| Perennial ryegrass is vital cool season gramineous forage and turf grass,which is widely distributed in temperate zone,its leaves are smooth,soft,beatiful,of long period of green color,strong tillering and high formation speed,therefore,it is a good turf grass.Mammalian metallothioneins (MTs) are a sort of low-molecular-weight, cysteine-rich and metal-binding proteins, which mainly participated these processes such as the metabolism of essential metal ions and the detoxification of toxic heavy metals in animals.Mice's MT-1 hasαandβtwo domains, which are not only interrelated but also independed on each other in the structure and function. They have different affinity toward the heavy metals, a-domain binding Cd2+, Hg2+ preferentially andβ-domain favoring to Cu2+,Zn2+.In this study, the author used mature perennial ryegrass of variety "Matilda" as experimental material and initiated the research of the genetic transformation regarding mouse metallothionein (MT) gene and its mutant geneααby Agrobacterium-mediated transformation. The research aims at utilizing the MT's characteristics of the conjunction and detoxification of heavy metals to increase perennial ryegrass's resistance and accumulation toward heavy metals, such as Cd, thus developing a new type of plant, which is used for the treatment of heavy metal pollution in the environment. The results are as follows:1.Establishment of plant regeneration system of perennial ryegrass.Perennial ryegrass mature seeds were used as explant.The result showed the induction rate were high on 8mg/L 2,4-D combining with 0.025mg/L 6-BA,56.42%.Callus were differentiated after 2～3 generation,the highest differentiation rate,32.25%,was achieved in the medium contained MS medium with 2mg/L 6-BA, and the differentiation rate was affected by the callus condition after proliferation obviously. The root inducing medium, containing 0.5mg/L NAA and MS medium with half macro, gave 98% root inducing rate.2.A Histological Study on Somatic Embryogenesis of perennial ryegrass.Paraffin slice are utilized to study the characteristics of morphogenesis and anatomical structure during the occurrence and development of somatic embryo in tissue culture of perennial ryegrass.The results show:the somatic embryogenesis and development inducing by 2,4-D experience all the stages of zygote embryo;There is distinct boundary between the different stages of somatic embryo and the cells around;Most of embryoids generate from the surface of the callus through the indirect process;somatic embryogenesis originated from one single embryogenic cell,thus avoiding chimeras in the regenerated plants.And an increased regeneration rate,even from long-term culture.3.Optimization of concentration of PPT and hygromycin.Concentration of PPT(Basta) and hygromycin,the screening agent,including herbicide resistance gene(bar gene) or hygromycin phosphotransferase gene(hpt gene) were tested for perennial ryegrass.The results showed that PPT 1 mg/L, hygromycin 30 mg/L is efficient for select concentration in genetic transformation of perennial ryegrass.4.According to GUS transient detection explore the system of Agrobacterium-mediated genetic transformation. Favourable embryogenic calli after 3 days culture in advance and 5 to 10 minutes evacuate were immersed in agrobacterium suspension for 15 min in the dark.Excess bacteria on the calli were removed and then transferred into co-cultivation medium (subculture medium containing 100μmol/L As)in the dark for 3 days.After co-cultivation, the infected calli were rinsed with sterile water containing 200mg/L cefotaxime sodium for three to four times,and calli were ransferred into selection medium supplemented with regeneration medium,and subcultured at 2-week intervals.Then some survived callus analysis in GUS histochemical stain, GUS positive incidence rate 51.61%.others were transferred into shoots inducing medium to induce shoots.Shoots points but no shoots had been founded.
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