Agrobacterium MEDIATED TRANSFER OF RICE CHITINASE GENE FOR FUNGUS RESISTANCE IN CHRYSANTHEMUM (Dendranthema grandiflorum Tzvelev cv. Snow Ball)
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2010
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ABSTRACT
An investigation was conducted to standardize the protocols for fungus resistance gene (chi 11) transfer studies in chrysanthemum cultivar Snow Ball using Agrobacterium mediated gene transfer technique. Agrobacterium tumefaciens strain containing a binary vector pCAMBAR:chi 11 having a marker genes (hpt and bar) and fungus resistance (chi11) gene was used for genetic transformation studies. Plant regeneration studies were carried out using two regeneration systems viz. through callus cultures and direct adventitious shoot regeneration from leaf and internode explant. The regeneration protocols were standardized which were utilized during genetic transformation experiments. Hygromycin, cefotaxime and kanamycin sensitivity experiments were conducted to study the effect of antibiotics on relative growth of tissues of chrysanthemum and hygromycin, cefotaxime and kanamycin concentration of 10 mg l-1, 300 mg l-1 and 10 mg l-1, respectively was optimized for selection of transformed tissues. The explants were dipped in the Agrobacterium suspension having 108 cells/ml (OD-0.521at 540 nm) and 20 and 15 min dipping duration was optimum for leaf and internode explants, respectively. The highest transformation frequency with leaf and internode explant was achieved with their pre-conditioning for 48 h followed by 96 h co-cultivation on selective medium inducing callus (10 mg l-1 kinetin + 0.5 mg l-1 NAA + 10 mg l-1 hygromycin + 300 mg l-1 cefotaxime and 0.5 mg l-1 BA + 0.5 mg l-1NAA +10 mg l-1 hygromycin + 300 mg l-1 cefotaxime, respectively). On the other hand, highest transformation frequency from explants inducing direct adventitious shoots was achieved with 48 h pre-conditioning followed by 96 hr co-cultivation on selective medium (2.0 mg l-1 BA + 0.5 mg l-1 NAA + 10 mg l-1 hygromycin + 300 mg l-1 cefotaxime for leaf explant and 2.0 mg l-1 BA + 0.25 mg l-1 NAA + 10 mg l-1 hygromycin + 300 mg l-1 cefotaxime for internode explant). The transformed calli from leaf explant was differentiated on selective medium containing 0.5 mg l-1 BA + 0.1 mg l-1 IAA + 10 mg l-1 hygromycin + 300 mg l-1 cefotaxime, wheras, the transformed calli from internode explant was differentiated on selective medium containing 0.5 mg l-1 BA + 0.25 mg l-1 IAA + 10 mg l-1 hygromycin + 300 mg l-1 cefotaxime. The putative transgenic shoots were multiplied on selective medium (0.5 mg l-1 BA + 0.1 mg l-1 IAA + 1 mg l-1 GA3 + 10 mg l-1 hygromycin + 300 mg l-1 cefotaxime) by repeated subculture to remove the escapes. The shoots were rooted on selective medium (1/2 MS + 0.2 mg l-1 IBA + 5 mg l-1 hygromycin) and acclimatized with 60-90% success. The putative transformed shoots were analyzed by PCR for the presence of chi11 gene with specific primers and by southern blot analysis for the integration of chi11 gene in plant genome. Only 7 plants out of 20 randomly chosen hardened plants showed the presence of chi11 gene in PCR analysis. PCR confirmed plants were further analyzed for the integration of chi11 gene in the genome of chrysanthemum plant and only 2 out of 7 PCR positive showed the integration of gene in southern hybridization experiment. The 2 plants were multiplied and hardened and were analysed for the expression of chi11 gene through bioassays which were carried out by spraying Septoria obesa spore suspension on the transformed plants kept under warm and humid conditions. Preliminary studies with the spore suspension of Septoria obesa indicated a significant expression of chitinase (chi11) gene in the transformed plants with no development of symptoms on the transformed plants in comparison to control plants.
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chrysanthemum cultivar,Agrobacterium