Prospects for using transgenic resistance to insects in crop improvement
Keywords : Biotechnology, Insect resistance, Novel genes, Transgenic plants.
There is a continuing need to increase food production, particularly in the developing countries in Africa, Asia, and Latin America. And this increase has to come from increased yields from major crops grown on existing cultivable lands. One practical means of achieving greater yields is to minimise the pest associated losses, which are estimated at 14% of the total agricultural production. The losses are far more significant in food crops, e.g., 52% in wheat, 83% in rice, 59% in maize, 74% in potato, 58% in soybean, and 84% in cotton. In addition to direct losses caused by insects, there are additional costs in the form of pesticides applied for pest control, which is currently valued at over US $10 billion annually. Pesticide application not only affects the non-target organisms, but also leaves harmful residues in the food, and results in environmental pollution.
With the advent of genetic transformation and recombinant DNA techniques, it has become possible to clone and insert genes into the plant genome that confer resistance to insects. Genes from bacteria such as Bacillus thuringiensis (Bt) and Bacillus sphaericus have been the most successful group of organisms identified for use in genetic transformation of crops for pest control on a commercial scale. Protease inhibitors, plant lectins, ribosome inactivating proteins, secondary plant metabolites, vegetative insecticidal proteins from Bacillus thuringiensis and related species, and small RNA viruses can also be used alone or in combination with Bt genes to generate transgenic plants for pest control. The search for alternatives to toxins from Bacillus thuringiensis has concentrated, with a few exceptions, on genes derived from the plants. Plant derived genes target different sites in insects than the synthetic chemicals, and may be deployed in combination with the exotic genes and insecticides. Retardation of insect development, slower rate of insect population growth, and reduced fitness of the surviving insects would allow a much wider window within which intervention with insecticides can be successfully employed. This will help to generate greater confidence in integrated pest management (IPM) by the farmers, who normally prefer complete insect control based on chemicals.
Recombinant DNA technology offers the possibility of developing entirely new biological insecticides that retain the advantages of classical biological control agents, but have fewer of their drawbacks. Biotechnology has provided: 1) access to novel molecules, 2) ability to change the level of gene expression, 3) capability to change the expression pattern of genes, and 4) develop transgenic plants with different insecticidal genes. The basic requirements for genetic transformation are: 1) a target genome, 2) a candidate gene, 3) a vector to carry the gene, 4) modification of the foreign DNA to increase the level of gene expression, 5) method to deliver the transgene into the cell, 6) protocols to identify the transformed cell, and 7) tissue culture and procedures to recover the viable plants from the transformed cells.
In addition to widening the pool of useful genes, genetic engineering also allows the use of several desirable genes in a single event, and reduces the time to introgress novel genes into elite background. However, transgenic plants are not a panacea for solving all the pest problems. The major limitations of transgenics are: 1) secondary pests may not be controlled in the absence of sprays for the major pests, 2) need to control the secondary pests through chemicals will kill the natural enemies, and thus offset one of the advantages of transgenics, 3) cost of transgenics may be very high, 4) proximity to sprayed fields and insect migration may reduce the effectiveness of transgenics, and 5) development of resistance in insect populations may limit the usefulness of transgenics.
Efficient deployment and management of transgenic plants in an effective manner will be an important prerequisite for sustainable use of biotechnology for crop protection. The most effective system for delivering insecticidal genes is through the production of transgenic plants. The system is economic, environmental friendly, and cost-effective. The transgenic plants provide season long protection against the target pests, while the pesticides need to be applied several times during the growing season. Also, only the insects feeding on the crop are exposed to the toxin, and this overcomes the difficulty of targeting pesticide application at the site of insect feeding. As a result of advances in genetic transformation and gene expression during the last decade, there has been a rapid progress in using genetic engineering for crop improvement, of which protection of crops against the insects is a major goal.
Entomologists, breeders, and the molecular biologists need to determine how to deploy this technology for pest management, and at the same time avoid or reduce possible environmental risks. To achieve these objectives, it is necessary to have an appropriate understanding of the insect biology, behaviour, its response to the insecticidal proteins, temporal and spatial expression of insecticidal proteins in the plants, strategy for resistance management, and impact of insecticidal proteins on natural enemies and non-target organisms. Equally important are the issues concerning the transfer of technology to the resource poor farmers. Development and deployment of transgenic plants with insecticidal genes for pest control will lead to: 1) reduction in insecticide sprays, 2) increased activity of natural enemies, and 3) IPM of secondary pests.
Bacillus thuringiensis was discovered in 1901 from diseased silkworm (Bombyx mori) larvae, and later isolated from diseased larvae of Ephetia kuehniella, and designated as Bacillus thruringiensis. Bacillus thuringiensis is a gram-positive bacterium, which produces proteinaceuos crystalline inclusion bodies during sporulation. There are several subspecies of this bacterium, which are effective against lepidopteran, dipteran, and coleopteran insects. Bacillus thuringiensis based formulations are the most important biopesticide world-wide with annual sales of nearly $90 millions, and there are 67 registered Bt products with more than 450 formulations. Bt also produces cyto-toxins that synergise the activity of Cry toxins. The identification of kurstaki strain provided the mush needed boost for commercialisation of Bt.
The Bt toxin gene was cloned in 1981, and the first transgenic plants were produced by mid-1980s. Since then, several crop species have been genetically engineered to produce Bt toxins to control the target pests. Genes conferring resistance to insects have been inserted into crop plants such as maize, cotton, potato, tobacco, rice, broccoli, lettuce, walnuts, apples, alfalfa, and soybean. The first transgenic crop was grown in 1994, and large-scale cultivation was taken up in 1996 in the USA. Since then, there has been a rapid growth in the area under transgenic crops in the USA, Australia, and China. Transgenic plants with insecticidal genes are set to feature prominently in pest management in both developed and the developing world in future. The majority of the counties have now accepted a biosafety protocol for the production and deployment of transgenic plants during a recent meeting at Montreal, Canada.
Expression of first modified genes in tobacco and tomato provided the first example of genetically modified plants with resistance to insects. The first transgenic plants with Bt were produced in 1987. A number of vectors have been developed for bacterial plasmids. These contain resistance to antibiotics as a selectable marker, a replication gene, and a multiple cloning site, with several restriction sites for DNA insertion. Selectable markers such as bar gene associated with resistance to herbicide phosphoinothricin (PPT) are incorporated to facilitate the identification of transgenic plants. Delivery of the vectors into the nucleus has been achieved by using Agrobacterium mediated transformation and biolistic method. Genes conferring resistance to insects have been inserted into crop plants such as maize, cotton, potato, tobacco, rice, broccoli, lettuce, walnuts, apples, alfalfa, and soybean. Genetically transformed crops with Bt genes have been deployed for cultivation in USA, China, and Australia.
Considerable progress has been made in developing transgenic crops with resistance to the target pests over the past decade. Successful control of pink bollworm (Pectinophora gossypiella) has been achieved through transgenic cotton. Field trials of transgenic maize with Cry type toxins have shown that they are highly effective against the European corn borer (Ostrinia nubilalis), and can withstand up to 50 larvae per plant at the whorl leaf stage and about 300 larvae at the anthesis stage. Maize plants with CryIA(b) gene have shown resistance to the sugarcane borers (Diatraea grandiosella and Diatraea saccharalis) (damage rating 2.4 - 2.6 compared to 10.0 in the susceptible control with 50 larvae per plant at the 6-leaf stage). However, only a slight reduction in damage has been recorded in case of fall armyworm (Spodoptera frugiperda) (leaf damage rating 8.0 - 8.7 compared to 9.5 - 10.0 in the controls). Transgenic maize expressing Cry9C gene, an insecticidal crystal protein from Bacillus thuringiensis subsp. tolworthi, effectively controlled both generations of the European corn borer. A truncated CryIA(b) gene in transgenic sugarcane plants has shown significant larvicidal activity against neonate larvae of sugarcane borer (D. saccharalis) despite low expression of CryIA(b).
Transformation of high-quality rices of group V is a feasible alternative to sexual hybridisation, and transgenic plants caused 100% mortality of the yellow stem borer (Scirpophaga incertulas). The transgene, Cry IA(b), driven by different promoters showed a wide range of expression (low to high). Insect bioassays have shown enhanced resistance to yellow stem borer. Successful expression of Bt genes has also been obtained in tobacco (Helicoverpa armigera), potato (Phthorimaea opercullela), brinjal (Leucinodes orbonalis, and Leptinotarsa decemlineata), and broccoli (Plutella xylostella, Trichoplusia ni, and Pieris rapae). A codon-modified Cry IA(c) gene introduced into groundnut indicated various levels of resistance to the lesser corn stalk borer (Elasmopalpus lignosellus). Chickpea cultivars ICCV 1 and ICCV 6 have been transformed with Cry IA(c) gene (Helicoverpa armigera).
Vegetative insecticidal proteins (VIPs), isolated from the clarified culture supernatant fluids collected during the vegetative growth (log phase) of Bacillus spp, can be used for genetic transformation of crops for resistance to insects. Bacillus cereus fluids show acute toxicity to Western and Northern corn rootworms (VIP 1 and VIP 2). Bacillus thuringiensis fluids show insecticidal activity against black cutworm, fall armyworm, and beet armyworm (VIP 3). Their acute toxicity is in the range of h g/ml (same as d -endotoxins). VIP 3 induces gut paralysis followed by complete lysis of gut epithelium cells, resulting in larval death. Symptoms resemble those of d -endotoxins, but action is delayed.
Enzymes with potential for use in genetic transformation of plants include alpha amylase inhibitors (e.g., from wheat against Agrotis spp. and from Phaseolus against Collasobruchus), polyphenol oxidases (Spodoptera exigua), insect chitinases (Spodoptera exigua), lipoxigenase from pea (Nilaparvata lugens), and cholesterol oxidase from Streptomyces culture filtrate (Anthonomus grandis). Proteinase inhibitors includes serine proteinase inhibitors and trypsin inhibitors [Kunitz type (soybean) and Bowman-Birk type (soybean, cowpea, pigeonpea], cysteine proteinase inhibitors (oryzacystatin against Spodoptera littorallis), and proteinase inhibitor I and II from tomato (Manduca sexta). Transgenic tobacco plants expressing trypsin inhibitor gene at nearly 1% (derived from cowpea via CaMV35S constitutive promoter) resulted in increased mortality, reduced insect growth, and reduced plant damage by Heliothis virescens and Helicoverpa zea. Transgenic tobacco has also been shown to enhance protection against Spodoptera littoralis, and Manduca sexta. Sweet potato cultivar Tainong 57 trypsin inhibitor gene introduced into tobacco cultivar W38 retarded larval growth of Spodoptera litura as compared to control plants. Several protease inhibitor gene constructs have been introduced into different transgenic crops. However, the observed effects have not been considered to be sufficiently convincing to lead to a serious attempt at commercialising these genes. Deployment of protease inhibitors for insect control requires a detailed analysis of the particular crop-insect interactions. The range of dissociation constants (Kd) for different PIs (protease inhibitors) with specific proteases is large, and this can be used to select the most effective inhibitor for gene transfer in a particular situation, e.g., transgenic tobacco expressing high levels of Kunitz type of trypsin inhibitor from soybean (SBTI) performs better than the tobacco plants expressing cowpea trypsin inhibitor (CpTI) against Heliothis virescens. Proteolysis by gut extracts is 40-fold more susceptible to inhibition by SBTI than to CpTI. However, CpTI is considered to be more useful for transfer, because unlike many SPIs, it is not deleterious to mammals. Also, many SPIs are toxic to beneficial insects such as honeybees, but CpTI is not.
Lectins bind to glycosylated proteins in the insect midgut, and are classified mainly on the basis their sugar-binding properties. They are active at µg levels (at least 1 order of magnitude higher than Bt), and result in larval growth inhibition with very little mortality. Lectins with biological activity have been isolated from snowdrop (GNA lectin - active against Nilaparvata lugens, Nephotettix virescens, and Myzus persicae), pea (against Callosobruchus maculatus), wheat germ (agglutinin), rice (cystanin), soybean (toxic to mammals), Phaseolus vulgaris (arcelin), castor (ricin - highly toxic to insects), garlic, and chickpea.
During the course of evolution, plants have developed effective counter measures to withstand the herbivores. Many classes of plant proteins and secondary plant substances have been shown to have toxic or antimetabolic effect on insects, and have been proposed as possible candidates for genetic engineering. A common feature of many of these compounds is that they have a chronic rather than an acute toxicity on insects, and their effects are less dramatic than those of the synthetic insecticides. Plant secondary metabolites include peptide hormones, foliar phenolic acid esters (rutin and chlorogenic acid), foliar enzymes (polyphenol oxidases and peroxidases), pyrethrins, alkaloids, terpenoids, steroids, non-protein amino acids, and flavonoids. These compounds (phytoalexins) are also produced in response to insect feeding. Peptide hormone is systemic, and has been implicated to mediate the induction of proteinase inhibitors. Systemic-induced responses are mediated through synthesis and action of jasmonic acid via its lipid precursor linolenic acid in tomato. Application of exogenous jasmonate induces synthesis of proteinase inhibitors. Arabidopsis mutants deficient in linolenic acid cannot synthesise jasmonates, and are highly susceptible to the fungal gnat (Bradysia impatiens). Foliar phenolic acid esters from Arachis paraguariensis (chlorogenic acid and rutin) inhibit development of Spodoptera litura. Chlorogenic acid (5 - caffeoyl quinic acid, CQA), and its precursors 5-CQA, 3-CQA, and 1- CQA severely impair the larval growth. Foliar enzymes such as polyphenol oxidases and peroxidases increase the inhibitory effect of 5-CQA by oxidising dihydroxy groups to ubiquinones that covalently bind to nucleophilic (-SH and -NH2) groups of proteins, peptides, and amino acids. Inhibitory effects on larvae feeding on plants are more severe than on larvae feeding on the isolated compounds.
Considerable progress has been made over the past decade in genetic engineering of crop plants for providing resistance to insects. The ideal transgenic technology should be commercially feasible, environmentally benign (biodegradable), and easy to use in diverse agroecosystems. It should also be harmless to the natural enemies, target the sites in insects that have developed resistance to the conventional pesticides, flexible enough to allow ready deployment of alternatives (if and when the resistance is developed by the pest), and preferably produce acute rather than chronic effects on the target insects. The value of chronic effects of plant derived genes on insects need to be emphasised. Transgenic crops may satisfy many of these requirements. Some of the criteria can be achieved by exploiting genes that are based on antibody technology. Single chain antibodies can be used to block the function of essential pest proteins. This approach of controlling insects would offer the advantage of allowing some degree of selection for specificity effects, so that pests, but not the beneficial organisms, are targeted. The development of a delivery system from transgenic plants to the insect haemolymph will remove a key constraint in the transgenic approach to crop protection. While several crops with commercial viability have been transformed in the developed world, very little has been done to use this technology to increase food production in the harsh environments of the tropics. Equally important is the need to make this technology available to farmers, who cannot afford the high cost of seeds marketed by the private sector. International research centres, advanced research institutions, and the national agricultural research systems can play a major role in promoting biotechnology for food production in the developing world.
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