Optimization of in vitro shoot regeneration and Agrobacterium-mediated transformation of Brassica napus

Abstract

Rapeseed (Brassica napus L.) is one of the most cultivated oil crops in the world. Its genetically engineered varieties harboring new characteristics rely significantly on establishment of an efficient in vitro tissue culture and transformation. In this study the potential of shoot regeneration of two B.napus cultivars, RGS003 and PF7045-91, and their Agrobacterium-mediated transformation using three methods were investigated. Hypocotyl and cotyledonary explants of 5, 10 and 15 day-old seedlings were used for indirect and direct shoot regeneration, respectively. The hypocotyl explants showed best callus formation in 0.5 mg/L 2,4-D. The calli and cotyledonary explants were regenerated in 3 to 4.5 mg/L BAP, successfully. The regenerated shoots were maturated and then rooted in 0.5 mg/L IBA. For genetic transformation, both explants were infected by Agrobacterium using immersing, incubation and well-based methods. None of hypocotyl explants get transformed. The transformation frequency of cotyledonary explants in well-based method was higher than the others, showing an increase of 15%. The obtained putative transgenic plantlets maintained green in presence of kanamycin. The transformation confirmed by PCR and histochemical GUS assay. The well-based inoculation method appeared promising to enhance the efficiency of cotyledon-based transformation.