Calcium changes in ovules and embryo sacs of Plumbago zeylanica L.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell. Received: 30 December 1999 / Revision accepted: 24 March 2000     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 2. Quantitative cell and organelle dynamics of the male reproductive cell lineage

Quantitative cell and organelle dynamics of the male gamete-producing lineage of Plumbago zeylanica were examined using serial transmission electron microscopic reconstruction at five stages of development from generative cell inception to sperm cell maturity. The founder population of generative cell organelles includes an average of 3.88 plastids, 54.9 mitochondria, and 3.7 vacuoles. During development the volume of the pollen grain increases from 6,200 μm³ in early microspores to 115,000 μm³ at anthesis, cell volume of the male germ lineage decreases more than 67% from 362.3 μm³ to 118.4 μm³. By the time the generative cell separates from the intine, plastid numbers increase by >600%, mitochondria by 250%, and vesicles by 43 times. A cellular projection elongates toward and establishes an association with the vegetative nucleus; this leading edge contains plastids and numerous mitochondria. When the generative cell completes its separation from the intine, organellar polarity is reversed and plastids migrate to the opposite pole of the cell. Cytoplasmic microtubules are common in association with cellular organelles. Plastids accumulate at the distal end of the cell as a linked mass, apparently adhered by lateral electron dense regions. Before division of the highly polarized generative cell, plastids decrease in number by 16%, whereas mitochondria increase by ∼90% and vacuoles increase by ∼140% from the prior stage. After mitosis, the resultant sperm cells differ in size and organelle content. The sperm cell associated with the vegetative nucleus (S_vn) contains 62.7% of the cytoplasm volume, 87% of the mitochondria, 280.4 vesicles (79% of those in the generative cell), and 0.6% of the plastids. At maturity, the S_vn mitochondria increase by 31% and the cell contains an average of 0.4 plastids, 158.9 vesicles, and 0.36 microbodies. The mature unassociated sperm (S_ua) contains 39.8 mitochondria (up 3.3%), 24.3 plastids (down 31%), 91.1 vesicles (up 54.9%), and 3.18 microbodies. The small number of organelles initially in the generative cell, followed by their rapid multiplication in a shrinking cytoplasm suggests a highly competitive cytoplasmic environment that would tend to eliminate residual organellar heterogeneity. Cell and cytoplasmic volumes vary as a consequence of fluctuations in the number and size of large vesicles or vacuoles, as well as loss of cytoplasmic volume by (1) formation of “false cells” involving amitotic cytokinesis, (2) “pinching off” of cytoplasm, and (3) dehydration of pollen contents prior to anthesis.     

Freeze-fracture of sperm of Plumbago zeylanica L. in pollen and in vitro

 Sperm of Plumbago zeylanica are dimorphic with regard to numbers of mitochondria and plastids. In most cases examined, the plastid-rich sperm fused with the egg while the sperm with fewer plastids fused with the central cell. However, plastids cannot be directly responsible for fusion because fusion occurs between the plasma membranes of egg and sperm. The question is whether sperm cell membranes are distinctive and possibly dimorphic. Sperm in whole pollen grains and isolated sperm were freeze-fractured. In pollen, freeze-fractured sperm appeared only in cross fractures. No extended membrane fracture faces of sperm were found. Among isolated sperm, two sizes of sperm with different organelles were observed. Isolated sperm were assigned to two categories based on cell diameter and on size and density of organelles. Membrane particles on most sperm were arranged without distinctive pattern. Some hexagonal arrays were observed. In sperm that had been maintained at 4°C, particle-free areas, a probable consequence of lipid phase separations, appeared on plasma membrane fracture faces. No unique fracture patterns and no patterns of dimorphism were detected on freeze-fractured plasma membranes of Plumbago sperm. Received: 14 January 1997 / Revision accepted: 6 June 1997     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

Juxtaposition of the generative cell and vegetative nucleus in the mature pollen grain of amaryllis (Hippeastrum vitatum)

Summary Computer-generated, three-dimensional reconstructions from serial ultrathin sections were used to investigate the spatial organization and extent of association between the generative cell and vegetative nucleus within the mature pollen grain of amaryllis. In all cases examined, the highly lobed vegetative nucleus was found in close proximity and positioned laterally to the elongated, oval shaped generative cell. Numerous projections of the vegetative nucleus come to within 53 nm of the inner vegetative cell plasma membrane which surrounds the generative cell. These areas of close association may continue transversely around the generative cell for a distance of up to 4 m. Although an association exists between the generative cell and vegetative nucleus of the mature pollen grain, it is apparent that several changes must take place after pollination in order to achieve the high amount of close contact that occurs between the vegetative nucleus and the numerous terminal cell extensions of the leading sperm in the pollen tube of amaryllis (Mogensen 1986). Thus, this study demonstrates that the spatial organization among components of the male germ unit in the mature pollen grain does not necessarily reflect relationships that ultimately exist among these components within the pollen tube.     

Structural characteristics of the mature embryo sac of Camellia oleifera

The ovule is the most important reproductive organ in the pistil of phanerogamae. Camellia oleifera (Theaceae) is an important woody plant producing edible oil in southern China, and its embryo sac structure has a positive effect on seed breeding. In this study, the microstructure, ultrastructure and three‐dimensional structure of the ovule and embryo sac of C. oleifera were observed and described based on a combination of advanced microscopy techniques (SEM, TEM, CLSM). The ovule comprises the inner and outer integument. Large quantities of secretions in the micropylar canal exit and may participate in the guidance of the entry of the pollen tube into the embryo sac. The synergids have a dense cytoplasm, abundant organelles, and strong polarity. Little cytoplasm is present in the egg cell, yet there are many vacuoles. The center of the cell is taken up by a large vacuole, and the cytoplasm is pushed towards the edges to form obvious cytoplasmic cords. The two polar nuclei are large and conspicuous. The antipodal cells degenerate to fulfill a nutritional function.     

Isolation of the male germ unit: organization and function in tobacco (Nicotiana tabacum L.)

Sperm cells are released from pollen tubes of tobacco as linked cells, associated with the vegetative nucleus in an assemblage known as the male germ unit (MGU). Using light microscopy, the MGU assemblage appears to be ensheathed by cytoplasmic material of the pollen tube, which may stabilize their association. Following their release, the shape of the sperm cells and vegetative nucleus changes from an ellipsoidal to a more spheroidal morphology. When most of the cytoplasmic material is dispersed, a boundary remains around the two sperm cells. Using scanning electron microscopy, the cytoplasmic material surrounding the MGU appears filamentous, sometimes twisted and rope-like. Based on these observations, the function of the MGU of tobacco is discussed. Received: 23 February 1998 / Revision received: 21 May 1998 / Accepted: 1 June 1998     

Sperm cells in pollen tubes ofNicotians tabacum L.: three-dimensional reconstruction,cytoplasmic diminution,and quantitative cytology

Summary The organization of the sperm cells and vegetative nucleus (male germ unit) ofNicotiana tabacum was examined 18 h after semivivo pollination using transmission electron microscopy, computerassisted serial section reconstruction and quantitative cytology. Based on a measurement of 11 cellular parameters in nine reconstructed sperm cell pairs, there are no statistically significant differences between the two cells. The S_vn is characterized by a strapshaped cytoplasmic extension that is physically associated with the surface of the vegetative nucleus. The nucleus is located adjacent to the sperm crosswall, with sperm organelles being distributed between the nucleus and the extension. The S_ua is a tapered cell with cytoplasmic areas at both poles and deep axial invaginations near the crosswall. This cell has a centrally-located nucleus and a largely polar distribution of organelles. Three mechanisms for cytoplasmic diminution were observed that appear to contribute actively to the loss of cytoplasmic volume and organelles: (1) enucleated cytoplasmic body production in the S_ua; (2) vesiculation at the tip of the cytoplasmic projection of the S_vn; and (3) vesicle-containing body accumulation in the periplasm of both the S_vn and S_ua.Abbreviations 3-D three-dimensional - ECB enucleated cytoplasmic body - MGU male germ unit - S_vn leading sperm cell - S_ua trailing sperm cell - TEM transmission electron microscopy - VCB vesicle-containing body     

The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L.)

 Sperm cells released from in vivo-in vitro grown pollen tubes of tobacco are associated in pairs and initially enclosed by the plasma membrane of the pollen tube. When the sperm cells are placed together, using glass microinjector needles, in an enzymatic solution, up to half undergo cellular fusion with subsequent nuclear fusion. The frequency of sperm cell fusion decreases with time during the elongation of the pollen tube, suggesting that mechanisms inhibiting self-fusion of sperm cells may develop as the pollen tube elongates through the style toward the ovule. This tendency may play an important role in inhibiting fusion of the two sperm cells inside the calcium-rich synergid where the male germ unit dissociates and sperm cells are transported to their target cells - the egg and central cell. Received: 25 August 1997 / Revision accepted 16 March 1998     

Symplastic communication between the central cell and the egg apparatus cells in the embryo sac of Torenia fournieri Lind. before and during fertilization

 Various membrane-impermeable, water-soluble fluorescent tracers with different molecular weights were microinjected into the central cell of the embryo sac of Torenia fournieri Lind. before and during fertilization. Before anthesis, there was high symplastic permeability between the central cell and the egg apparatus cells. In this stage, fluorescent tracers up to 10 kDa could pass from the central cell into the egg apparatus cells, whereas those with larger molecular weight remained in the central cell. As the embryo sac matured, symplastic permeability decreased such that 2 d after anthesis only tracers less than 3 kDa could spread from the central cell into the egg cell. There appeared to be no symplastic permeability between the primary endosperm and zygote after fertilization, since tracers as small as 521 Da could not pass into the zygote in about half of the microinjected embryo sacs. This is the first report of a change in cell-to-cell communication among the cells of the female germ unit before and after fertilization. Received: 16 December 1999 / Accepted: 4 February 2000     

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.     

Associations among components of the male germ unit followingin vivo pollination in barley

Summary The male gametophyte of barley has been studied during post-pollination stages from the time of pollen activation through pollen tube growth to the level of the ovule. Techniques included the use of serial thick sectioning, serial ultrathin sectioning of re-embedded thick sections, and computer generation as well as manual production of three-dimensional reconstructions. It was found that after pollination, but before the sperms exit the pollen grain, an intimate association is formed between the vegetative nucleus and the two sperms, and between the sperms themselves. The latter association is maintained through the duration of pollen tube growth observed in this study. These results indicate that the male germ unit of barley, a grass, is ultimately similar to that of other angiosperms studied thus far at the electron microscope level and that the concept of the male germ unit may have even greater significance and application within flowering plants than previously thought.     

Origin of sperm cell association in the “male germ unit” ofBrassica pollen

The association of the two sperm cells inBrassica napus pollen following the generative cell division was investigated. The generative cell during division is located in the center of the pollen grain, within the vegetative cell. The space present between the two cells is slightly irregular as seen following standard glutaraldehyde fixation. After completion of mitosis vesicles appear in the equatorial plane, coalescing centripetally to form a cell plate which fuses with the membrane of the generative cell, dividing it in two sperm cells. They are isolated from the vegetative cell by the space between the two cell membranes and are separated from each other by a similar space resulting from the cell plate formed during cytokinesis.     

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.     

Preliminary observations on the formation of the male germ unit inCatananche caerulea L. (Compositae: Lactuceae)

Summary We have used the freeze fracture and cytoplasmic maceration technique to study microsporogenesis inCatananche caerulea L. and here we present preliminary observations of the male germ unit (MGU)in situ. The technique has the advantage of enabling the complex structure of the MGU to be seen in a single fracture plane whereas previously this was only possible by the reconstruction of transmission electron micrographs of serial thin sections. The arrangement of the two generative cells and the vegetative nucleus closely resembles that described from other species includingPlumbago zeylanica.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Ultrastructure of sperm cells and the male germ unit in pollen tubes ofNicotiana tabacum

Summary The structure of sperm cells and their association with the vegetative nucleus in pollen tubes ofNicotiana tabacum grown in styles were observed with the electron microscope, demonstrating the existence of a male germ unit. The two sperm cells are arranged in tandem and are closely associated with the vegetative nucleus, which always takes the lead. The leading sperm cell (SC 1) has a long and narrow cytoplasmic projection which lies within the enclaves of the much lobed vegetative nucleus, thus forming a physical association. The trailing sperm cell (SC 2) and the SC 1 are not only joined by a common transverse cell wall but also are surrounded by a periplasm bounded by the plasma membrane of the sperm cells and that of the vegetative cell, thus forming a structural connection. The sperm cells are elongated, with cytoplasmic projections at the anterior end of the SC 1 and at both ends of the SC 2. The cytoplasm of both sperm cells includes mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, small vacuoles and axially oriented microtubules. No plastids were observed.Abbreviations DAPI 4,6-diamino-2-phenylindole - MGU male germ unit - MT microtubule - SC 1 the leading sperm cell physically associated with the vegetative nucleus - SC 2 the trailing sperm cell     

Preliminary results of a fertilization-irrigation experiment in a Quercus ilex L. forest in relation to leaves and twigs characteristics

A multifactorial ANOVA experiment was designed for a Quercus ilex forest in the Prades mountains in Tarragona (NE Spain). The purpose of this study was to clarify the relative importance of water, nitrogen and phosphorus as factors controlling primary production.Eight plots comprising all possible combinations of the three factors (including a control) were laid out in three replicate blocks. Fertilizers were applied to the appropriate treatments in March 1989 with a dosage of 125 kg of P/ha and 250 kg N/ha. Irrigation was applied with a rate of 20 mm per week during the warm season.To evaluate the effects on canopy structure we measured specific leaf weight (mg/cm²), average leaf weight (mg), average twig height and average twig length. These variables were selected as they are the most likely to manifest changes over short time periods following changes in environmental conditions.The values of these parameters before initial treatments (autumn 1988) and after treatments (summer 1990) were analyzed. The results obtained show that water and nitrogen have a greater effect than phosphorus on tree canopy structure, at least during initial development.     

Steroid transport through the surface of the prothoracic gland cells in Galleria mellonella L.

Steroid transport through the cell surface of the giant polyploid prothoracic gland cells of Galleria mellonella L. was studied by an ultracytochemical method. The alkaloid digitonin, known to form a complex with all sterols having a free-OH radical in position 3, proved to be suitable for studying the interiorisation of moulting hormone precursors and the release of synthesized hormones. The results suggest that cholesterol uptake in the last larval instar occurs by macropinocytosis during the feeding period, while the release of the steroids produced by the gland occurs by reverse micropinocytosis mostly on days 5-7 of the instar. The two processes are not simultaneous. The intracytoplasmic localisation of the reaction product confirms the steroidogenic role of the prothoracic gland.