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.     

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.     

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.     

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.     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 2. Quantitative cell and organelle dynamics of the male reproductive cell lineage

Quantitative cell and organelle dynamics of the male gamete-producing lineage of Plumbago zeylanica were examined using serial transmission electron microscopic reconstruction at five stages of development from generative cell inception to sperm cell maturity. The founder population of generative cell organelles includes an average of 3.88 plastids, 54.9 mitochondria, and 3.7 vacuoles. During development the volume of the pollen grain increases from 6,200 μm³ in early microspores to 115,000 μm³ at anthesis, cell volume of the male germ lineage decreases more than 67% from 362.3 μm³ to 118.4 μm³. By the time the generative cell separates from the intine, plastid numbers increase by >600%, mitochondria by 250%, and vesicles by 43 times. A cellular projection elongates toward and establishes an association with the vegetative nucleus; this leading edge contains plastids and numerous mitochondria. When the generative cell completes its separation from the intine, organellar polarity is reversed and plastids migrate to the opposite pole of the cell. Cytoplasmic microtubules are common in association with cellular organelles. Plastids accumulate at the distal end of the cell as a linked mass, apparently adhered by lateral electron dense regions. Before division of the highly polarized generative cell, plastids decrease in number by 16%, whereas mitochondria increase by ∼90% and vacuoles increase by ∼140% from the prior stage. After mitosis, the resultant sperm cells differ in size and organelle content. The sperm cell associated with the vegetative nucleus (S_vn) contains 62.7% of the cytoplasm volume, 87% of the mitochondria, 280.4 vesicles (79% of those in the generative cell), and 0.6% of the plastids. At maturity, the S_vn mitochondria increase by 31% and the cell contains an average of 0.4 plastids, 158.9 vesicles, and 0.36 microbodies. The mature unassociated sperm (S_ua) contains 39.8 mitochondria (up 3.3%), 24.3 plastids (down 31%), 91.1 vesicles (up 54.9%), and 3.18 microbodies. The small number of organelles initially in the generative cell, followed by their rapid multiplication in a shrinking cytoplasm suggests a highly competitive cytoplasmic environment that would tend to eliminate residual organellar heterogeneity. Cell and cytoplasmic volumes vary as a consequence of fluctuations in the number and size of large vesicles or vacuoles, as well as loss of cytoplasmic volume by (1) formation of “false cells” involving amitotic cytokinesis, (2) “pinching off” of cytoplasm, and (3) dehydration of pollen contents prior to anthesis.     

The Development and Structure of the Generative Cell Wall in Polygonatum simizui

The developmental structure and components of the generative cell wall in Polygonatum sirnizui Kitag were studied by means of cytochemical and electron microscope observation. The early generative cell wall separating the generative and vegetative cytoplasm contains callose and cellulose. From the time when the generative cell detaches from the intine untill it is freely suspended in the cytoplasm of the vegetative cell, the wall becomes progressively thinner and does not show the specific fluorescence when stained with aniline blue and cai- cofluor white although it remains PAS positive. At later developmental stage when the generative cell moves into the pollen tube but before its initiation of mitosis, an envelope with weak PAS positive reaction appears on the surface of the cell. Its morphological nature is similar to that of the sperm cell discribed as the "periplasm”. This study proves that a cell wall is present in the generative cell of Polygonatum simizui throughout the developmental process, althrough changes in structure and components of the wall may occur. The properties of the generative cell wall at different stages, its significance in differentiation between generative and vegetative cytoplasm and translocation of nutrient materials, and the possible mechanism of the detachment of the generative cell from the intine are the subjects to discussion.     

栽培甜菜花粉发育过程的超微结构

利用透射电镜技术对栽培甜菜(Beta vulgaris)花粉发育过程进行了超微结构观察。结果表明, 在小孢子母细胞减数分裂期间, 细胞内发生了“细胞质改组”, 主要表现在核糖体减少, 质体和线粒体结构发生了规律性变化。末期I 不形成细胞板,而是在2个子核间形成“细胞器带”。“细胞器带”的存在起到类似细胞板的作用, 暂时将细胞质分隔成两部分。四分体呈四面体型, 被胼胝质壁包围。小孢子外壁的沉积始于四分体晚期, 至小孢子晚期外壁已基本发育完全。单核小孢子时期, 细胞核大, 细胞器丰富。二细胞花粉发育主要表现在生殖细胞壁的变化上, 生殖细胞壁上不具有胞间连丝。成熟花粉为三细胞型, 含有1个营养细胞和2个精细胞。精细胞具有短尾突, 无壁, 为裸细胞, 每个精细胞通过2层质膜与营养细胞的细胞质分开。生殖细胞与精细胞里缺乏质体。     

栽培甜菜花粉发育过程的超微结构

利用透射电镜技术对栽培甜菜（Beta vuigaris）花粉发育过程进行了超微结构观察。结果表明,在小孢子母细胞减数分裂期间,细胞内发生了“细胞质改组”,主要表现在核糖体减少,质体和线粒体结构发生了规律性变化。末期1不形成细胞板,而是在2个子核间形成“细胞器带”。“细胞器带”的存在起到类似细胞板的作用,暂时将细胞质分隔成两部分。四分体呈四面体型,被胼胝质壁包围。小孢子外壁的沉积始于四分体晚期,至小孢子晚期外壁已基本发育完全。单核小孢子时期,细胞核大,细胞器丰富。二细胞花粉发育主要表现在生殖细胞壁的变化上,生殖细胞壁上不具有胞间连丝。成熟花粉为三细胞型,含有1个营养细胞和2个精细胞。精细胞具有短尾突,无壁,为裸细胞,每个精细胞通过2层质膜与营养细胞的细胞质分开。生殖细胞与精细胞里缺乏质体。     

Male reproductive cell development in Nicotiana tabacum: male germ unit associations and quantitative cytology during sperm maturation

Generative and sperm cells were examined at four stages of development from generative cell formation to sperm cell maturation using serial transmission electron microscopy. The generative cell and vegetative nucleus are associated in a male germ unit association during pollen maturation and tube elongation, except for generative cell mitosis. At late stages of prophase, this association loosens; the generative cell separates from the vegetative nucleus at metaphase. Slender, unbranched, or occasionally branched projections may be found at one or both ends of the generative cell, or they may be single, blunt, and short. Slender projections are rare during anaphase and telophase. The vegetative nucleus moves back into apposition with one sperm cell at the end of mitosis. During the re-establishment of the association, the vegetative nucleus first touches the end of the leading sperm cell and then moves next to the middle of the sperm nucleus. As the sperm cells enter interphase, a conventional association is re-established between one cell and the vegetative nucleus through one or more long and slender cytoplasmic extensions; these associations are maintained throughout later passage in the pollen tube. During maturation, a significant increase occurs in the surface area of the sperm cells (particularly in the sperm cell in association with the vegetative nucleus), and a lesser increase in nuclear volume and surface area. Other sperm cell parameters, including those of heritable organelles, remain unchanged during sperm cell maturation.     

Preliminary observations on the formation of the male germ unit in pollen tubes ofCyphomandra betacea sendt.

Summary The structure of the generative cell and its association with the vegetative nucleus in the pollen tube ofCyphomandra betacea Sendt. were observed with the electron microscope. The generative cell, bounded by its own plasma membrane and the inner plasma membrane of the vegetative cell, possesses the cytoplasmic extension which lies within the embayments of a vegetative nucleus. The generative cell contains the normal complement of organelles and, especially, microtubules which cluster into several groups adjacent to the plasma membrane, oriented along the longitudinal axis of the cell. In the pollen tube reaching the lower end of the style aftersemivivo pollination, both of the sperm cells are elongated and polyribosomes and microtubules are the outstanding feature in the cytoplasm. The two sperm cells are connected by a common transverse cell wall, while cytoplasmic channels exist in both the periplasm of the two sperm cells and the transverse wall. The leading sperm cell (S_vn) is closely associated with the vegetative nucleus. Thus the present study demonstrates the existence of the male germ unit in the pollen tube ofC. betacea. The possible cytoplasmic continuity between the sperm cells and between the gametes and vegetative cell is considered.Abbreviations S_vn sperm cell physically associated with the vegetative nucleus - S_ua sperm cell unassociated with the vegetative nucleus - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

The male germ unit in the pollen and pollen tubes ofPetunia hybrida: Ultrastructural,quantitative and three-dimensional features

Summary The male germ unit ofPetunia hybrida was examined quantitatively and qualitatively at the ultrastructutral level. Three-dimensional reconstructions, the determination of nuclear and cytoplasmic volumes and surface areas, and organelle counts were obtained from serial ultrathin sections and computer analysis. In the mature pollen grain, an elongated generative cell is found in direct physical association with and partially surrounded by the vegetative nucleus. The mature generative cell lacks plastids and has mitochondria equally distributed at both of its tapering ends. In the pollen tube, the sperm cells are physically associated by cytoplasmic connections to each other and to the surrounding vegetative cell membrane. At full style length, the lobed vegetative nucleus and sperm pair are found in close association near the end of the pollen tube. The two sperms of a pair are not strongly dimorphic.     

FINE-STRUCTURAL STUDY ON THE FORMATION OF THE GENERATIVE CELL WALL AND INTINE-3 LAYER IN A GROWING POLLEN GRAIN OF LILIUM LONGIFLORUM

At the end of mitosis in the lily pollen microspore, the fan-shaped cell plate gives rise to a cell wall delineating a hemispherical cell. At first, the cell wall of the newly formed generative cell and the intine-3 layer of the pollen grain wall are inseparable. Gradually, the wall of the generative cell near the pollen grain wall becomes thicker and wall segments are formed between the thickened zones, and these make a network system by which the generative cell becomes suspended and separated from the pollen grain wall. After the separation, the intine-3 layer is formed inside the intine 2. The generative cell wall and the intine-3 layer are formed by coated vesicles, polysaccharide particles and rough ER.     

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.     

Generative Cell Division and Sperm Cell Association in the Pollen Grain of Sambucus nigra

Generative cell division in tricellular pollen grains of Sambucusnigra L. (Caprifoliaceae) has been examined with light and electronmicroscopy. During division the generative cell is located inthe centre of the pollen grain, near to the nucleus of a surroundingvegetative cell. Conventional mitosis of the generative cellis followed by cytokinesis through centrifugal cell plate formation.Two sister sperm cells remain in spatial contact with each otherand are surrounded, as formerly their progenitor cell was, bythe vegetative cell. From the changes of shape of the generativecell during division and of the sperm cells it may be assumedthat the space between these cells and the vegetative one containsa labile, non-rigid, wall material. No plastids have been observedin the generative cell and its mitochondria appear to be unequallydistributed between the two future sperm cells during division. Sambucus nigra L., generative cell division, pollen, sperm cell association     

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.     

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.     

Isolation of generative cells from pollen of Phoenix dactylifera

Summary Generative cells in pollen of Phoenix dactylifera had a convoluted surface and a small cytoplasmic volume, and condensed chromatin in the nucleus. Treatment of hydrated pollen with pectinase followed by grinding in a hypotonic medium released the generative cells from pollen. Convolutions of the plasma-membrane surface in vivo were retained by generative cells in vitro. Generative cells had a much lower solute concentration than did vegetative cells, a property that explains why hypotonic shock was effective in releasing intact generative cells from pollen.     

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.     

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     

土麦冬离体萌发花粉管中生殖细胞与营养核的动态变化

主要报道了土麦冬人工培养萌发花粉管中生殖细胞与营养核的动态变化。多数花粉管中,生殖细胞与营养核贴合后,开始进行有丝分裂,贴合时,营养核略呈弥散状态。在分裂早中期,生殖细胞与营养核分开,从贴合到分开大约经历３－５ｈ,精子形成后,不与营养核连接。ＤＡＰＩ对生殖细胞的有丝分裂有抑制作用。少数花粉管中,生殖细胞核进行无丝分裂,有缢裂和劈裂两种方式。生殖细胞核发生缢裂的花粉管中,未观察到生殖细胞与营养核的贴