Establishment And Verification Of Hairy Roottransformation System Of Common Vetch (Vicia Sativa L.)

Common vetch?Vicia sativa?is an annual forage and green manure crop of the genus Vicia of the Leguminosae family.It has high nutritional value,wide distribution,strong adaptability,short growth cycle and outstanding cold resistance.However,there is no report on the genetic transformation system of it at home and abroad,which has greatly hindered the research of gene function verification and molecular breeding.Therefore,the genetic transformation system of common vetch needs to be established and optimized.Agrobacterium rhizogenes can induce hairy roots in plant wounds.The hairy root system mediated by it has been successfully applied to leguminosae such as soybean?Glycine max?,Medicago truncatula,and alfalfa?M.sativa?.But there is no related hairy root system of common vetch.In this paper,for the first time,a genetic transformation system of A.rhizogenes-mediated hairy roots of common vetch was established by three experimental protocols.And on this basis,the hairy root transformation systems for 13 leguminous forages,such as V.villosa,were successfully established.The main results are as follows:1.Root cutting infection protocol:This protocol chooses to cut a three mm root tip from a 4-day-old Lanjian NO.3 seedling,and the remaining part was used as an explant.A.rhizogenes strain was used to infect the explant via wrapping the wound site.Transgenic hairy roots were generated around the wounds of the explants after three to five days.This method can detect GUS,GFP and YFP reporter genes in hairy roots.Both ARqua1 and K599 strains can induce hairy root production.The hairy root induction rates of Lanjian NO.3 were 90%and 99%,and positive transformation rates were 60%and 80%;In addition,according to the GUS staining result screened out the ARqua1 strain which was most suitable for infecting Lanjian NO.3.This method was also successfully applied to the other 3 subspecies of common vetch.The transformation rate of positive hairy roots were 66%?68%and 82%,respectively.2.Piercing and infection protocol:This protocol used a disposable syringe needle to pierce the hypocotyl of a 5-day-old common vetch seedling,and apply the A.rhizogenes paste to the wound of the hypocotyl for infection.After infection,transgenic hairy roots can be generated in about seven days,and the induction rate and positive transformation rate of this protocol can reach 43%.3.Immersion infection after scratch protocol:Using the cotyledons and hypocotyls of the isolated and scratched common vetch seedlings as explants,infects the explants with A.rhizogenes bacterial solution for infection.Two days after induction and co-culture,the hypocotyl part become enlarged and hairy roots appeared nearby.Through comparation and analysis,the induction efficiency of hairy roots was highest under the conditions that the OD₆₀₀ value of the ARqua1 bacterium solution was 0.4 and the co-culture time was three days.The hairy root induction rate and the positive transformation rate were 60%and 55%,respectively.4.According to the research results of common vetch,the hairy roots induction efficiency of ARqua1 strain and root cutting infection protocol was the highest.Based on this,the hairy roots transformation system of 13 legumes including Vicia.villosa,Lathyrus sativus,Melilotus officinalis,Trifolium repens,Onobrychis viciaefolia,T.pratense,Lupinus luteus,Lotus corniculatus,Astragalus sinicus,A.adsurgens,M.sativa,Caragana intermedia and Hedysarum scoparium were established.The positive transformation rate of hairy roots of 13 legumes was 10%⁷5%.These three protocols mentioned in this study have the advantages of high transformation efficiency and short transformation cycle.They are of great significance for the research on the development of root system,symbiotic nitrogen fixation and ion absorption of common vetch.In addition,it can also provide technical support for the molecular breeding of common vetch and 13 other legume pastures.