Development And Optimization Of Highly Efficient Genetic Transformation And Genome Editing System For Alfalfa(Medicago Sativa L.)

Alfalfa(Medicago sativa L.)known as "the king of forage",is the most important forage legume species in China.With the rapid development of graziery,it is still an urgent problem to cultivate new alfalfa varieties to meet the personalized environmental needs of different regions in China.Since gene editing strategy can shorten breeding cycle andachieve accurate improvement of crop traits,it has been widely used in molecular breeding of different plants.Highly efficiency of genetic transformation is the premise of gene editing system,and genotype is one of the important factors affecting genetic transformation of plant.At present,the genetic transformation system of alfalfa has been reported,but the genetic transformation efficiency of alfalfa varieties "Gongnong 1 " and "Longmu" suitable for planting in Northwest China is relatively low,only about 20%;And whether the reported editing technology can be well used in these two varieties has not been verified.Therefore,the use of gene editing is still a challenge for the improvement of these two local varieties.In this study,genetic transformation systems of the two local alfalfa varieties "Gongnong-l" and "Longmu" were increased by optimizing various parameters,and gene editing systems of them were also established.The results showed that:(1)Infection conditions and explant selection could affecte the infection efficiency of Agrobacterium tumefaciens.The leaves at the top of the stem treated with ultrasound for 15 min could achieve the highest infection rate,70%.It was found that the regeneration efficiency of "Longmu" was significantly different among different plants in this experiments.It is planned to improve the regeneration efficiency by selecting plants with high regeneration efficiency as explants for subsequent transformation.Finally,the transformation efficiency of "Gongnong 1" and "Longmu" mediated by Agrobacterium tumefaciens reached 52.78%and 35.21%respectively;(2)In addition,the genetic transformation system mediated by Agrobacterium rhizogene of alfalfa was established through four different explants,roots,stems and leaves of sterile seedling and leaves of soil cultured seedling,among which,hairy root induction efficiency of leaves of soil cultured seedling was the highest in this study.On this basis,hairy root induction efficiency of "Gongnong l" and"Longmu" were further improved by optimizing the screening agent concentration.The results showed that 1 mg/L glufosinate screening pressure could screen efectively and caused less damage to explants.Finally,the transformation efficiency of "Gongnong 1" and "Longmu" mediated by Agrobacterium rhizogenes reached 51.19%and 51.5%,respectively in the present study;(3)On the basis of the highly efficient genetic transformation system,MsPALM and MsCOMTwere selected as the target genes for gene editing to generate new germplasm resources with high yield and high quality alfalfa.In order to establish the gene editing system of "Gongnong-1" and "Longmu" quickly,hairy root platform established previously was employed to detect the editing efficiencies of four gene editing systems,tRNA system,ribozyme system,casl2a system and pHSE6401 system in this study,and pHSE6401 was proved be the optimal editing system in the two local alfafa varieties,by which the editing efficiency could achieve 61.9%.Meanwhile,the homozygous edited hairy root lines of MsPALM were obtained.In addition,in order to further improve the gene editing efficiency of alfalfa,several MsU6 promoters sequences of alfalfa were cloned to improve the expression of guide RNA.In conclusion,the genetic transformation systems mediated by Agrobacterium tumefaciens and Agrobacterium rhizogene of two local alfafa varieties,"Gongnong-1" and "Longmu",were optimized or established,respectively.In addition,gene editing system suitable for them was screened out.By which,homozygous edited hairy root lines of MsPALM which had potential to improve alfalfa yield were obtained.In all,the results in present study can pave the way for the rapid and accurate improvement of important agronomic traits of local alfalfa verieties through gene editing in the future.