Identification Of Elite Genotypes For Genetic Transformation And Creation Of Genetically Modified Soybean

Soybean is one of the most important oil crops in the world,providing abundant plant proteins and oils to humans.Breeding new soybean varieties with saving cost and increasing efficiency is the main goal of breeders.The transgenic CP4-EPSPS glyphosate herbicide-tolerant soybean produced by Monsanto has become the most widely used crop in the world,and has promoted the new soybean cultivation and production technology system.However,there is not any glyphosates-resistance soybean cultivars with independent intellectual property rights for productionin China.CRISPR/Cas9 system has been used in soybean genetics and breeding research,but the genetic transformation efficiency of soybeans is still relatively low(generally less than 5%),which greatly limits the application of gene-edit technology.Therefore,this study intends to use the EPSPS+GAT gene with independent intellectual property rights to carry out genetic transformation experiments on the newly-developed soybean varieties(lines),and to screen the receptors with high genetic Transfomation efficiency.At the same time,glyphosate-resistant transgenic materials with good background of soybean varieties were obtained,from which excellent germplasms with high resistance to glyphosate were found.In addition,soybean GmMLO02 gene,which is homologous to an important gene Mildew resistance locus O(MLOwith resistance to powdery mildew,was edited using CRISPR/Cas9 genetic editing technology to obtain new disease-resistant germplsm.The new genetic resources can be used for soybean herbicide resistance and disease resistance breeding.The main results are as follows:1.Five receptor varieties with high efficiency of genetic transformation were screened out.The EPSPS+GAT gene was transformed into 138 soybean cultivars.The results showed that 57 genotypes could generate rooting seedlings,the average regeneration rate was 44.28%,and the maximum value was 97.37%of the foreign introduction accession PI341257.The average values of elongation and rooting rate were 10.31%and 6.17%respectively,and the maximum values were 69.23%for NJ17-33and 45.65%for NJ17-45 respectively.The average transfomation rate is 1.58%,and the maximum value is 19.57%for NJ17-45.Ten lines including NJ17-45,ZZJ13264,NJ17-33,L1419,F13A3,J098098,NJ17-38,PI647962,Y13-B9,Y1511 with higher transfomation rates(4.76%-19.57)in comparison with the control TL1H were initially screened out.Further transformation experiments were carried out on the five materials(L1419,NJ17-33,J098098,NJ17-38 and F13A3)under two different transformation vectors and screening agents.It was found that L1419 performed high transfomation rates for both EPSPS+GAT gene and dmo gene(21.19%,23.38%respectively),much higher than that of the controls TLIH and Williams 82.J098098 has higher efficiency when dmo gene was transformed by using glufosinate as a screening agent,and the efficiency of F13A3 and NJ17-38 is high for transformation of EPSPS+GAT gene vector.2.Transgenic soybean plants from 37 receptor varieties with glyphosate resistance were obtained and two lines with extreme high-resistance to glyphosate were identified.In T0 generation.300-500 ml/mu of glyphosate was sprayed on the regenerated seedlings,and 279 glyphosate resistant plants from 37 varieties were obtained.Some materials T2 and T3 were screened by spraying with 500 ml/mu glyphosate,and 39 transfonnants were further screened out from 18 genotypes.Among them,T4 pure lines from the 11 resistant transformants have been developed.Among them,PI654356-1-5 transformant plants can grow normally under the treatment of 2000 ml/mu glyphosate,and NJ17-33-1-7 transformant progeny can grow normally after treatment of 1200ml/mu glyphosate concentration.3.New soybean plants with GmMLO02 gene editing were createdThe gene editing vector for soybean GmMLO02 was constructed,and was transformed into three genotypes Williams 82,TL1H and Jack.The herbicides and sequence detection were performed on the transf-ormed progeny.Total 29,40,43 T0 plants edited with GmMLO02 gene sequences were obtained from Williams 82,TL1H and Jack respectively.T1 homozygous plants of target GmMLO02 gene with different sequence variation were also identified.There are differences in the editing efficiency of the three genotypes,and Williams 82 and Jack are more efficient than the TL1H as a receptor.Among the 27 strains,Williams 82 and Jack had 4、3,and 2、3 respectively,homozygous editing types of 2 and 1,respectively,and in the TL1H 9 lines,from the T1 plant editing type.There were no cases where both homologous chromosomes of T0 were edited,and only three lines were edited for a single chromosome.The results laid the foundation for the functional study of MLO gene and improvement of resistance to powdery mildew in soybean.