Establishment Of High Efficient Regeneration System And Study Of Influence Factors On Transformation By Agrobacterium Tumefaciens Of Lolium Perenne

Perennial ryegrass is an excellent cool-season grass of Gramineae,which is widely distributed in Europe,the Americas and Asian temperate regions and is a common forage grass.The rapid development of plant molecule biology has supported a new way to create new varieties of Perennial ryegrass and the utilization of genetic transformation to improve the trait and quality of Perennial ryegrass has become practicable.Agrobacterium tumefaciens as a cartier is low-cost and effective which is available for transformation of extrinsic gene.Perennial ryegrass is not sensitive to Agrobacterium tumefaciens because it is a kind of monocotyledons,so we should explore an efficient way to transform extrinsic gene into Perennial ryegrass.Based on the high efficient transformation system,the experiment uses callus induced by the mature seed of Perennial ryegrass as transformation material and pIG121(without extrinsic gene)as transformation carrier to establish an acceptor system of Agrobacterium-mediated transformation of Perennial ryegrass.1.Establish a high regeneration systemThe optimum medium to induce the generation and differentiation of callus was screened out by use of resection part of the endosperm of mature seeds of Perennial ryegrass as explants and the MS medium as the basic medium in this experiment,which were added in different kinds of plant hormones.The results showed that the best medium for callus induction was MS+2,4-D6.0 mg/L+6-BA0.1 mg/L,and the ratio of induction was 86.3%.The optimum medium for callus differentiation was MS+NAA1.0 mg/L+6-BA1.0 mg/L,which could be 76.5%.The best medium for rooting was 1/2MS+NAA0.8 mg/L,where the ratio of rooting could be 100%.2.The preliminary establishment and optimization of genetic transformation system of Perennial ryegrassWhen selective concentration of G418 and Cef was confirmed and GUS gene was shifted into callus by means of agrobacterium-mediated genetic transformation by use of callus as explants,the genetic transformation system of Perennial ryegrass was preliminarily established.By detecting the transient expression of GUS gene,this study probed into different factors effected on Perennial ryegrass' transformation,which were pre-culture,drying treatment,different types of infectious liquid,concentration of agrobacterium,time of infection,means of infection,time of co-culture, different light for co-culture and addition of AS.The results showed that these were important factors in Perennial ryegrass' transformation.In Perennial ryegrass' transformation system mediated by Agrobacterium tumefaciens EHA105-pIG121,the selective concentration of G418 was 200 mg/L,and Cef was 200mg/L.The optimal method of transformation was as follows:the callus was cut the size of the diameter of 3ram,and pre-cultured in the medium MS+2,4-D3.0 mg/L+6-BA0.1 mg/L for 3～5 days.After the bacterial liquid whose OD₆₀₀ was 0.6～0.8 by the second activation was centrifuged,thalli were collected.The OD₆₀₀ of bacterial liquid should be about 0.7 after thalli was resuspended in liquid MS+AS100 um/L.Then the callus that had been pre-cultured was put into the resuspended bacterial liquid to be infected for 20min in the shaking bed.After putting the callus with agrobacteria into the Petri dish with sterile filter paper for drying 1 day,these callus were transferred into the medium MS+2,4-D3.0 mg/L +6-BA0.1 mg/L +AS100 mg/L in the darkness for 2 days.Later,it was time to wash bacteria off on the surface of callus in the liquid MS+Cef500 mg/L for 20min.And next,after bacterium on the surface of callus was soaked up by aseptic water,which had been washed by aseptic water 5 times,the callus was selectively cultured in the medium MS+NAA0.1 mg/L+6-BA1.0 mg/L+G₄₁₈ 200 mg/L+Cef200 mg/L.Under these optimum conditions,the rate of transient expression of GUS gene in Perennial ryegrass' could be 60%.