Study Of Transgenic Sugarcane With Cry1C And Cry2A Genes

Sugarcane is the important sugar crops in our country. In the longer growing time, sugarcane was vulnerable to the threat of pests and weeds harm in the field and finally was caused the lower production. Therefore, to breed the high resistant sugarcane varieties was a efficient way to solve the problem.Because of genetic background of sugarcane was very complex, it was hard to concentrate all excellent characters of sugarcane by general hybridization breeding. Plant gene engineering provided the simple and feasible technique for transgenic breeding to obtain higher resistant sugarcane varieties.In the study, the Ⅱ type embryo callus induced by sugarcane leaf were used as the receptor materials, and herbicide resistance gene (bar gene) as selected marker gene, Cry1C and Cry2A were respectively transferred into sugarcane (ROC22and GT21) by agrobacterium-mediated method. The results of screening with PPT and PCR detection showed that the foreign genes had been integrated into sugarcane genome successfully. The main results were as following:1. Tissue culture technology of sugarcane has been optimized. Sugarcane lobus cardiacus were used as explants after sugarcane tip were disinfected with75%ethanol. More than90%callus induction rate,2times higher than blank control (CKO), were obtained by the means of immersing the leaf in the sterile water with200mg/L PVP solution for5min before being cultured. And the high quality callus and the stronger differentiation were observed.2. The PPT screening system of two sugarcane varieties (ROC22and GT21) were established. To ROC22,60mg/L PPT were determined as the optimal screening concentration for leave daub and direct spray, and the screening time was7days. It was the suitable screen conditions for in vitro leaves to be immersed into30mg/L PPT for5days. To GT21,70mg/L PPT were determined as the optimal screening concentration for leave daub and direct spray, and the screening time was7days, and to be immersed into30mg/L PPT for5days was the optimal screening for in vitro leaves.3. It was the efficient detection method to reduce false positive rate of transformed sugarcane plants and screening workload by means of directly spraying PPT to leaf combined with immersing the leaf into PPT solution. Finally, the transgenic plants were further confirmed by PCR detection with Bar and Bt gene. In the study,14of transformed plants (ROC22) with Cry1C gene were obtained and the transformation frequency was5.2%.17of transformed plants (ROC22) with Cry2A gene were obtained and the transformation frequency was5.8%.18of transformed plants (GT21) with Cry1C gene were obtained and the transformation frequency was4.7%.13of transformed plants (GT21) with Cry2A gene were obtained and the transformation frequency was3.9%.