Agrobacterium mediated transformation of Brassica juncea with Arabidopsis annexin (AnnAt1) gene

Annexin (AnnAt1) gene from Arabidopsis thaliana was introduced into Brassica juncea by Agrobacterium tumefaciens mediated transformation. Hypocotyls from 5-day-old seedlings of Brassica juncea cv RLM 198 with 2 day preculture were infected with Agrobacterium strain GV2260 and GV3101 harboring a binary vector containing Annexin (AnnAt1) gene. It was observed that the Agrobacterium strain GV3101 transformed the Brassica hypocotyls with more efficiency than the strain GV2260. A total of 1716 hypocotyls of Brassica juncea cv RLM 198 co-cultured with GV3101 strain, showed average callusing of 57% and shoot regeneration of 13% in MS medium supplemented with 5.37 μM NAA, 4.44 μM BAP, 20 μM AgNO₃ and 86μM kanamycin as selective agent. A total of 108 kanamycin resistant plantlets were regenerated. PCR was used to identify and characterize the transformed plantlets. Finally, five T₀ plants were hardened in transgenic glass house and scored for incidence of Alternaria blight. At least one T₀ plant showed a decrease in area and number of necrotic lesions when compared with untransformed control plant. This has suggested that presence of Arabidopsis annexin (AnnAt 1) which is anti-oxidative in nature, modulated defense response in Brassica plants. The heritability analysis of T₁ progeny of a T₀ plant for annexin gene by PCR has further suggested that the T₀ plant had mosaics of transformed and untransformed sectors at organ level.