Agrobacterium-mediated transformation and regeneration of transformants of Brassica juncea

A protocol for Agrobacterium-mediated genetic transformation was developed using hypocotyl and cotyledonary leaves of Brassica juncea varieties RSPR 01 and Varuna. Agrobacterium strain LBA4404 used in the study contained a binary plasmid pBI121 having nptII and GUS as marker gene and reporter gene, respectively. In order to optimize parameters that influence transformation efficiency, the explants of both varieties were suspended in bacterial suspension for 20 minutes and inoculated on MS medium (Murashige and Skoog, 1962) supplemented with growth regulators and antibiotics for selection of transformants. Maximum transformation was achieved when the explants were immersed in bacterial suspension having an OD of 0.6 at 600 nm and co-cultivated for 48 hrs. Optimum concentration of kanamycin for selection of transformants was 25 mg/l. Putative transformants were confirmed by histochemical GUS assay. It was observed that hypocotyls displayed better transformation efficiency in comparison to cotyledons. Regeneration of plantlets from transformed calli of Varuna was obtained after 1 month of culture on MS medium augmented with BAP (4 mg/l) and NAA (0.5 mg/l). It was concluded that RSPR-01 showed higher transformation efficiency while Varuna had higher regeneration response.