Factors Enhancing Agrobacterium tumefaciens mediated Gene Transfer in Chilli (Capsicum annum L.)

A reliable and highly efficient system of transforming shoot tips mediated by Agrobacterium tumefaciens was developed in Capsicum. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. Agrobacterium tumefaciens strain EHA 105 harbouring the pIG121 Hm plasmid containing �-glucuronidase (GUS) gene driven by CaMV 35s promoter, with an intron from castor catalase gene. The plasmid was also contains two selectable marker genes, hygromycin resistance genes (HPTII) and kanamycin resistance gene (NTPII). An efficient protocol for direct plant regeneration from the shoot tip explant of Capsicum cultivar PLR 1 was standardized. The effect of various factors like preculture, bacterial concentration, co cultivation period and optimal concentration of selection agents on transformation efficiency were evaluated. The precultured shoot tip explants (1-2 days) co cultivated for days after infection with Agrobacterium having a concentration of 1. 0 OD at A600 for 10 min were showed better transformation efficiency. For regeneration of transformed shoots 100 mg l-1 kanamycin was chosen as the optimum concentration, where as for rooting it was reduced to 50 mg l-1. Five transgenic lines were developed and confirmed by gus assay, PCR and southern analysis. A transformation efficiency of 2.5% was achieved using the optimized transformation procedure.