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Accueil du site → Doctorat → Chine → 2008 → Research of Gene Transformation and Drought-resistant Genetic Improvement of Perennial Ryegrass (Lolium Perenne L.)

Beijing Forestry University (2008)

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

Li Xue

Titre : Research of Gene Transformation and Drought-resistant Genetic Improvement of Perennial Ryegrass (Lolium Perenne L.)

Auteur : Li Xue

Grade : Doctoral Dissertation 2008

Université : Beijing Forestry University

Résumé partiel
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.

Mots clés : Perennial ryegrass (Lolium perenne L.) regeneration system genetic transformation transgenic filial generation transgenic biosafety

Présentation (Dissertationtopic)

Page publiée le 3 mai 2013, mise à jour le 10 janvier 2018