Large-scale Agrobacterium-mediated Genetic Transformation And Breeding Of Glyphosate Resistant/Stress Tolerance Cotton

Cotton is an important economic crop which is widely cultivated in the whole world.Its yield and quality play a pivotal role in the development of national economy.The breeding of good varieties is limited due to the species isolation during the traditional cross breeding technology.However,this isolation is broken by the rapid development of modern plant gene engineering.This technology can change the biological genetic traits orientation which can make up for the deficiency of the conventional breeding.In this study,the genetic transformation system of cotton was successfully set up by Agrobacterium tumefaciens-mediated method.The further study on G10eve glyphosate resistant cotton and AmCBFl stress resistant cotton was carried out.The detailed research results are as follows:1.In this study,the genetic transformation system of cotton was successfully set up by Agrobacterium tumefaciens-mediated transformation method using hypocotyl as explants.The high frequency regeneration system of R15 was obtained,and the regenerated plants from the embryogenic callus which was used as the receptor based on the transformation system were gotten.2.36 strains were transformed,including 10 stress tolerance related genes,1 herbicide resistance gene,16 insect-resistant genes,5 disease-resistant genes and 4 other genes.A lot of genetically modified plants were acquired.The test of embryonic callus lines showed that the transformation efficiency has a big difference among different strains,the lowest transformation efficiency was 7.37%,while the highest was 75%.3.G10eve gene is a self-developed high glyphosate-tolerant gene by our country.Through the Agrobacterium tumefaciens genetic transformation system,the gene was transferred into C312 with the transformation efficiency of 49.3%.PCR,qRT-PCR and southern blot experiment results showed that G10eve gene was stably integrated into the cotton genome,but the relative expression and copy number of G10eve gene were different among different transgenic lines.4.Transgenic cotton varieties were analyzed for glyphosate tolerance both indoor and in the field.The results showed that the transgenic L91 strain had the highest glyphosate tolerance,and the inheritance of exogenous gene and glyphosate resistance was basically stable in different generations.The field experiment results showed that the L91 strain could tolerate a maximum of 20 mL L-1 concentration of glyphosate at seedling stage and anthesis stage.Its resistant levels of glyphosate were similar to that of Monsanto's resistance materials and reached the spraying requirement of glyphosate in the field.5.The AmCBFl gene cloned from Ammopiptanthus mongolicus was transferred into R15.The transformation efficiency was 22.45%,and three transgenic homozygous lines were obtained.The transgenic plants were identified by PCR,qRT-PCR,southern blot experiment and proved that AmCBF1 gene has been successfully integrated into cotton genome.Some flanking sequences have been obtained and mapped to the corresponding chromosomes.Using the Tail-PCR technique.The physiological and photosythetic indexes of the transgenic AmCBFl gene homozygous plants were studied under low temperature and drought treatment.The results showed that overexpression of AmCBFl gene in transgenic cotton lines increased the cold and drought tolerance.In summary,Agrobacterium tumefaciens-mediated transformation platform was built and optimized for the cotton transformation.By this way,we obtained the G10eve glyphosate resistant cotton and AmCBFl stress resistant cotton respectively,which improved the tolerance against the glyphosate and resistant factors.This study provide the certain experimental data and germplasm resources.