Genetic engineering for crop improvement
- 1. GENETIC ENGINEERINGGENETIC ENGINEERING FOR CROP IMPROVEMENTFOR CROP IMPROVEMENT PLANT TRANSFORMATION WITHPLANT TRANSFORMATION WITH A.TUMIFACIENSA.TUMIFACIENS BY T.NIVETHITHA III-BIOTECHNOLOGY BON SECOURS COLLEGE FOR WOMEN.
- 2. Plant Transformation with the TiPlant Transformation with the Ti-- Plasmid ofPlasmid of A.tumefaciensA.tumefaciens Agrobacterium tumefaciens are gram-negative organisms responsible for transfer of target DNA(T- DNA). These phytopathogens, transforms plant cells (trasngenic plants), which genetic transformation(trasngenic plants), which genetic transformation leads to the formation of Crown gall tumors. As it helps in the transformation of plants, Agrobacterium sp. are known as Natural Genetic engineers. The involvement of Agrobacterium sp. was discovered by Smith and Townsend in 1907.
- 3. TiTi--PlasmidPlasmid Ti-plasmid carries about approximately 10-30kbp of T-DNA. The virulent strains of A.tumifaciens harbor large plasmids of about 140-plasmids of about 140- 235 kbp, known as Ti- Plasmids. It consists of vir region, origin of replication, region enabling conjugative transfer, o-cat region for catabolism of opines and most importantly T-DNA.
- 4. STRUCTURE OF TSTRUCTURE OF T--DNADNA T-DNA is a part of the “tumor-inducing” (Ti) plasmid, that is carried by most strains of A.tumefaciens. T-DNA is defined by its left and right borders and includes genes for the biosynthesis of auxin,cytokinin and opine.genes for the biosynthesis of auxin,cytokinin and opine.
- 5. Process Involved:Process Involved: The initial step in the infection is the attachment of A.tumefaciens to a plant cell at the site of an open wound, often at the crown(base), which produces a network of cellulose fibrils that bind the bacterium tightly to the plant cell surface. These bacteria respond to certain plant phenolic compounds, acetosyringone and hydroxyacetosyringone, excreted by wounded plants. These small molecules act to induce the virulence (vir) genes located on 35kb region of Ti-plasmid.
- 6. After Ti-plasmid carrying A.tumifaciens attaches to a host plant, the vir genes are induced and becomes integrated into the chromosomal DNA. The T- DNA also contains iaaM and iaaH genes.iaaH genes. The iaaM codes for the enzyme tryptophan 2- monooxygenase which converts tryptophan to indole 3-acetamide hydrolase, which coverts it to indoleacetic acid.
- 8. The T-DNA also carries ‘tmr’ gene(ipt), that encodes isopentenyltransferase responsible for the synthesis of cytokinin isopentenyladenine. The opines are synthesized within the crown gall and then secreted, which can be used as a carbon source, and sometimes as nitrogen source by A.tumifaciens. The binary cloning vector system and cointegratedThe binary cloning vector system and cointegrated cloning vector system are being used in this transfer technique. Finally, the transformed cells are being tested by Kanamycin treatment, and if they grew the transformation is confirmed and those transgenic plants or crops are cultivated in a large scale.
- 9. Genetic Engineering usingGenetic Engineering using A.tumifaciensA.tumifaciens -- An OverviewAn Overview
- 10. Physical Methods of TransferringPhysical Methods of Transferring Genes to PlantsGenes to Plants MICROPROJECTILE BOMBARDMENT- (Biolistics) Used with a wide range of plants and tissues; easy and inexpensive. VIRALVECTORS- Not an effective way to deliver DNA to plant cells. DIRECT GENETRANSFER INTO PLANT PROTOPLASTS- Can be used only with the plant cell protoplasts that can be regenerated into viable plants.
- 11. ELECTROPORATION- Generally limited to plant cell protoplasts that can be regenerated into viable plants. LIPOSOME FUSION- Can be used only with plant cell protoplasts that can be regenerated into viable plants. MICROINJECTION- Has limited usefulness because only one cell can be injected at a time; requires the services of a highly skilled individual.
- 12. APPLICATIONS:APPLICATIONS: • Crop Improvement • Genetically Engineered Traits: The Big Six 1. Herbicide resistance 2. Insect resistance2. Insect resistance 3. Virus resistance 4. Altered oil content 5. Delayed fruit ripening 6. Pollen control
- 13. • Biotech Revolution: Cold and Drought Tolerance and Weather-guard genes 1. Genetically Engineered foods 2. Soybeans 3. Corn3. Corn 4. Cotton 5. Other crops
- 14. Reference: Molecular Biotechnology: Principles and Applications of Recombinant DNA(2010) – Bernard R. Glick, JackDNA(2010) – Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten - 4th Edition.