Study On The Establishment Of Regeneration Of Chrysanthemum And The Preliminary Functional Analysis Of FLC

Chrysanthemum(Chrysanthemum morifolium Ramat.)originates from China,which is an important potting,cutting and ground cover flower in the world.With a long cultivation history,chrysanthemum takes a key role in Chinese traditional culture and flower industry.Most of the chrysanthemum are short-day cultivars and come into bloom in autumn.Nevertheless,there are some day-netural cultivals which come into bloom in summer.Day-netural chrysanthemum are less sensitive to photoperiod,their flower bud's differentiation mainly rely on vernalization.It was reported that the blooming of summer chrysanthemum was related to low remperature inducing,however,the mechanism and critical genes in vernalization were not disclosed.According to studies on model plant(Arabidopsis thaliana)and other species,this study focused on the responding mechanism to vernalization in summer chrysanthemum,cloning and studying the function of FLC(FLOWERING LOCUS C)which was a vital gene in vernalization.The main results were as follows:1.Taking fourteen summer chrysanthemum cultivals as materials for esttablishment of regeneration system.Cutting their leaves from aseptic plants,respectively,various combinations of plant growth regulators(6-BA,NAA)were used to induce callus and adventitious buds to establish the regeneration system.The highest regeneration rate and shortest growth cycle which cultival and regeneration system was compared,which would be used in genetic transformation of summer chrysanthemum.The results showed that the best transgenic receptor was 'Yuuka',which had the highest callus rate,regeneration rate,regeneration buds and lowest browning rate.Taking 'Yuuka' as explants,the optimal regeneration medium was MS+6-BA 2.0 mg·L-1+NAA 0.5 mg·L-1.2.The full-length cDNA sequence of chrysanthemum 'Yuuka' CmFLC-likel was obtained by cloning.The cDNA was 945 bp in length with a 636 bp open reading frame which encodes a peptide of 211 residues.CmFLC-likel has a high homology to VvFLC(Vitis vinifera)and DlFLC(Dimocarpus longan)contained a typical MADS domain,with the homology of 55%and 52%,respectively.CmFLC-likel was located in the nuclear genome by subcellular localization.And transcription activation activity was not found in yeast system,while inhibitory activity was shown in the protoplast transformation system.CmFLC-likel has the highest expression levels in leaves,while lower expression in stems and shoot tip,followed by root.The expression of CmFLC-likel was inhibited by low temperature,especially at 4?.Furthermore,the longer the cold treatment,the more suppresser of CmFLC-likel expression.CmFLC-likel was transformed into Arabidopsis thaliana for further functional research.3.Transformation of AtFLC into chrysanthemum.The pMDC43-AtFLC vector were constructed,through agrobacterium-mediated transformation,3 transgenetic chrysanthemum plantlets were obtained respectively.