Optimization of regeneration and transformation of canola, Hyola 308 and RGS003 lines

The objective of the study was to develop an efficient method for shoot regeneration and Agrobacterium-mediated gene transformation into Brasica napus cultivars, Hyola308 and RGS003. Different concentrations of benzylaminopurine (BAP) (3.5, 4, 4.5, 5, 5.5 and 6 mg/L) were evaluated for shoot regeneration of petiol cotyledonary explants. Also, the effect of preconditioning and co-cultivation periods and acetosyringone concentrations were studied on the Gus reporter gene transformation. The experimental design was factorial based on completely randomized design with 3-5 replications. Maximum shoot regeneration was obtained using 3.5 mg/L BAP with 112% for Hyola308 and 120% for RGS003 cultivars. The transformed plants were screened on kanamycin containing medium and the presence and the expression of the reporter gene were confirmed by PCR and Gus assay, respectively. The highest effect on transformation efficiency was observed through 48 h preconditioning and co-cultivation period and 100 µM acetosyringone for Hyola308 (33%), and 38 h preconditioning period, 72 h co-cultivation period and 50 µM acetosyringone for RGS003 (38%).