水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻(Oryz a sativa)受精过程中雌雄性细胞融合时的形态特征及时间进程, 确定合子期, 为花粉管通道转基因技术的实施提供理论依据。结果表明: 授粉后, 花粉随即萌发, 花粉管进入羽毛状柱头分支结构的细胞间隙, 继续生长于花柱至子房顶部的引导组织的细胞间隙中, 而后进入子房, 在子房壁与外珠被之间的缝隙中向珠孔方向生长, 花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞, 释放精子。精子释放前, 两极核移向卵细胞的合点端; 两精子释放于卵细胞与中央细胞的间隙后, 先后脱去细胞质, 然后分别移向卵核和极核, 移向卵核的精核快于移向极核的精核; 精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下: 授粉后, 花粉在柱头萌发; 花粉萌发至花粉管进入珠孔大约需要0.5小时; 授粉后0.5小时左右, 花粉管进入一个助细胞, 释放精子; 授粉后0.5-2.5小时, 精卵融合形成合子; 授粉后约10.0小时, 合子第1次分裂, 合子期为授粉后2.5-10.0小时; 授粉后1.0-3.0小时, 精核与两极核融合; 授粉后约5.0小时, 初生胚乳核分裂。     

水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻（Oryza sativa）受精过程中雌雄性细胞融合时的形态特征及时间进程,确定合子期,为花粉管通道转基因技术的实施提供理论依据。结果表明：授粉后,花粉随即萌发,花粉管进入羽毛状柱头分支结构的细胞间隙,继续生长于花柱至子房顶部的引导组织的细胞间隙中,而后进入子房,在子房壁与外珠被之间的缝隙中向珠孔方向生长,花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞,释放精子。精子释放前,两极核移向卵细胞的合点端：两精子释放于卵细胞与中央细胞的间隙后,先后脱去细胞质,然后分别移向卵核和极核,移向卵核的精核快于移向极核的精核：精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下：授粉后,花粉在柱头萌发：花粉萌发至花粉管进入珠孔大约需要0．5小时：授粉后0．54,时左右,花粉管进入一个助细胞,释放精子：授粉后0．5—2．5小时,精卵融合形成合子：授粉后约10．0小时,合子第1次分裂,合子期为授粉后2．5-10．04,时：授粉后1．0-3．04,时,精核与两极核融合：授粉后约5．0小时,初生胚乳核分裂。’     

FERTILIZATION IN BARLEY

The mature embryo sac of barley consists of an egg, two synergids, a central cell, and up to 100 antipodal cells. At shedding the male gametophyte is 3-celled, consisting of a vegetative cell with a large amount of starch and two sperms having PAS+ boundaries. Before pollination the nucleus and cytoplasm of each synergid appear normal. After pollination the nucleus and cytoplasm of one synergid undergo degeneration. The pollen tube grows along the surface of the integument of the ovule, passes through the micropyle, and enters the degenerate synergid through the filiform apparatus. The pollen tube discharges the vegetative nucleus, two cellular sperms, and a variable amount of starch into the degenerate synergid. Soon after deposition the sperms migrate by an unknown mechanism to the chalazal end of the degenerate synergid. Sperm nuclei then enter the cytoplasm of the egg and central cell, ultimately resulting in the formation of the zygote and primary endosperm nucleus, respectively. Sperm boundaries do not enter egg or central cell, but it was not possible to determine the fate of other sperm components. Degenerate vegetative and synergid nuclei remain in the synergid after fertilization, constituting what are considered to be X-bodies in barley. The second synergid degenerates during early embryogeny.     

烟草雌、雄配子DNA 含量变化

采用显微分光光度法测定了烟草( Nicotiana tabacum) 精细胞和卵细胞的DNA 含量。烟草是二胞花粉, 花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45 h 花粉管到达子房, 在花粉管内的精细胞DNA 含量为1C。当花粉管在退化助细胞中破裂, 释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA 含量接近2C。随着精细胞的到达及合成DNA, 卵细胞也开始合成DNA, 融合前的卵细胞DNA 含量也接近2C。精、卵细胞融合后, 合子DNA 含量为4C。烟草雌、雄配子是在细胞周期的G2 期发生融合, 属于G2 型。     

烟草雌、雄配子DNA含量变化

采用显微分光光度法测定了烟草（Nieotiana tabacum）精细胞和卵细胞的DNA含量。烟草是二胞花粉,花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45h花粉管到达子房,在花粉管内的精细胞DNA含量为1C。当花粉管在退化助细胞中破裂,释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA含量接近2C。随着精细胞的到达及合成DNA,卵细胞也开始合成DNA,融合前的卵细胞DNA含量也接近2C。精、卵细胞融合后,合子DNA含量为4C。烟草雌、雄配子是在细胞周期的G2期发生融合,属于G2型。     

Electrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process,fusion of sperm cells with synergids and central cells,and cell reconstitution in maize

Summary Fusion products were created by the electrofusion of single sperm cells with single synergids and central cells. The synergid was also fused with the sperm cell, occasionally in the presence of adhering second synergids, egg cells, and central cells. Single egg cells were fused with single sperm cells in the presence of adhering synergids and the central cell. Cytoplasmic organelles were transmitted through the fertilization process by electrofusion using cytoplasts of maize mesophyll cells. Cell reconstitution was achieved by fusion of one or two sperm cells with single enucleated protoplasts, thus creating a haploid or a diploid cell.     

Transmission of male cytoplasm during fertilization in Nicotiana tabacum

Serially sectioned embryo sacs of Nicotiana tabacum were examined during fertilization events using transmission electron microscopy. After pollen tube discharge, the outer membrane of the sperm pair is removed, the two sperm cells are deposited in the degenerate synergid and the sperm cells migrate to the chalazal edge of the synergid where gametic fusion occurs. During fertilization, the male cytoplasm, including heritable organelles, is transmitted into the female reproductive cells as shown by: (1) the cytoplasmic confluence of one sperm and the central cell during cellular fusion, (2) the occurrence of sperm mitochondria (distinguished by ultrastructural differences) in the zygote cytoplasm and adjacent to the sperm nucleus, (3) the presence of darkly stained aggregates which are found exclusively in mature sperm cells within the cytoplasm of both female cells soon after cell fusion, and (4) the absence of any large enucleated cytoplasmic bodies containing recognizable organelles outside the zygote or endosperm cells. The infrequent occurrence of plastids in the sperm and the transmission of sperm cytoplasm into the egg during double fertilization provide the cytological basis for occasional biparental plastid inheritance as reported previously in tobacco. Although sperm mitochondria are transmitted into the egg/zygote, their inheritance has not been detected genetically. In one abnormal embryo sac, a pair of sperm cells was released into the cytoplasm of the presumptive zygote. Although pollen tube discharge usually removes the inner pollen-tube plasma membrane containing the two sperm cells, this did not occur in this case. When sperm cells are deposited in a degenerating synergid or outside of a cell, this outer membrane is removed, as it apparently is for fertilization.     

Changes in actin organization in the living egg apparatus of Torenia fournieri during fertilization

Changes in actin organization in the living egg apparatus of Torenia fournieri from anthesis to post-fertilization have been investigated using microinjection and confocal microscopy. Our results revealed that the actin cytoskeleton displays dramatic changes in the egg apparatus and appears to coordinate the events of synergid degeneration, pollen tube arrival and gametic fusion during fertilization. Synergid degeneration occurs after anthesis and is accompanied by actin fragmentation and degradation. The actin cytoskeleton becomes organized with numerous aggregates in the chalazal end of the degenerating synergid, and some of the actin infiltrates into the intercellular gap between synergids, egg and central cell, forming a distinct actin band. An actin cap is present near the filiform apparatus after anthesis and disappears after pollen tube arrival. In the egg cell, actin filaments initially organize into a network and after pollination become fragmented into numerous patches in the cortex. These structures, along with the actin in the degenerating synergid and intercellular spaces form two distinct actin coronas during fertilization. The actin coronas vanish after gametic fusion. This is the first report of changes in actin organization in the living egg apparatus. The reorganization of the actin cytoskeleton in the egg apparatus and the presence of the actin coronas during fertilization suggest these events may be a necessary prelude to reception of the pollen tube and fusion of the male and female gametes. Received: 11 November 1999 / Accepted: 31 January 2000     

向日葵胚囊的超微结构和雌性生殖单位问题

本文对向日葵胚囊中卵细胞、助细胞与中央细胞开花前和传粉后的超微结构变化进行了研究。着重报道了不同发育时期这三种细胞之间特定区域的界壁的消长动态。在此基础上结合现有文献资料探讨了由三者共同组成“雌性生殖单位”以适应受精作用的问题。     

Fertilization inNicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cell with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed coronas. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.Abbreviations ES embryo sac - FA filiform apparatus - Mf microfilament - Mt microtubule - PT pollen tube - RF-FS rapid-freeze freeze-substitution - TEM transmission electron microscopy We thank Gregory W. Strout for technical assistance in the use of the RF-FS technique and Dr. Hongshi Yu for providing Fig. 1. This research was supported by U.S. Department of Agriculture grants 88-37261-3761 and 91-37304-6471. We gratefully acknowledge use of the Samuel Robert Noble Electron Microscopy Laboratory of the University of Oklahoma.     

被子植物受精机制的研究进展

被子植物的受精是一个复杂而精巧的过程。花粉管到达子房,通过退化助细胞进入胚囊,释放出两个精细胞。原来在花粉管中相互联结的两个精细胞在退化助细胞中分开,一个与卵细胞融合,另一个与中央细胞融合,完成双受精。目前对双受精过程中有关雌、雄配子识别的机制还知之甚少。本文介绍了目前被子植物精、卵细胞融合前后的细胞周期变化、退化助细胞的功能、精细胞在退化助细胞中迁移的研究动态、精细胞的倾向受精和卵细胞的激活等被子植物受精生物学领域中的一些新的研究成果和发展趋势。     

The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L.)

 Sperm cells released from in vivo-in vitro grown pollen tubes of tobacco are associated in pairs and initially enclosed by the plasma membrane of the pollen tube. When the sperm cells are placed together, using glass microinjector needles, in an enzymatic solution, up to half undergo cellular fusion with subsequent nuclear fusion. The frequency of sperm cell fusion decreases with time during the elongation of the pollen tube, suggesting that mechanisms inhibiting self-fusion of sperm cells may develop as the pollen tube elongates through the style toward the ovule. This tendency may play an important role in inhibiting fusion of the two sperm cells inside the calcium-rich synergid where the male germ unit dissociates and sperm cells are transported to their target cells - the egg and central cell. Received: 25 August 1997 / Revision accepted 16 March 1998     

Synergid: a key link in fertilization of angiosperms

In over 80 % of the angiosperms, the female gametophyte is comprised of seven cells, two of which are the synergid cells. These cells are considered pivotal in assuring successful fertilization. The synergid cells direct pollen tube growth toward the female gametophyte, and facilitate the entrance of the tube into the embryo sac. Once the pollen tube enters the synergid cell, its growth is arrested, the tip of the tube breaks, and two sperm cells are released. This sequence of events is also synergid dependent. In addition, separation of the cells of the male germ unit, orientation of the two sperm cells in the degenerating synergid, and fusion of the egg and central cell with sperm cells may also be related to synergid cells. Synergid structure has been widely studied, but development and function of these cells during angiosperm fertilization remains elusive. Recent molecular approaches have provided an enhanced understanding of the role of synergid cells in fertilization. The present review summarizes the results of current studies regarding the role of synergids in angiosperm reproductive function.     

Observations on the Developmental Femal Gametophyte and the Process of Fertilization of Amcmum visllosum Lour.

The development of megarametophyte antl the process of fertnization are investigated in detail and the following results are obtained: 1. The development of the embryo sac conforms to Polygonmn type. The pollen tube reaches the base of style about 16 hrs., enters into the ovary, 16–18 hrs. and then it, to the embryo sae, 24–28 hrs. after pollination. 2. Pollezt grains are 2-celed at maturity. 3. When the pollen tube reaehes the mieropylar end, both synergids are intact; but their nueleolus become small. Pollen tube enter the synergid through the filiform apparatus and the synergid is degenerated. 4. Pollen tube discharges two sperms which move towards the egg cell and the secondary nucleus respectively. 5. One sprm fuses with the egg nucleus and another with the secondary nueleus. The proeess of syngamy is siniilar to that of secondary nucleus fusion. Primary endosperm nueleus s formed 30 hrs. after pollination. The zygote is formed 30–62 hrs. after pollination. 6. There is no distinct correlation between the syngamy and secondary nueleus fusion.     

Observations on Fertilization in Sugar Beet

The whole process of double fertilization in sugar beet has been observed, the main results are as follows: About 2 hours after pollination, the pollen grains germinate, the sperms in the pollen tube are long-oval. 15 hours after pollination, the pollen tube destroys a synergid and releases two sperms on one side or at the chalazal end of the egg cell. The sperms are spherical each having a cytoplasmic sheath. 17 hours after pollination, one sperm enters the egg cell, and the sperm nucleus fuses with the egg nucleus rapidly. 21 hours after pollination, the zygote is formed. In the meantime, the primary endosperm nucleus has divided into two free endosperm nuclei. 25 hours after pollination, the zygote begins to divide, forming a two-celled proembryo. The dormancy stage of the zygote is about 4 hours. In the meantime the endosperm is at the stage of four free nuclei. 17 hours after pollination, the sperm nucleus comes into contact and fuses with the secondary nucleus. The sperm nucleus fuses with the secondary nucleus, faster than the sperm with the egg. he first division of the primary endosperm nucleus is earlier than that of the zygote, it takes place about 20 hours after pollination, the dormancy stage of the primary endosperm is about 2 hours. The endosperm is free nuclear. The fertilization of sugar beet belongs to premitotic type of syngamy. From the stage of zygote to the two-celled proembryo, it can be seen that addition- al sperms enter the embryo sac, but polyspermy has not been observed yet.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

Pollen tube entry into the synergid cell of Arabidopsis is observed at a site distinct from the filiform apparatus

In higher plants, the double-fertilization process begins with the successful delivery of two sperm cells to the female gametophyte. The sperms cells are carried by a pollen tube that upon arrival at the micropylar end of the female gametophyte, bursts, and discharges its content into one of two specialized cells called the synergid cells. At their micropylar ends, both synergid cells form a thickened cell wall with a unique structure called the filiform apparatus. The filiform apparatus is believed to play a major role in pollen tube guidance and reception. It has also been assumed that the pollen tube enters the receptive synergid cell through the filiform apparatus. Here, we show that in Arabidopsis ovules, the arriving pollen tube appears to grow beyond the filiform apparatus to enter the synergid cell at a more distant site, where the tube bursts to release its contents. Thus, fertilization in Arabidopsis might involve two spatially and temporally separable stages, recognition and entry, with the latter apparently not requiring the filiform apparatus.     

Fertilization in maize indeterminate gametophyte1 mutant

Summary. Mature embryo sacs of the maize mutant indeterminate gametophyte1 displayed different cellular patterns compared to those of the wild type. About 40% of the ig1 embryo sacs contained three or more synergids and two or more egg cells at the micropylar end. During fertilization in embryo sacs with two synergids, both of them frequently degenerated and were penetrated by two pollen tubes. 75% of the embryo sacs containing three or more synergid cells were penetrated by two or more pollen tubes, although most of them had only one degenerated synergid. Multiple fusions between the sperm cells and eggs frequently occurred in the same embryo sac, which subsequently generated multiple embryos. There were two or more central cells in about 33% of ig1 embryo sacs. The largest central cell was usually adjacent to the egg apparatus and contained two unfused polar nuclei, while those extra central cells located at the chalazal end usually had a single nucleus. Fertilization occurred only between the male gamete and the largest binucleate central cell. The extra central cells eventually degenerated after fertilization.Present address: GI Basic Research Center, Mayo Clinic, Rochester, Minnesota, U.S.A.Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, Peoples Republic of China.     

Fertilization and Histochemical Investigation on Pulsatilla chinensis (Bung) Regel

Fertilization and variation of protein and starch grains in Pulsatilla chinensis (Bung) Regel have been studied at light microscopic level with histochemical test. Based upon the observations, the main conclusions are summarized as follows: The mature pollen grains are two-celled in which the generative cell shows the stronger protein staining than the vegetative cell. And vegetative cells are full of starch garins. When the pollen tube enters into the embryo sac, one synergid is destroyed, or in a few cases synergids are intact. Occasionally two synergids are disorganized as pollen tube penetrates. However, most of the remaining syuergids break down during fertilization, only in a few cases it remains till early stage of embryo development. The contents discharged by the pollen tube consist of two sperms, which stain intensely blue with protein dyes, a great amount of protein and starch grains. Mature female gametophyte (embryo sac) consists of an egg apparatus, central cell, which has a huge secondary nucleus, and antipodal apparatus which retain in course of fertilization. A few of embryo sac contain two sets of egg apparatus, a central cell with two huge secondary nuclei and two sets of antipodal apparatus. In some nucleoli of the central cell the comb-like structure pattern may be detected clearly. There are 1–2 small nucleoli in some egg cells and central cells. All the cells in embryo sac show protein positive reaction. According to the different shades of the color in cells, its may be arranged in the following order: antipodal cells, synergids, central cell and egg cell. Only a few small starch grains are present near nuclei of central cell and egg cell before fertilization, but no starch grains remain in most of the central cell, the synergids and antipodal cells. The fertilization is of the premitotic type. The fusion of the sexual nuclei progresses in the following order: 1, sperms approach and lie on the egg nucleus and secondary nucleus; 2, sperm chromatin sinks themselves into female nucleus, and male nucleolus emerges with the sperm chromosome; and 3, male nucleoli fuse with the nucleoli of egg nucleus and central cell nucleus, and finally forming the zygote and the primary endosperm cells respectively. Nevertheless, as it is well known, the fertilization completes in central cell obviously earlier than that in egg cell. Though it has been explained in cereals and cotton, in Pulsatilla chinensis the main reason is that nucleolar fusion of the male and female nucleoli in egg nucleus is slower than that in secondary nucleus. And the dormancy of the primary endosperm nucleus is shorter than that of the zygote. In the process of fertilization, histochemical changes are considerably obvious in the following three parts: 1, from the begining of fusion of male and female nuclei to form zygote and primary endosperm cell, Protein staining around female nucleus appears to increase gradually; 2, no starch grains are detected in embryo sac. Though only starch grains are carried in by pollen tube, they are completely exhausted during this period; and 3, near completion of fertilization starch grains appear again in zygote, however, not yet in primary endosperm nucleus till its dividing for the first time. The present study reveals that antipodal cells and synergids seem to play a significant role in nutrition of the embryo sac during the fertilization.