Microtubule organization of in situ and isolated generative cells in Zephyranthes grandiflora Lindl

Summary Microtubule organization in the generative cells of Zephyranthes grandiflora was investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. The experimental materials used were generative cells located within pollen grains and tubes (i.e., in situ) as well as those artificially isolated after osmotic shock or grinding treatments of the pollen grains. Diverse microtubule organization patterns were revealed. In situ, the generative cells appeared spindle-shaped and contained mainly longitudinally oriented microtubule bundles, although other types were found as well. After isolation, as the alteration in microtubule patterns took place, the spindle-shaped generative cells became ellipsoidal and then spherical. In the ellipsoidal cells a transitional form consisting of a mixture of microtubule bundles and meshes could be found. In spherical cells the mesh structure appeared to be the predominant pattern. These results indicate that the microtubule cytoskeleton of the generative cells can change easily from one structural form to another in accordance with environmental conditions and may play an important role in determining the cell shape.     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Ultrastructure and microtubule organization of isolated generative cells ofAllemanda neriifolia at interphase and prophase

Summary The ultrastructure of isolated generative cells ofAllemanda neriifolia at interphase and prophase was studied. The microtubule organization of the isolated cells was also investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. After the generative cells had been isolated from the growing pollen tubes by osmotic shock, most of the cells were at prophase and only a few were at interphase. The interphase cell is spindle shaped and contains an ellipsoidal nucleus. In addition to the usual organelles, the cytoplasm of the interphase cell contains numerous vesicles (each measuring 40–50 nm in diameter) and two sets of longitudinally oriented microtubule bundles — one in the cortical region and the other near the nucleus. Most of the prophase cells are spherical in shape. Based on the ultrastructure and the pattern of microtubule cytoskeleton organization three types of prophase cells can be recognized. (1) Early prophase cell, which contains the usual organelles, numerous vesicles, and a spherical nucleus with condensed chromosomes. Longitudinally oriented microtubule bundles can no longer be seen present in the early prophase cell. A new type of structure resembling a microtubule aggregate appears in the cytoplasm. (2) Mid prophase cell, which has a spherical nucleus containing chromosomes that appear more condensed than those seen in the early prophase cell. In addition to containing the usual organelles, the cytoplasm of this cell contains numerous apparently randomly oriented microtubules. Few vesicles are seen and microtubule aggregates are no longer present. (3) Late prophase cell, typified by the lack of a nuclear envelope. Consequently, the chromosomes become randomly scattered in the cytoplasm. Microtubules are still present and some become closely associated with the chromosomes. The changes in the ultrastructure and in the pattern of microtubule organization in the interphase and prophase cells are discussed in relation to the method of isolation of the generative cells.     

Confocal laser scanning microscopy of microtubule organizational changes in isolated generative cells of Allemanda neriifolia during mitosis

Summary Organizational changes in the microtubules of isolated generative cells of Allemanda neriifolia during mitosis were examined using anti--tubulin and confocal laser scanning microscopy. Due to an improved resolution and a lack of out-of-focus interference, the images of the mitotic cytoskeleton obtained using the confocal microscope are much clearer than those obtained using the non-confocal fluorescence systems. In the confocal microscope one can see clearly that the spindle-shaped interphase cells contain a cage-like cytoskeleton consisting of numerous longitudinally oriented microtubule bundles and some associated smaller bundles. At prophase, the shape of the cells invariably becomes spherical. The microtubule cytoskeleton inside the cells concomitantly changes into a less organized form — consisting of thick bundles, patches, and dots. This structural form is not very stable, and soon afterwards the cytoskeleton changes into a reticulate network. Then the nuclear envelope breaks down, and the microtubules become randomly dispersed throughout the cell. Afterwards, the microtubules reorganize themselves into a number of half-spindle-like structures, each possessing a microtubule-nucleating center. The locations of these centres mark out the positions of the presumptive spindle poles. Numerous microtubules radiate from these centres toward the opposite pole. At metaphase, the microtubules form a number of bipolar spindles. Each spindle has two half-spindles, and each half-spindle has a sharply focused microtubule centre at the pole region. From the centres, kinetochore and non-kinetochore microtubules radiate toward the opposite half-spindle. At anaphase A, sister chromatids separate, the cells elongate, and the kinetochore microtubules disappear; the non-kinetochore microtubules, however, remain, and a new array of microtubules, in the form of a cage, appears. The peripheral cage bundles and the non-kinetochore bundles coverge into a sharp point at the pole region. Later, at anaphase B the microtubule cytoskeleton undergoes reorganization giving rise to a new array of longitudinally oriented microtubule bundles in the cell centre and a cage-like cytoskeleton in the periphery. At telophase, some of the cells elongate further, but some become spherical. The microtubules in the central region of the elongated cell become partially disrupted due to the formation of a phragmoplast-junction-like structure in the mid-interzone region. The microtubule bundles at the periphery are spirally organized, and they appear not to be disrupted by the phragmoplast-like junction. The microtubules in the spherical telophase cells (unlike those seen in the elongated telophase cells) are arranged differently, and no phragmoplast-junction-like structure forms in the spherical cells. The structural and functional significances of some of these new features of the organization of the microtubule cytoskeleton as revealed by the confocal microscope are discussed.     

Branch formation induced by microbeam irradiation ofAdiantum protonemata

Branches were induced in centrifugedAdiantum protonemal cells by partial irradiation with polarized red light. Nuclear behavior and microtubule pattern change during branch formation were investigated. A branch formed at any part where a red microbeam was focused along a long apical cell. The nucleus moved towards the irradiated area and remained there until a branch developed. The pattern of microtubules changed from parallel to oblique at the irradiated area and then a transverse arrangement of microtubules appeared on both sides of the area. It appeared as if the nucleus was suspended between two microtubule rings. This nuclear behavior and the changes in microtubule pattern were different from those observed during branch formation under whole cell irradiation. From the results of this work we suggest that there is an importance for precise control of experimental conditions.     

Kinetochore behavior during generative cell division inTradescantia virginiana

Summary Previous observations indicate that division of the generative cell inTradescantia virginiana is characterized by several unusual features, including persistence of surrounding microtubule (Mt) bundles during karyokinesis, lack of a distinct metaphase plate and direct contribution by mitotic Mts to the cytoskeleton of young sperm. We have further probed karyokinesis in these cells using additional antitubulin and chromosome staining, as well as kinetochore visualizations with CREST serum. The CREST antibodies reveal kinetochores as paired and single fluorescent dots similar to those seen in other species stained with this preparation. Double localizations show that the dots are located at the ends of Mt bundles previously identified as kinetochore fibers (Palevitz and Cresti 1989). Before anaphase, paired kinetochores are distributed along the length of the cell. They also tend to be located at the cell periphery or are directly connected to peripheral Mt bundles by their kinetochore (K)-fibers. Twelve pairs of dots can be counted per cell, equal to the expected number of chromosomes. During anaphase, kinetochore separation starts at various positions along the length of the cell, producing single, relatively uniformly distributed kinetochores in the crotches of forks formed by K-fiber trunks and elongating Mt branches attached to the base of the trunks. Eventually, K-fibers with attached kinetochores aggregate in stepwise fashion on thick Mt bundles at both ends of the cell. This pattern is reflected in the cytoskeleton of young sperm. These results further document the unusual distribution of chromosomes and kinetochores inTradescantia generative cells and the origin of the Mt cytoskeleton in sperm cells.Abbreviations CREST Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia - K-fiber kinetochore fiber - Mt microtubule Dedicated to the memory of Professor Oswald Kiermayer     

Organization of the cytoskeleton in the coenocytic algaVaucheria longicaulis var.macounii: an experimental study

Summary The organization of the cytoskeleton of vegetative filaments ofVaucheria longicaulis Hoppaugh var.macounii Blum is investigated by fluorescence microscopy using monoclonal anti -tubulin antibodies and fluorescein (FITC)-labelled phalloidin. Confocal laser scanning microscopy observations give further information on the distribution of the cytoskeletal elements. Phalloidin labelling reveals F-actin bundles in the cortical cytoplasm of both fixed and unfixed vegetative filaments of this alga. In addition a more diffuse fluorescent component, seen at higher magnification to be made up of thinner F-actin bundles, can also be detected in unfixed cells. The distribution of the F-actin bundles resemble that of filamentous structures observed with differential interference contrast (DIC) microscopy in living cells. These structures seem to correspond to the microtubule associated reticulum (MAR) described in literature and overall the evidence suggests that actin and MAR elements are co-distributed. F-actin bundles are always found in association with focal masses (foci) of phalloidin-positive material. Foci are also observed by DIC microscopy associated with the cytoplasmic filamentous structures in living cells.Depolymerization of F-actin with cytochalasin D and the subsequent repolymerization that occurs on transfer ofVaucheria vegetative filaments to cytochalasin-free medium suggest that these foci are involved in the organization of the F-actin array. Immunofluorescence for -tubulin reveals microtubule bundles that are shorter in length and straighter in configuration than microfilament bundles. Microtubule bundles are associated with spot-like focal structures that, in many instances, show a close relationship with respect to nuclei. Oryzalin and cold temperature cause the depolymerization of the microtubule bundles and suggest, in conjunction with repolymerization studies, that these fluorescent spots associated with the ends of the microtubule bundles are involved in their organization; hence, they represent microtubule organizing centres or MTOCs. The importance of both microfilament and microtubule bundle focal regions is discussed with respect to the apical growth exhibited by the vegetative filaments of this alga.     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

Organization of the mitotic apparatus during generative cell division inNicotiana tabacum

Summary In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes ofNicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information onTradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA ofTradescantia GCs.Abbreviations GC generative cell - MA mitotic apparatus - Mt microtubule     

Microtubule organization in sperm cells in the pollen tubes ofBrassica oleracea L.

Summary Microtubules tightly cross-linked into bundles are described in the sperm cells ofBrassica oleracea pollen tubes. The sperm cells are lobed and tailed and the microtubule bundles are often located in these parts of the cells. In the present paper we suggest that the cross-linked microtubule organization could determine an intertubular sliding, probably generating a motility system that propels the sperm cells through the tube.Abbreviations GC generative cell - Mfs microfilaments - Mts microtubules - SC sperm cell - VC vegetative cell - VN vegetative nucleus     

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.Abbreviations GMC guard mother cell - IMB interphase microtubule band - Mt microtubule - PPB preprophase band     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。     

Studies on the development of the air pores and air chambers ofMarchantia paleacea

Summary The preprophase-prophase initial aperture (IA) cells ofMarchantia paleacea undergo a particular sequence of protoplasmic changes, which reflects the establishment of an unusual premitotic polarization. The marking feature of preprophase-prophase thallus cells is the shape of the nucleus which becomes spindle-shaped. This phenomenon accompanies the organization of an extranuclear microtubule (MT) sheath, nucleated and/or organized by distinct polar MT organizing centres (MTOCs).The interphase MTs disappear after activation of polar MTOCs. In preprophase IA cells incomplete preprophase MT bands (PMBs) are organized. They consist of PMB portions which traverse only small portions of the cell cortex at the level of the future cytokinesis and do not form a complete ring. In the same cells other MT bundles, independent of the incomplete PMBs terminate in the cortical cytoplasm abutting on the lower part of the intercellular spaces (ISs) or the surface cavities (SCs). Almost complete or complete PMBs are organized in IA cells in which the plane of PMB formation coincides with that passing through ISs of the same growth.The observations suggest that in preprophase-prophase IA cells ofMarchantia paleacea cortical MTOCs function in regions distant from each other: One region is the PMB cortical cytoplasm, probably that covering the wall edges, and the other is the one adjacent to the lower part of the wall facing the IS(s) or that underlying the SCs. The competition between the cortical MTOCs as well as between them and the polar ones may be responsible for the organization of incomplete PMBs.     

Mitotic Microtubule Development and Histone H3 Phosphorylation in the Holocentric Chromosomes of Rhynchospora Tenuis (Cyperaceae)

In the present work we report the phosphorylation pattern of histone H3 and the development of microtubular structures using immunostaining techniques, in mitosis of Rhynchospora tenuis (2n = 4), a Cyperaceae with holocentric chromosomes. The main features of the holocentric chromosomes of R. tenuis coincide with those of other species namely: the absence of primary constriction in prometaphase and metaphase, and the parallel separation of sister chromatids at anaphase. Additionaly, we observed a highly conserved chromosome positioning at anaphase and early telophase sister nuclei. Four microtubule arrangements were distinguished during the root tip cell cycle. Interphase cells showed a cortical microtubule arrangement that progressively forms the characteristic pre-prophase band. At prometaphase the microtubules were homogeneously distributed around the nuclear envelope. Metaphase cells displayed the spindle arrangement with kinetochore microtubules attached throughout the entire chromosome extension. At anaphase kinetochoric microtubules become progressively shorter, whereas bundles of interzonal microtubules became increasingly broader and denser. At late telophase the microtubules were observed equatorially extended beyond the sister nuclei and reaching the cell wall. Immunolabelling with an antibody against phosphorylated histone H3 revealed the four chromosomes labelled throughout their entire extension at metaphase and anaphase. Apparently, the holocentric chromosomes of R. tenuis function as an extended centromeric region both in terms of cohesion and H3 phosphorylation.     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Fine structure study on the association of the caulerpalean plastid with microtubule bundles in the siphonalean green algaChlorodesmis fastigiata (Ducker,Udoteaceae)

Summary In the dichotomously branched caulerpalean green algaChlorodesmis fastigiata long range cytoplasmic streams run through the siphon and form a network of shorter strands in the region of the bulbous enlargement of the dichotomies. Continuous transport of organelles occurs along these streams, which are contructed of a central bundle of microtubules, around which the organelles are grouped. Both chloroplasts and amyloplasts exhibit a unique dorso-ventral symmetry: their flattened ventral side is closely apposed to the surface of the microtubule bundles. The concentric lamellar system (CLS) at the tip of the plastids invariably points in the direction of movement.These findings are discussed in relation to microtubule based motility. It is suggested that the unique plastid architecture serves as an efficient differentiation facilitating long range transport along the microtubule bundles.     

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The microtubule organizational changes in the isolated generative cells of Allemanda schottii were followed using immunofluorescence and confocal laser scanning microscopy. Due to the improved resolution and the lack of out-of-focus flares, the microtubule cytoskeleton of the generative cells could be visualized more clearly than using conventional epifluorescence systems. Immediately after isolation the microtubule cytoskeleton of the generative cells was cage-like composed of longitudinally oriented microtubule bundles. Later, some bundles began to depolymerize and at the same time some smaller bundles appearred. The smaller bundles unlike the longitudinal bundles crisscrossed throughout the cell. Later still, the cells became spherical. Both the longitudinal and the smaller bundles disappearred. At the same time some of the microtubules began to aggregate around the nucleus. These perinuclear microtubules were apparently not very stable, because soon afterwards,they started to disintegrate. By the time the cells became completely spherical,the cytoplasm became filled with diffuse fluorescence indicating that the tubulin was no longer existing in a polymerized form but in a monomeric form inside the cell. After the fuberlin had completely depolymerized the microtubules started to reform. The sequence of events leading to the reformation of the microtubule cytoskeleton in the spherical cells was as follow: A few nucleating centres began to form first. Then the nucleating centres gave rise to microtubule bundles. The bundles extended and aggregated to form a reticulate network. This cytoskeletal network appearred stable and well organized. It also had a lot of microtubule-bundle junctions. The network persisted after Triton X-l00 extraction.     

The organization of the cytoskeleton in the generative cell and sperms ofHyacinthus orientalis

Summary The microtubular cytoskeleton of the generative cell (GC) ofHyacinthus orientalis has been studied until the formation of the sperm cells (SCs). Immunofluorescence procedures in combination with confocal laser scanning microscopy (CLSM) has enabled the visualization of the organization of the microtubular cytoskeleton. Chemical fixation and freeze-fixation electron microscopy have been used to investigate the cytoskeleton and the ultrastructural organization of the GC and SCs. During pollen activation the GC is spindle-shaped. Microtubules (MTs) are organized as bundles and distributed in proximity of the GC plasmamembrane, forming a basket-like structure. Following migration through the pollen tube, the basket-like structure becomes more intertwined. During the nuclear division the MTs are involved in the segregation of the chromosomes and kinetochores are clearly discernible. Association with organelles is also observed. The chromosomes of the GC remain condensed until they separate in two sperm nuclei. The pre-prophase band was never observed. At the end of the GC division the microtubular network reorganizes in the two SCs.Abbreviations CLSM confocal laser scanning microscopy - DAPI 46-diamidino-2-phenyl-indole - F-S freeze-substitution - GC generative cell - MT microtubule - PBS phosphate buffered saline - R-F rapid freeze-fixation - SC sperm cell - TBS tris buffered saline - VN vegetative nucleus     

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.     

Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.