Optimization of Genetic Transformation System in Brassica napus via Agrobacterium

Efficient callus induction and plant regeneration are critical steps in plant tissue culture and genetic transformation. This study focused on optimizing nutrient media, plant growth regulators (PGRs), sterilization protocols, and transformation parameters to enhance regeneration efficiency in Brassica species. Murashige and Skoog (MS) medium supplemented with specific concentrations of cytokinins (BAP, KN) and auxins (NAA, IAA) proved most effective for callus induction and shoot regeneration. The optimal combination of 5.0 µM BA and 0.5 µM IAA yielded the highest regeneration rate (~90%), while Treatments with 5.0 μM BA and 0.5 μM IAA showed reduced efficiencies at 70% and 75%, respectively. Sodium hypochlorite (NaOCl) was the most effective surface sterilant, with eight rinsing cycles post-treatment leading to maximal regeneration by minimizing residual toxicity. Explant type significantly influenced outcomes; cotyledonary nodes and immature embryos demonstrated superior regenerative potential compared to leaf or hypocotyl tissues. Environmental conditions such as a temperature range of 25–28°C and controlled light intensity further improved culture performance. Sucrose at 2–3% served as the optimal carbon source, balancing energy supply and osmotic pressure. These findings support an integrated protocol for efficient genetic transformation in Brassica, facilitating future applications in functional genomics and crop improvement.