AGROBACTERIUM TUMEFACIENS MEDIATED TRANSFORMATION OF INDICA RICE AND MOLECULAR ANALYSIS OF REGENERATED PLANTSN. MANDAL, R. MAJUMDAR AND J. TARAFDAR
An attempt was made to Agrobacterium mediated transformation in indica rice for developing abiotic stress tolerance. Two plasmids pEXSOD10 and pGV2260 each containing riptIl selectable marker were used for genetic transformation. Fe-SOD gene (954 bp) was housed in pEXSOD10 to transfer into cultivar Annada to impart resistance to superoxide radical damage. On the other hand, osrnotin gene (750 bp) was housed in pGV2260 to dispense into Pusa Basmati providing tolerance to excess salt (NaC1) and drought. Somatic embryogenic calli of Annada (562) and Pusa Basmati (336) were co-cultivated with Agrobacterium strains C58C1 and GV2260 separately, harbouring pEXSOD10 and pGV2260, respectively. Out of 562 calli of Annad a co-cultivated for transformation with Fe-SOD, only 370 survived on the kanamycin selection medium. Similarly in respect of osmotin, out of 336 only 257 calli survived in Pusa Basmati. But only 26 transformed calli in Annada and 14 in Pusa Basmati were regenerated into plantlets on regeneration medium. Finally 19 plantlets in Annada and 11 in Pusa Basmati were grown to maturity after selection in kanamycin for 14 days. Nineteen tillers one from each of 19 lines of Annada were screened for high level of superoxide clismutase activity through methyl violagen test. Only three lines showed positive result and expected to reveal tolerance to photooxidative damage / superoxide radical toxicity. The presence of riptll gene in regenerated plants of Annada and Pusa Basmati were also demonstrated by PCR analysis. Integration of the transgene Fe-SOD in three putative transgenic plants of Annada (ATP5, ATP14 and ATP20) and osmotin in three plants of Pusa Basmati (BTP8, BTP10 and BTP11 were confirmed by Southern hybridization. This method of gene transfer holds great promise to improve indlca rice with the incorporation of alien genes in the genome.
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