A Single Step Agrobacterium-Mediated Transformation System For The Generation Of A Large-Scale T-DNA Insertional Mutant Population Of Rice (Oryza Sativa L.)

A single step transformation system for the generation of a large-scale T-DNA insertional mutant population of rice was developed. Mature embryo-derived calli of japonica rice (Oryza sativa L.) cultivars Nipponbare were transformed using Agrobacterium tumefaciens strain AGL1 carrying a binary vector pCAS04 harboring the marker gene, neomycin phosphotransferase gene (NPTII), driven by a promoter from the ubiquitin gene in Maize, a promoterless p-glucuronidase gene near to the left border of T-DNA for trapping gene and a strong promoter, rice actin-GB promoter, near to the right border of T-DNA as activation tagging.In this system, co-cultivation was simplified, special selection stages and pre-regeneration stage were omitted, the whole process was almost under continuous light at 30 癈 except co-cultivation and transgenic plants began to generate only after 7 weeks calli were induced. Totally 99 transgenic rice plants from 125 resistant calli of 191 calli were obtained and PCR assay showed that 80.3 % of them were positive. The result of southern blotting analysis for primary plants revealed that each transgenic plant contained a average of 2.5 copy of T-DNA . During generation of rice mutant population, the efficiency of gene trap system was tested. Histochemical GUS assays were carried out in the leaves, flowers, immature seeds and roots from independent transgenic lines obtained using this T-DNA tagging system, and the rate of GUS-positive organs were 7.7%(76/985), 34.3%( 134/391), 10.4%(23/221) and 12.4% (50/404) respectively and the frequency of preferential expression of gus gene were 23.3%, 85.8%, 43.5% and 44% respectively. To confirm the efficiency of activation tagging, the model and activation of GUS expression, which was driven by actin-GB promoter in rice, were tested and the result revealed that actin-GB promoter was a constitutive and strong promoter in rice. Additionally, different rice mutants were obtained during generation of rice mutant population, such as partial-sterility plant, most -sterility plant, complete sterile plant and so on.