Studies On The Genetic Transformation Method By Cotton Shoot Tip-Agrobacterium Medium Based On Glyphosate Resistant Gene

As one of the most important economic crops, cotton hold the balance for development of national economy. Weeds are one of the main factors which is affecting cotton production. With the development of biotechnology, it is an effective way to germplasm innovation and breeding in biotechnology. It is important to control weeds in cotton field, reduce investment and improve production efficiency by breeding herbicide resistant transgenic cotton varieties. In this study, Glyphosate-resistant EPSPS-G6gene, which was cloned by Zhejiang University, was introduced into upland cottons culitivars, using YZ-1, coker201and CCRI-49as the receptors by cotton shoot tip-agrobacterium medium method, and glyphosate-resistant transgenic cotton germplasms were obtained, which provided the new germplasm for development of glyphosate-tolerant cotton cultivars with our own intellectual property rights. The major results in this study are as follows:1. A genetic transformation system via cotton shoot tip-agrobacterium medium based on glyphosate resistant gene was established through the optimization of the glyphosate concentration during screening, infection period and infection concentration of the agrobacterium, period of co-culture with the agrobacterium, and the conditions of recover culture etc., used CCRI49, Coker201, and YZ-1as the materials, and their transgenic herbicide resistant cotton plants with EPSPS-G6were obtained in this experiment as well. As the results shown that, the optimal glyphosate concentration in screening for transgenic cotton shoot tips was10mg/L, the optimal agrobacterium density was0.9-1.0in OD600and infect period was about20min, co-culture period with agrobacterium was48hours, and using SH medium with0.5g/L activated carbon as the recovery medium for transgenic shoot tip to grow. Based on the explants numbers, the successful of transformation in this experiment was6.4%. From the transformation of time, the method from the aseptic seedling culture begins to calculate, obtain regenerated plantlets in vitro for only2months,4.5months can blossom,6months can obtain T1seeds,which show that the present transformation system is of good practical value. 2. Using the transformation system established in this experiment,60glyphosate resistant plantlets were obtained from the360cotton shoot tips of3upland cotton cultivar or germplasms. After the PCR detection and23plants with glyphosate resistant gene were survived after transplanting.3. PCR detection for the regenerated plants (To) genomic DNA,26regenerated plants could be amplified the same size with the plasmid DNA. The result show that the exogenous gene has been successfully integrated into the chromosomal DNA of the regenerated plants. Southern blot analysis results show that the exogenous genes are single-copy transgenic plants. The results of the main agronomic traits of transgenic To plants show that the To transgenic plants can grow normal,all can blossom and bear fruit, and obtain T1seed. In addition, there are no significant difference between the23transgenic plants and the control plants, and no significant variation between different transformants. The results show that the creation of glyphosate-resistant transgenic cotton germplasms have better utilization value in breeding.