| Recently, with increasing care to surroundings by people, turf-grass cultivation has developed progressively as a new industry. Poa pratensis L., Festuca arundinacea, Agrostis palustris Huds, Lolium multiflorum, Lolium perenne, Zoysia japonica, Festuca rubra L. etc have been planted in Northwest, Northeast and Southwest area of our country. They can be used in beautifying city environment, building playground or as forage grasses.Disease is main factor affecting growth of plants. Turf-grass diseases are mainly arosed by fungus including rust, leaf spot ect. Modern biotechnology has developed rapidly in recent 20 years , it has been applied in quality improvement and mannual culture of plants including turf-grasses. Genetic engineering could be used to promote the adversity, disease and insect resistance of turf-grasses. Genes with resistance function could be introduced into calluses or protoplasts, and transgenic plants with modified resistance could be regenerated.Tall fecue is not only a kind of high quality turf-grass but also a kind of nourishing cool-season gramineous herbage. It possesses the characteristics of drought resistant, barrenness enduring, disease resistant and broad adaptability. However, with prolonging of planting age and expansion of planting area, many disadvantages sush as coarse vane, growth slowness in summer, weed invasion easily and no creeping stem ect all need to be improved urgently by modern biotechnology means.In this work, tissue culture and plantlet regeneration system of tall fescue was established with MS medium (Murashige & Skoog 1962) as basal medium and mature seeds were used as explants. Effect of different concentration of 2,4-D on callus induction time, callus induction frequency and formation of embryonic calluses were analyzed. It has shown that 9.0mg/L 2,4-D was optimal for callus induction. On MS medium supplemented with 9.0mg/L 2,4-D, frequency of callus induction reached 68.63% and frequency of embryonic callus formation was 9.80%. Embryonic calluses could regenerate green plants if they were transferred onto MS medium containing KT (kinetin). The frequency of plantlet regeneration was changeable with different concentration of KT. 0.2mg/L KT was optimal to plantlet regeneration of tall fescue, and the frequency of regeneration reached45.83%. Effect of agar concentration on maintenance of embryonic calluses were also analyzed. In comparison with 0.8% agar concentration, the frequency of embryonic callus maintenance increased 16.67% when 1.0% agar was supplemented in MS medium.Embryonic calluses of tall fescue were used as target materials of transformation. After precultured for 5h on osmotic medium, rice chitinase gene was transferred into embryonic calluses by microprojectile bombardment. Two green plants were finally regenerated from 149 embryonic calluses bombarded with plasmid pYAO24 containing rice chitinase gene (RC24) and selective marker gene (nptâ…¡) on medium supplemented with 50mg/L kanamycin. Five green plants were finally regenerated from 123 embryonic calluses bombarded with plasmid pARN6 containing rice chitinase gene and pD containing reporter gene (gus) and selective marker gene (bar) on the medium supplemented with 2.0mg/L PPT. These seven green plants were verified with PCR method. Among 7 green plants, only A2 green plant was identified to be transgenic plant integrating the target genes, other 6 plants were escapes.Effects of different quantities of microprojectile and times of bombardment on transient expression of gus gene were analyzed by GUS staining of some embryonic calluses bombarded with plasmid pARN6 and pD. It had shown level of transient expression of gus gene would enhance with heightening of quantities of microprojectile and times of bombardment.A 2.75Kb fragment carrying the chitinase gene from rice, Actinl promoter and NOS terminator was isolated by Kpn I digestion from plasmid pYAO24. It was inserted into the multi-cloning sites in the expression vector pCAMBIA1301, yielded a new expression vector pCAMBIA1301-RC24 harbouring chitinase gene. This expression vector was transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and gus expression were identified in tobacco leaf segments after infection.
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