Creation of Fragrant Rice by Means of CRISPR/Cas9 Technology

Objective With the continuous improvement of people’s living standards，high-quality rice with fragrance is increasingly welcomed by consumers.Fragrance in rice is mainly controlled by the BADH2 gene on chromosome 8，the mutation of this gene resulted in the accumulation of 2-acetyl-1-pyrroline in rice to produce fragrance，which accelerated the cultivation of fragrant rice. Method The japonica variety Tainan11 adapted to Jiangxi Province was used as the material and the targeted sequence was desigend according to the sequence of exon 3 of the BADH2 gene to create BADH2 knockout lines via CRISPR/Cas9，the positive transgenic plants were obtained by the screening of hygromycin resistance，and the stable marker-free gene-edited lines carrying the mutation on BADH2 gene were obtained by sequencing and PCR.The content of 2-acetyl-1-pyrroline in wild-type and T3 generation mutant materials was determined by GC/MSD.The yield and quality of wild-type and T3 generation mutant materials were investigated and analyzed. Result Rice lines with mutations at BADH2 were successfully generated and identified，four mutation types were obtained，including 1 insertion mutation and 3 deletion mutations.The content of 2-acetyl-1-pyrroline in mutants was significantly higher than that in the wild type.Compared with the wild type，the number of effective tillers，spikelets per panicle and seed setting rate of the mutants showed significant differences，and the average yield decreased by 8.7%.Compared with the wild type，the protein content，head rice rate，chalkiness degree and Length/width of the mutants showed significant differences too，and the quality of the mutants was improved. Conclusion Fragrance rice plants can be generated by BADH2 disruption by CRISPR/Cas9，which significantly increases the accumulation of aroma substances in rice，and it would provide theoretical technology and material support for further breeding of fragrant rice，and shorten the time for the cultivation of fragrant rice.