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Plant transformation is an indispensable tool both for the experimental investigation of gene function and for the improvement of plants either by enhancing existing traits or introducing new ones (Hansen et al., 1999; Birch RG, 1997; Komari et al, 1998 ). It is now possible to introduce and express DNA stably in nearly 150 different plant species. Plant transformation research reflects expectations that the technology can rapidly produce plants with improved or novel traits for the benefit of mankind. These improvements would be difficult or impossible to achieve with conventional breeding alone. The results of this research are already available and include herbicide-tolerant plants, plants showing resistance to insect pests and viral, bacterial, and fungal diseases (Birch RG, 1997).
Control of insect pests represents one of the major input costs of the world agricultural production and consumes billions of dollars annually, predominantly in chemical pesticides and lost production. Insect control with pesticides is however, becoming more problematic in both developed and third world countries. Farmers and consumers are increasingly aware of the environmental and ecological impacts of excessive pesticide use. As the target insects are repeatedly exposed to the same pesticide chemistries, they often develop heritable resistance to those pesticides and require the use of higher and higher doses or more toxic insecticide blends to achieve economic levels of pest control. Plant breeders, seed companies, and farmers are now turning to biotechnological solutions to these important pest problems in agriculture because of the environmental and economic advantages of deploying inbuilt insect control strategies as opposed to external applications. Over the past 10 years, transgenic crops protected against insects have been generated in some of the important broadacre crops, including cotton and corn, as well as in horticultural crops such as potato, and have been released commercially (Peferoen M, 1997).
It is estimated that about 15% of world's crop yield is lost to insect or pests. A selected list of common insects and the crops damaged is given in table 1.
LIST OF COMMON INSECT PESTS ALONG WITH THE MAJOR CROPS DAMAGED BY THEM (TABLE 1)
COMMON NAME OF THE PEST
Rice, maize, cotton, tobacco
Tobacco, tomato, potato
European corn borer
Cowpea seed beetle
Brown plant hopper
The damage of crops is mainly caused by insect larvae and to some extent adult insects. That damage crops belong to the following orders (with examples), Lipidoptera (bollworms, coleopteran (beetles), orthoptera (grasshoppers), homoptera (aphids) (satyanarayana U, 2008)
PROBLEMS OF THE RESISTANCE OF INSECT PESTS TO INSECTICIDES
Now a day the major problem of using insecticide is, resistance occurs to the insect when it is exposed to insecticide, so not all insects are killed by them. This is because the insect pests produce resistant to the insecticide. The offspring of these survivors will carry same genetic information of their parents. The offspring has ability to tolerate and survive to the exposure of insecticide. Therefore many new generations of insect pests will take place in few weeks, so in the short period of time the entire population of insects will be resistance to the insecticide. Thus the resistance develops in same manner in all other insect pests against insecticides. Therefore this is the main failure case in using pesticides (Robert G. Bellinger, 1996). It is a fortunate that scientists have been able to discover new biotechnological alternatives to chemical pesticides thereby providing insect resistance to crop plants. Transgenic plants with insect resistance transgenes have been developed. About 40 genes obtained from the microorganisms of higher plants and animals have been used, to provide insect resistance in crop plants.
NATURAL PLANT RESISTANCE TO INSECTS
RESISTANCE GENES FROM HIGHER PLANTS
Certain genes from higher plants were also found to result in the synthesis of products processing insecticidal activity, they are called as non-Bt insecticidal proteins. A selected list of plant insecticidal (non Bt) genes used for developing transgenic plants with insect resistance is given in table 2. Some of them are explained below.
PROTEINASE (PROTEASE) INHIBITORS:
Proteinase inhibitors are the proteins that inhibit the activity of proteinase enzymes. Certain plants naturally produce proteinase inhibitors to provide defence against herbivorous insects. This is possible since the inhibitors when injected by insects interfere with the digestive enzymes of the insect. This results in the nutrient deprivation causing death of insects. It is possible to control insects by introducing proteinase inhibitor genes into crop plants that normally do not produce these proteins.
COWPEA TRYPSIN INHIBITOR GENE:
It was observed that the wild species of cowpea plants growing in Africa were resistant to attack by wild range of insects. Research findings revealed that insecticidal protein was trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera, Orthaptera and Coleoptera. Cowpea trypsin inhibitor (CpTi) has no effect on mammalian trypsins; hence it is non-toxic to mammals. CpTi gene was introduced into tobacco, potato and oilseed repe for developing transgenic plants. Survival of insect and damage to plants were much lower in plant processing CpTi gene.
the insect larvae secrete a gut enzyme α-amylase to digest starch. By blocking the activity of this enzyme by α-amylase inhibitor, the larvae can be starved and killed. Α-amylase inhibitor gene (α=Al-Pv) isolated from the been has been successfully transferred and expressed in tobacco. It provides resistance against Coleoptera (e.g.Zabrotes subfasciatus).
Lectins are the plant glycoproteins and they provide resistance to insects by acting as toxins. The lectin gene (GNA) from snowdrop (Galanthus nivalis) has been transferred and expressed in potato and tomato. The major limitations of lectin are that it acts only against piercing and sucking insect, and high doses are required.
SELECTED LIST OF PLAT INSECTICIDAL (NON BT) GENES USED FOR DEVELOPING TRANSGENIC PLANTS WITH INSECT RESISTANCE (TABLE 2)
RESISTANCE TO INSECTS
Potato, apple, rice, sunflower, wheat, tomato
Tobacco, oilseed rape
Potato, rice, sugarcane, sweet potato, tobacco
INSECT RESISTANCE THROUGH COPY NATURE STRATEGY
Some of the limitations experienced by transferring the insecticidal genes (particularly Bt) and developing transgenic plants have promoted scientists to look for better alternatives. The copy nature strategy was introduced in 1993 (by Boulter) with the objective of insect pest control which is relatively sustainable and environmentally friendly. The copy nature strategy for the development of insect-resistant transgenic plants as follows.
The first step in the copy nature strategy is to identify the plants that are naturally resistant to insect damage. The protein with insecticidal properties (from the resistant plant) is isolated and purified. The sequence of the protein is determined, and the gene responsible for its production identified. The activity of the protein against the target insects is determined by performing a bioassay. It is necessary to test the toxicity of the protein against mammals. By the conventional techniques of genetic engineering, the isolated gene corresponding to the protein is introduced into the crop plants. The developed transgenic plants should be tested for inheritance and appropriate expression of transgene. The efficiency of the insecticidal protein to destroy insects is also evaluated. Field trials have to be conducted to evaluate the crop yield, damage to insects, influence on the environment with respect to the transgenic plant (satyanarayana U, 2008).
SELECTABLE MARKER GENES
The selectable marker genes are usually an integral part of plant transformation system. They are present in the vector along with the target gene. In a majority of cases, the selection is based on the survival of the transformed cells when grown on a medium containing a toxic substance. This is due to the fact that the selectable marker gene confersmresistance to toxicity in the transformed cells, while the nontransformed cells get killed (Wilmink et al., 1993)
AGROBACTERIUM-DERIVED PROMOTERS AND TERMINATORS
The genes coding for napaline synthase (nos) in the Ti plasmid of Agrobacterium are frequently used as promoters and terminators in plant transformation vectors. Originally derived from bacteria, the genes coding for opine synthesis are well adapted to function in plants. In fact, nos promoter is regarded as constitutive by many plant biotechnologists.
METHODS FOR INTRODUCING INSECT RESISTANCE INTO CROP PLANTS
Here both Agrobacterium tumefaciens- mediated gene transfer and direct DNA transfer methods have been used to produce transgenic plants with new genetic properties for the plants resistance to insects. Several genes for insect resistance have been transferred through genetic engineering techniques. Use of delta-endotoxin coding sequences originating from Baccilus thuringinesis (BT) and the use of plant derived genes, such as those encoding enzyme inhibitors or lectins are the two main approaches to produce such genetically engineered plants ( Mohan Babu et al., 2003)
Agrobacterium tumefaciens is a gram-negative soil bacterium responsible for crown gall disease, a neoplastic disease of many dicotyledonous plants characterized by the appearance of large tumors (galls) on the stems. Virulence is conferred by a large tumour- including plasmid (Ti plasmid) containing genes encoding plant hormones (auxins and cytokinins) and enzymes that catalyze the synthesis of amino acid derivatives termed opines (Van Larebeke et al., 1974). The plant hormones are responsible for deregulated cell proliferation that accompanies crown gall growth, while the opines are secreted by the plant cells and used by bacteria as food. These genes are contained on a specific region of the Ti plasmid, the T-DNA (transfer DNA), so called because it is transferred to the plant nuclear genome under the control of virulence genes carried elsewhere on the Ti plasmid (Zupan et al., 1997). It is this natural gene transfer mechanism that is exploited for plant transformation.
DEVELOPMENT OF Ti-PLASMID VECTORS
The earlirst indication that t-DNA could be used in plant transformation vector was the demonstration that DNA from Escherichia coli plasmid (the Tn7 transposon) could be stably transferred to the plant genome by first incorporating into the T-DNA (Hernalsteens et al., 1980). However transgenic plant could not be recovered from the transformed cells, either by regeneration or by grafting onto normal plants, because the hormones encoded by the T-DNA oncogenes causes unregulated and disorganized callus growth (Hernalsteens et al., 1980). In rare cases, shoots were derived from such callus tissue, and analysis showed that much of the T-DNA (including the oncogenes) had been deleted from the genome (Wullems et al., 1981). An important of T-DNA vectors was the realization that the only requirements for T-DNA transfer to the plant genome were the vir genes and the 24-bp direct repeat structures marking the left and right boders of the T-DNA. No genes within the T-DNA were necessary for transformation and any sequence could be incorporated therein. This allowed the development of disarmed Ti plants (Zambryski et al., 1983) lacking all the oncogenes, facilitating T-DNA transfer to plant cells without causing neoplastic growth.
Once suitable selectable markers has been incorporated into the TDNA, Ti plasmids became very powerful gene delivery vectors. However, wild type Ti plasmids were unsuitable for the task due to their large size, which made them difficult to manipulate in vitro (large plasmids have a tendency to fragment and they lack unique restriction sites for sub cloning). An early strategy to overcome this problem was the development of intermediate vectors, where the T-DNA was subcloned in to a standard E.coli plasmid vector, allowing in vitro manipulation by normal procedures, and then integrated into the T-DNA sequence of a disarmed Ti plasmid resident in A.tumefaciens by homologous recombination (Matzke et al., 1981). This system was simple to use but relied on a complex series of conjugative interactions between E.coli and A.tumefaciens requiring three different bacterial strains (triparental matings). However, because the vir genes actin trans to mobilize the T-DNA, it was soon discovered that use of large natural Ti-plasmids was unnecessary. Intermediate vectors have been largely superseded by binary vectors (Bevan M, 1984), in which vir genes and the T-DNA are cloned on separate plasmids. These can be introduced in to A.tumefaciens by conjugation with an E.coli donar or by freeze-thaw cycles or electroporation. Most contemporary Agrobacterium-mediated transformation systems employ binary vectors.
Agrobacterium mediated transformation was achieved by co cultivating a virulent A.tumefaciens strain containing a recombinant Ti plasmid with plant protoplasts and then obtaining callus from the protoplasts from which fertile plants were regenerated. This strategy was widely replaced by a simpler method in which small discs were punched from the leaves of the recipient species and incubated in medium containing A.tumefaciens prior to transfer to solid medium (Horsch et al., 1985). Infection can be promoted under conditions that induce virulence (presence of 10-200µM acetosyringone or α-hydroxyacetosyringone, acedic pH, and room temperature), although these need to be optimized for different species. After co culture for several days, the discs are transferred to a medium containing selective agents to eliminate non transformed plant cells, antibiotics to kill the bacteria, and hormones to induce shoot growth. After a few weeks, shoots develop from transformed callus cells. These can be removed and transferred to rooting medium or grafted onto seedling root stock. Most current protocols for the Agrobacterium-mediated transformation of solanaceous plants are variations on the leaf disc theme, although different tissue explants are suitable transformation targets in different species. Alternative methods are required for transformation of monocots. Rapidly dividing embryonic cells (eg.. immature embryos or callus induced from scutellar tissue) all required for transformation of rice (Hiei et al., 1994) and other cereals. These are cocultivated with Agrobacterium in the presence of acetosyringone.
FIGURE 1: Strategies for the transformation of higher plants.
EXAMPLE FOR INSECT RESISTANT TRANSGENIC PLANT BY Agrobacterium-MEDIATED TRANSFORMATION
The BT gene of a bacterium, Baccilus thuringinesis has been found to encode the toxins called endotoxin which pose cidal effect on certain insect pests. These toxins are of different types such as beta-endotoxin and delta-endotoxin. The cry genes of Baccilus thuringinesis (commonly called BT gene) was found to express proteinaceous toxin inside the bacterial cells. when specific insects (specius of Lepidoptera, Diptera, coleopteran, etc.) inject the toxin, they are killed. Toxin denatures the epithelium of gut by creating many holes at alkaline pH (7.5-8). The insecticidal toxin of Baccilus thuringinesis has been classified in to four major classes: cry I, cry II, cry III, cry IV, based on insecticidal activities against many insects. These toxins affect to the specific group of insects. They do not kill the silkworm and butterflies or other beneficial insects.
Figure 2:Transgenic cotton (A) and non-transgenic cotton (B)
The approach has been the isolation of the Bt gene and its introduction into Ti-DNA plasmid of A.tumefaciens. The genetically modified A.tumefaciens was allowed to infect the desired plant. Thus the Ti-plasmid transformation of several plants has been done, for example tobacco, cotton, tomato, corn, etc. Field experiments has also been done with manducta sexta which is a serious pest of tobacco. It has been found that 75-100% larvae of manducta sexta died when chewed the leaves of transgenic tobacco, where as the control plants (that was not transgenic) were severly damaged by the insect. Besides when tobacco plant was crosses with normal (control) plant, the resistance gene was inherited as per mendelian principle. Using biotechnological approaches too many transgenic crops having cry gene that is Bt-genes have been developed and commercialised. Some examples of Bt-crops are brinjal, cauliflower, cabbage, canola, corn, cotton, eggplant,, maize, potato, tobacco, rice, soyabean, etc (Dubey R.C, 2009)
This technique was based on a device that used gun powder to accelerate small tungsten particles to a velocity of approximately 400 m/s. The particles were coated with DNA and could penetrate plant cells without killing them. Particle bombardment was an efficient method for stable integrative transformation (klein et al.,1988).
ADVANTAGES AND DISADVANTAGES OF PARTICLE BOMBARDMENT
In this technique the plasmid DNA is prepared by standard methods and precipitated on to tungsten or gold particles using CaCl2. Spermidine and PEG are included to protect DNA during replication, and the particles are washed and suspended in ethanol before drying on to a mylar aluminized foil. This is fired against a retaining screen that allows the microprojectiles through, to strike the target tissue. Particle bombardment is widely used because it circumvents two major limitations of Agrobacterium system. First, it is possible to achieve the transformation of any species of the cell type by this method because DNA delivery is controlled entirely by physical rather than biological parameters. Second, particle bombardment allows the stable and heritable introduction of many different genes at once using different plasmids, as these tend to concatemerize to form one DNA cluster that integrates at a simple locus. Conversely, multiple transformation using the Agrobacterium system requires the co integration of all the genes in the same T-DNA (Christou p, 1994; Vain et al., 1993).
Insect control through biotechnology is an outstanding example of the use of gene technology to enhance the efficiency and sustainability of production of broadacre crops. It is not without its risks, such as the development of resistance by the target insects and potential nontarget impacts in the environment. There must be a concerted effort to develop new insect tolerance genes to ensure that the existing technologies are not lost by overuse in the short term. However, finding highly potent insecticidal genes that are as effective as the first generation of genes based on delta endotoxins from Bacillus thuringiensis is not a simple matter. The new class of vegetative insecticidal genes from Bacillus species holds the most promise for the control of difficult lepidopteran and coleopteran pests, but they have yet to be
Expressed effectively in transgenic plants.