Creation Of Tomato Germplasms With Less Lateral Branches Via CRISPR/Cas9 Techinque

Tomato（Solanum lycopersicum）is one of the vegetables with large cultivated area in China.The growing of lateral branches is an important agronomic character,which directly determines the morphological structure of plants.Excessive branches often compete for nutrient distribution and lighting,which will results in the reduction in land utilization,negatively affects the number,yield and quality of crop fruits,and be easy to cause various diseases.As tomato is a kind of vegetable with more branches,it is inevitable to carry out regular management of lateral branches,whether with high-tech information or traditional cultivation techniques,which makes it become time-consuming and labor-intensive.Selecting and breeding tomato varieties with fewer lateral branches is one of the important ways to solve this problem.However,currently,there are no varieties with fewer or no lateral branches found in the tomato germplasm resource bank.Previous studies have found that mutations caused by deletions of Cs PIN3 in cucumber,Md WUS in apple,and Sl Sde1A in tomato can inhibit the growth of lateral branches respectively.In this study,CRISPR/Cas9gene editing technology was used to knockout Sl PIN4（PIN-FORMED3）,Sl WUS（WUSCHEL）,（the homologous genes of Cs PIN3 and Md WUS in tomato）,and Sl Sde1A（Super determinant1A）genes to prevent the growth of tomato lateral genes,so as to develop tomato germplasms with fewer branches.The main results obtained from this research are listed as following.1.The growth of tomato lateral branches can be inhibited by the knockout of both Sl PIN4 and Sl Sde1A genes,but not by the knockout of Sl WUS gene.2.Compared with the wild type（WT）,the number of lateral branches of tomato mutants of T₀ generation and T₁ generation decreased significantly,and tomato germplasm materials with fewer lateral branches were obtained,achieving the expected effect.By observing the growth of slwus mutant T₀ generation tomato,it was found that the number of lateral branches did not decrease,the internodes became shorter,the plants were short,and the style was flat without pollen after flowering.3.By detecting and sequencing the T₁ generation of tomato slpin4 mutant,a tomato strain without Cas9 protein was obtained,and the number of lateral branches of this plant line is lower than that of wild type,which was a non-transgenic gene-edited tomato germplasm material.