| Kentucky bluegrass (Poa pratensis L.) is a typical cool-season perennial grass, commonly used for forage and turf in temperate zone. In northern China, it is widely maintained on lawns, parks, public grounds and so on, for the characters of beautiful colour, high shoot density, cool tolerence and better shear-bear. But it is sensitive to drought and heat stress, and is easily aggrieved from diseases and insects. These defects influenced Kentucky bluegrass to popularize in Torrid Zone. Due to its facultative apomictic nature of reproduction, genetic improvement by conventional breeding methods has been difficult; genetic engineering manipulation may be used as a method in the improvement of its heat tolerance and other qualities. The published Kentucky bluegrass transformation system has many disadvantages including the diffculty of regeneration, high frequency of somaclonal, long-time transformation period and low efficiency of transformation.In this research, an in vitro proliferation system of mutiple bud clumps was esablished first. Shoot apices from sterilized seedlings were cultured on MS medium supplemented with 0.5mg/L 2,4-D and 3.0mg/L 6-BA for base enlargement, then the enlarged basements were transferred to MS medium supplemented with 2.0mg/L 6-BA for bud clumps formation, and multiple bud clumps were obtained on poliferation medium containing 0.07mg/L 2,4-D and 3.0mg/L 6-BA.hsp101 gene from maize encoding heat shock protein 101 was introduced into naked bud tips of two cultivars of Kentucky bluegrass, Nassau and Mardona, mediated by Agrobacterium tumefaciens LBA4404. Apical meristems were exposed by cutting out leaves from multiple bud clumps subcultured for 5-7 days for the infection at 0.5SMPa pressure for S minutes and co-cultured for another 3 days. Then the bud clumps were transferred to medium supplement with 100mg/L cefotaxime to inhibit the growth of Agrobacterium for 20 days, and further 30 days subculture on... |
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