菊花再生体系的建立及用PPMADS5基因进行遗传转化的研究

【作者】 王蔚林；

【导师】 吴忠义；

【作者基本信息】 首都师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 本研究以传统名贵菊花(Dendranthema morifolium Ramat．)品种“碧玉钩盘”和“绿松针”为试验材料，研究了不同激素浓度配比对菊花不定芽直接再生诱导和根诱导的影响。优化了菊花再生条件，获得了菊花叶片和花蕾的高效再生体系。试验结果表明，以“碧玉钩盘”的无菌苗叶盘为外植体，在芽诱导培养基上直接诱导植株再生，筛选出了能直接诱导芽再生的培养基(MS+NAA0.1m/L+6-BA 0.5mg/L)，平均叶盘再生率达到35.4％，平均芽点诱导率达到77.1％。再生的不定芽在生根培养基(1/2MS+0.1mg/L)上诱导生根，生根率为100％，生根的小苗长势长良好。以“绿松针”的花蕾为外植体，在芽诱导培养基上直接诱导植株再生，筛选出了能直接诱导芽再生的培养基(MS+NAA1.0mg/L+6-BA 2.0mg/L)，平均花蕾再生率达到100％，增值系数达到7.9。再生的不定芽在生根培养基1/2MS+NAA 0.1mg/L上诱导生根，生根率为100％，生根的小苗长势良好。在建立了菊花品种“碧玉钩盘”再生体系的基础上，利用农杆菌介导法进行遗传转化的研究。试验结果表明，叶盘预培养、农杆菌菌液处理、共培养时间均对菊花再生率有较大影响。把农杆菌侵染过的叶盘转入MS+NAA0.1mg/L+6-BA0.5mg/L+Km20mg/L培养基上进行分化筛选培养，未得到再生苗。将筛选压Km降到10mg/L，获得再生苗6棵，再生率为3.7％。但PCR扩增表明，没有检测出有目的基因的存在，说明目的基因尚未插入到菊花的基因组中。在建立了菊花品种“碧玉钩盘”再生体系的基础上，利用基因枪转化法进行遗传转化研究。叶盘预培养1d后，进行基因枪轰击转化。转化后经过Km 20mg/L抗性筛选培养30d，获得再生苗13棵，取其中长势旺盛的4棵再生苗进行PCR扩增检测。结果表明，在3个转基因植株中检测出有目的基因存在，初步证明目的基因插入到菊花的基因组中。更多还原

【Abstract】 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.更多还原