Improvement Of Grain Shape And Fragrance In Japonica Rice By Using CRISPR/Cas9 System

Rice（Oryza sativa L.）is one of the most important food crops in the world,and rice is the staple food for more than half of the population.As the largest and most important japonica rice production area in China,Heilongjiang Province,the planting area accounts for more than 40%of the country’s japonica rice planting area.The creation of new japonica rice germplasm in Heilongjiang Province has important practical significance.With the improvement of people’s living standards,consumers are increasingly demanding high-yield and high-quality rice.In recent years,long-grain fragrant rice is gradually being welcomed by the market.In traditional breeding,crossing and backcrossing methods are usually used to achieve the aggregation of excellent genes,but there are disadvantages such as long breeding cycles and uncertain mutation directions,which are time-consuming and laborious,resulting in low breeding efficiency.Gene editing technology can target and edit genes more quickly,effectively and accurately,providing a new way to improve rice varieties.In this study,the round non-scented japonica rice variety Longjing 11 in Heilongjiang Province was used as the experimental material,and the negative-regulatory genes GS3,GS9 and the negative aroma-regulatory gene Badh2 were used as target genes.The CRISPR/Cas9 gene editing technology was used to construct a knockout vector,p YLCRISPR/Cas9-GS3/GS9/Badh2-g RNA,genetically transformed Longjing 11 through Agrobacterium-mediated method,and successfully obtained the three-gene homozygous mutant materials which without the T-DNA elements.The specific research contents and results are as follows:（1）A target was designed in the first exon of GS3,the first exon of GS9,and the second exon of Badh2,and a CRISPR/Cas9 knockout vector（p YLCRISPR/Cas9-GS3/GS9/Badh2-g RNA）was constructed,Longjing 11 was genetically transformed by Agrobacterium-mediated method,and 4 positive mutant plants were obtained in the T₀generation.（2）In the T₁generation,Cas9-F/R primers were used to screen out mutants without the T-DNA elements,and then specific primers were used to screen to obtain two three-gene homozygous plants with different mutation types,55-2 and 58-2,respectively.Mutant plants have1-2 bp frameshift mutations at the targets of GS3,GS9,and Badh2,which obtaining a truncated protein lacking a large number of amino acids,resulting in functional the loss of GS3,GS9,and Badh2 genes.（3）In the T₂generation,compared with the wild-type Longjing 11,the grain length of 55-2and 58-2 lines increased by 27.01%and 26.43%,the grain width decreased by 13.4%and 24.6%,and the thousand-grain weight increased by 18.34%and 41.36%.There was no significant change in the seed setting rate,the yield per plant was increased by 10.82%and 12.11%,and the aroma of rice changed from no aroma to strong aroma,providing a theoretical basis for creating long-grain fragrance germplasm resources.