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用酶解-研磨法分离烟草与大叶烟草受精后的胚囊,继之以显微解剖分离合子与二胞原胚。将3—5个合子或二胞原胚置于微室的琼脂糖小滴中,以预先培养3—4天、分裂1—2次的烟草、大叶烟草或黄花烟草叶肉原生质体饲养。培养基为KM8p与其它附加成分。在25℃与黑暗下静置培养。培养3—4天,约60%合子完成第一次分裂。多数行不等分裂产生大小两个细胞。12天后形成少数原胚或多细胞团。二胞原胚培养亦分裂为多细胞原胚。研究了合子分离方法、合子发育时期、饲养细胞种类与培养天数等因素对合子离体发育的影响。
The embryo sacs of tobacco and big-leaf tobacco after fertilization were separated by enzymolysis-grinding, followed by microdissection of zygotes and two-pronuclei. 3-5 zygotes or proembryosides are placed in micro-chamber agarose droplets and cultured in mesophyll protoplasts of pre-cultured 3-4 day, 1-2 day split tobacco, big leaf tobacco or yellow tobacco. The medium is KM8p with other additional ingredients. Let stand culture at 25 ° C and darkness. After 3-4 days of culture, about 60% of the zygotes complete the first division. Most lines split equally between two cells of size. After 12 days to form a few embryos or multicellular groups. Diploid embryo cultures also divide into multicellular proembryos. The effects of zygote isolation, zygotic development, type of feeder cells and days of culture on the in vitro development of zygotes were studied.