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.     

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 Structure of Pollen Wall and Its Relation to Po41en Aggregation in Cymbidium goeringii (Rchb. F.) Rchb. F.

The pollen wall of tetrads located in different positions of a mature pollinium of Cymbidium goeringii was examined with the electron microscope, and the compositions of wall materials were also tested with different histochemical methods. In all tetrads of a pollinium, the pollen wall can be distingished into an exine and an intine, but the exine may be varied greatly according to the tetrad position in a pollenium. The part of the pollen wall (the outer wall) of the external tetrads, lying close, to the tapetum, is composed of two layers, i.e. the exine, and the intine. Theexine consists of tectum, granulate ectexine and endexine, without foot layer. The intine is cellulose in nature. In the outer wall between different groups of: tetrads and in the inner wall within an individual tetrad, the structure of ectexine becomes simple and the deposition of sporopollenin is roduced The degree of reduction of ectexine nicreases from the outer to inner tetrads in several external layers of a pollinium, and even the internal tetrads have a reduced ectexine or lack of it. The present study also demonstrates that the mechanism of pollen aggregation into a pollinium is built on a combined effect of the following features: (1) connected bridges formed' by intine between two pollens within a tetrad, (2) formation of cytoplasmic channels between two pollens within a tetrad, (3) incomplete cell wall formation within a tetrad, (4) little size of tetrads and compact arrangement of mature tetrads and (5) a sticky viscin material surrounded on the outside of a pollinium.     

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.     

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.     

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 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.     

Cotton embryogenesis: The pollen tube in the stigma and style

Summary The ultrastructure and composition of the pollen tube of cotton (Gossypium hirsutum) growing in the tissues of the stigma and style of the flower were examined. The distal portion of the tube is densely cytoplasmic and contains the vegetative nucleus and the two sperms. The vegetative nucleus is highly convoluted and the membrane contains many pores and connections with the ER. No organized nucleolus is present but 4–6 membrane-bound, protein containing bodies are found in the nucleus. Mitochondria containing numerous cristae are abundant in the cytoplasm. Dictyosomes are also plentiful and are engaged in the production of many large vesicles. Rough ER is conspicuous and polysomes are found in the cytoplasm. Plastids are few in number, poorly developed, and contain little starch. Many uniform, small vesicles are found throughout the cytoplasm. Lipid bodies frequently with small vesicles associated with them are found in the tube. In the proximal region vacuoles form and the cytoplasm becomes pressed against the wall. In the transition zone the ER frequently becomes distended and filled with protein. The wall has two distinct layers: one strongly PAS positive, the other faintly PAS positive. The inner wall is apparently formed by the deposition of large dictyosome vesicles. Plug structure and development were studied.     

Structure and development of sieve cells in the primary phloem ofPinus resinosa

Summary The primary phloem consists mostly of sieve cells. Procambial cells and very young sieve cells contain all the components characteristic of young nucleate cells. Increase in wall thickness, which is relatively limited, constitutes the first indication of sieve-cell differentiation. During the period of wall thickening, the plastids develop starch grains and then fibrillar inclusions. Eventually the internal lamellae of the plastids collapse. The plastids do not form crystalline inclusions. As the sieve cell approaches maturity, an extensive network of smooth, tubular endoplasmic reticulum (ER) appears and then becomes mostly parietal in distribution. At maturity, large aggregates of this ER occur at the sieve areas. These aggregates are interconnected longitudinally by the parietal network of ER. In addition to the ER, the mature, plasmalemma-lined primary sieve cell contains a degenerate nucleus, with intact nuclear envelope, plastids, and mitochondria. Dictyosomes, ribosomes, and vacuoles are lacking. P-protein is not present at any stage of development.This work was supported by U.S. National Science Foundation grants GB 8330 and GB 31417 to R. F.Evert.     

Immunocytochemical localization of the allergenic proteins in the pollen of Cryptomeria japonica

Applying an immunocytochemical method, a localization of the protein Cry j I in the Cryptomeria japonica pollen, which is the major allergen responsible for Japanese cedar pollinosis, is investigated with the monoclonal and polyclonal antibodies produced from the protein. The protein that reacts to the polyclonal antibody localizes on the sexine, nexine, between nexine and intine layers, orbicles, cell wall of a generative cell, Golgi body and Golgi vesicles. The allergenic protein contained in the exine and orbicles of Japanese cedar pollen can diffuse or dissolve easily from there into the mucus covering of the eye and nose, causing a response in less than 1 min after exposure. Since the orbicles have a diameter of about 430 nm, they can pass easily through the pores of most protective masks to reach the sensitive tissues of the patient. The proteins react to the monoclonal antibodies (J1BO1 and J1BO7) and localize on the Golgi body, sexine, nexine and orbicles (but not between the nexine and intine layers), and on the generative cell wall. In the young pollen grain, numerous allergenic protein particles contained in the orbicles and sexine layer, but there is only a small amount of the protein between the nexine and intine layers, since the intine layer is not yet complete at this stage. More will be accumulated there during developmental maturation. The allergenic protein is also found on the tapetal materials remaining in the young anther. Since the materials forming the exine layer and orbicles come from tapetal tissue, it is assumed that some of the allergenic protein is produced in the tapetum and localized in the orbicles and pollen wall during maturation, and that the rest of the allergenic protein is produced in the Golgi body in the mature pollen grain.     

Pre-fertilization ovule development inCapsella: ultrastructure and ultracytochemical localization of acid phosphatase in the meiocyte

Summary Pre-meiotic and prophase I ovules ofCapsella bursa-pastoris (L.) Medic.(monosporic,Polygonum type of gametophyte development) were fixed routinely or incubated in a modified Gomori medium containing -glycerophosphate as a substrate. Prior to the beginning of meiosis the potential meiocyte is ultrastructurally similar to the other cells of the nucellus and is distinguished only by its size and position. At the initiation of prophase I dramatic ultrastructural and ultracytochemical changes take place in the female meiocyte. These include the sudden appearance of cytoplasmic structures composed of single and multiple concentric cisternae, distinctive changes in plastids and mitochondria, and the blebbing of 0.3 m double-membraned vesicles from the nuclear envelope. The concentric cisternae encapsulate portions of cytoplasm containing ribosomes, plastids, mitochondria, ER fragments and vesicles. Both single and multiple concentric cisternae localize high levels of acid phosphatase and function as autophagic vesicles (AVs) that sequester ribosomes and organelles for destruction during meiosis. Plastids stop dividing and become more spherical during prophase I. Some plastids localize acid phosphatase and many show continuities between the outer membrane and the plastid envelope and acid phosphatase-rich RER cisternae. Mitochondria appear as dense, contracted spheres or rods. Some mitochondria localize acid phosphatase but they do not show membrane confluencies with the ER. Some of the plastids and mitochondria that are segregated into the functional megaspore at meiosis II are destroyed but others apparantly survive meiosis and give rise to the plastid and mitochondrial populations of the young gametophyte (Schulz andJensen, unpublished). The lateral and end walls of the meiocyte show patches of intense aniline blue fluorescence and the chalazal end wall of the cell is perforated with large numbers of plasmodesmata.Research supported by NSF Grant PCM-79-11018. The authors gratefully acknowledge the valuable assistance of David Lee Ivans in this project.     

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.     

Unequal distribution of plastids during generative cell formation in Impatiens

Summary This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts.     

An ultrastructural study of carpospores in the red alga <Emphasis Type="Italic">Chondrus pinnulatus</Emphasis> (Harvey) okamura, 1930 (Rhodophyta: Gigartinaceae) from Vostok Bay,the Sea of Japan

The morphological features of carpospores in the red alga Chondrus pinnulatus have been studied using methods of transmission and scanning electron microscopy. Rounded mature carpospores are assembled into groups. Each carpospore is surrounded by a two-layered mucilage wall. The electron-dense cytoplasm contains numerous starch grains, fibrous vesicles, and large clusters of fibrous vesicles. The plastids have well-developed thylakoids and the cell nucleus occupies a nearly central position. The nucleolus is large and loose and is localized near the nuclear membrane. Dictyosomes, small fibrous vesicles, osmiophilic granules, and plastids are localized at the periphery. Mitochondria are arranged near the dictyosomes, plastids, and around the nucleus. A generalized scheme of the fine structure of the carpospore has been proposed for red algae on the basis of our own and literature data.     

Investigation on the Development of Male Cametophyte in Anemarrhena Asphodeloides

Anemarrhena asphodeloides is a monotypic genus of Liliaceae, endemic to China and Korea. This genus is characterized by possessing three stamens. From development of male gametophyte, three features of the species are noteworthy. (1) During meiosis of the micros- pore mother cells, the Golgi vesicles are immediately incorporated into the formation of the material of callose wall; The latter lying at the outer tangential is about 4 gm in thickness dining formation of the tetrad. In the outer tangential callose wall there are certain cytoplasmic canals, which are about 0.6 to 1 μm in diameter. During the development of pollen grains, there are a number of other vesicles dispersing in the cytoplasm of the microspores. The activity of these vesicles seems to be involved in accumulation and formation of lipid bodies. But the above vesicles, which were derivxed from Golgi or endoplasmic reticulum, have not been known in this genus. (2) By two-celled stage of pollen grains, the unequal distribution of lipid bodies is very prominent, and they are singular in being placed on the boundary between the plasmalemma of vegetative and generative cells. While the generative cell is delached from the intine of pollen grain, the generative cell is surrounded by the lipid bodies which had been called the corona of them. By the observation of TEM, these lipid bodies come from the cytoplasm of vegetative cell and did not remain a constant surrounding layer. Towards the stage of pollen maturation, the lipid bodies lying oppositely to the nucleus of vegetative cell were gradually dispersed in the cytoplasm. Their function is unknown but the observation shows that some of them move to the plasmalemma of the pollen grain. (3) An important feature of the mature pollen grain in Anemarrhena is that the generative cell does not contain plastids during polle development. On the basis of cytological mechanisms of the plastid inheritance, Hagemann (1983) has classified the angiosperms into four groups of species, of which the Lycopersicum type, Solanum type, and Triticum type belong to the mode of a uniparental maternal inheritance of plastids; while the Pelargonium type represents the mode of biparental inheritance of plastids. Our studies have confirmed that the mode of plastid inheritance in Anemarrhena asphodeloides is similar to Gasteria verrucosa, both show the same mode of plastid inheritance of Lycopersicum type.     

LIGHT AND ELECTRON MICROSCOPIC STUDIES OF THE FUSION CELL IN ASTEROCOLAX GARDNERI SETCH. (RHODOPHYTA,CERAMIALES)12

The fusion cell in Asterocolax gardneri Setch, is a large, multinucleate, irregularly-shaped cell resulting from cytoplasmic fusions of haploid and diploid cells. Subsequent enlargement takes place by incorporating adjacent gonimoblast cells. The resultant cell consists of two parts—a central portion of isolated cytoplasm, surrounded by an electron dense cytoplasmic barrier, and the main component of the fusion cell cytoplasm surrounding the isolated cytoplasm. The fusion cell contains many nuclei, large quantities of floridean starch, endoplasmic reticulum, and vesicles, but few mitochondria, plastids and dictyosomes. The endoplasmic reticulum forms vesicles that apparently secrete large quantities of extracellular mucilage which surrounds the entire carposporophyte. The isolated cytoplasm also is multinucleate but lacks starch and a plasma membrane. Few plastids, ribosomes and mitochondria are found in this cytoplasm. However, numerous endoplasmic reticulum cisternae occur near the cytoplasmic barrier and they appear to secrete material for the barrier. In mature carposporophytes, all organelles in the isolated cytoplasm have degenerated.     

Cotton embryogenesis: The sperm

Summary The sperm of cotton were observed in the pollen tube in the style. They are true cells but relatively simple in organization. The nuclei are small and each contains a single, very small nucleolus. Nuclear pores are common and heterochromatin lines the nuclear membrane. The plastids and mitochondria are so reduced in internal structure that it is impossible to separately identify them. The suggestion is put forward that only mitochondria are present in the sperm. Dictyosomes are few but appear to be producing large numbers of vesicles. Single membrane vesicles of a large range of sizes are common. ER is scarce but polysomes are numerous.     

The reticular substance of the medulla oblongata of the albino rat

Summary The region of the reticular giganto-cellular nucleus, perfused with formalin and postfixed in osmium tetroxide, was studied with histochemical and electron microscopic techniques. The perikarya of the neurons have two zones. The peripheral cytoplasm contains Nissl bodies, mitochondria, and free RNP particles. The juxtanuclear cytoplasm contains the Golgi complex, mitochondria, RNP particles and dense bodies. The nucleus is indented and has a prominent nucleolus and a paranucleolar body. Dense bodies are found along the axon and dendrites as well. Three different types of synapses are described and two types of synaptic vesicles (spherical and ellipsoidal) are shown.The capillary endothelium shows microvilli and marginal flaps. The endothelial cytoplasm contains vacuoles, micropinocytotic vesicles, and a few dense bodies. Processes of pericapillary cells, surrounded by a basement membrane, also contain dense bodies. The dense bodies found in the neurons and endothelial cells show acid phosphatase activity. On the basis of their morphology and their enzymatic activity these bodies are identified as lysosomes.Partially supported by a school grant No RF 62051 from the Rockefeller Foundation, New York, USA, and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Fellows of the Consejo National de Investigaciones Científicas y Técnicas, Argentina. The authors wish to thank Dr. Mario H. Burgos for his constant interest and criticism, and to Dr. Eduardo Rodriguez Echandia and Dr. Fabio L. Sacerdote for their valuable assistance.     

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.     

Studies on the Relation Between the Ultrastructure of Secretory Cells and Their Synthesis and Secretion of Lacquer in Laticiferous Canals of Rhus verniciflus

Secretory cells of laticiferous canals contain many plastids and endoplasmic reticulum (ER) in Rhus verniciflua. The electron microscopy suggests that osmiophiiic Lacquer component is mainly synthesized in the plastids and ER. They may be eliminated from the protoplasts to the space between the plasmalemma and the cell wall in three ways: (1) by ER elements, (2) by vesicles approaching the plasmalemma and fusing their membrances with the latter, and (3) by their becoming surrounded by plasmalemma invaginations, and then they traverse the wall through the channels of plasmodesmata which became disconnected during the schizogenous development of the canals and percolate through the wall that faded into an even looser mesh of fibrillar material toward the canal lumen. More or less, nucleus, mitochondria, Golgi bodies and ground cytoplasm also take part in the above-mentioned process.