Agriculture is the backbone of the economy of many of the developing nations. Also they have an important role in the food and food processing areas of life. In order to satisfy the growing populations and their food needs, many new revolutions have been made in the Agricultural industry to increase the yield of crop plants. The yield of the plants is increased by incorporating a resistance to abiotic or biotic factor that affect the growth of the plants. Many crop plants of this kind were successful and also proved useful. Without losing the nutritive value of the crop plants, these resistances are being engineered into them using genetical transformations. The major resistance engineered into plants is against insects, pests and microbes as they cause serious damage to the crop plants. One such method, widely used, is transformation using Agrobacterium tumefaciens. Also widely used another method is with the bacterium Bacillus thuringenisis. Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduce directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant. Agrobacterium tumefaciens is the causal agent of Crown Gall disease (the formation of tumors) in over 140 species of dicot. It is a rod shaped Gram negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for 'transfer DNA') into the plant cell, which is incorporated at a semi-random location into the plant genome.
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Agrobacterium tumefaciens (or A. tumefaciens) is an alphaproteobacterium of the family Rhizobiaceae, which includes the nitrogen fixing legume symbionts. Unlike the nitrogen fixing symbionts, tumor producing Agrobacterium are parasitic and do not benefit the plant. The wide variety of plants affected by Agrobacterium makes it of great concern to the agriculture industry (Moore et al., 1992).
Introducing insect resistance to plants
Many crop plants have been under the plant breeding technologies for years that have helped us to attain some resistance against the nematodes, microbes and insects. But the classical methods have failed to control few losses of the cropplants. So inorder to increase the yield, new techniques have been designed. The method of introducing novel genes into nuclear and chloroplast genomes by molecular genetics leads to introduction of new plant varieties. There are a wide range of genes available, among the genetic weapons used buy the insect species against their own kind. Several of these have been used and strategies have been designed to counter the inevitable evolution of resistance in crop plants. This has led to the development of transgenic plants carrying novel genes conferring resistance to insects. There are serious consequences with the environment mainly with the lack of bio degradation when insecticides are used to control insects. Protein toxins are used efficiently because they are completely degradable and due to their specificity to insects. Thus proteins those are toxic to insects but not to mammals are used. One such kind is using toxins derived from varieties of Bacillus thuringiensis. This spore forming soil bacterium produces a Crystal shaped inclusion bodies composed of the delta- endotoxin proteins 'Crystal' (Cry) and 'Cytolytic' (Cyt) coded by more than 50 Cry genes where Cry I- Cry IV are specific for insects and Cry V and Cry VI for nematodes. The protein of about 5Kda has two domains. The first domain, the toxic portion of the molecule, a seven numbered Î±-helix bundle with a central amphibatic helix thought to form pores in susceptible membranes, resulting in loss of ion selectivity and subsequent cell death. The second domain is required for receptor binding and it is this domain that restricts the toxicity of Cry proteins to certain cells in the midgut of insects. Expression of more than one toxic of Cry and Cyt toxins simultaneously may have a synergistic effect on resistance to insects. Although the Bt toxins are usually specific foe one order of insects, the toxins from the b bacterium Photorhabdus luminescens affects all groups of insects and have an effect similar to that of Bacillus thuringiensis. (Setlow, 2000).
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The use of gene transfer technology to introduce to crop plants provides an effective alternative to the use of chemicals. This technique has proved to be useful in the crops like Canola, Corn, Cotton, Papaya, Squash and Soya bean.
Insect resistance in Rice
Transgenic plants that have the ability to express Bt Cry endotoxins were found to be resistance against those insects that feed by chewing or biting. The simultaneous introduction of three genes expressing insecticidal proteins, Cry IAC, Cry 2A and gna into indica rice to control the pests have been successfully demonstrated (Magbol, et al, 2001). The Cry genes target the leaf folder and stem borer and the gna gene targets the planthoppers. This type of transgenic plant proved more useful and efficient than other binary transgenic plants.
A novel way of producing insect resistant plants which exhibit higher level of resistance against diamond back moths in Broccoli by combining both genetic transformation and conventional methods of breeding were demonstrated (Cao, et al, 2002). They generated independent transgenic Broccoli plants containing Bt Cry IAC and Cry IC genes in Broccoli and were pyramided into a single plant using hybridization techniques. The lines carrying Cry I AC and Cry I C Bt genes were selected by screening with Kanamaycin and Hygomycin and confirmed by PCR analysis.
Chitinolytic enzymes are interesting candidates for plant protein as they are active against many pests and because of their non-toxicity against non target organisms that lack Chitin. Thee enzymes act on the peritrophic membranes of pests feeding plants that express these protein and impair the gut physiology. One such Chitinolytic enzyme called Chitinase proved to be effective in enhancing plant resistance with other known insecticidal proteins and toxins (Jain, et al, 2009).
Problems of the resistance of insect pests to insecticides
Even though there are many advantages to creating the resistance of plants to insects the process that has its own disadvantages also. The use of synthetic toxic insecticides has created a greatest threat to the environment and health in their persistence and negative effects to non target organisms (Ishaaya, et al, 2007). There are problems linked with the resources of air, water and soil pollution, directly. There are also problems that are counter parts of the soil which include loss of crop, wild plant and animal genetic resources, elimination of natural enemies of pests, Pest resurgence and genetic resistance top pesticides, chemical contamination and complete destruction of natural control mechanisms (Conway, et al, 1991).
One of the important pesticide included problem is the development of resistance by the insect pests. The use of these insect resistance plants has led to the development of cross resistance in insect pests. Thus once the resistance is developed by the pests, they become resistance to all other crops in future. Globally about 504 insects and mites, 150 plant pathogens are known to have developed resistance. Resurgence is yet another problem being faced. They occur in two ways firstly rapid resurgence of pests populations exposed to the pesticide, nextly, minor pests or unimportant pests or target species developing into major pests as a result of decreased competition for food and shelter. Also it has led to social and health problems, such as birth defects, nerve damage, cancer and other effects that might occur over a period of time (Peshin, et al, 2009).
Natural example of plant resistance to insects
Plants also have the ability to fight against the insects by its own. The studies on these have led to the development of insect resistance to plants. Mechanisms of genetic resistance to insects have been divided into three categories, namely,
Antixenosis in which plant colonization by insects is deterred or reduced, primarily by altering the insect's behavior.
Antibiosis in which insects development is altered after colonization
Tolerance in which the resistance cultivar sustains as much as damage as a susceptible cultivar but without loss in quantity or quality of field.
Antixenosis is based upon physical characteristics such as thickness of cell walls, proliferation of wounded tissues, solidity of stems etc. Trichomes and Bract structures have proved important for boll Weevil resistance in cotton and Trichomes ion potatoes. Plant surface waxes also proved a deterrent effect on selected insects in Sorghum, Brassica Spp., Apple, Rice and Cotton. Also the resistance offered by the plants can be classified as vertical and horizontal resistance. Some example of vertical resistance also called as Qualitatively inherited insect resistance are Green Bug resistance in Barley and Wheat, Fall Armyworm and Corn Earthworm in Pearl Millet, Gall midge in rice, etc,. The Quantitatively inherited resistance or horizontal resistance includes first brood European corn borer in Maize, brown planthopper in rice and Mexican bean beetle in soya beans (Carozzi, et al, 1997).
Development of new methods for deterring insect herbivory
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Normally leaves release low levels of volatile chemicals, but when a plant is damaged by herbivorous insects, the emission of volatile compounds increases. The volatile photochemical can serve as airborne semiochemicals deterring or promoting interaction between plants and insect herbivores. For example, moths are repelled by herbivore induced volatiles released from tobacco plants at night such as odor cues and allow females to avoid oviposition on previously damaged plants. One important hypothesis is that the insect herbivores feed preferentially on stressed plants and on vigour sly growing plants as they are useful to them in many ways (Gullan, et al, 2010).
Mass production techniques and methods for applying S.carpocapsae are being developed by commercial producers. Steinernematid infective stages can be stored for a period of five years under cold conditions. They can be used along with pesticides for spraying to control pests and Nematodes. Heterorhabditiis Spp and S.glasesis have proved useful Steinernema for control of certain pests. But unfortunately the development of mass production and application techniques are not developed for their process (Capinera, 2008).
Techniques available for breeding
The same methods used to breed for any heritable characteristic are also used for breeding for insect resistance and depend on the mating system of the plant. Breeding for insect resistance however is complicated as the resistance can be assayed by making the plant to be attacked by the insect. Breeding for resistance may not be stable as they may loss certain conditions. Also recurrent selection or back crossing, a desirable but susceptible variety of a crop is crossed with another cultivated or wild relative that has the resistance to the insect. Other techniques available includes the use of F1 hybrids of two different genes of resistance, use of naturally or artificially (by UV light, x rays) that show increased resistance and altering the number of chromosomes by adding chemicals like Colchine and by radiation (Agrios, 2005).
Breeding for resistance using tissue culture and genetic engineering techniques
The process of breeding to produce insect resistance to plants using tissue culture is effective as they include meristerm tip propagation, callus and single cell culture, haploid plant production and protoplasm isolation, culture, transformation, fusion and regeneration to whole plants. The potential of these techniques is further augmented by molecular technologies. Tissue culture of insect resistance plant is useful on plants like strawberries, apple, bananas, sugarcane and potatoes. The plants generated from the culture are isolated and are tested for the insect resistance. These cultures were processed for the production of resistant dihaploids from Haploid plants by addition of chemicals like Colchicine. The insect resistance of the plants can be increased by protoplast fusion. This results in the production of Cybrid cells containing the nucleus of one cell and the cytoplasm of the other which are produced by the fusion of hybrid cells containing the nuclei and cytoplasm of both protoplasts. These cells produced are genetically transformed into the plant cells. There are various methods available for the same. This includes direct DNA uptake, microinjection of DNA, liposome mediated delivery of DNA, use of plant viral vectors and by the use of natural gene vector system of A. Tumefaciens. When DNA are introduced into plant cells, DNA gets integrated into the plan chromosomal DNA. When this DNA carries appropriate regulatory genes recognized by the plant cells, the DNA is expressed. So far only micro projectile bombardment and Agrobacterium system have been used successfully to introduce into plants specific new genes that were then expressed by the plant (Agrios, 2005).
There are at least four different ways of transferring the gene into the plants namely viral transduction, bacterial gene delivery, chemically and physically directed DNA transfer. And Agrobacterium mediated transformation. Agrobacterium mediated transformation is widely used and is most efficient in dicotyledonous plants. Agrobacterium tumefaciens (Figure 1) is a plant pathogen that induces the formation of tumours and is responsible for crown Gall disease. This crown gall tissues formed represents pure oncogenic transformation. The metabolism of the opines is a central feature of crown gall disease. The type of opine synthesised is determined by the bacterial strain. The ability to induce tumour into the plants is controlled by a Ti plasmid found only in virulent Agrobacterium strains. Since the presence is not required, it is clear that tumour inducing principle is transferred from the bacterium of the plant at wounded site (Zaenen, et al, 1974).
Figure 1: Plant infected with Agrobacterium tumefaciens (Brown, 2010).
Virulent strains of Agrobacterium tumefaciens harbour large plasmids (140-235 Kbp) and experiments involving the transfer of such plasmids between various octopines and nopaline utilising strains established that virulence and the ability to use and induce the synthesis of opines are plasmid borne traits (Zaenen, et al, 1974). Thus these plasmids that induced the tumours in the plants are known to be called as Ti- plasmids. They specify the type of opine that is synthesized in the transformed plant tissue and the opine utilized by the bacterium. The figure 2 describes the Ti-plasmid gene maps. Complete Ti-plasmid DNA is not found in plant tumour cells but a small significant segment of about 23Kbp is found integrated to the plant DNA. This DNA segment is called as T-DNA and carries gene that are responsible for unregulated growth and ability to synthesize opines.
Figure 2: Ti-Plasmid gene maps with the octopines and Nopaline maps (Primrose, et al, 2006)
Experimental protocol with Agrobacterium tumefaciens
The bacterium Agrobacterium tumefaciens were cultured with the Ti-plasmid containing the gene of our interest and are allowed to grow in a suitable medium. Once the bacterium is fully developed, they were allowed for co-cultivation with the explants obtained from the crop plants. The foreign DNA being inserted contains a chimeric 'neo' gene conferring resistance to antibiotic Kanamycin. Theses explants were cultured for two days and are selected for the transferred neo gene in a medium containing Kanamycin. The ex-plants containing the gene of interest are transferred into a suitable medium with required hormones to develop the plant parts. Rooted plantlets were transplanted to soil about 4-7 weeks from the inoculation step (Figure 3). Thus the process of transformation of genes into the plant can be achieved using Agrobacterium tumefaciens (Zaenen, et al, 1974).
Figure 3: Leaf disk transformation by Agrobacterium tumefaciens (Primrose, et al, 2006)
This is now evident that the various methods available to insert the insect resistance to the crop plants. These steps are under development in many cases in order to improve the efficiency and the quantity of the yield that is being produced.
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