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。     

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。     

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。     

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     

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。     

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。     

天竺葵雌性生殖单位的超微结构

应用透射电镜研究了临近受精时天竺葵(Pelargonium hortorum Bailey)胚囊中的卵细胞、助细胞和中央细胞的结构。证明了卵细胞与助细胞以及助细胞与助细胞之间从合点端至珠孔端有很大的面积以质膜分界,仅珠孔端少部分以壁分隔。卵细胞与中央细胞之间同样缺乏细胞壁。在卵细胞的合点端,两质膜不同程度地分离形成宽窄相间的间隙。在间隙的絮状基质中存在小泡,这些小泡的产生似与卵和中央细胞中周质内质网的活动有关。推测小泡为多糖性质,可能为合子新壁的建造提供物质。卵细胞质中含巨大线粒体,质体和内质网也较丰富。基于超微结构的特征,可认为卵细胞具高度的生理合成活动的潜能。中央细胞极核位于珠孔端与卵器细胞毗邻,有利于在双受精作用中同时发生精细胞与卵细胞和精细胞与中央细胞核的融合。中央细胞的侧壁在珠孔端形成内突,具传递细胞的特点,表明这是雌配子体向孢子体摄取营养的重要部位。助细胞的细胞质含丰富的细胞器,这与多数植物中的相似,但具几个明显的特征,即核中存在微核仁,内质网形成圆球体或脂体,线粒体富集在丝状器的附近。传粉后花粉管进入胚囊之前,两个助细胞中一个退化。     

扁豆成熟胚囊的超微结构

本文对扁豆（Dolichos lablab)成熟胚囊的超微结构进行了研究,在成熟胚囊中,卵细胞和助细胞仅在珠孔端1／3有细胞壁,靠近合点端,卵细胞一助细胞,卵细胞－中央细胞,助细胞－中央细胞之间没有细胞壁存在,相邻细胞的质膜靠在一起,在卵细胞和中央细胞的质膜间,有些地方存在中等电子密度的物质,卵细胞的细胞质中含有很多的线粒体和质体,内质网和高尔基体较少,助细胞的珠孔端有一复杂的丝状器,靠近珠孔端的细胞质中有很多管状的内质网,表明助细胞可能具有分泌功能,在助细胞的合点端,含有丰富的粗糙内质网,助细胞和卵细胞的质膜之间有很多囊泡状的结构,中央细胞内含有丰富的线粒体,高尔基体和内质网,中央细胞的壁向内形成突起,在周缘细胞质中含有丰富的脂滴。     

扁豆成熟胚囊的超微结构

本文对扁豆（Dolichos lablab)成熟胚囊的超微结构进行了研究,在成熟胚囊中,卵细胞和助细胞仅在珠孔端1／3有细胞壁,靠近合点端,卵细胞一助细胞,卵细胞－中央细胞,助细胞－中央细胞之间没有细胞壁存在,相邻细胞的质膜靠在一起,在卵细胞和中央细胞的质膜间,有些地方存在中等电子密度的物质,卵细胞的细胞质中含有很多的线粒体和质体,内质网和高尔基体较少,助细胞的珠孔端有一复杂的丝状器,靠近珠孔端的细胞质中有很多管状的内质网,表明助细胞可能具有分泌功能,在助细胞的合点端,含有丰富的粗糙内质网,助细胞和卵细胞的质膜之间有很多囊泡状的结构,中央细胞内含有丰富的线粒体,高尔基体和内质网,中央细胞的壁向内形成突起,在周缘细胞质中含有丰富的脂滴。     

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.     

Isolation of Viable Embryo Sacs and Their Protoplasts of Nicotiana tabacum

Isolation of fixed and fresh embryo sacs has been reported. However,the isolation of protoplasts of embryo sac elements is reported here for the first time.The protoplasts of egg cell, synergids, central cell and antipodal cells have been isolated with the retaining of their viability. Though this is a preliminary work, it indicatesthe potentiality of isolation of naked female gametes of angiosperms, which may beused in genetic manipulation and plant biotechnology. Nicotiana tabacum was grown in the greenhouse of the Department of Biology,Peking University. From opened and unpollinated flowers, the ovaries were removedand sterilized with 70% alcohol. The ovules were dissected out from those ovaries andfollowed by incubation (4–8 hrs. 28℃) in anenzyme solution containing 2% driselase, 0.65 M mannitol and 0.25% potassium dextran sulfate. Ovules from 3 4 ovariescould be incubated with 1 ml of enzyme solution in a 3 cm petri dish. All these manipulations and the following procedures were carried out under sterile conditions. Afterincubation, ovules were washed 3 times with a washing solution of 0.65 M mannitol.The isolated embryo, sacs and their protoplasts were obtained by gently squashing digested ovules in a small volume of washing solution on a slide. When the fresh ovules were incubated 3–3.5 hrs in the enzyme solution, the embryosacs may be successfully isolated in an intact manner, either for mature or immatureembryo sacs. The isolated embryo sac looked plump, viable and very distinct in itsstructure. If the isolated embryo sacs were incubated in 0.01% fluorescein diacetate(FDA) used as a test for the viability of the embryo sac, and observed under fluorescein microscope, the cytoplasm of all embryo sac elements, including egg cell, synergids,central cell and antipodal cells, showed strong fluorescence. It is proved that these iso-lated embryo sacs are still viable. When the incubation of ovules was prolonged as to 8 hrs in certain cases, theboundary wall of the embryo sac may be partially digested and the protoplasts of embryo sac elements came out from micropylar or chalazal end after squashing. The difference of the protoplasts derived from different embryo sac elements could be recognized by their relative size and other characteristics. The egg protoplast is smallerthan that of the synergid. However, the protoplasts of antipodal cells were. obviouslysmaller than that of egg. But the central cell protoplast was the largest among theseprotoplasts and possessed two polar nuclei and a very large central vacuole. All theseisolated protoplasts of embryo sac elements were also proved viable with FDA method. The importance of isolated protoplasts of embryo sac elements is discussed withrespect to genetic manipulations.     

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.     

Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump

AIMS: To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. SCOPE: An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. CONCLUSIONS: The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary.     

Isolation and preservation of the living embryo sac ofCrinum asiaticum L. var.japonicum baker

The enzymatic maceration method was used to isolate an intact embryo sac ofCrinum asiaticum and its component cells. Best results were obtained when using enzyme solutions that contained pectinase hemicellulase, cellulase and pectolyase. Aseptic ovules were incubated in the enzyme solution for 1.5 hr at 25 C. This allowed the isolation of embryo sacs to yield up to 20% of the amount present. An isolated embryo sac usually consists of an egg cell, synergids, antipodals and a central cell. Some embryo sacs can be digested as gametophytic protoplast. The size, shape and position of the isolated embryo sac seemingly possessed similarities with those of the fixed embryo sac in the ovary. An isolated embryo sac can be in a living state when the result of the fluorochromatic reaction (FCR) and protoplasmic streaming is positive. When cultured in proper media, 68% of the isolated gametophytic protoplasts were observed to have sustained their positive FCR for more than 1 month.     

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.     

Video microscopic observations of living,isolated embryo sacs of Nicotiana and their component cells

Summary Living embryo sacs and megagametophytic cells of Nicotiana alata and Nicotiana tabacum were obtained using enzymatic maceration and microdissection. The yields of isolated embryo sacs, egg apparatus and central cells were up to 35%, 40% and 35%, respectively. Vectorial movement of organelles and undulations of tubular structures, presumably endoplasmic reticulum, were observed in eggs, synergids and central cells using video-enhanced microscopy. Despite evident viability using the fluorochromatic reaction, the egg displays much less organelle movement and therefore appears to be quiescent. The large vacuole of the central cell is traversed by mobile strands of cytoplasm through which organelles migrate. A polygonal network is located at the periphery of the central cell, which may contribute to anchorage of the cell with the embryo-sac wall. The observation of organelle movement provides direct evidence of the condition of the cell and may be a useful approach for assessing cell vigor.     

甜菜无融合生殖单体附加系M14成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14（Betavulgaris,2n=18＋1）为实验材料,利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明：M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态：（1）极性正常的卵细胞,细胞核位于合点端,细胞质含有大量核糖体、线粒体、内质网等细胞器;（2）细胞核位于细胞中央;（3）细胞核位于珠孔端,且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近;未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显,细胞器的种类与数量多,呈现旺盛代谢活动特征,成为二倍体孢子无融合生殖过程中,卵细胞与次生核自发分裂的细胞学标志。