Establishment Of Agrobacterium-Mediated CBF1 Gene Efficient Transformation System Of Agapanthus Praecox

The full-length genes encoding transcription factor CBF1 was cloned by PCR from genomic DNA of wild type Arabiopsis thaliana. Then the fragment was inserted into cloning, which was transferred into E. coli DH5a. The vector pMD18-T was digested by two restriction enzyme Ncol and Bgl II and then was joined with pCAMBIA 1304 plasmid which was digested by NcoⅠand BglⅡtoo. Then the outcome was transformed into E. coli DH5a. By cheeking the transformed bacterial colonies, we had filtrated the positive clone successfully. The gene fragment was inserted into the plasimad between NcoⅠand BglⅡsites, behind the CaMV 35S promoter. Then transferred the expression vector into the Agrobacteria tumefaciens EHA105.In the meantime, one test which aimed to select the most favorable range of Hyg in Agapanthus praecox callus differentiation and its seedlings rooting,according the varieties of differention and rooting, was performed. Then transferred Agapanthus praecox callus and its seedlings into differentiation and seedlings rooting mediums of Agapanthus praecox callus respectively, which contained different level of Hyg (hygromycin)(0 mg/L,5 mg/L, 10mg/L,15 mg/L,20 mg/L,25 mg/L). The optimum level of hygromycin was determined as 20 mg/L for differention and 15 mg/L for rooting.As establishmented high-efficient genetic transformation system in Agapanthus praecox, optimize five conditions (pre-culture time, Agrobactrium cell concentration, transformation time, the level of AS in pre-culture medium, the level of AS in Agrobactrium liquid) by orthogonal design according hygromycin resistant callus efficiency. The optimized result were 5 days of pre-culture, (OD600)=0.9,25min, AS 50μmol/L in pre-culture medium, AS 100μmol/L in Agrobactrium liquid. This test was feasible by histochemical methods of the GUS, because the GUS reporter gene can express in Agapanthus praecox callus.Above all, after transformating the vector into Agapanthus praecox embryogenic callus by agi-bacterium, transfer the transfromedcalli of Agapanthus praecox into selection medium which contained 20 mg/L hygromycin. The regenerated buds was selected on the rooting medium for 4-5 weeks, which contained 15 mg/L hygromycin. Then remove the resisitant seedlings into substrate to grow. Resistance seedlings were obtained. The intergration of AtCBF1 gene into the genome of transgenic plants was confirmed by PCR analysis and analysis of GUS expression in lamina explants. The intergration of AtCBF1 gene were found eleven in seventeen. PCR analysis and analysis of GUS expression showed that the AtCBF1gene was co-integrated into the plant genome.