An improved method for Agrobacterium-mediated genetic transformation from cotyledon explants of Brassica juncea

An efficient Agrobacterium-mediated genetic transformation method was established for Brassica juncea by investigating several factors responsible for successful gene transfer. Four-day-old cotyledon explants from in vitro grown seedlings were co-cultivated with Agrobacterium strain GV3101 harboring the binary vector EnPCAMBIA1302-YCF1, which contained the hygromycin phosphotransferase (HPT) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene. Two days co-cultivation period on shoot induction medium (MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid, 1.0 mg l−1 6-benzyladenine, and 2.0 mg l−1 silver nitrate) containing 20 mg l−1 acetosyringone and five days delaying exposure of explants to selective agent enhanced transformation efficiency significantly. A three-step selection strategy was developed to select hygromycin resistant shoots. Hygromycin-resistant shoots were subsequently rooted on root induction medium. Rooted plantlets were transferred to pot-soil, hardened, and grown in a greenhouse until maturity. Using the optimized transformation procedure, transformation efficiency reached at 16.2% in this study. Southern blot analysis was performed to confirm that transgenes (HPT and YCF1) were stably integrated into the plant genome. All transgenic plants showed single-copy of transgene integration in the host genome. Segregation analysis of T1 progeny showed that the transgenes were stably integrated and transmitted to the progeny in a Mendelian fashion.