葱卵细胞的分离

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。     

洋葱卵细胞的分离

将洋葱的胚珠置于酶液中酶解50-110 min后剥去其珠被,可清楚地看到珠心中的胚囊轮廓。用解剖针将珠心从中部横切,然后挤压其珠孔部位,卵器细胞从胚珠的切口处逸出。再用显微操作仪的玻璃针将卵细胞和两个助细胞分开,达到分离洋葱卵细胞的目的。酶对分离卵细胞具有重要作用,在最佳的酶液浓度[0.02%果胶酶Y23、0.08%果胶酶（Serva）、0.05%纤维素酶和0.05%半纤维素酶]下酶解胚珠110 min后,解剖1 h可从24个胚珠中分离出10个卵细胞（41.67%）。随着胚囊的发育,两个助细胞的体积出现明显的二形性。洋葱生活卵细胞的分离为开展洋葱离体受精建立了基础,也为研究洋葱卵器细胞的发育创造了条件。     

洋葱卵细胞的分离

将洋葱的胚珠置于酶液中酶解50~110 min后剥去其珠被,可清楚地看到珠心中的胚囊轮廓。用解剖针将珠心从中部横切,然后挤压其珠孔部位,卵器细胞从胚珠的切口处逸出。再用显微操作仪的玻璃针将卵细胞和两个助细胞分开,达到分离洋葱卵细胞的目的。酶对分离卵细胞具有重要作用,在最佳的酶液浓度[0.02%果胶酶Y23、0.08%果胶酶(Serva)、0.05%纤维素酶和0.05%半纤维素酶]下酶解胚珠110 min后,解剖1 h可从24个胚珠中分离出10个卵细胞(41.67%)。随着胚囊的发育,两个助细胞的体积出现明显的二形性。洋葱生活卵细胞的分离为开展洋葱离体受精建立了基础,也为研究洋葱卵器细胞的发育创造了条件。     

胡萝卜精、卵细胞、助细胞和中央细胞的分离

用两个解剖针挤压胡萝卜花粉使其破裂释放出精细胞。用酶解-解剖方法分离胡萝卜胚囊中的卵细胞、助细胞和中央细胞。胡萝卜胚珠先在酶液中酶解40~50min,然后将其转移到不含酶的分离液中用解剖针解剖胚珠。将胚珠的合点端切破,轻轻挤压胚珠的珠孔,卵细胞、助细胞和中央细胞即可逸出。在最佳条件下,20min可从20个胚珠中分离出5个卵细胞。对分离胚囊细胞的渗透压和酶液成分进行了筛选。分离出的卵细胞用显微操作仪收集。胡萝卜精、卵细胞的成功分离为在双子叶植物中进行离体受精探索创造了条件。     

蓝猪耳卵细胞和合子的分离

蓝猪耳(Torenia fournieri)胚囊部分裸露出胚珠,在光学显微镜下能清楚观察到卵细胞和助细胞的形态结构.用解剖和酶解-解剖两种方法都能分离出生活卵细胞.用前种方法机械分离出的卵细胞数量较少(5%),但避免了酶对配子识别研究的干扰.在后种方法中加入0.1%纤维素酶和0.1%果胶酶既能使分离更加容易操作,又对卵细胞没有致命伤害,能在短时间内分离出较多的卵细胞(18%).用酶解-解剖方法也可分离出授粉14 h后的合子细胞.     

莴苣胚囊细胞分离

用酶解和解剖方法分离了莴苣的卵细胞,助细胞,中央细胞和合子。莴苣子房先在酶液中酶解40～50min,然后在不含酶的分离液中用解剖针解剖子房。在解剖出的胚囊中,可看到卵细胞,两个助细胞和中央细胞的轮廓。将胚囊的合点端切破,轻轻挤压胚囊的珠孔端,四个细胞即可逸出。在最佳条件下,90min可从40个子房中分离出29个胚囊,进一步从中分离出11个卵细胞。分离出的胚囊细胞用显微操作仪收集备用。莴苣卵细胞的成功分离为进行离体受精探索创造了条件。     

莴苣胚囊细胞分离

用酶解和解剖方法分离了莴苣的卵细胞,助细胞,中央细胞和合子。莴苣子房先在酶液中酶解40～50min,然后在不含酶的分离液中用解剖针解剖子房。在解剖出的胚囊中,可看到卵细胞,两个助细胞和中央细胞的轮廓。将胚囊的合点端切破,轻轻挤压胚囊的珠孔端,四个细胞即可逸出。在最佳条件下,90min可从40个子房中分离出29个胚囊,进一步从中分离出11个卵细胞。分离出的胚囊细胞用显微操作仪收集备用。莴苣卵细胞的成功分离为进行离体受精探索创造了条件。     

颠茄胚囊细胞原生质体的酶法分离和观察

分离被子植物雌配子原生质体对发展植物受精工程有着重要意义。最近几年,虽然用酶法分离生活的胚囊已有一些成功的报道。但对分离组成胚囊细胞的原生质体前人还没有进行过尝试。1984年我们首次从烟草生活胚珠中分离出卵细胞、中央细胞、助细胞和反足细胞的原生质体。最近我们又用颠茄胚珠为材料,采用酶解压片技术进行分离胚囊细胞的原生质体,也取得预期的结果。获得了有活性的卵细胞、中央细胞和助细胞的原生质体。从而证明这项技术是有推广应用价值的。在观察中发现用酶法分离生活胚囊细胞的原生质体,能     

蝴蝶兰合子和胚的分离

该文用酶解一振荡法分离蝴蝶兰受精后的胚囊,然后显微解剖出合子、早期原胚及接近成熟的球形原胚。酶解液由O．7％-1．3％的纤维素酶、0．6％-1．0％果胶酶和10％甘露醇组成,pH5．8,酶解时间20-30min。分离的蝴蝶兰早期原胚和接近成熟的球形原胚均发现有发达的胚柄吸器。     

金鱼草胚囊的人工分离

用果胶酶与纤维素酶的混合液离解金鱼草的胚珠,从中分离出完整的胚囊。由固定材料分离的胚囊,经透明染色后可借助干涉差显微装置观察由受精前直至受精后具上百个胚乳细胞时期的胚囊的内部结构。由新鲜胚珠分离胚囊也已初步突破,并对其作了简易的显微化学观察。讨论了活胚囊分离的意义与胚囊离体培养的前景。     

Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge

Egg cells of Torenia fournieri were isolated from embryo sacs 1 day after anthesis using enzymatic digestion or mechanical dissection. About 5% of the egg cells and zygotes (2-3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added to the mannitol isolation solution, about 18% of the egg cells (8-10 from 50 ovules) could be isolated within 2 h. The egg cells isolated by mechanical dissection could be used for in vitro fertilization studies without any of the potentially deleterious effects of the enzymes on the plasma membrane of egg cell. The egg cells isolated using enzymatic digestion could be used in the study of the molecular biology of female gamete because more egg cells could be isolated with this technique. Using enzymatic digestion, over 10 zygotes from 50 ovules (over 20%) were isolated from the pollinated ovules. Coupled with our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible in a dicotyledon plant.     

蓝猪耳合子和二胞原胚细胞的分离(简报)

合子是被子植物个体发育的第一个细胞.合子分裂的规律性是植物早期胚胎发生的种属甚至是科的特征,也是控制早期个体发育的关键阶段.     

Isolation and Identification of Viable Embryo Sacs in Several Angiosperm Species

Since the enzymatic technique for isolating embryo sac (ES) has been established on fixed materials of several angiosperms as well as on fresh ovules of Antirrhinum majus in our lab, further works on isolation of viable ESs were carried on. Fresh ovules were macerated in a solution of enzymes, sucrose with or without potassium dextran sulphate by a microshaker at 28–30℃ for several hours. The enzymes included pectinase, cellulase, snailase and pectolyase Y-23, the combination and concentration of which varied with the plant species and the developmental stages of ESs. To date the mature ESs of Helianthus annuus, A. majus and Nicotiana tabacum and the ESs after fertilization with proembryo and endosperm cells in the two former species were well isolated. Nomarski interference contrast and Hoechst 33253 fluorescence microscopical observations revealed that the ESs retained their cell structure and were rich of ergastic substances. Fluorochromasia induced by fluorescein diacetate further proved that they were really viable.     

黄花木本曼陀罗卵细胞分离(简报)

采用分离的精、卵细胞体外融合并诱导人工合子长成植株的离体受精方法可在没有其他组织影响的单细胞水平上探索受精事件的发生过程．为研究高等植物的受精机理、探索配子识别和合子激活等问题提供有效手段。分离的卵细胞不仅可以用来开展离体受精研究．也提供了用分子生物学方法研究被子植物卵细胞发育和合子发育机理的实验基础.     

A New Method for Embryo Sac Isolation and In Situ Fusion of Egg and Synergid Protoplasts in Nicotiana tabacurn

A new method combining enzymatic maceration with osmotic shock was developed for isolation of living embryo sac and its protoplasts in Nicotiana tabacum L. The principle of this method was that the ovules submitted to enzymatic treatment and osmotic shock could release embryo sacs along with some internal ovular cells through either the funicle cut end or the micropyle. Factors affecting embryo sac isolation were investigated, including concentration of mannitol as a shock osmoticum and in enzymesolution ,duration of enzymatic maceration,and duration of osmotic shock. As a result a procedure was established: Ovules at mature embryo sac stage were macerated for 2. S h in 1 %–1.5% cellulase R-10 and 0. 5% macerozyme R-10 (or 1% Pectinase,Serva) dissolved in 13% mannitol solution using microshaker,followed by osmotic shock for 15–30 min with enzyme free 8% mannitol solution and gentle agitation using a pipette. Using a capillary,50–70 embryo sacs could be collected manually in one hour. The embryo sacs thus isolated could be kept viable from which protoplasts of egg cell and other componcnt cells could be further isolated. An additional interesting phenomenon was that osmotic shock often caused in situ fusion the protoplasts of egg cell and synergids. The rate of fusion ranging 9%—71.9% could be controlled by modification of the procedure. This phenomenon merits further attention both from basic and practical point of view. The present method gives the advantages of faciliting isolation and promoting good harvest of viable embryo sacs/female protoplasts within a relative short time.     

In vitro fertilization of single,isolated gametes of maize mediated by electrofusion

Summary Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.