Isolation And Transformation Of DgDREB1A In Cut Chrysanthemum

Cut chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitam) is one of the four main cut flower in the world,have the advantage of abundant colors,various flowertypes,and long vase life et.al.,and it is the highest value flower type in gross output value of international commodity flower. However,lack of water and high production cost seriously limit the scale of production and extent of supply. To solve the problem of enegy-consumable in the production of chrysanthemum in winter,It will be signaity to breed new cultivar that have low-temperature tolerance with modern molecular biology technology and perfect the system of all year production and sdeady supply technology.With the method of PCR, we coloe DgDREB1A gene from chrysanthemum, sequence alignment with reported sequence,the homology reach to 98%, then we built transgene expression vector pBIG-DREB1A and transform it into Agrobacterium LBA4404 and EHA105.Using cut chrysanthemum 'jinba' as the test material,we cultivated sterile culture system, using young leaves of the sterile plant as explant,we optimize the adventitious shoot regeneration system, the leaf explants were cultured 15 days in MS medium supplement with 0.25mg/L 2,4-D,1mg/L BA, then transferred onto development medium with 0.25mg/L NAA,1mg/L BA,after 3 weeks, the regeneration rate reach to 100%.Based on the highly efficient regeneration,we optimized the genetic transformation system,and have got the positive lines,we found that 2 days preculture,OD600=0.05-0.07 engineer agrobacterium,10min.affection time and 2 days co-cultivation were necessary for the thansformation of chrysaanthemum,In our study,after 8 weeks selection under pressure,the resistent shoots were transferred to root medium which contain 7.5mg/L kan.to take the second selection,extract DNA of the rooted lines,PCR result showed that DgDREB1A was detected in 8 lines,the transformation ratio was 1%.In this study, we use the method of genetic transformation mediated by agrobacterium tumefaciens,obtain new cut chrysanthemum lines that have good tolerance to low temperature,lower the cost for production in winter,supply the fundamental material for breeding new chrysanthemum cultivar that have good synthesized tolerance.