Agrobacterium Mediated Perennial Ryegrass Transformation

Turfgrass plays an important role in human daily life. To improve the quality of turfgrass is becoming a significative research in the world. There are some restrictions in traditional breeding methods. However, it is convenient and practical to adopt biotechnology in developing and improving new turfgrass varieties, and it will become a new trend in turfgrass breeding study. Perennial ryegrass (Lolium perenne L.) is one of the major cool-season turfgrass utilized worldwide. Improving herbicide-resistant and insect-resistant with plant genetic engineering is so significant that it can reduce the usage of chemical pesticide or herbicide, protect environment, save the maintenance of turfgrass, and also keep the turf clean and tidy. Up to date, Transgenic plants of ryegrass have been reported, almost of them were obtained through microprojectile bombardment, silicon-carbide fibre-mediated transformation, direct DNA uptake by protoplast inosculation or Agrobacterium-mediated transformation.In this study, a high efficiency ryegrass regeneration system was established, the Agrobacterium-mediated transformation system for ryegrass was optimized and transgenic plants which contain herbicide-resistant gene or insect-resistant gene were obtained. The main results are as followed:1. Establishment of ryegrass regeneration system .The ryegrass(Dt808011) mature embryos were used as explants in this study. When the embryos were cultured on MS culture medium supplemented with 5mg/L2,4-D, the embryogenic callus Induction efficiency was 15.1%. The good quality calli and high differentiation efficiency could be reached when 1mg/L 6-BA was present in regeneration medium.2. The factors that influenced the efficiency of ryegrass transformation mediated Agrobacterium were studied in this experiment. Embryogenic callus obtained after about 5-10 days to rounds of subculture were used as recipient material for transformation , the concentration of Agrobacterium tumefaciens suspension was 1.0(λ=660) added with AS of 100mg/L, treated with 5 min in 0.5×105Pa negative pressure, and then infected within 30 min. GUS transient expression assay showed that the most effective transformation occurred and around 54.32% calli are positive.3. Transgenic plants were obtained. The binary vectors of p3301-Ubi8c ,p3301-Ubi8e and p3301-Ubi8g which are harboring insect-resistant genes(cry8C,cry8E and cry8G) were constructed. The plasmid pDM805 harboring herbicide-resistant gene (bar); the p3301-Ubi8c,p3301-Ubi8e and p3301-Ubi8g were transformed into Agrobacterium tumefaciens AGL1 respectively. Transgenic plants were obtained via the transformation of Agrobacterium tumefaciens AGL1 into Lolium perenne calli. 61 out of 117 were positive transgenic plants after amplification the bar gene with PCR. GUS staining and Basta resistance results showed that the around 52.1% transgenic plants were positive. PCR results also indicated that the insect -resistant genes were integrated to transgenic plants chromosomes.