Arabidopsis transformation.ppt

拟南芥实验操作Arabidopsis Laboratory Operation 植物生物工程研究室 王冬冬 2006.7.13 拟南芥（Arabidopsis thaliana ） 生长周期短 体积小，占地少 核基因组小 自交植物，遗传稳定性高 易产生突变体 农杆菌直接转化 功能基因保守 Arabidopsis Laboratory Operation How to Grow Arabidopsis Mutants (Obtaining ;Genetic, Phenotypically Analysis) How to Transform Arabidopsis How to Study Gene Expression and Gene Function Where to Find Information on Arabidopsis How to Grow Arabidopsis Seed Collection and Storage Cultivation of Arabidopsis General Consideration Growth temperature Ideal temperature 16~25 ℃ . If adequate water is available , the survive temperature up to 34 ℃ . 22~23 ℃ is optimal for rapid , robust doevlopment . Light The optiumum light intensity is approximately 120~150 μmol / m2sec . 1 μmol / m2sec = 0.217W /m2 = 53.8 lux Photoperiod 8~24 hours . Long days （12 hr）accelerate the reproduction cycle . Short days （12 hr）favor growth of vegetative tissues . Seed germination It is necessary to maintain high humidity (with a plastic dome) until the seedlings are 1 week old . Seeds dormancy can be overcome by imbibing seeds at 4℃ 2~4 days . Humidity The range for humidity must be 25~75% . Sterility can result form very high humidity (90% ) . Cultivation of plants in soil 草炭土:蛭石=1:1 , 1:2 , 2:1 (营养土:蛭石:草炭=3:1:1) Pack the soil into pots , filling them to approximately ? inch(1cm) from the top . 4-inch square pots can accommodate 4~8 plants . Suspending the seeds in sterile water or 0.1% agar and pipetting the directly onto soil . When seed stocks are limited , individual seeds can be placeed onto soil using a fine forceps . Watering 拟南芥遗传转化 In Planta Transformation of Arabidopsis Plant Growth 种子置于1.5mlEP管中，4℃春化3~4天； 种植土按营养土:蛭石:草炭=3:1:1混匀，分装至营养钵中； 用牙签点播种子至土壤上层，每钵5~7处； 覆膜保湿3~4天，于70%湿度培养箱中，22±1 ℃恒温 9~12小时光照培养（花序长出后16～24小时光照培养）。 遗传转化植株 Agrobacterium-mediated transformation of Arabidopsis Floral dip Vacuum infiltration Procedure Culture of Agrobacterium tumefac