摘要
以酶解－振荡、酶解－解剖及酶解－研磨３ 种方法分离出烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）受精后生活胚囊。其中以第三种方法效果最好。将分离的胚囊经再次酶解并结合显微解剖，进一步分离出合子、胚乳细胞及其原生质体。以微室饲养法培养离体合子，启动了第一次分裂。
Three methods were established to isolate fertilized embryo sacs in Nicotiana tabacum,i.e.enzymatic maceration combined either with shaking,microdissection or grinding respectively.Living fertilized embryo sacs of various developmental stages after fertilization could be isolated successfully by these methods.Each method had its own adoptation to the materials of different developmental stages.Among them the method of enzymatic maceration combined with grinding was the best:Ovules were first treated in enzymatic mixture (1% cellulase R 10,0 5% macerozyme R 10,12% mannitol,pH 5 7) for about 30 min.Then droplets of the ovule suspension were gentlely grinded by a flat headed glass rod.After grinding several droplets of mannitol solution (8%～10%) were added for releasing and washing embryo sacs.Compared with the other two methods this method was more convenent and had higher isolation efficiency.Isolation of fertilized embryo sacs offered a good means for microscopic observation on the postfertilization development including synergid degeneration,endosperm formation and zygotic changes without interference by the surrounding sporophytic tissue.Living zygotes and endosperm cells could be further isolated by a second enzymatic maceration procedure followed by brief micromanipulation.Several characters had been found to distinguish the protoplasts of free zygotes from those of other cell sources.Isolated zygotes were cultured in microchambers (Millicell CM) feeded with macrocultured mesophyll protoplasts.The first division of zygotes was induced,resulting in proembryos consisting of two cells.
基金
国家自然科学基金
高等学校博士点基金
关键词
受精
烟草
胚囊
合子
分离
Fertilized embryo sacs
Zygote
Isolation
Microculture
Nicotiana tabacum
