Research Of Gene Transformation And Drought-resistant Genetic Improvement Of Perennial Ryegrass (Lolium Perenne L.)

Based on the construction of high frequency regeneration system of Perennial ryegrass (Lolium perenne L.), DREB1A and BADH-CMO gene were transformed into embryogenic calli via biolistic bombardment and Agrobacterium infection transformation. All the transgenic plants grew naturally, advanced abiotic stress-tolerance and developed into sexual stage. Transgenic first filial generation (T1) were harvested by artificial pollination and isolated cross. Their characters of germination period, pot culture in the greenhouse and cultivated in the field were studied. Meanwhile, a quick identification method of transgenic Perennial ryegrass hygromycin (hyg) resistance and a selection system for drought-resistant filial generation were created. Biosafety of transgenic Perennial ryegrass was discussed synthetically. Keeping on studying Perennial ryegrass genetic engineering breeding for more than 5 years, conclusions showed as followings:1. A high frequency regeneration system of Perennial ryegrass with proper varieties using mature embryo as explants was constructed. The rate of callus induction, embryogenic callus and regeneration could reach to 97.9%, 19.4% and 90.9%, respectively.2. Establishment of a genetic transformation selection system for Perennial ryegrass: add 100mg/L hyg at callus cultivation period for 30d while 50mg/L at generation period until the bud come out. Cefotaxime (cef) was used as the bacterioatatic in Agrobacterium infection transformation and it was 300mg/L at callus cultivation stage while 250mg/L at generation stage.3. A biolistic gene deliver system of Perennial ryegrass was established. It included using 0.5-1mm embryogenic calli as target tissue, 1μm diameter gold powder as the vector of plasmid DNA which coated by Ca(NO3)2+PEG4000 and twice bombardment at 6cm distance. Transgenic plants were obtained containing 68 transgenic Accent plants (ATR), 7 transgenic Delaware plants (DTR), 18 transgenic Juventus plants (JTR) with DREB1A gene and 15 transgenic Accent plants with BADH-CMO gene (ABC). The average transformation rate of the three varieties was 11.1%.4. Agrobacterium-mediated Perennial ryegrass foreign gene transformation system was established. The embryogenic calli precultured on fresh medium for 5d were infected with Agrobacterium tumefaciens (OD600=0.1) for 10 min with discontinuous vacuum treatment and shake. After wiping off the Agrobacterium, they were co-cultivated for 4d. Then, the calli were washed with 500mg/L cef and transferred to selected medium. 14 transgenic Accent plants with DREB1A (AGR) were obtained by this system.5. ATR, AGR and ABC grew slower than ACK in pot while ATR, AGR, JTR and DTR showed no growth difference with their control plants except ABC appeared a notable growth inhibition. The foreign genes in this study not only advanced the heading time of transgenic plants, but also shortened the spike length especially of ABC. DREB1A gene increased the heading quantity a little while BADH-CMO decreased it dramatically. Compared to the control plants, transgenic plants represented a better drought, cold and salty tolerance, a worse disease resistent and equal pest tolerance. All the transgenic plants could seed except ABC.6. Transgenic filial generation were gained by artificial pollination and isolated free-cross. T1 seeds expressed a high germination potential and rate. The seeds came from JTR as pollen and female parent showed a dominant germination potential. Similarly, the seeds using AGR as female parent and the seeds from DTR as pollen parent both exhibited the lowest average germination potential. The average bud length of all T1 seeds was 3.9cm while the root length was 3.2cm at 7th. The highest average bud length was from the seeds using JTR as pollen and female parent while the shortest was from the one using AGR. The T1 from JTR presented best in the field and both of the seeds from AGR-12 and DCK as famail parent exhibited 100% potential and rate of emergence. The free-cross seeds showed higher potential and rate of emergence, height of seedlings and tillering number than artificial pollination. T1 seeds presented different germination abilities even if they were from the same female parent.7. PEG and hyg exhibited a distinct influence on Accent seeds germination. The germination potential declined to 44.8% and kept the germination rate on 61.5% when hyg was 75mg/L, so hyg was not fit to be the screen agent. Only a little seeds could germinate when PEG was 15%, but none when PEG was 20%. The separated law conjectured by 15% PEG selection was below the mondelism.8. A quick identification method of transgenic Perennial ryegrass hyg tolerance was created. The brown rate of the isolate leaf sector at 8th with 1mg/L 6-BA+50mg/L hyg could be the selection evidence. Pot cultured T1 plants after drought selection and hyg leaf sector screen showed light demanding that concluded elementary the foreign gene could steady heredity in plants.9. The transgenic Perennial ryegrass with DREB1A and BADH-CMO gene were I class transgenic plants. Both of them were in the middle test confirmed by State Forestry Administration, P. R. China.