Construction Of Dwarf Cucumber Lines By CRISPR/Cas9 Technique

Cucumber(Cucumis sativus)is the largest facility vegetable crop of China,which is widely cultivated in the world.The CRISPR/Cas9 system is an.RNA-mediated genome-directed editing technique with high mutation efficiency,low cost and ease of operation.Compared with the traditional transgenic technology,CRISPR/Cas9 technology can be derived by hybridization of exogenous fragments,leaving no traces of transgenic,so it has a broader application prospects in crop genetic improvement and breeding research.CRISPR/Cas9 technology has been successfully applied in directed editors of different plant genomes so far,including Arabidopsis thaliana,tobacco,wheat,rice,maize and tomato.However,the genetic transformation technology of cucumber is not mature enough and limit the application of CRISPR/Cas9 gene editing technology in cucumber breeding.Plant height is an important agronomic trait that affects the yield of cucumber,and VFB1 gene involved in regulating the high traits of cucumber.At present,the production of main varieties of cucumber are mostly sprawl,with long of joints,need to manually take the vine and pruning.Dwarf cucumber varieties,compact plant type,suitable for close planting,take full use of land and light resources,not only improve the population production,but also simplify the cultivation techniques and agronomic facilities,saving manpower,material and financial resources.In this study,CU2,South China cucumber species was used as the material,and the VFB1 gene was knocked out by Agrobacterium tumefaciens-mediated method.The dwarf cucumber plants were obtained by CRISPR/Cas9 technique.The main results are as follows:1.CRISPR/Cas9 vector was modified by inserting GFP reporter gene for the screenong of transgenic plants.2.The transient transformation system of cucumber hypocotyl explants was established to detect the mutation efficiency of the target.3.Using cotyledons as explants,cucumber genetic transformation system was optimized,including the selection of Kanamycin screening pressure,antibiotic selection and so on.4.VFB1 gene in cucumber was successfully knocked out by CRISPR/Cas9 and the TO transgenic plants of dwarf cucumber were obtained.