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Transgenic plants
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- 2. NORMAL TOMATO E.coli Desired gene vector Modified vector Normal tomato plant GM PLANT
- 3. METHODS OF GENETRANSFER IN PLANTS GENE TRANSFER HORIZONTAL GENE TRANSFER VERTICAL GENE TRANSFER SOMATIC HYBRIDISATION GENETIC TRANSFORMATION DIRECT GENE TRANSFER AGROBACTERIUM MEDIATED GENE TRANSFER VIRUS MEDIATED GENE TRANSER
- 4. MECHANISM OF TRANSFORMATION There aremainly 3 methods for genetic transformation :- Agrobacterium mediated gene transfer Ti plasmid Ri plasmid Virus mediated gene transfer Direct gene transfer/physical method microinjection , electroporation , chemical transformation, particle bombardment
- 5. AGROBACTERIUM MEDIATED GENE TRANSFER Most classical example of HGT in plants is the infection of dicot plant cells with Agrobacterium. It has 2 species that are useful in gene transfer , Agrobacterium tumifaciens & Agrobacterium rhizogenes. These are gram negative soil bacteria responsible for causing crown gall disease and hairy root in plants respectively. The plasmids carrying for these characters are known as Ti (tumour inducing) plasmid & Ri (root inducing) plasmid respectively.
- 6. Ti plasmid :-Plasmid conjugation Virulence region Right borderLeft border T - DNA Over drive ORI Norpaline / octopine catabolism (2,00,000 nucleotide pairs)
- 7. Ti plasmid vector:- (onco genes deleted) They are of 2 types based on whether the DNA to be introduced is physically linked to vir gene (co-integrating or cis-vectors) or not (binary or trans vectors).
- 8. Ri plasmid :- Both Ti and Ri plasmid have similar appearance except for that, the Ri plasmid lack the cytokinine synthesis region and have homology only for auxin and opine synthesis sequences. Mechanism of transfer for Ti & Ri plasmids are the same.
- 9. Method of agrobacteriummediated gene transfer:-
- 10. Virus mediated genetransfer :- Of all the viruses cauliflower mosaic virus (CaMV) is considered a most potential vector for plant transformation as its genome is composed of double stranded DNA. Brisson et al. (1984) introduced methotrexate resistant dihydrofolate reductase (DHFR) gene of E. coli into turnip plants using CaMV as the vector.
- 11. Limitation of virusmediated transformation :- It is not expected to bring about heritable transformation as neither the gene gets integrated into plant genome nor is the virus transmitted through seeds. As a plant transformation vector is that genes larger than a few hundred bp (DHFR is 234 bp) interfere with the packaging of the genome into the virion particle.
- 12. Direct gene transfer:- (i) Chemical transformation :- Many chemicals such as polyamines (poly-L- ornithine and poly-L-lysine) and dextran sulphate stimulate DNA uptake into protoplasts. However, they also highly reduce cell viability. Of the many chemicals tested for their ability to stimulate DNA uptake into protoplasts polyethylene glycol (PEG) has proved most effective. Treatment of protoplasts with MgC12 (5-25 mM) significantly improves transformation rates.
- 13. (ii) Electroporation :- Apopular physical method for introducing new genes into protoplasts is the use of electric field which makes the protoplasts temporarily permeable to DNA.
- 14. (iii) microinjection :- Directinjection of DNA into plant protoplast or cells using fine tipped pipette has also been tried to transform plant cells.
- 15. (iv) Particle bombardmentmethod:- Particle bombardment (or Biolistics), wherein microscopic tungsten or gold particles (1-3 ttm) coated with genetically engineered DNA are explosively hurled into plant cells with high velocity.
- 16. Application :- Herbicide resistantplants :- Several classes of herbicides are quite effective for broad spectrum weed control. However, as they act by inactivating target proteins (mostly enzymes) essential for vital functions in plants, such as photosynthesis, shared by both crop and weed plants, they are either nonselective and kill the crop plants or cause significant injury to them at the application rates required to eliminate the weeds. Production of herbicide resistant/tolerant crop plants can considerably reduce these losses.
- 17. Three approaches havebeen followed to obtain herbicide resistant transgenic plants:- (1) modification of the plant enzyme or other sensitive biochemical target of herbicidal action to render it insensitive to the herbicide (2) overproduction of the unmodified target protein permitting normal metabolism to occur even in the presence of the herbicide (3) introduction of an enzyme or enzyme system to degrade and/or detoxify the herbicide prior to its action.
- 18. Insect resistance :- Insectsare serious pests of agricultural products in the field and during storage. Insects belonging to the orders Coleoptera, Lepidoptera and Diptera are the most serious plant pests. Bacillus thuringiensis (Bt), employed as an insecticide for over 20 years and its demand is continuing to increase because of its specificity towards lepidopteran pests and being environmentally safe. Insect resistant transgenic plants have also been created by introducing trypsin inhibitor gene.
- 19. (i) Bt toxingene expression:- Sporulating Bt produces non-toxic proteinaceous endotoxin (protoxin) of 130-140 kDa which upon hydrolysis, under highly alkaline conditions (pH 9-12) of the insect gut, produces smaller insecticidally active peptides of 65 -70kDa.These polypeptides specifically attack the brush bordermembrane of midgut epithelial cells paralysing the insect and eventually killing it. The first Bt gene was cloned and characterized in 1985. The first transgenic plants showing field resistance to insects were produced by introducing the truncated Bt toxin gene.
- 20. (ii) Trypsin inhibitorgene expression :- The occurrence of certain proteins in plants which inhibit the common insect digestive protease have been implicated in natural mechanism of plant defence against insect attack. For example, the leaves of a number of plants, including tomato, synthesize protease inhibitors in response to insect attack. Similarly, cowpea trypsin inhibitor (CpTI), a 80 amino acid polypeptide, has been identified as an insecticidal component of cowpea seeds, which is active against a range of economically important field and storage pests, including bruchid beetle, a major pest of stored cowpea.