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。     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism     

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。’     

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。     

Light Microscope Study of Pollen Tube Growth, Fertilization and Early Embryo and Endosperm Development in the Avocado Varieties Fuerte and Hass

Pollen tube growth, fertilization and early embryo and endospermdevelopment were studied using light microscopy in the avocadovarieties Fuerte and Hass. The ovule was penetrated by a pollen tube by 24 h after pollination.On reaching the ovary, the pollen tube grew along the surfaceof the inner ovary wall. It then grew around the funicle, throughthe micropyle in the inner integument and between the papillatecells at the apex of the nucellus. It entered the embryo sacvia a synergid. Sperm nuclei were present in the embryo sacat 48 h after pollination and fusion of the polar and spermnuclei took place before fusion of the egg and sperm. The endospermnucleus was the first to divide and cell wall formation occurredfollowing division. The first division of the zygote occurredat 5 or 6 days after pollination. In the variety Fuerte less than 20 per cent of the 1- and 2-day-oldembryo sacs had been penetrated by a pollen tube although tubeswere often observed in the integument or nucellus. In the varietyHass over 60 per cent of the embryo sacs were penetrated. Inwas concluded that low yields of the variety Fuerte may be partlyattributable to the failure of the pollen tube to penetratethe embryo sac. Persea americana Mill, avocado, pollen tube, fertilization, embryo, endosperm     

Gamete attachment process revealed in flowering plant fertilization

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule (Fig. 1). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants.Open in a separate windowFigure 1.Illustration of the fertilization process in flowering plants. First, each pollen tube accesses an ovule containing egg and central cells. Next, the 2 sperm cells face the female gametes in the ovule after the pollen tube bursts. Finally, each sperm cell simultaneously fuses with either egg or central cell.     

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.     

枸杞雌、雄配子DNA含量与细胞周期变化的初步研究

枸杞是二胞花粉植物。花粉在柱头上萌发后生殖细胞在花粉管中分裂形成精细胞。在花粉管中,精细胞开始合成DNA,随着花粉管生长,精细胞DNA含量持续增加。当花粉管在退化助细胞中破裂,释放出的2个精细胞DNA含量达到1．92倍（1．92C）。在开花当天,卵细胞已合成了约1／3DNA,并持续增加。开花后30h,卵细胞中的DNA含量达到1．63C。在受精前,卵细胞中的DNA含量达到1．83C。精、卵细胞融合后,合子DNA含量为3．53C。去雄排除花粉管影响后,己合成1／5DNA的过熟卵细胞停止合成,表明卵细胞持续合成DNA需要花粉管的刺激。枸杞雌、雄配子在融合前合成DNA,在细胞周期的G,期发生融合,属于G2类型。     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一,是大多数被子植物完成受精作用的关键环节之一。在受精过程中,助细胞吸引花粉管向雌配子体生长,并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外,雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚,但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果,尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果,并对助细胞的生殖功能进行了分析。     

Early post-pollination events in hexaploid wheat × maize crosses

Summary Cytological events in the first 12 h after pollination were studied in crosses between the hexaploid wheat genotype Chinese Spring and the maize genotype Seneca 60. A pollen tube was first observed in the embryo sac 4 h after pollination, and maize sperm nuclei were first observed in the embryo sac after 5 h. On 29 occasions two, and on 1 occasion three, pollen tubes penetrated the embryo sac. Four categories of aberration limiting the frequency of fertilization were identified: (1) in 20% of florets no pollen tube reached the embryo sac; (2) in at least 1.9% the pollen tube severely damaged the wheat egg cell and polar nuclei; (3) in 33% the maize sperm nuclei were not released from the pollen tube; and (4) in 16% the sperm nuclei were released into the embryo sac but failed to move to either of the wheat gametes. In the remaining 29% sperm nuclei were more often found in the egg cell than at the polar nuclei. The results suggest that karyogamy occurs with very high efficiency when a sperm nucleus reaches the egg cell, but with only about 50% efficiency when a sperm nucleus reaches the polar nuclei.     

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。     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一, 是大多数被子植物完成受精作用的关键环节之一。在受精过程中, 助细胞吸引花粉管向雌配子体生长, 并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外, 雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚, 但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果, 尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果, 并对助细胞的生殖功能进行了分析。     

Centenary on S.G.Nawaschin's Discovery of Double Fertilization: Retrospects and Prospects

A review on the double fertilization in angiosperm is addressed at its centennial discovery by S.G. Nawaschen. Studies in the first 50 years mainly by light microscopy had defined this process of double fertilization as a general characteristic in angiosperms. In the later 50 years research works in this field have been greatly advanced on account of the developing new techniques especially the electron-microscopy. The topics in this review include: (1) The growth of pollen tube entering the embryo sac: role of the synergid in the pollen tube receiption and signals from the degenerated synergid. (2) The arrival of male gametes to female gametes: structure and function of the male germ unit, the function of cytoskeleton in the delivery of sperm cells. (3) Gametic fusion: the structure and function of the female germ unit, gametic membrane fusion, karyogamy, DNA contents in sperm and egg nuclei, the relationship between the karyogamy and cell cycle, sperm dimorphism and preferential fertilization, and spermegg recognition. Future directions for the research of double fertilization are also recommended.     

Fertilization in Arabidopsis thaliana wild type: developmental stages and time course

We describe some previously uncharacterised stages of fertilization in Arabidopsis thaliana and provide for the first time a precise time course of the fertilization process. We hand-pollinated wild type pistils with wild type pollen (Columbia ecotype), fixed them at various times after pollination, and analysed 600 embryo sacs using Confocal Laser Scanning Microscopy. Degeneration of one of the synergid cells starts at 5 Hours After Pollination (HAP). Polarity of the egg changes rapidly after this synergid degeneration. Karyogamy is then detected by the presence of two nucleoli of different diameters in both the egg and central cell nuclei, 7-8 HAP. Within the next hour, first nuclear division takes place in the fertilized central cell and two nucleoli can then be seen transiently in each nucleus produced. In a second set of experiments, we hand-pollinated wild type pistils with pollen from a transgenic promLAT52::EGFP line that expresses EGFP in its pollen vegetative cell. Release of the pollen tube contents into the synergid cell could be detected in living material. We show that the timing of synergid degeneration and pollen tube release correlate well, suggesting that either the synergid cell degenerates at the time of pollen tube discharge or very shortly before it. These observations and protocols constitute an important basis for the further phenotypic analysis of mutants affected in fertilization.     

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.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

ORCHID EMBRYOLOGY: MEGAGAMETOPHYTE OF EPIDENDRUM SCUTELLA FOLLOWING FERTILIZATION

The megagametophyte of Epidendrum scutella, an orchid, was examined with the electron microscope after the entrance and discharge of the pollen tube. The pollen tube enters the embryo sac by growing through the filiform apparatus of a synergid and discharges through a terminal pore into the degenerating cytoplasm of the synergid. The synergid nucleus appears pushed to one side by the discharge of the pollen tube. What is believed to be the remains of the vegetative nucleus has been found in the degenerate synergid, but no trace of the sperm cytoplasm has been seen. The zygote is approximately the same size as the egg. The ribosomes become grouped into polysomes. Both the egg and the zygote apparently completely lack dictyosomes. The polar nuclei partially fuse before fertilization, but fusion of the sperm nucleus with the polar nuclei does not occur and no endosperm is produced. Polysome formation occurs in the central cell and large amounts of tubular, smooth ER are seen. The antipodals remain following fertilization, undergoing ultrastructural changes similar to the central cell.     

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.