Optimization of transformation in canola (B.napus L.) with control of ethylene production and gradual increase in concentration of selection element

Canola (Brassica napus L.) is one of the most important oil producing plants in the world. In recent years, in spite of transformation in canola via Agrobacterium, the efficiency of this method is still low. To consider the effect of plant genotype and cultivation medium composition on transformation and regeneration in canola and the high toxicity of kanamycin for growth of this plant, it is necessary to develop more appropriate and efficient method for transformation of this oily plant. Here we used cotyledon explants and Agrobacterium tumefaciens strains LBA4404, GV3850, GV3101 harboring mutated gene EPSPS (pBI121+EPSPS) for transformation of canola PF7045-91 cultivar. After induction with agrobacterium, explants were placed in different cocultivation medium under dark condition at 25 degree centigrade. In regeneration process, to prevent necrosis of explants, the fixed amount of silver nitrate (5mg/L) was used for blocking ethylene production. The regeneration and selection procedure were started in medium containing silver nitrate without kanamycin and this carried on for one week. Subsequently, the kanamycin was added (5 mg/L) and its concentration was increased up to 15 mg/L in a ten days period. For root induction, the regenerated shoots were transferred to medium containing the MS salt with 15 g/L sucrose and 0.2 mg/L IBA. Transformation was confirmed by molecular analysis. Our data showed that gradually increasing kanamycin concentration and blocking ethylene production could improve the transformation of B. napus.