Selection Of Efficient Genetic Transformation System Of Japonica Rice And Study On Genome Editing Of OsBADH2

In recent years,the aroma quality of rice is an important character in rice breeding.Osbadh2 gene is an important gene that affects the flavor of rice.To select high-quality japonica rice varieties,gene editing based on CRISPR / cas9 technology is expected to create japonica rice materials with excellent flavor and quality.At present,the research of gene editing in rice based on CRISPR / cas9 technology has made progress in stages,but the safety and efficiency of gene editing still need to be fully optimized and improved.The ultimate goal of this study is to establish a rapid and efficient genome editing system for japonica rice.We carried out a study on the knockout editing of osbadh2 gene.In this study,the regeneration system of Japonica rice tissue culture was optimized.The feasibility of high resolution dissolution curve(HRM)technology in the detection of plant genome editing progenies was verified,and the advantages of HRM technology in screening plant gene editing materials were analyzed.In the selection process of tissue culture regeneration system of Japonica rice,this study focused on reducing the time cost,and analyzed the time required for callus induction,callus proliferation,callus differentiation,regeneration and seedling rooting by different media components;two commonly used gene editing vector transformation schemes(Agrobacterium mediated transformation,gene gun mediated transformation)for plants)The time cost and conversion probability per unit time of the two conversion schemes are compared.In this study,we first analyzed the conservation of osbadh2 gene exon sequence,selected three specific targets,and constructed different target driven gRNA expression cassettes driven by U6 promoter,and connected them into plant gene editing expression vector.Comparing the transient transformation system and stable transformation system mediated by Agrobacterium transformation and gene gun transformation,12 non fragrant Japonica Rice Varieties(jd1-jd12)were genetically transformed in the selected japonica rice regeneration system,and 847 regenerated materials were obtained in t0 generation.The results of HRM analysis and sequencing showed that different types of editing of osbadh2 gene editing targets occurred in 24 t0 generation materials.Seven kinds of gene editing progeny materials were obtained from T1 generation materials without transgene components,and osbadh2 gene target was homozygous.The results showed that based on the transient transformation system mediated by gene gun,the resistance screening process was omitted in the transformation and regeneration process,and the highly efficient HRM detection was used to obtain the gene editing progeny materials with homozygous editing and no transgenic components in T1 generation.The progeny materials of "Jijing 88" and "JD(1-10)" edited by osbadh2 gene obtained in this study also provide new materials for Breeding Japonica Rice Varieties with improved flavor and quality.