Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

Sperm cells of the pollen tubes of Brassica: Ultrastructure and isolation

Summary Sperm cells of pollen tubes grown both in vivo and in vitro form a male germ unit. Extensions from both sperm cells of each pollen tube are closely associated with the tube nucleus. A high yield (2.7 × 10⁴. 20 mg^–1 pollen grains germinated) of intact sperm cells was obtained following release by osmotic shock from pollen tubes grown in vitro. Structural integrity of isolated sperm was maintained by isolation at low temperature in an osmotically balanced medium. At 4° C many isolated sperm pairs were still enclosed within the pollentube inner plasma membrane. Sperm cells not enclosed within this membrane no longer remained connected as a pair. During isolation vesicles formed on the sperm cell surface from disruption of the fibrillar components bridging the periplasmic space. Both in the pollen tube and after isolation the sperm nucleus is in close association with at least one region of the sperm plasma membrane. Sperm isolated at room temperature showed the presence of nucleopores, and nuclei were euchromatic, instead of heterochromatic as in intact sperm in the pollen tube.     

The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

Semi in vivo pollen tube growth of Aechmea fasciata

With semi in vivo pollen tube growth assays, stigmas are pollinated in vivo and, after a fixed time interval, the styles are isolated from the ovary and placed on culture medium in vitro. Semi in vitro pollination includes isolation of the stigma and style complex, followed by pollination and placing the stylar end on nutrient medium. After semi in vivo pollination more and longer pollen tubes protruded from the cut end of the styles into medium, in comparison to semi in vitro pollination. Medium with 3 g l^–1 agar was better than that with 6 g l^–1 agar for pollen tube growth after the tubes emerged from the cut style. Semi in vitro pollination of the reversed style indicated that pollen tube growth was not influenced by the direction of the style. Fructose and glucose inhibited pollen tube growth compared to sucrose. Swollen tips characterized tube growth inhibition. After semi in vivo pollination all generative nuclei had divided to give two sperm nuclei. The average distance between the last sperm nucleus and the pollen tube tip as well as the distance between the two sperm nuclei diminished in growing pollen tubes between 24 and 48 h after pollination. The arrangements between the vegetative and the generative nuclei did not differ in semi in vivo and in vitro cultured pollen tubes of Aechmea fasciata. This information is important to explain why fertilization rate is low after placental pollination in comparison to placental grafted style pollination of Aechmea fasciata. The data may also contribute to the improvement of in vitro fertilization methods in Bromeliaceae and other higher plants.     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.     

Pollen tube penetration and fertilization in Lilium longiflorum (Liliaceae)

The ultrastructure of the embryo sac, nucellus, and parts of the micropyle of Lilium longiflorum were studied both before and after pollen tube penetration to examine the interactions between ovule and pollen tube, using transmission electron microscopy and light microscopy. Before pollen tube penetration the egg cell and two synergids are similar. No filiform apparatus was detected and no synergid degeneration occurs prior to pollen tube penetration. The polar nuclei do not fuse until fertilization. No differences in embryo sac ultrastructure were detected between pollinated ovules unpenetrated by pollen tubes and unpollinated flowers of a comparable age. Shortly after the discharge of the pollen tube two enucleated cytoplasmic bodies with different ribosome densities were observed in the degenerated cytoplasm. These structures border both on the central cell and the egg cell as well as each other and are interpreted as remains of sperm cytoplasm after transmission of sperm nuclei. In the central cell both the sperm nucleus and the polar nuclei are associated with endoplasmic reticulum (ER). ER is thought to be a transport mechanism to achieve contact between the haploid polar nuclei and the sperm nucleus. In the egg cell sperm nucleus alignment is not visibly achieved by ER. The persistent cells of the egg apparatus and the central cell appear to become more metabolically active after pollen tube penetration. Pollen tube penetration already occurs despite the absence of a filiform apparatus and a low level of differences between the cells of the egg apparatus.     

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     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.     

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

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

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

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

The male germ unit of Rhododendron: quantitative cytology,three-dimensional reconstruction,isolation and detection using fluorescent probes

Summary The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.     

Requirements for division of the generative nucleus in cultured pollen tubes ofNicotiana

Summary Production of sperm cells by division of the generative cell occurs during growth ofNicotiana (tobacco) pollen tubes through the sporophytic tissue of the style, and is associated with transition to the second phase of pollen-tube growth. WhenNicotiana pollen tubes are grown in liquid culture, the extent of generative-nucleus division and the timing of this division depend on the chemical composition of the medium. Addition of reduced forms of nitrogen, either as mixed amino-acids (0.03% w/v of an acid hydrolysate of casein) or as 1 mM ammonium chloride, induces division of the generative nucleus in over 90% of the tubes; 3 mM calcium nitrate does not stimulate division. Individual amino-acids differ in their ability to induce this division. Contaminants in some batches of poly(ethylene glycol), which is a major component of pollen-tube growth media, inhibit generative-nucleus division; this inhibition is greater in the absence of nitrogen, which increases the observed nitrogen-dependence of division. Reduced forms of nitrogen are also required for growth of pollen tubes after division, when callose plugs are deposited. In the absence of nitrogen, growth continues until the point where sperm cell production would normally occur, then ceases. Addition of amino-acids or ammonium chloride thus allows cultured pollen tubes ofNicotiana to progress to their second phase of growth. WhenNicotiana pollen is germinated in a complete culture medium at 25–26°C, sperm nuclei are first observed in the growing tubes after about 10 h, and by about 16 h most of the tubes have undergone division; at lower temperatures, division is delayed. The timing of division also varies between species ofNicotiana, but division occurs similarly in self-compatible and self-incompatible species. Anaphase in an individual pollen tube is calculated to take less than 4 min. The resultant sperm nuclei usually trail behind the vegetative nucleus, but a variety of arrangements of the three nuclei are observed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - PEG poly(ethylene glycol) - OG ordinary grade of PEG - SP Specially Purified for Biochemistry grade of PEG     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

ORCHID EMBRYOLOGY: MEGAGAMETOPHYTE OF EPIDENDRUM SCUTELLA FOLLOWING FERTILIZATION

The megagametophyte of Epidendrum scutella, an orchid, was examined with the electron microscope after the entrance and discharge of the pollen tube. The pollen tube enters the embryo sac by growing through the filiform apparatus of a synergid and discharges through a terminal pore into the degenerating cytoplasm of the synergid. The synergid nucleus appears pushed to one side by the discharge of the pollen tube. What is believed to be the remains of the vegetative nucleus has been found in the degenerate synergid, but no trace of the sperm cytoplasm has been seen. The zygote is approximately the same size as the egg. The ribosomes become grouped into polysomes. Both the egg and the zygote apparently completely lack dictyosomes. The polar nuclei partially fuse before fertilization, but fusion of the sperm nucleus with the polar nuclei does not occur and no endosperm is produced. Polysome formation occurs in the central cell and large amounts of tubular, smooth ER are seen. The antipodals remain following fertilization, undergoing ultrastructural changes similar to the central cell.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Fine structure of sperm cells in pollen grains ofBeta

Summary The structure of sperm cells in mature trinucleate pollen grains ofBeta vulgaris L. was studied with the electron microscope. The ellipsoidal sperm cell nuclei and cytoplasm are products of mitosis and cytokinesis of the ellipsoidal generative cell. Each sperm cell is separated from the vegetative cytoplasm by two contiguous membranes which enclose its cytoplasm and nucleus. Microtubules present in the sperm cell cytoplasm may be responsible for sperm cell motility.Approved as Journal paper Nr. 846, Utah Agricultural Experiment Station, Logan, Utah.     

Early post-pollination events in hexaploid wheat × maize crosses

Summary Cytological events in the first 12 h after pollination were studied in crosses between the hexaploid wheat genotype Chinese Spring and the maize genotype Seneca 60. A pollen tube was first observed in the embryo sac 4 h after pollination, and maize sperm nuclei were first observed in the embryo sac after 5 h. On 29 occasions two, and on 1 occasion three, pollen tubes penetrated the embryo sac. Four categories of aberration limiting the frequency of fertilization were identified: (1) in 20% of florets no pollen tube reached the embryo sac; (2) in at least 1.9% the pollen tube severely damaged the wheat egg cell and polar nuclei; (3) in 33% the maize sperm nuclei were not released from the pollen tube; and (4) in 16% the sperm nuclei were released into the embryo sac but failed to move to either of the wheat gametes. In the remaining 29% sperm nuclei were more often found in the egg cell than at the polar nuclei. The results suggest that karyogamy occurs with very high efficiency when a sperm nucleus reaches the egg cell, but with only about 50% efficiency when a sperm nucleus reaches the polar nuclei.     

The spatial association of the sperm cells and vegetative nucleus in the pollen grain ofBrassica

Summary In mature viable pollen ofBrassica oleracea, the pair of sperm cells and the nucleus of the vegetative cell are linked to form a structured unit we term the male germ unit. The sperm cells are held within a common periplasm and have no cell walls. Each sperm cell has a central globular body containing the nucleus surrounded by several evaginations which provide the means for linkage between them. One sperm cell, usually that closest to the nucleus of the vegetative cell contains most of mitochondria profiles (plastids are absent). This sperm cell appears to be linked by its protoplasmic evaginations to the envelope of the vegetative nucleus. The role of this unit in interactions with the female gametic complex is considered.