Tissue Culture And Genetic Transformation On Several Burfgrasses

Turfgrasses industry is developing rapidly in our country in as much as it plays more and more important roles in modern day age. As the limits of traditional method breeding new and good varieties of turfgrass by advanced biotechnology has becoming an inevitable trend. Plant genetic transformation bases on the establishment of plant tissue culture and regeneration system. This paper using several turfgrass as materials conducted a series of tests on pre-sowing seed treatments, callus induction and plant regeneration, inhibition of Agrobacterium tumefaciens by antibiotics and some traditional Chinese medicine and these antibacterial affecting callus growth and differentiation, transformation of Lolium perenne L. ect. The primary results are summarized as follows:1.In experiment 1, most turfgrasses have low rate of germination in the lab. Several pre-sowing seed treatments were applied to improve their germination, including dry and wet in turn, magnetic field, low temperature, KNO₃, culture in darkness and so on. The results showed that dry and wet in turn, dry in 35℃,magnetic field, low temperature and KNO₃ can increase seed germination. The good effects were obtained from dry in 35℃, magnetic field with 1000GS and 16h wet in dry-wet in turn. However, wet in 35℃ ,culture in darkness and H₂O₂ had adverse effects on the seed germination.2.In experiment 2, tissue culture and plant regeneration have been done on four types of turfgrasses. The results indicated that there is no identical concentration of 2,4-D for all plant materials used. Nevertheless, the concentration of 2,4-D for these callus inductions was higher than the other gramineous plants. N₆ was the optimal basic medium among three basic media tested. The quality of callus on the medium supplemented with 500mg/L praline,300mg/L casein hydrolyzed and 100mg/L inositol was better than that of callus on medium without these organic additive, with thecallus multiplication fasten structure compacter and colour of light yellow. The effects of three cytokinins on differentiation of turfgrass callus were different.3. In experiment 3, six antibiotics (cefradine,ceftriaxone sodium, cefazolin, ampicillin,carbenicillin and benzylpenicillin sodium) were used to inhibit Agrobacterium tumefaciens . The results demonstrated that the best was ceftriaxone sodium, then carbenicillin and cefazolin. Although antibiotics did check callus growth and differentiation to some extent, cefazolin , ceftriaxone sodium and carbenicillin can be used for callus of Festuca arundinacea L. and Lolium perenne L. .respectively.The optimal concentration range was 300 500mg/L when ceftriaxone sodium as antibacterial.4.1n experiment 4, traditional Chinese medicine was applied to plant genetic transformation by Agrobacterium tumefaciens. Fourteen traditional Chinese medicines including Rhizoma Coptidis Jiadix Scutellariae, Hypericum Perforatum L. and so on were choosen to replace of antibiotics for elimination of Agrobacterium tumefaciens . The results revealed that Rhizoma Coptidis,Hypericum Perforatum L.,Taraxacum monglicum Hahd-Mazz and Radix Scutellariae have bacteriostasis in varying degree. With the exception of Rhizoma Coptidis which reduced callus growth and differention sharply, the others had little influence on callus growth and differerntion. So in the Agrobacterium tumefaciens mediated genetic transformation on turfgrasses, Hypericum Perforatum L Jaraxacum monglicum Hahd-Mazz and Radix Scutellariae can be used as antibacterial.5. In experiment 5, gene transformation of Lolium perenne L. mediated by Agrobacterium tumefaciens is carrying on. The results showed that the optimal concentration of herbicide for selection was 6 mg/L. Transformed calli are being cultured on selective media for further research.