Establishment Of Genetic Transformation System For White Clover (T Rifolium Repens L.) And Chinese Zoysia (Zoysia Sinica Hance) Via Agrobacterium Tumefaciens Soaked Seeds

Genetic transformation is an important tool for studying genes function and breeding new varieties. Using transgenic could break species limit, realize interspecific genetic breeding and modify existing species character, apply to breed and improve new varieties. It was an convenient and effective way for screening cultivation fine varieties.White clover and Chinese zoysia were high quality grass species. They were graceful appearance, strong adaptability, strong resistance, trampling resistance, strong tolerance cutting. Root system of White clover and radiciform stolon of Chinese zoysia were developed, have very good effect of conserving soil and water, have been widely applied in landscaping; white clover also used as good pasture with rich nutrition and high yield.In this study, an high-frequency transformation system has been established by agrobacterium saked seeds transformation technology, White clover and Chinese zoysia germinating seed were used as transformation receptor. External factors that affect the efficiency of genetic transformation were studied and optimized, on the base of NPTⅡ. It was originally proved by the PCR examination that gene had been transferred into the genome.The major results as following:1. The best active period of internal cell splitting was found in 1-3h in White clover after accelerating germination. 20% NaOH immersion 20min was the best method to break dormancy of Chinese zoysia seed and the best active period of internal cell splitting was found on 5-9d of after accelerating germination.2. Effect of kanamycin on seed germination and growth were investigated. The results indicated that use 50mg/L kanamycin concentration and treatment 6-8 days were the best screening for White clover seed. Use 75mg/L kanamycin concentration and treatment 6-10 days were the best screening for Chinese zoysia seed.3. Gene transformation system was established. White clover seed was precultured for 1 hour, then was infected with Agrobacterium LBA4404 which OD600 was 0.9 for 24 hours with pumping 5min+ intermittent 1min+ pumping 5min+ intermittent 1min+ pumping 5min+ intermittent 1min vacuum treatment. After 1 day of co-cultivation, the bacterial liquid were washed off with cefotamixe, then transferred to culture containing kanamycin for selection. Chinese zoysia seed was precultured for 6-7 days, then was infected with Agrobacterium infection which OD600 was 0.9 and 100μmol/L acetosyringone was included for 48 hours with pumping 5min+ intermittent 1min+ pumping 5min+ intermittent 1min+ pumping 5min+ intermittent 1min vacuum treatment. After 2 days of co-cultivation, the bacterial liquid were washed off with cefotamixe, then transferred to culture containing kanamycin for selection..4. The kanamycin resistant plant were performed PCR examination with non-transformed plants as negative control and plasmid DNA as positive control, amplification NPT-Ⅱ,GUS gene part fragment. The results were part of transformed plants amplification the same band as plasmid and non-transformed plants had nothing band confirmed that gene had been transferred into the genome.