Optimization Of Agrobacterium-mediated Transformation System And Preliminary Establishment Of T-DNA Insertion Mutant Population In Rice

Generation of a large and saturated T-DNA insertion mutant population is an important and effective tool in rice functional genomic research. Therefore,it is important to establish stable and efficient methodologies for rice transformation. In order to increase the frequency of transformation,the major factors that influence rice transformation mediated by Agrobacteriwn tumifaciens was tested. Investigations were primarily done from the respects of the length of T-DNA,the different methods of infection,cultivation conditions for callus tissue regeneration,negative effects of selection agents ( antibiotics),and the effect of inhibitor (Cef) onfrequency of regeneration. Our results revealed that:1. If the length is less than 10kb,the length of T-DNA influences the transformation frequency in some extent. The longer the length is,the higher the transformation frequency we got. 2. The plasmids of pCAS series outgo those of pCAMBIA series in transformation frequency. S.Hygromycin showed more toxic to callus than G-418 and resulted in the reduction of transformation frequency. 4. Infection methods also influence the transformation and regeneration frequencies at a certain degree,within which the permeate-paper method is the simplest and most effective. 5. Inhibitors in regeneration media will repress the regeneration of callus tissue. Protocols for efficient and stable rice transformation mediated by A. tumifaciens is established. Of the 258 T-DNA insertion mutants,151 were subjected to PCR assay and 146 of them were showed positive. Further analyses,including Southern blotting to determine the copy number of T-DNA and Tail-PCR to determine the flanking sequence,are under way.