Establishment Of Regeneration System And High-efficiency Genetic Transformation System By Agrobacterium-mediated Of Lolium Perenne L.

Perennial ryegrass (Lolium perenne L.) (perenial ryegrass) is an excellent forage and turf-grass. Now, so many kinds of Perennial ryegrass varieties have been planted in all of the world. However, with the characteristic of self-incompatibility, it is hard to use traditional methods to improve its disadvantage properties .Biotechnology, especially transgenic technology can effectively solve this problem, and quickly improve the bad traits of the target gene as well.Partical-Bombardment was the main method of genetic transformation for monocotyledon in the past years. But now, more and more genetic transformation by Agrobacterium-mediated have been used in monocotyledons widely .In this experiment, first we established an regerneration system in vitro of perennial ryegrass, then focused on the high efficient genetic transformation system by Agrobacterium- mediated method. Results were as follows:(1) The mature embryoes and growing shoots of six kinds of perennial ryegrass varieties (YEYING, CHANGTONG, LVBAOSHI, DINGFENG, Nu Speed, A8393) were used to induce callus in MS medium with 6 kinds of 2,4-D concentrations. It was founded that the best concentration of 2,4-D was 5.0 mg/L, in which the highest frequency of callus and embryonic callus from mature embryo and growing point were 50%, 35% and 60%, 45% respectively.(2) There were different regeneration rate from different ryegrass callus. The highest differentiation rate was from YEYING, it was up to 98%. In addition, added with a certain concentration of 6-BA benefited embryogenic callus differentiation, the highest differentiation rate (87.5%) was obtained when 6-BA concentration was 1.0 mg / L.(3) the highest positive rate was obtained by GUS testing after the embryonic calli were cultivated 14 days and the OD650 of Agrobacterium was 0.8,it was up to 57.3 percent in this experiment of genetic transformation by Agrobacterium-mediated. Meanwhile, the highest frequency of resistant calli(56% )were received when adding 200 mg / L acetosyringone, infecting 20min and co-culturing 3 days. (4) The calli were treated with different concentrations of two antibio agents (benzylpenicillin and Timentin) after Co-culturing, the results showed that the 200mg / L timentin could restrain the bacterial activities completely, but the lowest concentration of benzylpenicillin was 250mg / L.(5) The best methods of selecting anti-callus had been obtained in this study. It was that the calli from co-culturing were treated with 80 mg / L hygromycin for two weeks, 50 mg / L hygromycin for two weeks, 25 mg / L hygromycin for two weeks. Then put the calli into differentiation medium under light treatment, and the translating plants were gained in 30 days.(6) A total of 28 transgenic plants had been gained, 8 plants showed positive reaction from 11 of YEYING by GUS assay. Furthermore , 4 plants of them had been proved containing Pcambia1305.1 by PCR testing.