Study On Callus Induction From Mature Embryos, Plant Regeneration And Genetic Transformation Of Wheatgrass

Agropyron is the one of the worldly important forage grasses. It is not only with high feeding value, but also is widely used as a fitted grass specie for improving deteriorated grassland and constructing artificial pasure. It is a considerable interest to genetically modify and improve growing properties of Agropyron. There are highly genetic differences among various Agropyron cultivars. Efficiency of regeneration and genetic transformation is markedly dependant on different species. An efficient and stable regeneration and transformation system had been one of key techniques in study of Agropyron molecular biology and genetic engineering. Although some studies on the Agropyron regeneration had been reported, they were mainly on the embryo culture. A effective genetic transgenic system need to be established with wheatgrass.The study in this paper is aimed to establish and optimize the wheatgrass regeneration and transformation system, using Hycrest-Mengnong as material. The factors that influence the wheatgrass callus inducing and difference and regeneration were primarily studied. Using the mature embryos as explants, tissue culture can not be limited by seasons. The results showed that with MS as basic medium, ABA promote inducing callus, mannitol with 0.2 M can markedly improve the quality of callus. In the differenting medium, the proper concentration of ZT can distinctly facilitate the callus shooting. In this paper, culturing in liquid medium was also explored to proliferate the callus and the conditions for improving the quality of callus were studied. Finally ,we established that the optimum medium for Induction of embryogenic callus was MS +mannite(0.2M)+2,4-D(0.2mg/L)+ABA(0.3mg/L),the medium for differentiation was MS+ZT(3.0mg/L)+NAA (1.0mg/L). PDS-1000He gene gun was used for transformation with calli derived from the. Hycrest-Mengnong mature embryo as explants. About 1000 calli was bombarded with bar gene as selection gene, P5CS as aimed gene. Under the selection of Glufosinate added in inducing, differentiating and rooting mediums, 42 green shoots were obtained two and half month later. PCR analysis of bar gene displayed that 6 plantlets showed positive results, which primarily indicated bar gene had intergrated into genome of these plantlet. The transformation rate is about 0.6%. Among these plantlets, two can be detected out as positive lines by P5CS PCR analysis. The co-transformation efficiency of both bar and P5CS is about 33.3%. In the paper, the reason of low transformation rate was discussed. Also, the possible solution was brought out. The herbicide(Glufosinate), which is the selection in transformation, maybe interference with the differentiation of calli. The expression construction had been modified by cutting out bar gene, replaced by hpt gene that resistant against hygromycin. The transformation with new constructed vector is under way.