Cotton embryogenesis: The pollen cytoplasm

Summary The ultrastructure and composition of cotton (Gossypium hirsutum) pollen, exclusive of the wall, was examined immediately before and after germination. The pollen grain before germination consists of two parts: the outer layer and a central core. The outer layer contains large numbers of mitochondria and dictyosomes as well as endoplasmic reticulum (ER). The core contains units made of spherical pockets of ER which are lined with lipid droplets and filled with small vesicles; the ER is rich in protein and may contain carbohydrate while the vesicles are filled with carbohydrate. Starch-containing plastids are also present in the core as are small vacuoles. The cytoplasm of the pore regions contains many 0.5 spherical bodies containing carbohydrate. After germination the ER pockets open and the lipid droplets and small vesicles mix with the other portions of the cytoplasm. With germination the pore region becomes filled with mitochondria and small vesicles. The vegetative nucleus is large, extremely dense and contains invaginations filled with coils of ER. A greatly reduced nucleolus is present in the generative cell which is surrounded by a carbohydrate wall. The cytoplasm of the generative cell is dense and contains many ribosomes, a few dictyosomes and mitochondria, many vesicles of several sizes, and some ER. No plastids were identified. The generative nucleus is also dense with masses of DNA clumped near the nuclear membrane. An unusual tubular structure of unknown origin or function was observed in the generative cell.     

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.     

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF THE SYNERGIDS OF COTTON

The composition and ultrastructure of the synergids of cotton were studied. The cells were found to be surrounded by a partial wall composed of cellulose, hemicellulose, and pectins. The structure of the wall was observed to consist of an unusual fibrillar arrangement. The filiform apparatus was demonstrated to be an extension of the wall at the micropylar end of the cell. Large amounts of ER surround the filiform apparatus. Also associated with the latter are large numbers of plastids and mitochondria. The nucleus is large and contains a single, large nucleolus and, frequently, 1 or more micronucleoli. The nuclear membrane contains membrane-bound vesicles but has few extensions into the cytoplasm. The ER is oriented parallel to the long axis of the cell and decreases in concentration from the micropylar to the chalazal end of the cell. Dictyosomes are common throughout the cell but are more numerous in the midportion where they are closely associated with the ER. The chalazal end of the cell is occupied by vacuoles rich in an inorganic compound which leaves a considerable residue of ash. Spherosome-like bodies are common throughout the cell. Both the plastids and mitochondria show evidence of division. Ribosomes are numerous and are both free and associated with the ER, nucleus, plastids, and mitochondria. The function of the synergids is proposed to be the absorption, storage, and transport of compounds from the nucellus. On the basis of this function, it is suggested that the synergids act by providing material to the egg and the developing embryo and endosperm and that they are involved in the growth of the pollen tube into the embryo sac.     

Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum

Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial slow and fast lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.Abbrevations CF chemical fixation - ER endoplasmic reticulum - GC generative cell - MF microfilament - MT microtubule - PD plasmodesmata - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution - VN vegetative nucleus     

Ultrastructure of the sperms ofPlumbago zeylanica

Summary Male gametes ofPlumbago zeylanica were examined in pollen grains and tubes using light and electron microscopy of chemically and physically fixed tissues, and Nomarski interference microscopy of isolated, living sperm cells. Male gametes are elongate, spindleshaped cells containing a nucleus, mitochondria, ER, ribosomes, vesicles, dictyosomes, probable microfilaments, and a variable number of plastids. In mature pollen grains ofP. zeylanica, the two sperm cells are directly linked; they share a transverse cell wall with plasmodesmata and are enclosed together by the inner vegetative cell plasma membrane. One of these two sperms is also associated with the vegetative nucleus as a consistent feature of pollen grain organization. The basis of this association appears to be a long, narrow projection of the sperm cell (averaging < 1 m wide and about 30 m long) which wraps around the periphery of the vegetative nucleus and occupies embayments of that nucleus. This association is maintained throughout pollen tube growth but becomes less extensive near the completion of tube growth and is severed following tube discharge. The consistent occurrence of the sperm-vegetative nucleus association in pollen grains, tubes and isolated pollen cytoplasm suggests that the two structures may be directly connected, but attempts to visualize this type of connection were unsuccessful. Possibly, the entwining nature and extent of complementary interfaces between vegetative nucleus and sperm may have a role in stabilizing their association. Functionally, the two sperms and vegetative nucleus appear to travel as a linked unit within the pollen tube, possibly increasing the effectiveness of gamete delivery and helping to ensure nearly simultaneous transmission of sperms into the receptive megagametophyte.     

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.     

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.     

Fertilization in Nicotiana tabacum: ultrastructural organization of propane-jet-frozen embryo sacs in vivo

Ovules of Nicotiana tabacum L. were cryofixed with a propane-jet freezer and freeze-substituted in acetone to examine technique-dependent changes in pre- and post-fertilization embryo sacs using rapidly frozen material. Freezing quality was acceptable in 10% of the embryo sacs in the partially dissected ovules, with ice-crystal damage frequently evident in vacuoles and nuclei. One of the two synergids begins to degenerate before pollen-tube arrival in cryofixed material, with breakdown of the plasma membrane and large chalazal vacuole delayed until the penetration of the pollen tube. Early synergid degeneration involved characteristic increases in cytoplasmic electron density and the generation of cytoplasmic bodies to the intercellular space through “pinching-off”. Upon pollen-tube arrival, the male gametes are released through a terminal aperture into the degenerate synergid. Sperm cells undergo morphological alteration before gametic fusion: their mitochondrial electron density increases, the endoplasmic reticulum dilates, cytoplasm becomes finely vacuolated and the surrounding pollen plasma membrane is lost, causing the sperm cells and vegetative nucleus to dissociate. Discharge of the pollen tube results in the formation of numerous enucleated cytoplasmic bodies which are either stripped or shed from sperm cells and pollen-tube cytoplasm. Two so-called X-bodies are found in the degenerate synergid after pollen-tube penetration: the presumed vegetative nucleus occurs at the chalazal end and the presumed synergid nucleus near the micropylar end.     

A re-examination of the morphology and reproduction of Nothocladus lindaueri (Batrachospermales, Rhodophyta)

The vegetative morphology and reproduction of the freshwater rhodophyte Nothocladus lindaueri Skuja [=Batrachospermum lindaueri (Skuja) Necchi et Entwisle] were examined by light and electron microscopy. It was confirmed that this alga has a typical batrachospermalean pit plug with two cap layers, the outer one of which is domed. During elongation of hair cells, the primary wall is broken, forming a basal collar. Hair cells have a single nucleus and abundant Golgi bodies, en-doplasmic reticula (ER) and vesicles. Dividing apical cells of the fascicles have a nucleus with art adjacent zone of exclusion, the latter containing a single polar ring. Branched trichogynes and fertilized carpogonia are shown for the first time in this species. Carpogonial branch and involucral cells contain a prominent axial nucleus, proplastids, ER and vesicles. The pit plugs disintegrate among these cells leaving open pit connections. Carpogonia have plentiful mitochondria and vesicles. The wall at the trichogyne apex is thickened and densely stained. The carposporophyte centre consists of a mass of fusion cells with open pit connections, and indeterminate gonimoblast filaments arise from this mass. The combination of a symmetrical carpogonial base, a carposporophyte centre consisting of a mass of fusion cells, and exclusively indeterminate gonimoblast filaments appears to be unique among the members of the Batrachospermaceae. The specimen of N. lindaueri contains epiphytic filaments of Audouinella meiospora producing both spermatangia and monosporangia. Spermatium formation in N. lindaueri remains unknown.     

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.     

The growth of the pollen tube wall in Oenothera organensis.

The growth of the pollen tube wall of Oenothera is effected by the expulsion of fibrillar material from the cytoplasm into the developing wall. This material may also be seen in the cytoplasm, contained in membrane-bound vesicles. It is not clear how the content of the vesicles is discharged, but it appears not to involve the participation of microtubules. The source of the cytoplasmic fibrillar bodies depends upon the stage of development of the pollen tube. The earilest growth is derived from the inclusion into the wall of vesicles containing pre-formed materials present in the grain on pollination. During the next stage of growth the wall is derived from the content of double-membraned inclusions also present in the pollen. The content of the former vesicles is not so similar to the wall as the latter, but intermediates between the 2 types of vesicle may be seen in the cytoplasm, indicating that the former are formed from the latter. Most of the tube wall is derived from the products of dictyosomes in the pollen grain or tube. These dicytosomes are few in number and they must be exceedingly active. This, and the observation that dictyosome vesicles are frequently associated with banked complexes of mitochondria, indicates that some steps in the metabolism of the vesicular content, perhaps phosphorylation, take place distant from the dicytosomes. These different sources of fibrillar material presumably permit the rapid starting of tube growth, without any attendant metabolism. However, it would be impossible to include enough pre-formed wall material in the grain to enable the full growth of the tube, so once started, it seems that the tube then relies on the elaboration of simple reserves for the contruction of its wall. These reserves are likely to be held in the pollen, and may be the large numbers of starch grains characteristic of the pollen cytoplasm.     

Light and electron microscopy studies of the oesophagus and crop epithelium in Aplysia depilans (Mollusca, Opisthobranchia)

The oesophagus and crop epithelium of Aplysia depilans consist in a single layer of columnar cells with apical microvilli, and some of them also possess cilia. Cell membrane invaginations, small vesicles, multivesicular bodies and many dense lysosomes were observed in the apical region of the cytoplasm. In most cells, a very large lipid droplet was observed above the nucleus and a smaller one was frequently found below the nucleus; glycogen granules are also present. Considering these ultrastructural features, it seems that these cells collect nutritive substances from the lumen by endocytosis, digest them in the apical lysosomes and store the resulting products. The cell bodies of mucus secreting flask-shaped cells are subepithelial in the oesophagus and intraepithelial in the crop. Histochemistry methods showed that the secretion stored in these cells contains acidic polysaccharides. Secretory vesicles with thin electron-dense filaments scattered in an electron-lucent background fill most of these cells, and the basal nucleus is surrounded by dilated rough endoplasmic reticulum cisternae containing small tubular structures. Considering the relatively low number of secretory cells, mucus production cannot be high. Moreover, since protein secreting cells were not observed in either oesophagus or crop, extracellular digestion in the lumen of these anterior segments of the digestive tract most probably depend on the enzymes secreted by the salivary and digestive glands.     

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.     

Paramural bodies of Albugo candida

The paramural bodies of Albugo candida were formed solely by elaboration of the plasmalemma. Two major forms were recognized: one consisting of plasmalemmal invaginations projecting into the cytoplasm; the other appearing like a pocket containing a number of vesicles and tubules. It is suggested that the first is the basic form of paramural body. In sporangia the paramural bodies break away from the plasmalemma and undergo autodigestion while in vegetative hyphae their tubules and lamellae break up into vesicles that are finally sequestered into the wall.     

地钱卵发育超微结构和细胞化学的研究

采用电镜和细胞化学技术对地钱(Marchantia polymorpha)卵发生过程进行了研究,根据卵发生过程中细胞化学和超微结构特征可将卵发育过程分为幼卵、中期卵和成熟卵3个阶段.幼卵阶段,卵细胞、腹沟细胞及颈沟细胞间有发达的胞间连丝,但卵与腹沟细胞间的胞间连丝很快退化,幼卵细胞内具大量透明的囊泡,均匀分布于细胞质中;卵发育中期,突出特征是卵细胞质内产生嗜锇性的脂滴,位于囊泡中,与此同时,腹沟细胞退化,其细胞质内产生大型囊泡,囊泡内分泌物与卵细胞外的物质类似,呈PAS反应阳性,表明该物质应为多糖类;卵成熟时,腹沟细胞和颈沟细胞完全退化,卵细胞外包被大量粘性多糖类物质,卵细胞核表面不规则,产生明显的核外突,众多的小泡围绕着细胞核,脂滴聚集成簇,卵细胞内其他细胞器不易区分.卵发育过程中,质体不含淀粉粒,线粒体退化,高尔基体相对发达.地钱卵发育的这些特征显著区分于蕨类植物.     

棉花花粉水合和萌发时超微结构的变化：着重论述内质网和被膜小泡

棉花(Gossypium hirsutum L.)花粉在授粉后水合至萌发时期的营养细胞中贮藏的大量淀粉粒和脂体被动用。超微结构的观察表明,首先是造粉质体中的淀粉粒降解,尔后是脂体。在花粉水合至萌发时期,营养细胞中内质网和高尔基体十分活跃,并含丰富的被膜小泡。内质网的构型发生明显的变化:花粉刚水合时内质网潴泡高度扩张,不同程度扩张的内质网潴泡连续成网状并折迭形成许多囊袋状结构单位,其中包含造粉质体、脂体和被膜小泡群;其后,内质网潴泡形成的囊袋状结构消失,变为分支互通的网状结构;至萌发时,内质网潴泡略为扩张,有些连续成简单的网状,有些呈游离的囊泡状。被膜小泡始终是成群地分布,并与脂体联结,当脂体降解时一些被膜小泡与之融合。根据棉花花粉在水合至萌发时期,营养细胞质中存在独特形态的内质网系统和含丰富的被膜小泡,它们的动态行为及与淀粉和脂体的转化和降解之间的密切关系,讨论了这两种细胞器可能的功能。     

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     

Ultrastructure of Characiochloris acuminata Lee et Bold

The ultrastructure of the vegetative cell and zoospore of Characiochloris acuminata Lee et Bold (Chlorangiellaceae, Tetrasporales, Chlorophyceae) is described.

The vegetative cell is distinctive in having numerous contractile vacuoles which are randomly distributed in the cytoplasm and visible through the fissures of the parietal chloroplast. A single pyrenoid, embedded in the chloroplast, is penetrated by cytoplasmic canals which are lined by the chloroplast envelope. The vegetative cell is attached to the substrate or host by two flagellar remnants (retained from the zoospore stage), each of which is ensheathed in a gelatinous tube through the cell wall at the cell base. The basal bodies are apparently abscissed from the flagellar shaft by a unit membrane which becomes continuous with the plasma membrane.

The zoospore is biflagellate, with the flagella equal in length, smooth and longer than the cell body. The flagellar sheath is characteristically undulate and the two flagellar bases are connected by a dense interflagellar fibre. The large nucleus has a conspicuously inflated nuclear envelope and the pyrenoid is similar to that of the vegetative cell.     

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.