Ipt Gene Introduced Into Kentucky Bluegrass Conferred Cold-tolerance And Cellular Engineering Lines Exhibited Improved Heat-stress Tolerance

Kentucky bluegrass(Poa pratensis L.)is a typical cool-season perennial grass, commonly used for forage and turf in temperate zone.It is widely maintained on lawns,parks,public grounds and so on,for its characters of beautiful color,high shoot density,and better shear-bear.Nevertheless it is sensitive to drought and heat stress. Due to its facultative apomictic nature of reproduction,genetic improvement through conventional breeding methods was difficult.Technical improvements in plant cellular and genetic engineering,such as genetic transformation,provide a promising approach for breeding new tolerant cultivars of this species.Generation of ipt transgenic Kentucky bluegrass and analysis of its cold toleranceIsopentenyl transferase(IPT)is the key enzyme in the biosynthetic pathway of cytokinins.Plants over-expressed ipt gene display increased cytokinins and delayed senescence.In this research,multiple shoots from sterilized seedling apices of Kentucky bluegrass were induced and proliferated in vitro on MS medium supplemented with 2,4-D and 6-BA.ipt gene on Ti plasmid of Agrobacterium tumefaciens LBA4404 was introduced into naked bud tips of three cultivars of Kentucky bluegrass.PCR assay and Southern analysis of the transformed plants demonstrated the integration of ipt gene into the genome of the transformed plants.The sense transgenic plants displayed evidently higher tillering ability compared to non-transgenic control,while the anti-sense lines showed no obvious differences from control.The detached leaves of sense lines showed better green-keeping ability in several different conditions than control.The green-keeping experiment of detached leaves at 4℃showed that the sense lines had better green-keeping ability than both anti-sense and control lines.The cold-tolerance of transgenic plants was studied by leaving outside under normal conditions in winter,together with control.The phenotype of sense transgenic plants showed improved cold-tolerance and prolonged green-keeping period.Physiological data indicated that transgenic plants and the control had no obvious differences at 10℃/1℃(day/night).But with the decline the temperature,transgenic plants had less membrane damage,higher chlorophyll content compared to the control.After maintained at—8℃for 5 d in chamber,transgenic lines had lighter injury,and the MDA content and the rate of ion leakage were still lower,compared to the control.Moreover,the cold-tolerance varied among these transgenic lines.Part of the sense lines displayed significant improved cold-tolerance, thus may be of wide application prospect in the future.Heat-tolerance analysis of cellular engineering lines of Kentucky bluegrassAfter a heat shock treatment of the multiple bud clumps at 49℃,survival buds which had better phenotype performance were selected,and several heat-tolerance lines were thus obtained.In this study,the heat-tolerance of these lines were studied.The cellular engineering and control lines were transferred into paper cups at 23℃for several days to establish.Then lines with uniform phenotypes were selected for further study.Plants were treated at 46℃for 5h or 40℃for 3d,respectively,and relevant physiological indexes were determined.After the heat shock at 46℃for 5h,cellular engineering lines had lower MDA content and ion leakage than control,indicating the more stability of the membrane of cellular engineering lines.The chlorophyll fluorescence(Fv/Fm photochemical efficiency of PSⅡ)and the content of chlorophyll indicated the worse damage of the photosynthesis system than control,suggesting that the selected lines had enhanced heat-tolerance after heat shock treatment.Of the physiological parameters measured, the photochemical efficiency(Fv/Fm)was much more sensitive to heat stress than the others,implying the possibility of using this for simple and rapid selection of heat-tolerant lines.In order to simulate the nature environment in summer,heat-tolerance of the lines were analyzed at 40℃for 3d.Results showed that damage in this condition was worse than that at 46℃for 5h.The data of Fv/Fm and the survival rate of plants suggested that the lines(except N55)had improved heat-tolerance than control.In this research,genetic improvement of elite varieties of Kentucky bluegrass was conducted via genetic and cellular engineering techniques,and new breeding materials with better cold tolerance and relative longer green-keeping period or improved heat stress tolerance were obtained,which can enlarge the growing area of Kentucky bluegrass and improve the quality of lawns,undoubtly with a wide application potential in the future.