The Study On Establishment Of Regeneration Systems Of Sorghum For Its Agrobacterium-medisited Transformation Of Bar Gene And Rice Transformation Of EIS Gene

Sweet sorghum, as an important feed and energy crop, has obtained widespread attention.In this study, three different explants of ten different sorghum varieties were cultured in vitroto establish their high efficient regeneration systems, and some were transformed byAgrobacterium to build their Agrobacterium-mediated genetic transformation system. Themain results were as follows:1. The results of mature seeds culture of two kinds of sorghum varieties showed that thecallus induction rates of Liaotian3and Lvneng2were56.34%and58.79%, and thedifferentiation rates of Liaotian3and Lvneng2were2.78%and0, showing significantdifference.2. The results of inflorescences culture of five kinds of sorghum varieties showed thatM81-E performed best in callus induction, with60.4%callus induction rate, followed by07-27, with51.23%callus induction rate, while the callus induction rates of BJ-285, Romeand Cowley were below50%, and that07-27performed best in callus differentiation, with13.13%callus differentiation rate and35regenerated green seedlings, followed by BJ-285,with0.71%callus differentiation rate and one regenerated green seedling, while M81-E,Rome and Cowley failed to differentiate plantlets. There were significant differences in callusinduction and callus differentiation. So comprehensive compared, the regeneration capabilityof07-27was the best, followed by BJ-285.3. The results of immature embryos culture of seven kinds of sorghum varieties showedthat Xinliang52performed best in callus induction, with72.97%induction rate, followed byRome, Tianza2and BJ-299, followed by07-27and M81-E, and the last was BJ-285, and that07-27performed best in callus differentiation, with20.56%callus differentiation rate and80regenerated plants, followed by Xinliang52, with11.43%callus differentiation rate, followedby BJ-285, M81-E and Tianza2, with the callus differentiation rate of1.53%,1.08%and0.84%respectively, while BJ-299and Rome failed to differentiate seedlings. So comprehensivecompared, the regeneration capability of07-27was the best, followed by Xinliang52,followed by BJ-285, M81-E and Tianza2.4., with high callus induction rate and callus differentiation rate, and and well growthwere selected to introduce into bar gene by Agrobacterium-mediated. The results of transformation of immature embryos and its calli of XinLiang52, and calli of M81-E showedthat17resistant calli were obtained from transformed immature embryos of XinLiang52, with5.67%transformation rate, and they differentiated one regenerated seedling on thedifferentiation media, with5.88%differentiation rate, while resistant calli from transformedcalli failed to differentiate seedlings.Rice is one of the most important food crops in the world, and its high efficienttransgenic system has been established. In this study, mature seeds of Jinyuan45were culturedin vitro to establish its efficient regeneration system and the plant expression vector of EIS(espA-intimin C300-stx2B) was conducted and introduced into rice cells, ultimately34regenerated transgenic plants were obtained. The main results were as follows:1. The results of mature seeds culture of Jinyuan45showed that the best callus inductionconditions were on NB basic medium with2.0mg/L2,4-D and16/8hours lighting and darkper day, with56.95%callus induction rate, and that the callus differentiation rate of callisubcultured for three weeks was the highest, up to90.48%. Finally, the high efficientregeneration system of mature seeds of Jinyuan45was established in vitro.2. According to EIS gene and vector sequences, specific primers were designed toamplify EIS gene sequences. The fragments were amplified by PCR amplification and clonedinto pUC19first, then constructed to plant expression vector pCAMBIA1300by enzymedigestion, ligation and transformation. Finally the EIS binary plant expression vector wassuccessfully constructed and introduced into the Agrobacterium EHA105.3. The results of transformation of rice showed that11resistant calli were obtained, with13.7%transformation rate, and five of them differentiated regenerated seedlings,with45.5%differentiation rate. Finally,34regenerated plants were obtained and19plants of themshowed positive by PCR detection.