Establishment Of Genetic Transformation System And Relative Technique For Disease Resistance Of Cymbidium Hybridum

The major objective of this study was to breed new Cymbidium hybridum varieties with improved resistant performance against various pathogens through transformation of the GAFP+NP-1 bivalent genes by using Agrobacterium-mediate transformation technology. For this purpose, A high frequency regeneration system of Cymbidium hybridum was established successfully, and factors that affecting the frequency of Agrobacterium-mediate transformation for Cymbidium hybridum was tested, and the transgenic plants were obtained and authenticated by a series of relevant molecular biology analysis and disease-resistance identification.1,PLB(Protocorm-like body) regeneration system of Cymbidium hybridum was established successfully, which used both segments of leaf and pieces of cuted plantlet base as explants. Using the MS medium as the basic medium, PLB is successfully induced from the leaf-segments of vitro-seedlings of Cymbidium hybridum. PLB can be induced only from the base of leaf in tests.2,Results showed that: untransformed PLBs were inhibited very sensitively by Km. When the concentration of Km was 7.5 mg/L, continuative inducement of PLB from cuted PLB of CAH and PET genotype was fully inhibited, and continuative inducement of PLB on CMM and CWI genotype was fully restrained at Km leval of 12.5mg/L; Cef has the most outstanding restrain efficiency on the engineering bacteria, while the disadvantageous influence of Cef on PLB is not evidence.3,In this study of genetical transformation for C. hybridum, with Agrobacterium tumefaciens strain LBA4404 carrying binary vector pBin35S-GAFP-NP1, a parameter-pool strategy was introduced to study on factors influencing efficiency of the transformation in two research steps. The data from first-hand tests show that: The efficiency of genetical transformation was advanced remarkably when AS was used in pre-culturing media, and optimum value of AS concentration was 100μmol/L; When agrobacterium inoculum liquor was at 0.2 density of OD600, while pH value of the inoculum liquor and co-culturing media was seted at 5.2, the Km-resistance positive rates was highest. At optimum level of AS and pH and inoculum density, the data from secend experiments show that: the optimum pre-culture time was 4 days; the optimum infect time was 20 minutes; and the optimum co-culture time was 6~9 days.4,By using improved method of SDS, genomic DNA from leafs of Cymbidium hybridum was extracted, and could be digested completely by restriction endonucleases and tested with random primer PCR. By using SDS or Guanidinum isothiocyanate, RNA extraction was tested respectively.The transformed lines which were positive for Km-resistance were identified with PCR screening and Southern blot. RT-PCR resault showed that the foreign genes cuold been transcripted to mRNA in cells of C. hybridum.5,By using extracted protain liquor of genetically transformed C. hybridum on media inoculated with the pathogy, and infecting in-vitro leafs of C. hybridum with pathogen inoculating, the pathogy-resistace ability of genetically transformed C. hybridum was validated affirmatively.