中国水仙花粉原生质体的分离与培养

取水仙花花葶中上部即将开放的花蕾,按常规方法消毒后,把花粉粒挤在含纤素酶0.5%、果胶酶1.5%、甘露醇12%的酶混合液中.保育5h 后离心、纯化,可获得2±0.04×10~5(个/朵)原生质体.花粉原生质体在附加 BA,2,4-D 分别为0.5mg/L,甘露醇5%,蔗糖10%的1/2 MS 或 KM8p 的液体培养基中,培养3d,原生质体开始分裂.每隔10d 换培养液一次,使培养基的渗透压逐渐降低至普通培养浓度.1个月后,形成肉眼可见的愈伤组织,转入以琼脂糖作固化剂的相同培养基中培养2个月后,细胞分裂减弱.愈伤组织的增殖和分化,目前正在进一步探索之中.

Isolation and Culture of Pollen Protoplasts in Narcissus tazetta var.chinensis

Fresh pollen grains,which of Narcissus tazetta var.chinensis Roem.pressed out from the sterile upper and middle alabastra in inflorcscences,were incubated in enzyme mixture consisting of 0.5% cellulase, 1.5% pectinase and 12% mannitol for 5h By substquent filtration and centrifugation,purified protoplasts were obtained with a high yield about 2±0.04×10~5 per alabastras.Then these purified protoplasts were cul- tured in a mordified MS(macroelements being 1/2 strengh) or KM8p liquid medium supplemented with 0.5 mg/L BA,0.5 mg/L 2,4-D,5% mannitol and 10% sucrose(while 4% sucrose in KM8p).The first cell di- vision was observed after three days and sustained division resulted in mass production of cell colonies and small calli a month later.During this period,original liquid medium was replaced with fresh one every 10 days to decrease osmaticum gradually to a normal concentration.The calli were transferred onto agrose me- dium.The cell division diminished in 2 months.The production and differentiation of calli is now in ad- vanced research.