| With rich colors and varied flower types,clematis has a broad market prospect at home and abroad and is favored by many horticulturists.However,it still faces many problems in the breeding.Based on the collected germplasm resources of clematis at home and abroad,the heat resistance of eight clematis varieties was evaluated based on the observed variation rules of chlorophyll fluorescence parameters.Some heat-resistant cultivars were selected as hybridization parents to observe the traits of their offspring.Meanwhile,embryo saving and seed dormancy breaking of clematis were also studied in order to solve the problem of embryo abortion at high temperature and long germination time of hybrid offspring.In addition,the in vitro regeneration system and genetic transformation of clematis ‘wang meng' were studied,which laid a foundation for the transgenic research of clematis.1.Variation rules of chlorophyll fluorescence parameters of clematis under high temperature stress were analyzed.The results showed that Fo in the Florida group had an upward trend under high temperature stress,but the clematis in the early-flowering and large-flowering group decreased as a whole,with an average decrease of 0.035 among the three tested varieties.Fo in the wild species group ' clematis wuxing 'and Texasgroup clematis ‘chrisba angel' were more stable.Fm decreased under high temperature stress in all varieties,and recovered to the initial level after 5 d was given to most varieties after high temperature stress.High temperature stress significantly reduced the potential photochemical efficiency of Florida group and "clematis wuxing",increased the early flowering group and the large flowering group,and the "crisba angel" in Texas group was relatively stable without significant fluctuations.The maximum photosynthetic efficiency and actual photosynthetic efficiency of clematis in the Florida group and 'wuxing clematis' all decreased,while the maximum photosynthetic efficiency of clematis in the early-flowering group increased slightly and the actual photosynthetic efficiency increased significantly.In terms of Y(NPQ)and Y(NO),varieties Y(NPQ)increased and Y(NO)decreased in Florida group under high temperature stress,while clematis Y(NPQ)decreased and Y(NO)did not change significantly in early flowering and large flowering group.In this experiment,there are significant differences in chlorophyll fluorescence parameters under high temperature stress in different species of clematis,which may be due to their different photosynthetic efficiency and adaptability to high temperature.Through the analysis of chlorophyll fluorescence parameters of clematis,it is concluded that the heat resistance of clematis from Texas group is similar to that of early flowering group.The heat resistance ability from high to low is: Texasgroup> wuxing clematis> Florida group.2.Through two years of hybridization,four hybrids plants with good ornamental traits were selected.At the same time,we found that clematis gardening kind of hybrid seed setting rate,germination rate was higher than in the group across a set of hybridization,Texas group with other groups of clematis hybrids ciuld have the effect of improved pattern,and clematis double petal gene might be dominant,single petal gene was recessive.The future breeding direction of clematis is cross-breeding between Texas group and Florida group with excellent disease resistance and flowering ability.3.The embryo saving experiment of Texas clematis group showed that the development and germination rates reached to 62% after the seeds were sterilized in 0.1% Hg for 12 minutes after inoculation with 0.5mg/L6-BA +0.05 mg/LNAA+10% coconut milk embryo salvage medium.The germination rate of seeds treated with 50 mg/LGA reached 77%,and that of seeds treated with 30 mg/L6-BA was 82%,and the germination time was 38 d shorter than that of the control group.4.Through different orthogonal experiments,MS culture medium was selected as the best culture medium for robust seedlings of clematis' wang meng 'from the Texas group,and the optimal culture medium for callus induction of stem segments was 0.5mg/LTDZ+0.3mg/LNAA,with an induction rate of 100% and good callus growth status.The optimalmedium for callus differentiation in the stem segment was 1/2ms +2 mg/L6-BA +0.05 mg/LNAA,with a differentiation rate of 46.6%.The optimal rooting medium was vermiculite + porous tissue culture bottle +1/2ms +0.1 mg/LNAA,with a rooting rate of 53.3% growing in natural temperature.5.Genetic transformation system of clematis.After 2 d's preculture procedure,the sterile stem segments of "wang mengs" of the Texas group were infected with agrobacterium sp.GV3101(OD600=0.5)with pBI121 expression vector for 15 min,and cultured in the co-culture medium of 0.5mg/LTDZ+0.3 mg/LNAA+100 mg/LAS for 4 d and then was transferred to the screening medium which was made of MS+0.5 mg/LTDZ+0.3mg/LNAA+300 mg/LCarb+20 mg/L Kan for 5 d.The resistant callus was sub-cultured every 15 d,and the resistant callus was stained with GUS dye after two sub generations.The final induction rate of resistant callus reached 63.3%. |
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