Efficient regeneration of Brassica napus L. hypocotyls and genetic transformation by Agrobacterium tumefaciens

An efficient system for shoot regeneration and Agrobacterium-mediated gene transfer into Brassica napus was developed through the modification of the culture conditions. Different concentrations of benzyladenine (1.5, 3.0 and 4.5 mg dm(-3)) and thidiazuron (0.0, 0.15 and 0.30 mg dm(-3)) were evaluated for shoot regeneration of 7, 14 and 21-d-old hypocotyl explants. Maximum shoot regeneration frequency was obtained in 21-d-old explants using 4.5 mg dm(-3) benzyladenine and 0.3 mg dm(-3) thidiazuron. Under above culture condition, the highest percentage of shoot regeneration frequency was 200 %. Agrobacterium-infected explants grown on the selection medium gave rise to transgenic shoots at a frequency of 11.8%. Transformed shoots rooted when cultured on a medium supplemented with 2 mg dm(-3) of indolebutyric acid and 10 mg dm(-3) kanamycin. The rooted plantlets were successfully established in the soil and developed fertile flowers and viable seeds. Evidences for transformation were confirmed by GUS assay and PCR analysis.