Agrobacterium mediated transformation in capsicum annuum L. cv.Mathania

A protocol for regeneration and genetic transformation has been established for chilli(capsicum annuum L. cv. Mathania). High frequency regeneration of shoot buds from cotyledonary leaves and hypocotyls was achieved with Murashige and Skoog's (MS) medium supplemented with 10 mg l⁻¹ BAP and 1 mg l⁻¹ IAA. Elongation of shoots bud and subsequent rooting was obtained on MS medium supplemented with 1 mg l⁻¹ IAA. A disarmed strain of Agrobacterium tumefaciens EHA 105 carrying a binary vector plasmid p35SGUSINT has been used for transformation. This vector contains neomycin phosphotransferase gene (nptII), whose expression confirms Kanamycin resistance in transformants. In addition to nptII, plasmid encodes β -glucuronidase, reporter enzyme used for studying the expression of foreign genes in plants. The cotyledonary leaf and hypocotyls explants from in vitro grown seeds were infected and co-cultivated for 10 min. in Agrobacterium solution was found to give maximum transient expression. Shoot buds were produced on the selective medium containing Kanamycin (50 mg l⁻¹) and Cefotaxime (700 mg l⁻¹). Frequency of transient GUS expression in leaves was (54.95%) and in hypocotyls was (59.83%). Conversion frequency of transient to stable transformation was 2.5% in both the explants. The transgenic nature of the regenerated plants was confirmed by the histochemical staining of GUS, and polymerase chain reaction (PCR) analysis of npt II gene.