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.     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

Divergent pollination systems in the cleistogamous species,Collomia grandiflora (Polemoniaceae)

Summary A structural study of pollination in the dimorphic flowers ofCollomia grandiflora, a cleistogamous species, reveals significant differences in stigma behavior during pollination, stylar structure, the timing of generative cell division, and pollen tube growth rate patterns. The cleistogamous flower shows a loss of protandry and the stigma is receptive only after reflexing and closing of its lobes. In contrast, the chasmogamous stigma is receptive when reflexed and closes when pollen has been deposited on the lobes. Pollen tube penetration of the dry stigma papillae and entry into the style is similar in the two morphs. The chasmogamous style is solid and the cleistogamous style partly hollow. The matrix of secretion produced by the transmitting tract cells is mainly carbohydrate with a trace of lipids. It is fibrillar in nature and appears to be partly comprised of wall material from the transmitting tract cells. In the chasmogamous pollen, the generative cell enters the tube before division, which occurs between 30 and 60 min after pollination. This division correlates with an increased growth rate for the pollen tube. In the cleistogamous pollen, contact with the stigma triggers generative cell division inside the hydrated pollen grain before germination. The two resulting sperm cells exit the grain 15–30 min after pollination when the pollen tube is in the stigma lobes. The cleistogamous pollen tube shows only one phase of growth which occurs at a rate similar to that of the slow, first phase of the chasmogamous pollen.Abbreviations CH chasmogamous - CL cleistogamous - DAPI 4, 6-diamidino-2-phenylindole     

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.     

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.     

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     

豌豆生殖细胞的发育—着重论述质体母系遗传的细胞学基础

超微结构的研究证明,豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）生殖细胞自形成直至成熟花粉时期,始终存在少量质体和较多的线粒体。ＤＮＡ 荧光的观察表明,在发育早期的生殖细胞中不含细胞质ＤＮＡ 类核,但在成熟花粉的生殖细胞中有许多的类核。在花粉离体萌发过程中,随着花粉管的生长,生殖细胞中的类核逐渐降解。在花粉培养２４ ｈ 后,生殖细胞的类核全部消失。研究结果确定了豌豆质体母系遗传的细胞学基础,支持遗传分析及ＲＦＬＰ研究的结论,阐明了过去在细胞学上认为是双亲遗传的判断不正确的原因     

Studies of Generative Cell Development in Pisum sativum--With Special Reference to Cytological Basis of Plastid Maternal Inheritance

It was proved by ultrastructural observations that few plastids and abundent mitochondria were ever present in the generative cell of Pisurn sativurn L. from its initiation to maturation. Fluorescence observations of DNA showed that many cytoplasmic DNA nucleoids were present in generative cell of mature pollen, but none in the early developing generative cell. During the germination of mature pollen in vitro, the cytoplasmic DNA nucleoids of the generative cell in the pollen tube degenerated gradually following the growth of the pollen tube and disappered completely 24 h after germination. The results provided a cytological basis for confirming the conclusion of plastid maternal inheritance in P. sativurn obtained from genetic study, and resolved the contradiction between results from cytological observation and genetic or RFLP analysis.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

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.     

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     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.     

Early events in division of the generative cell ofOrnithogalum virens

Summary Ornithogalum virens is a bicellular pollen species. In mature pollen, the generative nucleus is at advanced prophase. Mitosis of the generative cell is resumed just after pollen rehydration and prometaphase occurs within 10 min of germination. Prometaphase is manifested by nuclear envelope breakdown and the appearance of spindle microtubules in the nucleoplasm region. At this stage the number of cytoplasmic microtubules located in the generative cell periphery appears to decrease. Endoplasmic reticulum-like cisternae originating from the nuclear envelope tend to be spaced around the chromosomes, outside the area of the forming mitotic spindle. Some also begin to penetrate the spindle area. The results are discussed in terms of the generative cell cycle in bicellular pollen.     

Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize

Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.Key words: male gametophyte, generative cell, sperm, pollen tube, tubulin, fertilization, maize     

Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes

The germination and growth of pollen grains of Nicotiana tabacum and N. alata with the anti-microtubule drug oryzalin retarded significantly the movement of the vegetative nucleus (VN) and the generative cell (GC) from the grain to the tube apex but had no effect on pollen tube elongation. In N. tabacum, only 11% and 48% of the pollen tubes treated with oryzalin for 6 h and 12 h, respectively, had the VN and GC in the tube mainly in its middle part. In corresponding control materials, 79% and 99% of pollen tubes contained the VN and GC close to the apex. Indirect immunofluorescence microscopy and related studies of the tubes grown in the presence of oryzalin revealed complete absence of microtubules (MTs) but apparently intact microfilaments (MFs). These results suggested that the movement of VN and GC from the grain into the tube is possible when no MTs but only MFs are present, but the movement is then slow. In control tubes, the parallel orientation of MT bundles and extensions of VN were interpreted to represent the structural organization needed for the MT-dependent movement of VN.     

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     

Effects of Nifedipine on Pollen Germination,Pollen Tube Growth and Division of Generative Nucleus in Nicotiana tabacum

The deals with the effects of nifedipine (Nif), a Ca2+ channel blocker of rather high specificity, on pollen germination, pollen tube growth and division of generative nucleus (GN) in experimentlly germinated pollen tubes of Nicotiana tabacurn L. Pollen germination was inhibited by the addition of 10-4 mol/L Nif whereas no significant inhibition by 10-7~10-5 mol/L Nif was observed. The effects of Nif on pollen tube growth were related to its concentration and duration of treatment. At the earlier stage, tube growth was promoted at the lower concentrations (10-7~10-5 mol/L), but was significantly inhibited at a concentration of 10 4 mol/L Nif. With increasing time of culture, even the lower concentrations also became harmful; the stronger the concentration, the earlier the transition from promotion to inhibition. Generally, inhibition of tube growth occurred within 24 hours of culture with different extent in various concentrations. Moreover, higher concentrations also tended to disturb tube morphology and cytoplasmic streaming. Nif was observed to perturb GN division at various concentrations, either blocked it completely at 10-4 mol/L, or only delayed it at 10-7~10-5 mol/L. The dynamics of membrane-associated calcium in pollen tubes was tested with chlorotetracycline (CTC). With increasing time of culture and escalating Nif concentration, CTC fluorescence weakened gradually, indicating that the physiological effects of Nif is mediated by its in hibition on Ca2+ channel activities.     

Evidence for DNA repair after ultraviolet irradiation of Petunia hybrida pollen

Summary Ultraviolet irradiation of Petunia hybrida pollen led to an unscheduled labelling of pollen DNA by ³H-thymidine during the early stages of germination. Hydroxyurea increased this DNA labelling, while added boron, required absolutely for pollen germination, tube elongation and tube generative cell mitosis, was not needed for this repair — like DNA synthesis.     

Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

In vitro pollen germination and transient transformation of<Emphasis Type="Italic">Zea mays</Emphasis> and other plant species

To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such asPennisetum americanum andTradescantia species, and dicot species, such asArabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, andVicia faba. On average, reproducible germination rates ranging from 50–100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducibleLuc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin-and pollen-specificZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed. An erratum to this article is available at .