Ultrastructure of Male Gametophyte in wheat I. The Formation of Generative and Vegetative Cells

The present study of the formation of the generative and vegetative cells in wheat has demonstrated some cytological details at the ultrastructural level. The phragmoplast formed in telophase of the first microsporic mitosis extended centrifugally until it connected with the intine of the pollen grain. A new cell wall was then formed to separate the generative and the vegetative cells. By unequal cytokinesis the former is small and the latter large. In early developmental stage of male gametophyte, the organelles in the cytoplasm of the generaVive cell and the vegetative cells are similar, including mitochondria, dictyosomes, rough endoplasmic retieulum, free and clustered ribosomes and plastids, but microtubules were observed only in the early cytokinesis stage. In the further developmental stage of the male gemetophyte, the generative cell gradually detached from the intine of pollen grain and grew inward to the cytoplasm of the vegetation cell. When the generative cell became round and free in the cytoplasm of the vegetative cell, the wall materials between plasma membranes of the cytoplasm of the generative and the vegetative cells disappeared completely, so that it was a naked cell with a double-layer membrane at this time. The heterogeneity between both cells was then very conspiceous. The organelles in the cytoplasm of the generative cell have hardly any changed besides the degeneration of plastids, but in vegetative cytoplasm the mitochondria and plastids increased dramatically both in number and size. The rapid deposition of starch in the plastids of the cytoplasm of the vegetative cell made the most conspicuous feature of the vegetative cell in mature pollen grain. The significance of the presence of a temporary cell wall in generative cell and heterogeneity between generative and vegetative cells are discussed.     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 1. Descriptive cytology and three-dimensional organization

The generative cell is initiated as a small, lenticular, unpolarized cell with a cell wall traceable to two origins: the external segment originates as intine, while an inner callose positive cell wall forms de novo. As the lenticular generative cell begins its migration into the pollen cytoplasm, the generative cell becomes polarized both externally and internally, displaying a characteristic shape and patterns of organelle distribution oriented with respect to the vegetative nucleus and independent of pollen aperture location. Separation of the generative cell from the pollen wall begins at the end opposite the vegetative nucleus and results in an elongating protuberance at the opposite end of the generative cell; this becomes associated with a preformed groove located on the surface of the vegetative nucleus. The generative cell subsequently separates from the intine near the vegetative nucleus and moves progressively toward the opposite end of the cell; during this separation, the edge of the wall facing the intine becomes callose-positive and remains so until separating from the intine. The generative cell becomes a free cell within the pollen, which is in physical association with the vegetative nucleus. Generative cell organization and organelle content become increasingly polarized during maturation, with microtubules evident both in the elongating protuberance of the generative cell and in association with organelles. The generative nucleus migrates away from the vegetative nucleus and toward the plastid-rich end of the generative cell, whereas mitochondria are more generally distributed within the cell. Generative cell polarization is made permanent during mitotic division and cytokinesis, i.e., two sperm cells differing in morphology are formed: the larger cell associated with the vegetative nucleus (S_vn) contains a majority of the mitochondria, and the smaller, unassociated sperm cell (S_ua) receives the plastids.     

黄芪的胚胎学研究Ⅰ、雌雄配子体发育

通过光学显微镜对黄芪雌雄配子体的发育过程进行观察, 同时对花粉发育进行细胞化学实验, 其主要结果如下;1.花粉第一次有丝分裂形成一个较小的半球形生殖细胞和一个较大的营养细胞。2.生殖细胞发育过程中存在暂短的细胞壁, 经PAS反应和苯胺兰荧光显微反应鉴定均为负反应。即:生殖细胞壁并未显示出纤维素或胼胝质性质的壁。3.营养细胞与生殖细胞之间壁的解体及生殖细胞进入营养细胞的过程。4.成熟花粉中的生殖核为孚尔根反应强阳性, 营养核为弱的正反应。5.雌配子体发育起源于珠心组织亚表皮下的孢原细胞其中只有一个孢原直接发育成为蓼型胚囊。 文中对黄芪花蕾外部形态及其内部雌雄配子体的发育作了相关性比较。     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

The formation of the generative cell in Polystachia pubescens (Orchidaceae)

Summary The formation and nature of the generative cell wall and the detachment mode of the generative cell from the intine in Polystachia pubescens were observed by LM and TEM. Vesicles evenly positioned within the phragmoplast fuse to form a cell plate that divides the microspore into the generative and vegetative cell. This cell plate consists of callose. Before the generative cell leaves the intine, however, the callose is completely resorbed and is not replaced by any other substance. The generative cell becomes detached from the intine by moving towards the centre of the pollen grain. A constriction formed thereby gives the generative cell a bulb-like appearance and leads ultimately to the generative cell being pinched off. Plasma-filled vesicles originating from the generative cell remain between the intine and the plasma membrane of the vegetative cell.     

洋葱花粉发育过程中ATP酶的分布特征(简报)

在真核细胞中,除了线粒体和叶绿体ATPase的功能是合成ATP外,其余部位ATPase是水解ATP以获取生物能量的代谢酶,在生物体细胞内广泛存在。探索ATPase在细胞中的分布状态是研究细胞生理状态的一种重要手段。ATPase在细胞中的多少可反映出细胞当时的生活状态,这一特征已被初步用于探索小麦和水稻雄性不育的细胞生物学研究中,希望通过比较可育花药和不育花药中ATPase的分布差异寻找雄性不育的机理,发现     

Pollen ultrastructure in anther cultures of Datura innoxia. III. Incomplete microspore division.

During the microspore division in Datura innoxia, the mitotic spindle is oriented in planes both perpendicular (PE) and oblique (OB) to the spore wall against which the nucleus is situated. However, irrespective of polarity, the usual type of hemispherical wall is laid down at cytokinesis and isolates the generative cell from the rest of the pollen grain (type A). In PE spores the vegetative nucleus initially occupies a central position in the pollen grain, whereas in OB spores the vegetative nucleus lies at the periphery of the grain close to the generative cell. In anther cultures initiated just before the microspore division is due to take place, no marked change can be observed in either orientation or symmetry of the mitotic spindle when the spores divide. In some, however, cytokinesis is disrupted and deposition of the hemispherical wall arrested. In the absence of a complete wall, differentiation of the generative cell cannot take place and binucleate pollen grains are formed having 2 vegetative-type nuclei (type B). The 2 nuclei in the B pollens are always situated against the pollen-grain wall, suggesting that the disruption phenomenon is related to the OB spores. The incomplete wall always makes contact with the intine on the intine-side of the spindle. Wall material may be represented merely as short stubs projecting out from the intine into the cytoplasm, in which event the 2 nuclei lie close to each other and are separated by only a narrow zone of cytoplasm. In other grains the wall is partially developed between the nuclei and terminates at varying distances from the tonoplast; in these, the nuclei are separated by a wider zone of cytoplasm. The significance of these binucleate grains in pollen embryogenesis is discussed.     

Orchid embryology: Pollen tetrads of Epidendrum scutella in the anther and on the stigma

Summary The pollinium of Epidendrum scutella, both in the anther and on the stigma, was examined with the electron microscope. The sporoderm of the outer tetrads has a sexine and an intine while that of the inner tetrads lacks a sexine, and an intine is formed only after the pollinium is on the stigma. A fibrous wall layer apparently holds the tetrads together. The cytoplasm is filled with plastids, mitochondria, polysomes, vacuoles and vesicles of various sizes, and endoplasmic reticulum (ER) with narrow cisternae. The vegetative nucleus is oval in form and contains a large nucleolus. The generative nucleus is deeply lobed and contains a well-developed nucleolus. The generative cytoplasm lacks both plastids and mitochondria and has little ER. Dictyosomes are present as well as assorted vesicles. A pocket is present between the plasma membrane of the generative cell and the wall; it contains assorted membranes and ribosome-like particles. After the pollen is on the stigma the wall surrounding the generative cell begins to disappear and gaps develop in it.This investigation was carried out during the tenure of a research fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Research supported by grants from National Science Foundation (GB-3460) and the Miller Institute for Basis Science at the University of California, Berkeley. The authors would like to thank Miss Mary Ashton for her assistance in the research.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.     

Ultrastructural and Cytochemical Study on Mature Pollen of Pinus tabulaeformis

The pollen of Pinus tabulaeformis Cart. comprised two prothallial cells, a generative cell and a tube cell which degenerated at pollen maturation. The generative cell had its own cell wall, seperating from the intine of pollen, but with its side wall attached to the infine. Cytoplasmic channels were present on the side of the generative cell wall, which faced to the tube cell cytoplasm. The generative cell differed conspicuously from the tube cell. The main differences include: ( 1 ) The chromatin in the generative cell nucleus was condensed, but was dispersed and had numerous nueleare pores in the tube cell nucleus; (2)There was no microbody in the generative cell but many microbodies were present in the tube cell cytoplasm; (3)More inclusions were present in the tube cell than in the generative cell. Both the generative cell and the tube cells contained lipid bodies and amyloplasts in the cytoplasm, but there were more amyloplasts in the former. The tube cell also contained a few proteins which was absent in the generative cell. In addition, there were numerous mitochondria, polyribosomes, and a few endoplasmic reticulums and dictyosomes in the generative and tube cells. DAPI staining demonstrated numerous cytoplasmic DNA in both generative cell and tube cell. The mode of cytoplasmic inheritance, and the composition, structure and the nature of the pollen wall of P. tabulaefonnis are also discussed in this paper.     

Ultrastructure of Male Gametophyte in Wheat II. Formation and Development of Sperm Cell

Ultrastructural events in wheat sperm cell development were examined from the division of generative cell stage to the maturation of sperm cell in pollen grains. The results are smnmarized as follows: 1. The generative cell in forming microspore by mitosis goes through a series of changes including tile displacement and transformation. It finally becomes a spindle-shaped cell getting ready for another mitosis. The generative cell at this stage is naked. it is only surrounded by both membranes of its own and vegetative cell Most part of the generative cell is occupied by the conspicuous elliptical nucleus with highly condensed chromatin. With the exception of ribosomes, the organelles in the thin layer of generativc cell cytoplasm are obviously fewer and smaller than those in the vegetative cytoplasm. The mierotubules may also be seen in the cytoplasm of spindle-shaped generative cell parallel to the long axis of the cell. There is no amyloplast in generative cell. 2. When the generative cell has moved to the position close to the vegetative nucleus again, it begins to divide. The formation of sperm cells as the result of mitosis of generative cell, and the development of sperm cell involves the following main changes. The shape of the sperm cell tranforms from spherical to elliptical, finally it forms an elongated cell with a tail-like structure. At the sametime, the distribution of cytoplasm gradually concentrated at one end of the sperm cell to form the cytoplasmic extension, so that the so called "tail" of the sperm cell is formed. There are more organelles, especially the mitochondria, assembling in this part. The sperm cell just formed after mitosis is naked and the enclosed plasma membrane is discontinuous. The sperm cell membrane is enclosed by vegetative cell membrane, and the double membranes may be completed at a later stage. It is considered that the period which follows is very short, the deposition of wall material, the callose, occurs to fill up continuously the space between two membranes, but soon after this period the cell wall becomes discontinuous and the wall material is obviously decreased. The significance of the position of the generative cell before its mitosis and the morphological changes during the development of the sperm cell are discussed in this paper.     

An Ultrastructural Study on Pollen Protoplasts and Pollen Tubes Germinated From Them in Gladiolus gandavensis

Large quantities of protoplasts were isolated enzymatically from the mature pollen grains in Gladiolus gandavensis. Regeneration of cell wall and germination of pollen tubes were performed during culture of purified pollen protoplasts in Ks medium supplemented with 32% sucrose, 0.1 mg/1 2,4-D, 1 mg/1 NAA and 0.2 mg/1 6-BA, with a germination rate up to 47.7%. The materials were fixed gently with gradually increasing concentration of glutaraldehyde, followed by osmium, then preembedded in a thin layer of agar and surveyed under an inverted microscope so as to select desired specimens for subsequent procedure. Small agar blocks containing specimens were dehydrated through ethanal-propylene oxide series, embedded in Araldite and ultratomed. Electron microscopic observations show that the pollen protoplasts are surrounded by a smooth plasma membrane and with ultrastructurally intact cytoplasm, a vegetative nucleus and a generative cell. After 8h of culture, wall regeneration commences resulting in a multilayered, fibrillar wall structure which is different from the intine. No exine is formed. Numerous vesicles participate actively in the wall formation. The wall is uneven in thickness around its periphery; a thickened area somewhat resembling to germ furrow is formed, from which pollen tube emerges. The tubes contain abundant plastids, mitochondria and dictyosomes. Vesicles are released out of the plasma membrane and involved in tube wall formation. After 18h of culture, the vegetative nucleus and generative cell have migrated into the tube. Technical points of preparing pollen protoplast specimens for ultastructural studies and the fearnres of wall regeneration in pollen protoplast culture are discussed.     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.     

Intracellular motility and the evolution of the actin cytoskeleton during development of the male gametophyte of wheat (Triticum aestivum L.)

The uniaperturate pollen of wheat is dispersed in a partially hydrated condition. Amyloplasts are concentrated in the apertural hemisphere where they surround the two sperms, while vigorously moving polysaccharide-containing wall precursor bodies (P-particles) together with the vegetative nucleus occupy the other. This disposition is the product of a post-meiotic developmental sequence apparently peculiar to the grasses. During vacuolation of the spore after release from the tetrad, the nucleus is displaced to the pole of the cell opposite the site of the germination aperture, already defined in the tetrad. Following pollen mitosis, the vegetative nucleus migrates along the wall of the vegetative cell towards the aperture, leaving the generative cell at the opposite pole isolated by a callose wall. As the vacuole is resorbed, the generative cell rounds up, loses its wall and follows the vegetative nucleus, passing along the wall of the vegetative cell towards the aperture where it eventually divides to produce the two sperms. Throughout this period of nucleus and cell manoeuvrings, minor inclusions of the vegetative cell cytoplasm, including mitochondria, lipid globuli and developing amyloplasts, move randomly. Coordinated vectorial movement begins after the main period of starch accumulation, when the amyloplasts migrate individually into the apertural hemisphere of the grain, a final redistribution betokening the attainment of germinability. In the present paper we correlate aspects of the evolution of the actin cytoskeleton with these events in the developing grain, and relate the observations to published evidence from another monocotyledonous species concerning the timing of the expression of actin genes during male gametophyte development, as revealed in the synthesis of actin mRNA.     

Ultrastructural study of the relationship between generative and vegetative cells inMagnolia ×soulangeana Soul.-Bod. pollen grains

Summary InMagnolia ×soulangeana pollen grains the generative cell (GC) does not become totally free within the vegetative cell (VC), at least until the pollen tube emergence. Due to a deviation in its detachment process from the sporoderm, the opposing ends of the VC plasmalemma do not fuse themselves when the GC moves away from the intine. Consequently, the interplasmalemmic space surrounding the GC does not become isolated but rather maintains continuity with the sporoderm through a complex formation that we have called plasmalemmic cord. The real existence of this formation was confirmed through serial sectioning showing the plasmalemmic cord to consist of the VC plasmalemma. In its initial portion it is occupied by a reasonably accentuated wall ingrowth of the inner layer of the intine (intine 3). In the remainder portion, neither of the cytochemical tests used in this work have revealed the presence of a significant amount of wall material. However, ultrathin sections of samples processed either chemically or by cryofixation showed the existence of an intricate system of tubules and vesicles, some of which are evaginations of the VC plasmalemma. The hypothesis that the plasmalemmic cord may have a role in the complex interactions between the two pollen cells is discussed.     

Microsporogenesis,Megasporogenesis and the Formation of Male and Female Gametophyte in Coix lacryma-jobi L.

The development of the anther wall follows the monocotyledonous type. During meiotic stages of prophase I, some cytoplasmic channels are observed on the walls between meiotic cells. which divide synchronously. Cytokinesis in the microspore mother cell is of the successive type and gives rise to iscbilateral tetrad. The cell wall between the generative cell and the vegetative cell in early stage shows PAS positive reaction. The mature pollen grain is of 3-celled type. The development of the female gametophyte follows the polygonum-type; the antipodal cells proliferate to form a multicellular tissue mass. Many starch grains are present in the central cell. The nucleus of the mature egg cell is located at the micropylar end; a great deal of starch grains are in the cytoplasm surrounding the nucleus, while vacuoles of various size distribute throughout its cytoplasm but are more and larger at the chalaza,1 end. The nucleus of the synergid cell is located at the micropytar end where a filiform apparatus is formed and many small vacuoles are present at the chalazal part.     

水稻花药发育过程中腺苷三磷酸酶的分布

水稻花粉母细胞中的ATP酶反应颗粒很少,主要分布在细胞核中。组成花药药壁的4层细胞中只有绒毡层细胞核中有较多的ATP酶。减数分裂后,绒毡层细胞质中分化出许多内质网片层,但ATP酶反应颗粒仍很少,其它3层药壁细胞中质膜ATP酶明显增加。在花粉内、外壁中形成了大量的ATP酶反应颗粒,但花粉外壁在小孢子时期形成,ATP酶反应颗粒来自绒毡层细胞的鸟氏体。花粉内壁在二胞花粉时期形成,其中的ATP酶反应颗粒来自花粉营养细胞。二胞花粉的营养细胞比生殖细胞含有更多的ATP酶反应颗粒。     

Electron Microscope Observation of Microspore Division of Wheat in Vitro

First mitosis of wheat microspores in anther culture was studied by electron microscope. The division types of the most pollen grains were unequal (A pathway) and that of others were equal (B pathway). The characteristics of unequal division of microspores in vitro in contrast with in Vivo were as follows: (1) Phragmoplast and “phragmoplast-pla- smalemma complex” were of occurrence after nucleus division but new cell wall could not form between two daughter nuclei. (2) Generative cells were various in size, shape and amount of cytoplasmic organelles. (3) Generative cell could attach to intine at all times and underwent sporophyte division there. "Phragmoplast-plasmalemma complex" surrounding generative cell did not disappear even after generative cell detached from the intine, so that there was always an obvious demarcation line between derivative nuclei of generative and vegetative nucleus.     

ULTRASTRUCTURAL COMPARISION OF POLLEN GRAINS FROM 2n, 3n,AND 4n PLANTS OF EUPHORBIA DULCIS

Pollen development in plants with different ploidy levels of Euphorbia dulcis is similar but some ultrastructural differences do occur. In pollen of diploid plants large aggregations of rough endoplasmic reticulum [RER] are attached to the pollen wall near the young generative cell but such aggregations are not present in other karyotypes. Plastids are detected only in young generative cells of triploid plants. In diploid plants the generative cell becomes spindle-shaped, in triploid and tetraploid plants it remains round during the movement from the pollen wall to the center of the vegetative cell. The intine surrounding the generative cell in 3n plants is thinner than that found in 2n and 4n plants. Pollen grains in tetraploid plants are twice as large as those in diploid plants. Pollen viability is 90% in 2n plants, but only 10% in 4n plants.