Study on the conditions of transferring D32 gene into tobacco mediated by Agrobacterium tumefaciens.

By mediation of Agrobacterium tumefaciens C58C1,D32 gene derived from soybean was introduced into sterile leaves of tobacco'Zhongyan 99'.The key factors affecting the transformation were assessed and the transgenic plants were generated and further tested using PCR.The results showed that the optimal kanamycin concentration for tobacco leaves differentiation and regeneration was 150 mg/L and preculture had an impact on transformation efficiency.The optimized transformation conditions and procedures were as follows:explants were firstly immersed into liquid bacterium at OD600 of 0.7 for 5 min prior to pre-culture,followed by a two-day long co-culture before being transferred to and cultured in a differentiation medium for 50 days.A proportion 26.7% of the transgenic plants were kanamycin resistant.The PCR eventually detected that the D32 gene has preliminarily inserted into the tobacco genome.