The Optimization Of Stylo Genetic Transformation Via Agrobacterium Tumefaciens

Stylo(Stylosanthes spp.)is an important economic legume forage,mainly grown in subtropical regions.It has the capability of soil fertility restoration,soil physical properties improvement and acidic soil adaptation.The chilling-sensitive,fungi susceptible and heavy metal stress of stylo had limited its production severely.Those problems could be solved by the genetic transformation which relies on the efficient genetic transformation system of stylo.Molecular genetic improvement is an useful means to solve the above problems,this paper aims to establish Stylo efficient genetic transformation system,providing the basis for molecular breeding.This project used the Reyan 5 Stylo(S guianensis cv.Reyan No.5)as materials.the explants type(hypocotyl,cotyledon and growing point),concentration of Agrobacterium suspension(OD600=0.2,0.4,0.6,0.8,1.0),infected time(5,10,15,20,25 minutes),co-culture time(days 1,2,3,4,5),acetosyringone concentration(0,50,100,200,400?mol/L)and concentration of hygromycin(10,15,20,25 mg/L),were studied individually in the transformation.The results are as follows:Stylo was sensitive to hygromycin(Hyg).At the Hyg concentration of 15 mg/L,the callus induction rate was 9%without Agrobacterium disseminated;When Hyg concentration up to 17.5 mg/L,explants almost died.The explants Via Agrobacterium-mediaied after screening is used at Hyg concentration of 15 mg/1,The callus conversion efficiency is as high as 71%,so chose Hyg optimum concentration for the 15 mg/1 as the resistance callus and resistant shoots screening training.Agrobacterium bacterium concentration had a significant effect on the conversion efficiency,when Agrobacterium suspension at a concentration of OD600 = 0.6,The callus conversion efficiency was 67%;Infected time had a significant effect on the conversion efficiency,when the infected time was 15min,the allus conversion efficiency was 59%;Adding acetosyringone in co-culture medium had a significant effect on the conversion efficiency,when acetosyringone concentration 200 ?mol/L,the conversion callus efficiency was 18%;Co-culture time had a significant effect on the conversion efficiency,when co-cultured was 3 d,the callus conversion efficiency was 69%.The results showed that choose the cotyledons as conversion material,in a concentration of Agrobacterium OD600=0.6,infected time of 15 min,co-culture time of 3 days,as concentration of 200?mol/L,with all the conditions above,it obtain high transformation efficiency.On the use of resistant callus and resistance plant which obtained in the above-described transformation system,GUS staining and PCR assay was performed.Test results showed both the materials can be observed with blue spots,The PCR product after electrophoresis were appeared.This result indicated the GUS gene and the Hyg gene had been transferred into the Stylo.