Improved shoot regeneration protocol for canola explants and pre-assessment of salinity tolerance in canola transgenic plants

Regeneration of explants plays a significant role in plant transformation. Explant type, hormonal concentration, and pre-culturing period are important in transformation efficiency. To get an efficient transformation of canola and optimize regeneration conditions, different explants along with different culture media were studied. Four canola varieties were used to evaluate regeneration ability of hypocotyledonary and cotyledonary leaf explants. In addition, cotyledonary leaf explants were evaluated on the MS medium containing five different concentrations of BAP. Suitability of cotyledonary explants in transformation experiment was assayed by an exogenous gene, coding for P۵CS, the key enzyme in proline biosynthesis. The transformation of cotyledonary explants through Agrobacterium tumefacience-mediated gene transformation was used in a stepwise increased selection marker manner. PCR and proline content analysis confirmed the success of transformation. Cotyledonary explants displayed a higher regeneration efficiency than hypocotyledonary explants. In addition, BAP at ۵ mg/l in the MS medium increased the rate of regeneration. Results showed that pre-culturing explants for ۴۸ h increased the rate of transformation. Assessing the proline concentration further verified the expression and activity of the transformed gene. The P۵CS transformed plants were more resistant to salinity compared to the non-transgenic control plants

کلیدواژه ها:

Agrobacterium ، brassica napus L ، proline ، salt tolerance ، ∆۱-pyrroline-۵-carboxylate synthetase (P۵CS)