将大葱(AllIuM fISTulOSuM)胚珠置于酶液中30分钟可将其外珠被去掉。可清楚地看到由内珠被包裹的胚珠中胚囊的轮廓。将胚珠转移至不含酶的相同溶液中,用解剖针从胚珠中部切割,然后挤压胚珠的珠孔部位,卵器细胞从胚珠的切口处逸出。再用显微操作仪将卵细胞和2个助细胞分开,达到葱卵细胞分离的目的。酶对分离卵细胞具有重要的作用,经0.2%果胶酶y23、0.8%果胶酶、0.8%纤维素酶和0.5%半纤维素酶的处理,可在2小时内从30个胚珠中分离出18个卵细胞。随着胚囊的发育,2个助细胞的体积出现明显差异。生活的葱卵细胞的成功分离,为建立葱离体受精体系创造了条件。Viable egg cells of Allium fistulosum were isolated by enzymatic digestion and mechanical dissection.The ovules were incubated in an enzymatic solution containing 0-0.8% pectolyse Y-23, 0-1.2% pectinase (Serva), 0-1.2% cellulase (Onozuka RS), 0-1% hemicellulase (Sigma) and 6%-12% mannitol for 20-40 min to remove the outer integument.Then ovules were transferred into an isolating solution without enzymes for mechanical dissection.The ovules were cut from the middle part, and the micropylar part was gently extruded by use of a dissection needle.In general, the three cells of the egg apparatus spilled out from the cut end of the ovule.The enzymatic solution containing 0.2% pectolyse Y-23, 0.8% pectinase (Serva), 0.8% cellulase (Onozuka RS), 0.5% hemicellulase (Sigma) and 10% mannitol was best for isolating about 18 egg cells from 30 ovules in 2 h.The isolated egg cells could be used for in vitro fertilization to explore the fertilization mechanism and for egg development with molecular biological methods.国家自然科学基金(No.30670126
