Agrobacterium-Mediated Transformation Of Chrysanthemum×Morifolium With DFL, A LEAFY Homologue

Chrysanthemum is important flower of the world ornamental plants,and also is traditional famous flower in China.Exogenous genes are hard to be introduced into the chrysanthemum varieties cause of the restriction of species genes background with traditional breeding method.Transgenic breeding method for chrysanthemum can avoid this disadvantage and has highly economic benefit and social benefit.The progress of flowering transition will be accelerated by the heterologous expression of flowering time of flower's meristematic characteristic gene LEAFY.Thus,it's significant to change flowering time of chrysanthemum with introducing LFY by genetic engineering technologies..DFL,a LEAFY homologue had been cloned from Dendranthema lavandulifolium by our team in the prophase work.This paper used genetic transformation of chrysanthemum,with DFL gene mediated by Agrobacterium.Main research results as follows:1.The sense and anti-sense expression vectors with DFL gene had been built,pBI121 was selected as plant expression vector.DFL replaced GUS by double digested with restriction enzymes Bam HI and SacI.Then identified by PCR and double digestion,the result showed the reconstruct vectors pBI121-CaMV35S-Sense DFL and pBI121-CaMV35S-Antisense ADFL were successful built.Two reconstruct vectors were transformed into Agrobacterium tumefaciens C58C1 by freeze-thaw method.2.The highly efficiency and stable tissue culture regeneration system of chrysanthemum were built.This paper selected the traditional chrysanthemum 'Xiao Linjing','Zong Hongzhen' and 'Xiao Fenju'as the experimental materials Used 70%alcohol and the chlorine 1%of NaClO solution soaking stems explants.Through the experiment 'Xiao Linjing' and 'Zong Hongzhen' were selected for building vitro tube reproduction system..Though the three factors and two levels experiment of 6-BA and NAA, This paper did adventitious bud differentiation experiment with 'Xiao Linjing' and 'Zong Hongzhen'.'Xiao Linjing' was selected as a traditional chrysanthemum breed with highly frequency and highly efficiency regeneration system.Used the leaf as explant,put them on the differentiation medium,(MS+6-BA2.0 mg/L+NAA1.5 mg/L) The differentiation rate was 92.76%and the average number of shoots on each explant was 2.3767±0.76.Rooting medium was 1/2 MS solid medium.The rooting rate was 100%.The medium in the transplant was vermiculite with high temperature disinfection,the young plants was cultivated in the plug,the survivial rate of the plantlets can rise to ninety percent.3.Choose kanamycin as selection pressure,Benzyl carboxy as bacteriostatic antibiotic penicillin.The kan concentration of leaf explant selection differentiation was of 10mg/L,the rooting selection differentiation was 20 mg/L.Mediated by agrobacterium tumefaciens on 'xiaolinjing' genetic transformation experiment.The OD600 range was from 0.5 to 0.6.Infection time was 10 mins. Co-culture was dark culture about two days at 28℃..After this step put the leaf explant under the differentiation of antibiotic selection medium that contain kan10 mg/L and Carb 400 mg/L.When resistant shoots were formed,put them on the antibiotic selection medium for shooting,the medium contained kan10 mg/L and Carb 200～300 mg/L.The chrysanthemum 'xiaolinjing' genetic transformation system.was finally established.4.Mediated by agrobacterium tumefaciens on 'Xiao Linjing','Mohe' and 'Riqietaohong' experiment.Totally infected 1373 leaf explants,and got 64 resistant Kan shoots.Though many times screening antibiotic selection medium for shooting,finally obtained 20 resistant rooting shoots.The PCR results indicated that DFL gene had been successfully introduced and integrated into the genome.Two transformation seedlings were obtained,One line called df-1PD was 'Moju' that transformed sense expression vector,the otherline called df-3PAD was 'Riqietaohong' that transformed anti-sense expression vector.