Study On Tissue Culture Of Conventional Maize Inbred-lines And Sbeâ…¡b Gene RNAi Segments Transformation

Maize is important food,feed and important industry material crops. Maize highly efficient regeneration system always is the focus of scientist. And starch is the most component of maize seed,As maize starch was widely applied in energy sources,chemistry industry. Improving maize starch quality is the object of breeding scientists. Ten conventional maize inbred –lines regeration, constructing RNAi exoression vector of sbeⅡb and transformation mediated Agrobactirium tumefaciens have been detailedly researched . The results are as follows: 1. Ten conventional maize inbred –lines are researched in regeneration abilities. Maize regeneration abilities are highly genotype-dependent. Primary callus are easily induced in ten maize cultivars, but the inducive frequencies of embryogenesis callus are different. Four conventional maize inbred –lines have been regenerated and frequancy of them respectively is 78%(178), 45%(Luyuan92), 26% (Huang C), 20%(52106). 2. Maize immature embryo, mature embryo, radicel, germ layer, hypocotyl are served as explants to research their regeneration abilities. The results show the inducive frequency of primary callus achieve 100% and inducive frequency of embryogenesis callus achieve 78% in immature embryos. Inducive frequency of primary callus is high in mature embryos, but its ability of inducing embryogenesis callus is inferior and the frequency achieve 10%. Inducing frequency is very low and frequency of embryogenesis callus achieve only 1% in radicels, germ layers and hypocotyls. 3. Optimal culture condition has been developed in our mazie inbred –lines 178. Optimal inducing medium is N6B5 +2,4-D 2.0 mg/L+3% sucrose;optimal subculture medium N6B5 +2,4-D 1.0mg/L+6-BA0.1mg/L+4%sucrose+L-Pro 1000mg/L+CH800mg/L;optimal regeneration medium N6B5 +6-BA2.0mg/L+NAA0.5mg/L+3%sucrose;optimal rooting medium N6B5 +IBA1.0mg/L+3%sucrose. 4. Twentieth extron(155bp) from genome and twenty-first, twenty-second extron(280bp) from immature seed cDNA of sbeⅡb gene have been respectively amplified. Vector p1300+HMW+2F and p1300+bar+HMW+2F have constructed by utilizing pUCRNAi and pCAMBIA1300 vector which respectively contain hpt or bar selecting gene.The vectors will be used gene gun and A. tumefaciens transformation. 5. Twentieth extron(155bp) of sbeⅡb gene sequence have been analysed in four cultivars which has been regenerated. The results indicate sequences of three cultivars are completely accordant and sequence of 52106 only has a base diffirence. So vectors of construction is fit for transformation of the different genotypes maize 6. Through the tri-parental mating experiment, the RNAi expressing vector has been transformed into A. tumefaciens LBA4404. And transformation mediated A. tumefaciens indicated resistent callus frequency is high in OD 0.6, infection 20 min,and co-culture 3d. Meanwhile, AS can promote obviously ability of infection. Two transgenic maize are obtained by PCR analysis. Integrated studies demonstrated 178 conventional maize inbred –lines highly effective regeneration is been developeded by selecting in ten maize inbred –lines. RNAi expression vectors of sbeⅡb have been constructed to improve starch quality of mazie. Transgenic maize are obtained by transformation mediated A. tumefaciens. These research provide important means to transformation of maize and improving starch quality by gene engineering.