Brassica juncea is one of the most important oilseed crop. We have developed an efficient and reproducible protocol for Agrobacterium-mediated transformation of B. juncea variety NIFA RAYA with Wasabi defensin gene to produce transgenic plants. This gene was isolated from the leaves of Wasabia japonica. We find out that co-cultivation of explants with the transformed agrobacterium inoculum having an OD ranged from 0.6-1 for 15-20 minutes give high transformation efficiency. These explants were then regenerated on MS medium containing 1-6 mgL-1 BAP, 0.1-0.6 mgL-1 NAA and 300 mgL-1 cefotoxim sodium with subsequent application of 10-100 mgL-1 kanamycin. Our results indicated that medium containing 3 mgL-1 BAP and 0.3 mgL-1 NAA was found to be the best hormone combination for callus formation whereas BAP 3 mgL-1 and NAA 0.5 mgL-1 was found to be the best combination for shoot regeneration. In all cases supplementing the medium with 20 µM AgNO3 had positive effect on callus formation and shoot regeneration. The transgenic nature of the transformed callus and regenerated shoots was confirmed via PCR analysis.