitors:
Plants produce inhibitors of insect gut alpha-amylases, which are required for the digestion of starch, a major energy source, particularly for the weevils. The alpha-amylase inhibitors produced by plants have different types of structure and different mode of action and target specificity, and hence can be used for insect control in transgenic plants.
Transgenic tobacco plants expressing amylase inhibitors from wheat (wheat alpha-amylase inhibitor, WAAI) increase the mortality of lepidopteran larvae by 30-40 per cent. Similarly, transgenic pea seeds expressing alpha-amylase inhibitor derived from common beans (BAAI) were found to exhibit increased resistance against bruchid beetles, Callosobruchus spp. and pea weevil, Bruchus pisorum (Linnaeus).
Enhanced levels of resistance to the bruchids have been observed in transgenic adzuki beans expressing the alpha-amylase inhibitor of common bean. While even low levels of the amylase inhibitor were sufficient to provide resistance to the adzuki bean weevil, higher levels of the protein make the seeds resistant to the cowpea weevil, C. maculatus (Fabricus) and pea weevil, C. chinensis (Linnaeus) as well.
Two alpha-amylase inhibitors, α-AI-1 and α-AI-2, from the common bean inserted into pea, are effective in protecting peas against the pea weevil, B pisorum under field conditions. α-AI-1 provided complete protection from B. pisorum, by inhibiting alpha-amylase by 80 per cent, while α-AI-2 inhibits the enzyme by 40 per cent. α-AI-1 results in larval mortality, whereas α-AI-2 delays the maturation of the larvae.
3. Lectins:
Lectins are plant-derived proteins that bind to oligo-and polysaccharides, and cause agglutination and cell aggregation. Lectins have been isolated and characterized from a wide variety of plants such as pea, rice, wheat, snowdrop, castor, soybean, mungbean, garlic, sweet potato, tobacco, chickpea and groundnut.
A number of plant lectins exhibiting insecticidal characteristics are being evaluated as alternatives to Bt 8-endotoxins. The precise mode of action of insecticidal lectins is unknown. However, binding to specific carbohydrates and agglutination (fusion by adhesive substances) in the insect midgut has been clearly demonstrated.
Specifically, lectins may interfere with development and structural integrity of the midgut peritrophic membrane; bind to glycosated targets in the insect midgut, thereby inhibiting nutrient absorption or cell disruption in the midgut; or bind or block the peritrophic membrane protecting the insect midgut surface.
Lectins from wheat (wheat germ agglutinin, WGA) and the snowdrop plant (Galanthus nivalis agglutinin, GNA) are inhibitory to the sap-sucking homopteran pests such as aphids, leafhoppers and planthoppers, which feed on the phloem exudates and against which there are no known Cry proteins.
In addition, lectins have also exhibited inhibitory activity against several lepidopteran and coleopteran pests. A gene encoding the pea lectin (p-lec) has been expressed in transgenic tobacco and the plants expressing pea lectin upto 1 per cent of total protein reduced the larval biomass of Heliothis virescens (Fabricius) and leaf damage.
Transgenic tobacco plants containing both CpTi and and P-Lec were obtained through hybridization of two primary transformed lines. The plants expressing two insecticidal proteins reduced the insect biomass by 90 per cent as compared to 50 per cent reduction in plants expressing either CpTi or P-Lec.
This study demonstrated that not only the products of lectin genes could enhance resistance to insect attack in transgenic plants, but also showed that additive protective effects could be obtained from different plant-derived insect-resistant genes.
Survival of the brown planthopper, Nilaparvata lugens (Stal) decreased to 60 per cent in transgenic rice expressing GNA protein (2% of total protein), under the control of phloem tissue- specific sucrose synthase promoter which directs the expression of the gene in the phloem of leaves, stems, petioles and roots for protection from the phloem feeding pests. Wheat germ agglutinin (WGA) is antimetabolic, antifeedant and insecticidal to the mustard aphid, Lipaphis erysimi (Kaltenbach).
Bioassays using leaf discs showed that feeding on transgenic induced high mortality and significantly reduced fecundity of aphids. Thus, plant lectins have shown biological activity against a wide range of insects. However, consideration should be given with regard to their deployment in transgenic plants because of their known toxicity to mammals and humans.
4. Enzymes:
Transgenic expression of various enzymes has been proposed as a crop protection strategy. The most obvious candidate is chitinase, which is an important structural component of insects. Expression of an insect chitinase in transgenic tobacco enhances resistance to some lepidopterans. Similar marginal protective effects have been observed from expression of bean chitinase (BCH) in transgenic tobacco.
Transgenic potato plants expressing a gene encoding BCH were found to reduce fecundity of the glasshouse potato aphid, Aulacorthurn solani (Kaltenbach), though this reduction was not statistically significant. However, nymphs produced on these BCH expressing plants were significantly smaller compared to those on control, non-transformed plants.
Alarm Pheromones:
Alarm pheromones are volatile substances released by certain species of insects, which alert them about the potential danger from their predators. Many aphid species produce the sesquiterpene, (E)-β-farnesene (EBF) as the principal component of the alarm pheromone.
EBF is released when aphids are attacked by enemies and it leads aphids to undertake predator avoidance behaviours and to produce more winged offspring that can leave the plant. Many plants also release EBF as a volatile substance and this chemical could act to defend plants against aphid infestation by deterring aphids from settling, reducing aphid performance due to frequent interruption of feeding, and inducing the production of more winged offspring.
Scientists at Rothemsted Research (UK) have developed genetically modified (GM) wheat, by transferring EBF synthase gene from peppermint (Mentha piperita Linnaeus) to the genome of a spring wheat strain, Cadenza. Laboratory trials have shown that EBF emitting wheat not only repels aphids, but also attracts their natural enemies. This GM wheat is currently being evaluated in field trials at Rothamsted.
This is the world’s first GM crop which repels insects instead of killing them, reducing the chances of the pests developing resistance to it. Scientists have created GM wheat to combat aphid attacks that can cause loss of more than £120 million each year to the UK’s most important cereal crop, which has an annual value of more than £1.2 billion.
Other Novel Genes:
The genes derived from other sources such as chicken, scorpion and spider are also being screened for their insecticidal potential. Avidin, a glycoprotein found in chicken egg white, sequesters the vitamin biotin. Transgenic maize containing avidin gene has been produced.
The avidin at ≥ 100 ppm is toxic to and prevents development of insects that damage grains during storage. Insect- specific neurotoxin AalT from the venom of the scorpion, Androctonus australis (Linnaeus), in tobacco has shown insecticidal activity against H. armigera larvae (up to 100% mortality after 6 days).
Transgenic plants of tobacco have been obtained containing an insecticidal spider peptide gene, and some of these plants have exhibited resistance to H. armigera. The role of neurotoxins from insects and spiders need to be studied in greater detail before they are deployed in other organisms and plants because of their possible toxicity to mammals.