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.     

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.     

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.     

扇脉杓兰花粉超微结构及花粉管生长的观察

为明确扇脉杓兰花粉形态结构及雄性生殖特性,利用扫描电镜、透射电镜和荧光显微镜对花粉形态和超微结构及花粉管生长过程进行观察。结果表明:(1)扇脉杓兰单粒花粉长球形,表面光滑无特征纹饰,有少量胶黏物质,一些表面有2个或以上的深凹陷,凹陷内有球形突起的内容物。(2)花粉壁分为由棒状的基柱小单元组成的外壁和纤维素果胶组成的内壁,有覆盖层;生殖细胞近圆形,细胞核大而致密;营养细胞多弧形,核质分散;花粉粒细胞质含有大量的线粒体、质体和小泡等细胞器,淀粉、蛋白质和多糖含量丰富。(3)花粉管萌发后沿子房壁方向伸长,授粉20d花粉管伸长生长并不明显,授粉30d伸长的花粉管中出现大量胼胝质塞,并且伸长方向转为胚珠中间,花粉管逐渐接近胚珠,在授粉后50d基本完成受精作用。研究认为,扇脉杓兰花粉发育正常,不阻碍有性生殖过程。     

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     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

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.     

Ultrastructure and Germination Percentage of Crocus biflorus Miller subsp. biflorus (Iridaceae) Pollen

Pollen of Crocus biflorus Miller subsp. biflorus from natural habitats of Tusculum (Frascati, near Rome, Italy) has been studied in order to compare its structure and physiology to pollen of other Crocus species belonging to the Crocus sativus group. Mature pollen grains are rounded, 60 μm in diameter, in-aperturate (but with surface incisions where exine is lacking). DAPI staining reveals a spindle-shaped generative nucleus which is intensely fluorescent, and vegetative nucleus which is less fluorescent, and is elongated with numerous lobes. At anthesis the pollen is bicellular, but about 2% of tricellular grains occur among the pollen grains released from the anthers as well as on both naturally or handpollinated stigmas. Pollen germination is low in vitro, but higher in vivo. The pollen tubes are of normal shape. An electron-dense surface coat is sometimes visible on the exine, which in many cases, is detached from the exine. The vegetative cytoplasm is very rich in glycolipid bodies surrounded by endoplasmic reticulum. The generative cell has a lobed cell wall and is surrounded by the vegetative nucleus.     

Association of the Generative Cell and Vegetative Nucleus in Pollen Tubes of Rhododendron

Mixed fluorescence/bright field microscopy of Rhododendron pollentubes in the first 72 h after germination reveals a lens-shapedgenerative cell which divides to give two associated spermswithin the original cell boundary. The generative cell is closelyassociated with the vegetative nucleus which precedes it in92 per cent of pollen tubes. Three-dimensional reconstruction from serial thin sections ofa pollen tube fixed 24 h after germination shows that the associationbetween the generative cell and vegetative nucleus is extremelycomplex. Elongated tails of the generative cell physically enfoldthe vegetative nucleus and penetrate into enclaves within it.The association has been clarified by use of the periodic acid-phosphotungsticacid-chromic acid technique to enhance electron contrast ofthe plasma membranes surrounding the generative cell. In thisbicellular system, the male germ unit association is apparentlyinitiated after pollen maturity but prior to generative celldivision. Pollen tube, generative cell, male germ unit, plasma membrane, vegetative nucleus, Rhododendron, Ericaceae     

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 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.     

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.     

水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻（Oryza sativa）受精过程中雌雄性细胞融合时的形态特征及时间进程,确定合子期,为花粉管通道转基因技术的实施提供理论依据。结果表明：授粉后,花粉随即萌发,花粉管进入羽毛状柱头分支结构的细胞间隙,继续生长于花柱至子房顶部的引导组织的细胞间隙中,而后进入子房,在子房壁与外珠被之间的缝隙中向珠孔方向生长,花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞,释放精子。精子释放前,两极核移向卵细胞的合点端：两精子释放于卵细胞与中央细胞的间隙后,先后脱去细胞质,然后分别移向卵核和极核,移向卵核的精核快于移向极核的精核：精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下：授粉后,花粉在柱头萌发：花粉萌发至花粉管进入珠孔大约需要0．5小时：授粉后0．54,时左右,花粉管进入一个助细胞,释放精子：授粉后0．5—2．5小时,精卵融合形成合子：授粉后约10．0小时,合子第1次分裂,合子期为授粉后2．5-10．04,时：授粉后1．0-3．04,时,精核与两极核融合：授粉后约5．0小时,初生胚乳核分裂。’     

Autoradiographs of Pollen Tube Nuclei with Calcium-45

Autoradiography with Ca⁴⁵ has been used to obtain information about the relation between calcium and chromosomes. Labelled pollen from the Easter lily, Lilium longiflorum, was allowed to develop into pollen tubes between 5 and 6 cm. long in the styles of non-radioactive flowers. All of the nuclei, namely the tube nucleus and the two sperm nuclei, retain Ca⁴⁵ after this period of growth and development. Since the two sperm nuclei have formed during this interval by the mitotic division of the generative nucleus and growth of the tube has occurred under the influence of the tube nucleus, it is inferred that the calcium was bound in a stable nuclear component, the chromosomes.     

Western white pine (Pinus monticola Dougl.) reproduction: I. Gametophyte development

Male and female gametophyte development are described from light and transmission electron microscope preparations of ovules from first and second year Pinus monticola Dougl. seed cones. In the first year of development, pollen tubes penetrate about one-third the distance through the nucellus. The generative cell and tube nucleus move into the pollen tube. The megagametophyte undergoes early free nuclear division. First-year seed cones and pollen tubes become dormant in mid-July. In the second year, seed cones and pollen tubes resume development in April and the pollen tubes grow to the megagametophyte by mid-June. Early in June the generative cell undergoes mitosis, forming two equal-size sperm nuclei that remain within the generative cell cytoplasm. The generative cell has many extensions and abundant mitochondria and plastids. The megagametophyte resumes free nuclear division, then cell wall formation begins in early July. Cell wall formation and megagametophyte development follow the pattern found in other Pinaceae. Three to five archegonial initials form. The primary neck cell divides, forming one tier of neck cells. Jacket cells differentiate around each central cell. The central cell enlarges and becomes vacuolate; then vacuoles decrease in size and the cell divides, forming a small ventral canal cell and a large egg. Plastids in the central cell engulf large amounts of cytoplasm and enlarge. This process continues in the egg, and the peripheral cytoplasm of the egg becomes filled with transformed plastids. Mitochondria migrate around the nucleus, forming a perinuclear zone. The wide area of egg cytoplasm between these two zones has few organelles. A modified terminology for cells involved in microgametophyte development is recommended. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

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     

The effects of ethyl methanesulfonate treatment on <Emphasis Type="Italic">Eucalyptus</Emphasis> pollen behaviour in vitro

Treating pollen with mutagens prior to controlled pollination may facilitate the production of mutant trees for developmental studies and eventual plantation improvement. To establish a suitable dose of the chemical mutagen ethyl methanesulfonate (EMS) for the testing of this hypothesis, pollen of Eucalyptus globulus ssp. globulus and E. grandis was studied in vitro. Pollen germination, pollen tube elongation and generative cell division were examined after 48 h of culture, following exposure to between 0 and 1,000 ppm EMS. Doses of 600 to 1,000 ppm EMS reduced pollen germination in vitro in both species. Doses of up to 1,000 ppm EMS were not observed to significantly impact on either pollen tube length, or generative cell division in vitro of either species. A dose of 600 ppm EMS in paraffin oil is predicted to induce mutation in Eucalyptus species whilst impacting minimally on seed production based on the effect on pollen germination.     

Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii

Pollen tubes of Cyrtanthus mackenii, a species with bicellular pollen, were cultured in vitro to investigate nuclear phase changes during generative cell division and male germ unit (MGU) formation, using flow cytometric analysis. Results revealed that sperm cells were formed after 12 h of culture. During sperm maturation, the nuclei of sperm cells were not associated with the vegetative nucleus (unassociated sperm cells; Sua) and became longer than those of sperm cells associated with the vegetative nucleus (Svn). These findings indicate that the pair of sperm cells in the C. mackenii MGU is dimorphic in terms of nuclear shape. Dimorphism coincides with anti-α-tubulin antibody immunofluorescence, which was higher in the Sua than in Svn. Following treatment with oryzalin, triggering microtubule depolymerization, differences between nuclear shapes in the two sperm nuclei disappeared, suggesting that microtubule accumulation between sperm cells in the MGU correlates with differences in the nuclear shape.