Study On Improving Efficiency Of Cotton Transformation Using Virulence Genes Of Agrobacterium Strains

In terms of current plant transformation technology, most of transformation methods still have many constraints in cotton transformation.In this study,according to Agrobacterium-mediated plant transformation mechanism, toxic protein genes from Agrobacterium Ti plasmid promoting T-DNA transformation and target genes were transferred into cotton cells together by using pollen-tube pathway technique. Toxic protein genes express in cotton egg or zygote cells instantaneously and then produce toxic proteins with nuclear localization signal which can assemble with target genes automatically. Furthermore the target genes transfer into the nucleus because of assembling toxic protein with nuclear localization signal.In this way, exogenous genes transformation rate and descendants genetic stability after transformation. were increased .So an efficient cotton gene transformation technique was established.We assembled VirD₁, VirD₂ , VirE₂ genes and target genes together in vitro and then putthem into cotton cell through pollen tubles.The transformation rate was verified by poly-merase chain reaction(PCR) and agarose electrophoresis. Expression vectors named pblg-sm-gfp,pblg-pR1aS-gfp,,pblg-pa7-gfp,pblg-gfp were constructed to enhance repoter gene GEP expressing.The four vectors were tranfered into tobacco cells,and transgenic tobacco was obtained successfully.We detected the transgenic tobacco under fluorescence microscopeand found the transgenic tobacco harbored pBLG-pR1aS-GFP vector whose GFP emited more powerful blue light than transgenic tobacco with other three vectors.The comparison of Agrobacterium direct injection, Agrobacterium infecting pollen grain transfection and toxic protein gene assisting transformation shows: transformation rate of Agrobacterium direct injection is 0.6-1.8℅, , Agrobacterium infecting pollen grain transfection is 0.5-1.8℅,and toxic protein gene assisting transformation attached treatment with EMS,LW77,6BA,GA is 2.3-2.8%.Results shows that the study supplys a new way for cotton genetic transformation,and has significance for improvement of transgenic cotton breeding efficiency and increasing the speed of transgenic cotton breeding.