Genetic Transformation Of Maize Inbred Line18-599R With The Glyphasate-resistant Gene(2mg2-epsps) Mediated By Agrobacterimu Tumefaciens

As one of the three most important crops in China,maize plays an important role in food supply,feed crop supply and industrial production.Weed damage is an important reason for yield reduction in maize.Glyphosate is served as the world’s largest amount of a broad-spsctrum herbicide and played a great role in protecting weed damage but also affecting the growh of inaize.Transferring glyphosate-resistant gene into maize to cultivate glyphosate-resistant cron by genetic engineering,it has a very important significance for resolving these contradictions.In this study,a newly glyphosate resistance2mg2-epsps gene was cloned into binary vector based on pCAMBIA.3301vector and sereved as a seletablc marker and gene of interest.The recombinant plasmid transformed into immature embryo callus of maize inbred line18-599mediated by Agrobaeferiniu Tumefaciem.After selection on media supplemented with different concentration glyphosate,resistant plants were obtained.il wns demonstrated that the2mg2-epsps gene had been intergraded into genome and expressed in transgenic plants by PCR,RT-PCR and Quantitative PCR Analyses.The preliminary identification demonstrated that T2generations were capable of resisting glyphosatc’s impacts to some extent. The main results are as follows:1.We have successfully constructed plant expression vector p3301-2mg2-epsps. In (his study.the Gus reporter gene was replaced by the5-enolpyruvylshikimale-3-phosphate synthase gene from Pseudom onas tluorescens G2which confers resistance to glyphosate.The2mg2-epsps gene served two roles in this study—selectable marker and gene of interest. What’s important is that the pUC18multipe clonng site was retained and allowed insertion of any desired genes for transfotmation into plants.2.Thc recombinant plasmid was transfered into Agrobacterium tuinefaeiens by freeze thawing method,then transformed into embyronic callus of18-599R.139plants were regenerated from glyphosate-resistant callus and4positive transgenic individuals were amplified the. specific band.The sequencing results showed that the amplified target sequences from the maize-positive transgenic plants were on all fours with the 2mg2-epsps gene.3.A series of experments were designed for determing the selection pressure,specially for embryogenic callus which were induced by Inbred linesl8-599R.The results showed that two selected rounds of1mM and1.5mM glyphosate isopropylamine screening can be effectively inhibited non-transgenic maize callus tissue differentiation.4.Methods using PCR of56T2transgenic plants,33positive transgenic individuals were amplified the specific band,showed that the glyphosate-resistant gene was integrated into maize genome,with a positice fewquency of58.9%.RT-PCR and Quantitative PCR results showed that the glyphosate-resisant gene had be transcribed in stems,leaves and roots and inherited to next gengeration stably.5.Herbicide spray tests demonstrated T2transgenic plants could gown well when it was sprayed1.5mg/L glyphosate aqueous solution.Assay of detached leaves showed that transgenic leaves stayed green whereas non-transgenic leaves turned red.