Establishment Of Chrysanthemum Regeneration System And Genetic Transformation With Ppmads5 Gene

Materials used in this study are cultivars of two kinds of Chinese traditional famous chrysanthemum, "Biyugoupan" and "Lvsongzhen". The result showed that several plant growth regulators affected shoot and root inducement of chrysanthemum. The regeneration system of leaf discs from aseptic seedling of "Biyugoupan" has been established. Adventitious shoots were directly induced and regenerated from leaf discs of chrysanthemum on the medium (MS+NAA 0.1mg/l +6-BA 0.5mg/l). The mean regeneration rate of leaf discs reached 35.4%, while the mean shoots induction rate was 77.1%. All of the shoots were rooted on half-strength MS medium with NAA (l/2MS+NAA0.1mg/l). The rooted plantlets grew flourishing. Meanwhile, the regeneration system of buds from "Lvsongzhen" growing in the fields has been established. Adventitious shoots were directly induced and regenerated from buds of chrysanthemum on the medium (MS+NAA 1.0mg/l+6-BA 2.0mg/l). The mean regeneration rate of leaf discs was 100%, and the propagation coefficient reached 7.9. All of the shoots were rooted on half-strength MS medium with NAA (1/2MS+NAA 0.1mg/l). The rooted plantlets also grew flourishing.Based on the regeneration system of "Biyugoupan", agrobacterium-mediated transformation PPMADS5 gene into chrysanthemum was studied. Our results showed that the regeneration rate was affected by several factors, such as the time of pre-culture, treatment of agrobacterium, and the time of co-cultivation, etc. After infection by agrobacterium, leaf discs were transferred onto the selection medium (MS+ NAA0.1mg/L+6-BA0.5mg/L+Km 20mg/L) to regenerate transformants, but no resistant shoot was obtained. When the kanamycin concentration decreased to 10mg/L, then 6 regeneration shoots were obtained, and the regeneration rate of leaf discs was 3.7%. However, integration of target gene into chrysanthemum was not confirmed by PCR analysis in these seedlings. The transgenetic plants were not obtained through agrobacterium-mediated transformation. So we try the method of particle bombardment to introduce the PPMADS5 gene into chrysanthemum. After pre-culturefor 1 day, the leaf discs were bombarded with tungsten micro particles coated with the DNA of plasmid harboring PPMADS5gene using the Bio-Rad PDS-100/He biolistic device. After 30 days screening on the selection medium (MS+ NAA0.1mg/L+6-BA0.5mg/L+Km 20mg/L), 13 resistant shoots were obtained. Then, four putative transformants which grew flourishing were monitored by PCR analysis, The expected fragment was amplified from total DNA of all the four transformed lines except one line, which was showed that the PPMADS5 gene has been integrated into the genome of these lines.