Using CRISPR/Cas9 Gene Editing Technique To Transform Two Quality Traits Of Rice

In rice breeding of China,quality improvement has become a hot spot.Among rice quality traits,waxy and fragrance is the special ones of rice.The development of the CRISPR/Cas9 genomic editing technology provides an efficient technical means for the improvement of waxy and fragrance.In this study,we used CRISPR/Cas9 genomic editing technique to improve the waxy and aroma of the hybrid rice parental materials,209B and Y58S respectively.The negative regulation genes Wx and Badh2 for amylose content and rice flavor as were the target genes for edition to obtain superior hybrid rice parent material,respectively.The results of the study are as follow:1.The editing of Wx locus in rice material 209B.The target site of Wx gene was selected to construct sgRNA expression cassette and pYLCRISPR/Cas9 vector.The single target vector was transformed into rice material 209B by Agrobacterium tumefaciens.The edited site of T0 and T1 plants were analyzed by PCR and sequencing.The amylose content of the transformed rice was measured and then homozygous mutants were screened.The results showed that the mutation frequency on Wx site in T0 generation of 209B mutation treatment was as high as 66.7%,and the homozygous deletion mutation with amylose content below 4%amounted to 33.3%of the transformed plants.2.The editing of Badh2 locus in the rice material Y58S.The appropriate target site of the Badh2 gene was selected for the editing.The sgRNA expression cassette was ligated with the pYLCRISPR/Cas9 vector.The double target vector was used to transform the rice material Y58S by Agrobacterium tumefaciens.PCR and sequencing analysis of T0 and T1 transformed plants were carried out,the fragrant substances of the transformed plants were analyzed,and then the genetics of the aroma in T0 and T1 mutants was analyzed to see the T0-generation homozygous mutant could be stably inherited to T1 and the T0-generation double allele mutations were in accordance with Mendelian inheritance in the genetic process.The T-DNA-free mutants without exogenous gene elements were obtained from the genetic segregating mutation plants by analysis with agarose gel electrophoresis.The results showed that the mutation frequency of Badh2 locus was 69.7%in T0 generation of Y58S mutation treatment,and the homozygous deletion mutation with strong flavor phenotype was 31.3%of transformed plants.3.The investigation results of the T-DNA-free T1 homozygous mutants showed that the amylose content of most of Wx gene mutants from 209B significantly decreased up to the level of only 1 percent,the waxy quality was superior,and other agronomic and economic characters were not significantly different with the control variety,therefore,the transformed materials has practical value of importance.Most Badh2 T1 homozygous mutants of the Y58S had strong aroma substance and excellent quality.The other main agronomic characters such as plant height,panicle number,flag leaf length,flag leaf width,panicle length,grain number per spike and seed setting rate of these did not significantly differ with the control line Y58S.These transformed materials have practical significance.