| Zoysiagrass shows less ornamental value in north China because of its sensitivity to low temperature and short green period, which is possible to be improved by genetic transformation with cold responsive genes. In present study, we analyze the senescence and biochemical changes of zoysiagrass under the stress of low temperature either in the open field or in the controlled conditions; CBF1, a transcriptional factor induces cold-regulated genes expression in Arabidopsis, was delivered into tobacco to investigate the effects of CBF1 on cold tolerance and growth and development of cold sensitive plants; a plant regeneration system of Zoysia sinica Hance was established and used to investigat factors that may influence the Agrobacterium-mdiated transformation. The main results are follows.1) Cold sensitivity of zoysiagrass and biochemical changes induced by low temperature stress.The senescence of zoysiagrass was induced at chilling temperature lower than 15℃and was accelerated at temperature of 12/8℃(Light/dark), showing by lower water content, the accelerated decomposing of chlorophyll and soluble proteins, and the enhanced peroxidation of membrane lipids. Biochemical changes were induced by temperature of 12/8℃in leaf, including the accumulation of proline and water soluble sugars, levels of which were correlated with tolerance to low temperature. Both catabolism and biological adjustments were inhibited greatly at temperature lower than 5℃. Stolon of zoysiagrass was induced to accumulate higher level of proline, express specific peptides and accumulate soluble proteins without enhanced peroxidation of membrane lipids under low temperature stress.2) Cold tolerance and growth performance of transgenic tobacco plant expressing CBF1.Overexpression of Arabidopsis CBF1 in tobacco plants dose-dependently promoted surviving low temperature by increasing photochemical efficiency (Fv/Fm), soluble sugar content, and proline content and decreasing malondiadehyde (MDA) content under low temperature stress. Transgenic plants had shorter stature, thicker stems, thicker and glossier leaves, higher chlorophyll content, weaker apical dominance, and longer time to flowering than control tobacco plants.3) Plant regeneration system of Zoysia sinica HanceSeeds of zoysiagrass are usually in deep dormancy which can be broken up by churning up in solution of 30%(m/v) NaOH for 60min followed by sterilization with NaClO. The suitable culture for callus induction from mature embryo was modified MS ( 1mg/L VB1, 1/2×NH4NO3 ) supplemented with 20g/L glucose, 1mg/L VB2, 2,4-D 2.0mg/L, and 6-BA 0.1 mg/L. Callus of zoysiagrass varies greatly and could be divided into four types. Type I, yellowish and compact, could regenerate plant at a rate of 45-50%. Type II, milk white or white in color and soft, could regenerate plant at a rate of less than 10%. Type III, white in color, soft and watery, could not regenerate plants. Type IV, yellowish or yellow green in color, very compact with developed shoots on the surface, could regenerate plants at a rate of 80%. Callus type I losses regeneration ability after subculture twice because of its transition into other callus types.4) Factors influence genetic transformation of Z.sinica mediated by Agrobactrium tumefaciensA.tumefaciences strain EHA105 show high infection efficiency to Z.sinica callus. The profitable conditions for infection include bacterium suspension of OD600≈0.4, 4 days of co-culture time, 60 Kpa vacuum, and 100mg/L betain in the infection environment. But a successful A.t-mediated transformation system for Z.sinica is remained to be established. Results obtained in the present studies should provid useful references for accessment of cold tolerance and breeding by genetic transformation mediated by A.tumefaciens for zoysiagrass...
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