Pollen Tube Entry Research Articles

Evidence for Stigmatic Self-incompatibility, Pollination Induced Ovule Enlargement and Transmitting Tissue Exudates in the Paleoherb, Saururus cernuus L. (Saururaceae)

Saururus cernuus, a species belonging to the primitive herbaceous angiosperm family Saururaceae, exhibits high rates of self-sterility. We investigated the structural and functional aspects of pollen-carpel interactions following cross and self pollination to assess the tissue specific site and timing of self-sterility and factors important for successful cross pollen tube growth. Self-sterility was due to inhibition of self pollen germination at a dry stigma. Self pollination was associated with anomalous foot formation, reduced cell wall expansion and secretory activity of stigmatic papillae, and callose production in stigmatic papillae. Following germination, cross compatible pollen tubes entered a solid core of transmitting tissue and grew to the base of a short style. Entry of cross pollen tubes into the ovary was coincident with ovule enlargement which placed the micropyle in the proximity of cross pollen tube tips. Ovule enlargement also occurred following self pollination. Cross pollen tubes either entered an exudate-filled micropyle directly from the style, or growth in the ovary was localized to the epidermis of the locule and outer integument prior to entry into the micropyle. Prior to pollination, the transmitting tract was void of secretions except for exudate in the micropyle. Growth of pollen tubes on the locule and integument was associated with exudate apparently arising from transmitting cells adjacent to growing pollen tubes. The present study provides the first evidence in a primitive herbaceous species of stigmatic self-incompatibility (SI) in association with a dry stigma, pollination-induced signalling events affecting development of carpellary tissues, and micropylar exudates.

Sexual Reproduction of Interior Spruce (Pinaceae). I. Pollen Germination to Archegonial Maturation

Stages of normal sexual reproduction between pollen germination and egg maturation are described for interior spruce. These prezygotic stages were studied by light and electron microscopy in more detail than was possible in previous studies, and new observations have been made. Sperm (male gametes) formation and the organelles that accompany the sperm within the pollen tube are described. The pollen tube grows toward a lipid secretion that originates in the neck region of the archegonium. Megagametophyte development was followed from the early cellular stage through the series of mitoses that result in neck cells, the ventral canal cell, the central cell, and ultimately the egg. Maturation of the egg includes formation of modified plastids (formerly described as large inclusions) and small inclusions that are membrane‐bound regions of cytoplasm. At egg maturation, modified plastids are excluded from the mitochondria‐rich perinuclear zone that surrounds the nucleus. Small inclusions occupy the periphery of the cell so that the egg nucleus and perinuclear zone are centrally located. A second article in this series describes pollen tube entry into the archegonium, delivery of sperm to the egg, gamete fusion, and proembryo and embryo formation.

Differential ovule development following self‐ and cross‐pollination: the basis of self‐sterility in Narcissus triandrus (Amaryllidaceae)

Self-pollination results in significantly lower seed set than cross-pollination in tristylous Narcissus triandrus. We investigated structural and functional aspects of pollen-pistil interactions and ovule-seed development following cross- and self-pollination to assess the timing and mechanism of self-sterility. Ovule development within an ovary was asynchronous at anthesis. There were no significant differences in pollen tube behavior following cross- vs. self-pollination during the first 6 d of growth, regardless of style morph type. Double fertilization was significantly higher following cross- vs. self-pollination. Aborted embryo development was not detected following either pollination type up to seed maturity. Prior to pollen tube entry, a significantly greater number of ovules ceased to develop following self- vs. cross-pollination. These results indicate that self-sterility in N. triandrus operates prezygotically but does not involve differential pollen tube growth typical of many self-incompatibility (SI) systems. Instead, low seed set following self-pollination is caused by a reduction in ovule availability resulting from embryo sac degeneration. We hypothesize that this is due to the absence of a required stimulus for normal ovule development. If this is correct, current concepts of SI may need to be broadened to include a wider range of pollen-pistil interactions.

Erucastrum abyssinicum Brassica oleracea hybrids obtained by ovary and ovule culture

Pollen germination and pollen tube growth were studied in the cross Erucastrum abyssinicum × Brassica oleracea. 25 per cent ovules showed pollen tube entry into the micropyle 5 days after pollination. Embryos did not grow beyond the globular or heart-shaped stage. Thus post-fertilization barriers prevent hybridization in this cross. Sequential culture of ovaries and ovules 10 days after pollination helped rescue globular embryos. Three hybrids were obtained. All the hybrids flowered in vitro. Pollen fertility was 6.5 per cent. Leaf esterase and peroxidase isozymes confirmed the hybrid nature. Meiotic metaphase in the pollen mother cells showed 25 chromosomes. The presence of up to 9 bivalents and a quadrivalent indicates high homology between the two genera. The intergeneric hybrids have been multiplied using nodal segment culture. They are a source for obtaining cms cole crops and widening the gene pool of B. oleracea.

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca2+ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca2+ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate.

Overcoming prefertilization barriers in the crossDiplotaxis siettiana ×Brassica juncea using irradiated mentor pollen

Hybridization between the nearly extinct speciesDiplotaxis siettiana andBrassica juncea is prevented because of strong prefertilization barriers. Use of mentor pollen ofD. siettiana irradiated with 1000 Gy gamma radiation before the incompatible pollination led to fertilization. 5 d after pollination 17% ovules showed entry of pollen tubes, 10 d after pollination 27% ovules showed small globular embryos which grew no further. No embryos were found in control pollinations. Thus , use of irradiated mentor pollen brings about fertilization in this difficult cross and hybrids can be obtained if embryos are rescued.

Fertilization in Nicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cells with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed "coronas". This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.

Fertilization inNicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cell with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed “coronas”. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.

A scanning electron microscope study of the pollen tube pathway in pistils of Rhododendron

The pollen tube pathway was observed at regular intervals in pistils of four species of Rhododendron, with emphasis on Rhododendron fortunei. Rhododendron is characterized by a nonpapillate wet stigma, angled stylar canal, placentae with central clefts, and many unitegmic anatropous ovules. Receptive stigmas were hand-pollinated with self pollen 1 – 8 days after anthesis. The pollen, which occurs in permanent tetrads, started germinating during the 1st day. After crossing the stigma surface to one of the grooves leading into the stylar canal, pollen tubes grew straight through the style, and continued into the placental clefts from which they emerged onto the placental surface to grow among the ovules. Tubes reached the ovary in 5 – 10 days depending on the species and took several days after entering the upper ovary to reach the base of the placentae. Single tubes (rarely two or more) diverged from the interovular network and grew under the integument (which is close against the placental surface) to enter the slit-like micropylar opening of an ovule. The morphology of the micropylar slit and the direction of pollen tube entry showed variation among ovules. In R. fortunei ovule entries occurred first on the upper half of the placenta, though not at the top, and in ovules closest to the placental cleft. All portions of the pathway, from stigma surface to micropylar opening, are covered by exudate. Stigmatic exudate increased in amount and became more viscous after pollination, burying the pollen grains and tubes, then gradually dried. Exudate was produced in the style and ovary whether or not pollination occurred. Characteristics of the pollen tube pathway in Rhododendron are discussed relative to those in other angiosperm taxa. Key words: Rhododendron, pollen–pistil interactions, fertilization, transmitting tissues, pistil exudates, ovule entries.

Wheat x pearl millet hybridization: consequence and potential

Eight grain pearl millet (2n=14) accessions were crossed as male to hexaploid spring wheat cv. ‘Fukuho’ (2n=6x=42). An average of 80% wheat pistils showed pearl millet pollen tube entry in the ovules, compared to 56% in wheat x maize cv. ‘Seneca 60’ cross. Of the 15 embryos, obtained through in vitro immature seed culture from wheat x pearl millet crosses, 3 plantlets were produced and grown to maturity. These three were of the somatic chromosome constitution 2n=42, 21 and 22, respectively. Haploid wheat plant (2n=21) apparently originated from pearl millet chromosome elimination during embryogenesis. The 22 chromosome plant had retained a single pearl millet chromosome at tillering stage, but this chromosome was eliminated from pollen mother cells prior to and also during gamete formation. The significance and potential uses of this wide cross is discussed.

Organization of the early post-fertilization megagametophyte ofPopulus deltoides

Gynoecia ofPopulus deltoides, 48 to 60 h after pollination, were examined using transmission electron microscopy. The mature megagametophyte contains two synergids, an egg cell, a central cell with two unfused polar nuclei and three antipodals. Pollen tube passage between cells in the styloid, on the placenta and through the micropyle does not induce degeneration of surrounding cells. Pollen tube entry into the megagametophyte occurs through the degenerated synergid immediately adjacent to the filiform apparatus. The tube grows the length of the filiform apparatus 20–30 μm into the synergid, and then discharges its contents-two sperm cells, the vegetative nucleus, and pollen tube cytoplasm. Sperm cells appear progressively hypertrophied during their passage in the pollen tube. In the degenerate synergid, the sperm nucleus is located at one end of the cell and the remaining cytoplasm is diffuse, containing a large vacuole. Following transmission of sperm nuclei into the egg and central cell, one male nucleus fuses with the egg and the other fuses with the micropylar polar nucleus. Enucleate cytoplasmic bodies of apparent sperm origin were observed within the degenerated synergid. The gametic fusion mechanism in this plant appears to involve: (1) eccentric positioning of the nucleus within the sperm cell, (2) separation of cytoplasmic and nuclear portions of the sperm cell, and (3) fusion of the nuclear portion with the egg cell. This represents a previously undescribed mechanism by which male cytoplasmic organelles are eliminated from sperm cells prior to fusion.

The Significance of the Obturator in the Control of Pollen Tube Entry into the Ovary in Peach (Prunus persica)

Changes in the obturator of the peach (Prunus persica) have been investigated and related to pollen tube growth in this region. At anthesis, the cells of the obturator are active and rich in starch reserves. Twelve days after anthesis these cells produce a secretion that stains for carbohydrates and for proteins. As the secretion is produced, starch vanishes from these cells and they degenerate and collapse as callose is accumulated. Secretion is independent of pollination as it takes place in a similar fashion both in pollinated and in unpollinated flowers. Pollen tube growth along the obturator surface depends on this secretion for, although pollen tubes reach the base of the style seven days after pollination, they cannot grow on the obturator until five days later, when the secretion is produced. This discontinuous secretion taking place at the obturator may provide a mechanism that controls the entrance of pollen tubes into the ovary in the peach.

Interspecific hybridization between rice bean (Vigna umbellata) and its wild relative (V. minima): Fertility-sterility relationships

The pre-and post-fertilization barriers in the interspecific crosses between Vigna umbellata and V. minima were investigated. In the reciprocal crosses (V. minima as the ♂ parent) the entry of pollen tubes into the ovary was delayed by about 4 h, and no seed set was observed. However, no pre-fertilization barriers were encountered in crosses involving V. minima as the ⧫ parent and V. umbellata as the ♂ parent (normal cross). The delay/absence of divisions in the endosperm and the failure of embryo to divide were the post-fertilization barriers responsible for somatoplastic sterility in normal crosses which yielded a few hybrid seeds. The hybrid seeds showed poor germinability. The F1 hybrids were intermediate between the parents in most morphological characters, and are completely sterile for pollen. Backcrossing of F1 hybrid with both the parents did not restore fertility in the progenies. V. minima is considered as the tertiary gene pool of the rice bean.

Early events in the embryo sac after intraspecific and interspecific pollinations in Rhododendron kawakamii and R. retusum

Early events in the embryo sac of Rhododendron kawakamii and R. retusum have been studied after compatible self-pollinations and eight interspecific crosses, using sectioned ovaries, pistil squashes, and seed-set data. Ovules of Rhododendron kawakamii and R. retusum are anatropous, unitegmic, and tenuinucellate, with a typical eight-nucleate, seven-celled embryo sac. Fertilization normally occurs 4–5 days after pollination. The zygote lays down a callose wall but remains undivided during the first 13–15 days after pollination. The primary endosperm nucleus divides soon after fertilization, and development is cellular ab initio. Crosses of R. kawakamii (♂) with R. santapaui and R. retusum and crosses of R. retusum (♂) with R. kawakamii, R. santapaui, R. ovatum, and R. tashiroi showed apparently normal fertilization in a majority of ovules entered by pollen tubes. In crosses of R. kawakamii (♂) with R. quadrasianum and Kalmia latifolia entry of pollen tubes into ovules was delayed and frequently abnormal. Apart from compatible self-pollinations of R. kawakamii an R. retusum, only the cross of R. kawakamii (♂) with R. santapaui produced healthy seedlings. Of the remaining seven interspecific crosses only three showed significant embryo development in control pistils left to mature in situ. Similarities and differences in the breeding behaviour of R. kawakamii and R. retusum are discussed with reference to their taxonomic grouping within subsection Pseudovireya.

Megasporogenesis and Megagametogenesis in Soybean, Glycine max

Megasporogenesis and megagametogenesis were examined in Glycine max with light, fluorescence, and electron microscopy. Megasporogenesis results in a linear tetrad of four megaspores. Megagametophyte development is of the Polygonum type, with the functional chalazal megaspore undergoing three successive mitotic divisions to produce an eight-nucleate, seven-celled mature megagametophyte. The central cell becomes packed with starch. At fertilization, the antipodals are degenerate, the polar nuclei have fused, starch is diminished, and the egg occupies most of the micropylar portion of the megagametophyte. Several pollen tubes were occasionally observed at each micropyle, yet only one was involved in fertilization. Pollen tube entry occurs through a slightly reduced, viable synergid cell. Endosperm development precedes embryo growth. These results describing normal development allow important comparison with genetic mutants of soybean that affect female fertility.

MEGASPOROGENESIS AND MEGAGAMETOGENESIS IN SOYBEAN, GLYCINE MAX

Megasporogenesis and megagametogenesis were examined in Glycine max with light, fluorescence, and electron microscopy. Megasporogenesis results in a linear tetrad of four megaspores. Megagametophyte development is of the Polygonum type, with the functional chalazal megaspore undergoing three successive mitotic divisions to produce an eight‐nucleate, seven‐celled mature megagametophyte. The central cell becomes packed with starch. At fertilization, the antipodals are degenerate, the polar nuclei have fused, starch is diminished, and the egg occupies most of the micropylar portion of the megagametophyte. Several pollen tubes were occasionally observed at each micropyle, yet only one was involved in fertilization. Pollen tube entry occurs through a slightly reduced, viable synergid cell. Endosperm development precedes embryo growth. These results describing normal development allow important comparison with genetic mutants of soybean that affect female fertility.

Enzymic Removal of the Proteinaceous Pellicle of the Stigma Papilla Prevents Pollen Tube Entry in the Caryophyllaceae

Enzymic Removal of the Proteinaceous Pellicle of the Stigma Papilla Prevents Pollen Tube Entry in the Caryophyllaceae

Pollination and nucellus inAbelmoschus esculentus

Pollination induced dissolution of nucellar cells at the micropylar end forming a passage for pollen tube entry even when the pollen tubes are in the stylar region.

Influence of ovule position on pollen-tube entry and seed set.

Influence of ovule position on pollen-tube entry and seed set.