Study on the method for effecient and rapid genetic transformation of Kasalath mediated by <I>Agrobacterium</I>

Abstract

At present, Kasalath is a model Indica material for rice transgenic research. By using the technique of plant tissue culture and Agrobacterium-mediated genetic transformation, we optimized the condition for the callus induction and the performance of genetic transformation of Kasalath. The results showed that immature or newly harvested mature seeds as explants were more effective to generate callus than the seeds stored for 1 year. Among the induction media with 0.5,1,1.5,2,2.5,3,10,20 or 30 mg/L 2,4–D solutions, the medium with 2.5 mg/L 2,4–D had the best performance in callus induction;in callus infection by co-culturing with Agrobacterium Ag10 or EHA105 solution with OD600 nm being 0.05, 0.1, 0.15, 0.2,0.3 or 0.4, the efficiency of transformation reached the optimum when OD600 nm of the working solution of both Ag10 and EHA105 was 0.15, but EHA105 was superior to Ag10, its hygromycin-resistant callus reached up to 82.0% and 3 d co-culture with EHA105 was perfect. When the resistant calli suffered rapid dehydration for 6–8 h or slow dehydration for2–3 d, the regeneration frequency was evidently improved and the time needed for regeneration was shortened; while pre-culture before callus infection and pre-differentiation processing before hygromycin-resistant callus getting onto differentiation culturing had little effect on genetic transformation efficiency, so these procedures could be omitted. By the optimized procedures, the overall time from callus induction to regeneration of transformant plantlets was shortened to57–60 d, and 65% of transformation efficiency was attained.