Studies On Abiotic Stresses In Rosa Chinensis By Transforming Polymerization Of MtDREB1C And RcXET

Rose, as one of the three most popular cut-flowers in the world, has been widely cultivated in china for cut flower, pot and bedding plants. However, the yield and extension of Rosa are severely influenced because of the abiotic environmental stresses, such as drought, salinity, low and high temperature. What's more, many modern rose varieties have lost their excellent quality of fragrance and comprehensive resistance after a long time of cross breeding. Therefore, new rose varieties with nice qualities are urgently demanded to meet various markets. The successful application of transgenic technology in other important crops in 1990s has undoubtedly provided a new way for rose breeding. The low transformation rate has long been a major obstacle to promote the study of gene function in rose. Thus the establishment of a comprehensive system of regeneration and genetic transformation is critical for gene function research in rose.The China Rose (Rosa chinensis Jacq.) has been chosen as the experiment material. The efficiency of somatic embryogenesis induction and plant regeneration are greatly improved by improving the light conditions and the composition of culture medium. Moreover, the stress tolerance of transgenic rose has been advanced through construction and transformation of an aggregation expression vector of MtDREB1C and RcXET genes. In addition, the problem of hampered growth because of the expression of stress resistance gene in transgenic seedlings has been overcome as well. The major research results of this paper were list as follows.1. The micro-propagation system of China Rosa is optimized. The sterile leaves and young stems are used to induce callus. The suitable callus induction medium for leaves is SH basin medium with 30 g·L-1 sugar,3.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ. Callus induction efficiency can reach 100% when 2 mg·L-1 or 3 mg·L-1 2,4-D is added singly in SH basin medium in dark culture condition. The optimal callus induction medium for young stems is SH basin medium with 3.0 mg·L-1 2,4-D, the induction rate could reach 83.21%. The optimal medium for adventitious buds differentiation si MS basin medium with 1.0 mg·L-1 TDZ and 1.0 mg·L-1 6-BA. With this approach, adventitious buds can directly be induced through callus with a differentiate rate of 5%.2. The effect of red light and plant growth regulators on somatic embryogenesis in R.chinensis is researched. Embryogenic calli that had been induced by 2,4-D and TDZ in darkness are exposed to dark, red and white light treatments. Cultures subjected to red light treatment generated the greatest number of embryos, with one (SE1 embryos) or two (SE2 embryos) expanded cotyledons. The largest numbers of shoot-like embryos without cotyledons (SEO embryos) are produced in cultures subjected to dark treatment. The effects of different concentrations of abscisic acid (ABA) on the proliferation and germination of different types of somatic embryos are also evaluated. A concentration of 2.5 mg-L-1 is found to be the most effective in promoting the proliferation and germination of SE2 embryos. The higher the concentration of ABA (from 0 to 5.0 mg-L-1), the higher the percentage of abnormal polycotyledonary embryos produced. The highest percentage of regenerated plants is obtained from SE2 embryos, the rate can reach 41.8%. Therefore, TDZ and 2,4-D affect on the induction of somatic embryogenesis, red light affect leaf morphological of somatic embryo formation and ABA has an impact on the leaf number of somatic embryos.3. The expression vector of gene aggregation is constructed by PCR method with suitable primers. MtDREB1C and RcXET gene pyramid expression vector is constructed. The construction method of the double gene pyramidi expression vector is developed which is not dependent on enzyme digestion and connection. This approach greatly improves the efficiency of gene transfer and connection.4. An agrobacterium mediated transformation protocol is established for R.chinensis using callus as inoeulation material. Ten regenerated plants are obtained from those calli, and five seedlings are detected as positive transformed plants by Southern blotting and PCR. MtDREB1C and RcXET gene pyramid expression can simultaneously enhance \ the resistance and growth of rose transgenic seedling. It can overcome the problem of plant dwarf caused by the excessive expression of MtDREB1C.