Photoperiod Pathway Related Gene AtCO In Genetic Transformation Of Chrysanthemum Morifolium 'Huoyan'

Chrysanthemum morifolium is a new and important group of ground ornamental plant in world.According to the blossom period,it can be divided into summer or autumn and chrysanthemum.In gernal,summer flowering chrysanthemum blooms in the summer and shows less hybrids,smaller flowers in poor ornamental characteristic.Most hybrids of chrysanthemum are short-day flowering plants which bloosm in late autumn.The timing of flowering during the year strongly influences the application scope and commercial value of chrysanthemum.There are some conventional methods developed to regulate the timing of flowering,such as controlling temperature,light period,etc.But these methods are complicated,difficult to control,all of the most is that greatly increase the producing cost.Genetic engineering technology provides a high efficient and low cost approach to gain targeted plant trait.Some studies have shown that plant flower development is affected by many factors,and light cycle is particularly an important factor.CONSTANS(CO)gene in Arabidopsis is one of the critical factors in photoperiod genetic regulation pathway.The main objectives of this study are to obtain new varieties of chrysanthemum,especially for the early flower variety by screening the trangenetic lines introducing AtCO.Leaf explants from in vitro germinated plants of chrysanthemum 'Huoyan' were infected with Agrobacterium tumefaciens.The explants were selected on medium supplemented with a combination of mannose and sucrose as a carbon source at different concentrations.And further identified by PCR,Western blot molecular methods to confirm the positive lines.After surveyed and compared the difference in the flowering date,the earlier flowering varieties were screened to enrich different flowering period of chrysanthemum.This study can provide an efficient and east approach to obtain the different flowering period varieties.The overall results are summarized as follows:1.Construction of expression vector:the whole length of CO gene was amplified by RT-PCR from Arabidopsis thaliana.Based on Gateway technology AtCO was recombined with pCAMBIA1301-PM1 expression vector and transferred into Agrobacterium EHA105.2.Transformation:Leaf explants from in vitro germinated plants of chrysanthemum 'Huoyan' were infected with Agrobacterium tumefaciens EHA105 pCAMBIA1301.The explants were selected on medium supplemented with a combination of mannose and sucrose as a carbon source at different concentrations.Among the 5575 leaf explants were infected, there are 243 shoots surviving after selection by the medium containing 10 g·l1,mannose.PCR analysis confirmed that 17 independent transformed lines were positive for manA and AtCO gene.The efficiency of the transformation is 3.2‰.3.Molecular identification phenotypic observation of transgenic plants:Tested by GUS staining,TAIL PCR,western blot and qPCR determined the 17 transgenic lines were the positive lines.Furthermore,flowering phenotype were observed in 2014 and 2015.The date of bud,beginning flower and the bloosm flower shifted to an earlier date in most varieties.The average days of advanced days were 23.4,6.5 and 11 days in 2014 and 22.2,3 and 1.5 days in 2015,individually.On the whole,there are 88%and 72%transformed lines flowered in advanced.The results indicated that the transformed lines showed the distinct phenotype to flower in advanced.