A Genetic Transformation Method By Infecting Shoot Tips With Agrobacterium Tumefaciens For Cotton And Soybean

Cotton(Gossypium hirsutum L.)is one of the most important cash crops in the world.In our country genetically modified cotton has covered all cotton area and different variety types of conventional and hybrid cotton.Soybean(Glycine max L.)is an important grain and oil crop,China imports nearly ten millions of tons of genetically modified soybeans per year.Therefore,genetically transformation is an important way for cotton and soybean improvement.At present,it is a mature and widely used transgenic method for cotton and soybean to regenerate plants by using Agrobacterium to infect explants followed by tissue culture.However,this method is limited by the recipient material genotype,tedious operation,a long cycle to obtain transgenic plants,accidental microbe contamination during tissue culture,high death rate in seedling training and grafting process.etc.It is a great significance to develop a high-efficiency transgenic method indepent on tissue culture without recipient material genotype restriction for identification and utilization of functional genes,application of gene editing technology,and molecular breeding of cotton and soybean.In the current research,we tried to establish a genetic transformation method by infecting shoot tips with agrobacterium tumefaciens for cotton and soybean,verified the transgenic events,and used this method to introduce GhMAC3,a gene coming from cotton,into cotton and soybean,and obtain transgenic cotton and soybean plants.The main results are as follows:1.The Agrobacterium induced genetic transformation system was established by using shoot tips for cotton and soybeans receptor material.Plasmid pHellsgate-GhVLN4 and pBI121-GhVLN4 were transformed into shoot tips for cotton respectively by Agrobacterium LBA4404 strain.The results of PCR showed that obtained 12 plants ghvln4RN Ai and 5 plants 35S::Gh VLN4,The frequency of transformation was 18.18%and 6.58%respectively.2.The T0,T1 generation of cotton plants were identified by PCR Southern Blot,transgene copy number detection,vector Backbone PCR detection,RT-PCR and qRT-PCR,the results showed that the 5 transgenic cotton plants of 35S::GhVLN4 were positive.3.The plant expression vector pBI121-GhMAC3 was constructed by homologous recombination method.The recombinant plasmid pBI121-GhMAC3 was transformed into Agrobacterium EHA105 strain.The genetic transformation system based on plant shoot tips was used to transform upland cotton TM-1 and soybean(Nan Nong 34)induced by Agrobacterium.The results of PCR showed that 1 transgenic cotton and 3 transgenic soybean were obtained,and the was 1%and 3%respectively.Exogenous DNAs were successfully transformed into cotton and soybean by using different Agrobacterium stains to infect its tip with a considerable transformation efficiency,this method is simple and practicle that will greatly speed up gene function analysis and molecular breeding for cotton and soybean.