| The Dendranthema morifolium ’Bozhou’ mainly in Bozhou City, Anhui Province,which was authentic Chinese herbal medicine of Anhui, and was also one of China’s four famous chrysanthemums. Biologically active substances contained in its flowers, such as volatile oils, flavonoids and other ingredients could dispelling wind and heat cured, improve eyesight and detoxication. Indications hypertension, coronary heart disease and so on. It was also commonly used to add into beverage as one of the major flavorings,and used along with medicinal herbs and beverages.Its had high economic value.Its always been considered the best quality of chrysanthemums face tremendous risk due to the serious degeneration, vulnerable, weak resistance and other reasons,and the planting area was gradually reduced. According to statistics, drought caused cut production far more than the sum of other abiotic factors. Therefore, improving drought tolerance level in ’Bozhou’ was one of the key technologies, but the chrysanthemum was highly heterozygous, conventional hybridization breeding technology was difficult to be applied.In this experiment, Dendranthema morifolium ’Bozhou’ with stem explantswas usedas receptor material,resistanted to glyphosate herbicide as the filter gene and transfered the drought-resistant gene(EDT1) into ’Bozhou’ by using Agrobacterium Rhizogenes.The results were as follows:1 Works vector plasmid was introduced into Agrobacterium rhizogenes A4 by using transformed heat shock method,the results of Engineering bacteria growth curve showed that logarithmic growth phase at OD600=06.2 The selection results that the best of selection concentration of glyphosate for Dendranthema morifolium ’Bozhou’ tissue culture was 100 mg/L.3 Four factors influence efficiency of gene transformation for ’Bozhou’ were studied,Included the time of infection, the concentration of Agrobacterium bacterial andacetosyringone, the time of co-culture.And the best selected concentration of glyphosate.The concentration of Agrobacterium (A600) is 0.6-0.8,included AS 150 μmol/L,25 min of infection and co-culture three days,after 15 days’s sterilization culture,we get the maximum conversion efficiency 95.6%.4 Clipped the hair roots about 2 cm put into the medium of callus induction, we could get lots of callus by subculturing once every two weeks.5 The PCR technology initially proved that transgenic plants contain 800 bp aimed fragment in the 5 positive transgenic plants though the selection medium which add into 100 mg/L glyphosate, extracted the genomic DNA of plant leaf by using the method of CATB.6 Under drought stress, extracted and measured the chrysanthemum leaves’ superoxide dismutase (SOD), malondialdehyde (MDA), peroxidase (POD), catalase (CAT) contents, respectively. The results showed that the transgenic plants relative to non-transgenic plants with high drought tolerance. |
