The Transformation Of Cold Response Gene CBF1into Petunia(Petunia Hybrida Vilm.)and Pansy(Viola Tricolor L.)

Low temperature is one of the most adverse environmental factors that affects plant growth and development. When exposing to low temperature, the membrane of plants could percept the low-temperature signal, therefore the signaling networks which mainly composed of CBF signaling pathways are activated, and the expression of corresponding downstream functional proteins are further induced, which could endow plants with cold resistance. However, CBFs could also promote growth retardation by inhibiting GA synthesis. Accordingly, the accurate regulation of CBFs expression is very important. Whereas the RD29A promoter, with both DRE and ABRE elements, is an optimal stress-inducible promoter activated by dehydration, high salinity and low temperature. Therefore, in order to develop new floricultures with improved resistance to low temperature, we transformed the Arabidopsis RD29A-CBF1in eukaryotic expression vector pBI121into petunia(Petunia hybrida Vilm.) and pansy (Viola tricolor L.) using the Agrobacterium tumefaciens-mediated method in the present study. The main results are as follows:1. The establishment of genetic transformation system of petunia. We have successfully established the genetic transformation system of petunia by inducing callus, adventitious shoots and roots with leaves as explant. To petunia, the optimal regeneration medium was MS+0.5mg/L6-BA, with a regeneration rate of75%. The optimal root culture was MS and the regeneration rate was100%.2. The establishment of genetic transformation system of pansy. The terminal bud and petiole of pansy were chosen as explants to construct its regeneration systems. The optimal callus induction medium was1/2MS+0.2mg/L NAA+1.5mg/L2,4-D+3mg/L GA3with a regeneration rate of88.6%. The optimal differential medium was MS+2.0mg/L6-BA+0.5mg/L NAA, which successfully induced the embryoid of pansy. The optimal medium induced root was MS.3. The optimum selective pressure of Kan in petunia and the concentration of Cef in the suppression of LBA4404were determined. The optimum selective pressure of Kan was25mg/L to callus and5mg/L to roots. The optimum concentration of Cef in the suppression of LBA4404was determined at200mg/L, which could minimize the negative affection. 4. After the double selection of Kan in shoot germination and root regeneration, five pB1121-RD29A-L-DREB1B transplanted petunia and two pB1121-RD29A-L-DREB1B transplanted pansy were survived. The DNA of each transplants was extracted using CTAB method. Three pB1121-RD29A-L-DREB1B transplants were considered to be positive after PCR confirmation.5. The phenotypes of transplanted petunia were carefully observed and it was found that they were obviously increased in leaf size, decreased in leaf density and delayed in flowering compared to that of control. Cold treatment experiments were carried out:(1) The cold-pretreated wild and transplanted plants were treated for12h at-6℃and restored for3days at24-26℃, as a result, the wild plants died while the transplanted L4were still alive;(2) Non-cold-pretreated wild and transplanted plants were exposed to0℃for12h and restoration at24-26℃for6days, and it was found that both transplanted L4and wild plants died after cold treament and had not recovered;(3) After drought treatment, the transplants were still alive while the wild plants died. The results of RT-PCR identification confirmed that the RD29A gene conferred drought resistance of the transplants.From the results above we could see that the transformation of CBF1could change the phenotypes and resistance of petunia. The cold resistance of the transplants were enhanced by CBFs expression while the drought tolerance of transplants were enhanced by the RD29A gene.