Agrobacterium Mediated Genetic Transformation And Identification Of Bt Gene In Sea Island Cotton

This study was based on the island of cotton regeneration system in Xinjiang island cotton 33 cotton as a material,embryogenic callus was obtained,and then the genetic transformation of Bt gene was studied on embryogenic callus.The molecular detection of the obtained transgenic plants and the indoor bioassay,it was concluded that the Bt gene had been integrated into the genome of the island cotton cultivars and had a certain resistance to the cotton bollworm.The followings are the main results of this study:The hypocotyls of the sterile seedlings were cut into explants,and used 0.1mg·L^-11 2,4-D and 0.1mg·L^-1KT hormone combination induced callus,subculture,the embryogenic callus was differentiated.The expression vector pBI121 carrying Bt-Cry1A gene was transformed into embryogenic callus of Xinjiang Island cotton 33 by Agrobacterium tumefaciens-mediated transformation.The embryogenic callus was screened by medium containing Kan(50 mg·L^-1)and Cef(50 mg·L^-1).After the KNO3 doubled and NH4NO3 was reduced to 1/4 of the differentiation medium The resistant plants were obtained by dividing the somatic embryos into a large amount of 1/2 medium,and grafting method was used to improve the survival rate.Twelve resistant plants were obtained.The 12 resistant plants obtained by grafting were subjected to molecular detection,the results were as follows:the results of PCR test showed that the Bt gene specific primers,GUS gene specific primers and35S Promoter specific primers could amplify the matched fragments of 12 resistant plants,and the results were positive;The results of Southern blot showed that all the 7 positive samples were detected by the integration of the target genes;RT-PCR results showed that the target gene of 12 transgenic plants had been integrated into the genome and could reverse the mRNA;Bt-Cry1Ab/Ac transgenic test strips showed that there were two purple bands in 12 transgenic plants,indicating that the transgenic plants contained the target protein.The results of the indoor bioassay are as follows:The comprehensive evaluation results showed that the comprehensive resistance level of transgenic plants 4 10 and 12 was resistant,and the comprehensive resistance level of other transgenic plants was neutral.