Exploring The Method Of Quickly Obtaining Cas9-free Rice Plants By Agrobacterium Mutants

Rice is an important food crop in the world and a model monocotyledonous plant.The studies of rice gene function are of great significance for the improvement of rice yield and quality.CRISPR/Cas9 technology is a very important gene editing tool,which can realize site-specific editing of a genome.It plays an significant role in the research of crop genetic improvement and gene function studies.At present,the application of CRISPR/Cas9 technology in rice are very widespread.Many laboratories have implemented accurate editing of genes in rice by CRISPR/Cas9-mediated protocols.However,most techniques involve integration of T-DNA from Agrobacteria into the plant genome,which cause transformation of inheritable exogenous DNA elements into the host plants.In this study,a potentially very useful protocol for rapid screening of Cas9-free mutant plants was investigated.We reviewed the literatures of Agrobacteria and found that the ? domain of the avirulence protein VirD2 mediates the integration of exogenous DNA into the plant genome,whereas the deletion of the VirD5 gene affects the stable transformation of Agrobacterium,but does not affect its transient transformation.Therefore,we deleted the ? domain of the VirD2 gene of Agrobacterium tumefaciens EHA105.With an Agrobacterium strain lacking the VirD5 gene as a delivery agent,Cas9-free and mutated plants were obtained with one of the three test protocols.In scheme 1,we used three conditions for the screening process of tissue culture: the first was to add hygromycin in both screenings;the second was to add hygromycin for one week in the first screening,and hygromycin was omitted in the second screening;the third was not to add hygromycin in both screenings,no Cas9-free and gene-edited plants were found.The Scheme 2 was improved on the basis of the scheme 1,we employed three conditions for the screening process: the first was to add hygromycin for 3 days in the first screening,and without hygromycin in the second screening;the second was not to added hygromycin in both screenings.Additionally,new CRISPR plasmids were used,and the scale of the tissue culture was expanded.In scheme 2,we successfully obtained 8mutant plants out of 2688 plants,four of the eight DNA-edited plants were Cas9-free.However,the overall mutation efficiency of this experiment was ideally to be improved.We believed that after completing the tissue culture screening process,the proportion of mutant calli to total calli was too low,which was probably the main reason for the low mutation rate of the plants.Therefore,we designed the scheme 3,in which,we removed the screening process during tissue culture,and directly completed the differentiationexperiment after the steps of infecting,co-cultivating and washing.The results of protocol3 was not as desired,we obtained only one mutant plant carrying the Cas9 gene out of565 plants.The screening efficiencies of positive individuals still need to be further studied to improve the efficiency of this protocol.