Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. II. FERTILIZATION AND PROEMBRYO DEVELOPMENT

Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) ovules were used to study male gamete formation, insemination of the egg, and free nuclear and cellular proembryo development. Two male nuclei form as the pollen tube either reaches the megaspore wall or as it enters the archegonial chamber. No cell wall separates them. They are contained within the body-cell cytoplasm. A narrow extension of the pollen tube separates the neck cells and penetrates the ventral canal cell. The pollen tube then releases its contents into the egg cytoplasm. The two male gametes and a cluster of paternal organelles (plastids and mitochondria) migrate within the remains of the body-cell cytoplasm toward the egg nucleus. Microtubules are associated with this complex. The leading male gamete fuses with the egg nucleus. The zygote nucleus undergoes free nuclear division, but the cluster of paternal organelles remains discrete. Free nuclei, paternal and maternal nucleoplasm, maternal perinuclear cytoplasm, and the cluster of paternal organelles migrate en masse to the chalazal end of the archegonium. There, paternal and maternal organelles intermingle to form the neocytoplasm, the nuclei divide, and a 12-cell proembryo is formed. The importance of male nuclei or cells, the perinuclear zone, and large inclusions in cytoplasmic inheritance are discussed in the Pinaceae and in other conifer families. This completes a two-part study to determine the fate of paternal and maternal plastids and mitochondria during gamete formation, fertilization, and proembryo development in Douglas fir.     

FERTILIZATION IN PLUMBAGO ZEYLANICA: GAMETIC FUSION AND FATE OF THE MALE CYTOPLASM

Developmental phases surrounding the processes of gametic delivery and fusion were examined ultrastructurally in the reduced megagametophyte of Plumbago zeylanica, which lacks synergids. Gametic delivery occurs at the end of pollen tube growth and results in deposition of two male gametes, a vegetative nucleus, and a limited amount of pollen cytoplasm between the egg and central cell. Discharge of these materials from the tube is accompanied by loss of inner and outer pollen tube plasma membranes, loss of sperm-associated cell wall components, and disruption of the formerly continuous cell wall between the egg and central cell. The dispersion of egg cell wall components directly exposes female reproductive cell membranes to the unfused male gametes and pollen tube without disrupting gametic cell plasma membranes. Presence of unfused sperms within the female gametophyte appears to be a transitory phenomenon, lasting less than 5 min at the end of over 8½ hr of pollen tube growth. At the time of gametic deposition, plasma membranes of unfused sperm cells become directly appressed to plasma membranes of both the egg and central cell. Gametic fusion is initiated by a single fusion event between membranes of participating male and female cells, which is rapidly followed by subsequent, secondary fusion events between the same two cells at different locations along their surface. Gametic fusion results in the transmission of male gamete nuclei with co-transmission of nearly the entire sperm cytoplasmic volume and organellar complement, and it is possible to identify heritable male cytoplasmic organelles within both the incipient zygote and endosperm. Paternally originating plastids may be distinguished from maternal plastids by differences in morphology and staining characteristics, whereas paternal mitochondria may be distinguished from maternal mitochondria by populational differences in mitochondrial size which are statistically significant. Such observations further indicate that transmitted paternal mitochondria seem to remain viable, as judged by their ultrastructural appearance, and are transmitted exclusively by sperm cytoplasm rather than discharged pollen cytoplasm. The presence of anucleate, membrane-bounded cytoplasmic bodies between the egg and central cell are identifiable on the basis of their enclosed organelles and indicate that fragmentation of a small amount of the sperm cytoplasm associated with the vegetative nucleus commonly occurs. The presence and identification of sperm cytoplasmic organelles and associated membranes within female reproductive cells following gametic transmission represents strong evidence in support of the cellular basis of nuclear and cytoplasmic transmission during sexual reproduction in Plumbago.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

Generative cell division and sperm cell formation in barley

Summary Shortly before and during division, the generative cell of barley (Hordeum vulgare L.) is located near the vegetative nucleus, in the peripheral layer of the highly vacuolated vegetative cell at the aperture pole. This position is also characteristic of the two resulting sperm cells. Conventional mitosis of the generative cell is followed by cytokinesis through cell plate formation. Just after division, the two sperm cells are enclosed together within a common inner vegetative cell plasma membrane, and they gradually separate from each other only during pollen maturation. The space between the generative or sperm cell plasma membrane and the vegetative cell plasma membrane is very thin and appears to be devoid of a cell wall. Both the generative cell and the young sperm cells contain a normal set of organelles; plastids devoid of starch are only sporadically observed. Our data indicate that in Hordeum vulgare the generative cell divides after migrating inside the pollen grain. This follows the pattern of development well established for several species with tricellular pollen.     

AN ULTRASTRUCTURAL AND STEREOLOGICAL ANALYSIS OF POLLEN GRAINS OF HYOSCYAMUS NIGER DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Selected nuclear and cytoplasmic changes of pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development, induced by anther culture, were analyzed and compared ultrastructurally using stereological methods. Potentially embryogenic, uninucleate pollen could be identified within 6 hr of culture by an increased ratio of the volume density of the nucleolar granular zone to the volume density of the fibrillar zone and an increased ratio of dispersed to condensed chromatin in the nucleoplasm. Nonembryogenic pollen in vitro and in vivo possessed prominent nucleolar fibrillar zones and low ratios of dispersed to condensed chromatin. These differences may reflect changes in nuclear activity in potentially embryogenic pollen grains during early stages of culture. Following the first haploid mitosis, in potentially embryogenic pollen the generative cell maintained its large granular nucleolus and high ratio of dispersed to condensed chromatin through its first division to form a proembryoid. The volume fraction of the cytoplasm occupied by mitochondria and plastids and the area fraction occupied by RER and Golgi cisternae differed in the generative cells of potentially embryogenic and nonembryogenic pollen. Those changes only detected in generative cells of potentially embryogenic pollen include: increased area and complexity of cytoplasmic membranes, increased mitochondrial volume, and the presence of plastids at all stages of development. These results support the idea that embryogenic induction of H. niger takes place at the uninucleate stage of development and that subsequent nuclear and cytoplasmic changes are essential for continued sporophytic development.     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. I. POLLEN TUBE AND ARCHEGONIAL DEVELOPMENT

Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) ovules were used to study the method of pollen tube formation and penetration of the nucellus, the movement of the body cell down the pollen tube and development of the archegonia. No pollination drop forms but nucellar tip cells produce a minute secretion that may initiate pollen tube formation. Pollen tubes penetrate the nucellus causing degeneration of nucellar cells in contact with the pollen tube tip. The body cell becomes highly lobed and the tube cytoplasm forms thin sheets between the lobes. This may be the mechanism by which the large body cell is pulled down the narrow pollen tube. Body cell plastids and mitochondria remain unaltered during pollen tube growth, whereas tube cell organelles show signs of degeneration. The pollen tube penetrates the megaspore wall and settles in the archegonial chamber. During pollen elongation and pollen tube growth the egg matured. Egg cell plastids were transformed into large inclusions which filled the periphery of the egg while mitochondria migrated to the perinuclear zone. The neck cells, ventral canal cell and archegonial jacket cells are described. The significance of the body cell and egg cell ultrastructure is discussed in light of recent restriction fragment length polymorphism studies of plastid and mitochondrial inheritance in the Pinaceae.     

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.     

Unequal distribution of ubiquitinated proteins during Pinus bungeana pollen development

The production of gametogenesis is a charming and complicated event in higher plants, during that stage the protein population undergoes substantial alterations. But few attentions have been paid to the possible roles of the UPP in gymnosperm gametogenesis. In the present study, DNA-specific probe 4′,6-dimidino-phenylindole was employed to assess Pinus bungeana pollen developmental stage. It was revealed that the division of pollen mother cell occurred in late April. The uninucleate microspore then underwent three asymmetric divisions, forming a mature pollen grain including a tube cell and a generative cell together with two degenerated prothallial cells in early May. Immunofluorescence labeling of ubiquitinated proteins (UbPs) with an anti-ubiquitin antibody indicated that fluorescence signal was detected in both cytosol and nuclear of the microspore at the uninucleate stage. In the two-cell pollen grain, a brighter fluorescence was always detected in the first prothallial when compared with that in central cell. Similarly, unequal distribution of UbPs was observed again during the division of the central cell into the antheridial initial and the second prothallial cell. The high intensity of the fluorescence in the two degenerated prothallial cells remained in the mature pollen grain, but only a faint signal could be detected in the tube cell or the generative cell deriving from the division of the antheridial initial. The unequal distribution of UbPs was further unveiled by immunogold labeling among prothallial cells, generative cells and tube cells in mature pollen grains. Besides, Coomassie brilliant blue cytochemistry was also performed to illustrate the general subcellular distribution of total proteins in the two-cell and matured pollen grains. All these results indicated that the prothallial cells have high ratio of UbPs, and that the ubiquitin-mediated proteolysis might have an important role during pine pollen development.     

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

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.     

ULTRASTRUCTURE OF PLASTID INHERITANCE: GREEN ALGAE TO ANGIOSPERMS

1. Plastid inheritance in most green algae and land plants is uniparental. In oogamous species, plastids are usually derived from the maternal parent; even when inheritance is biparental, maternal plastids usually predominate. Only a few species of conifer are known to have essentially paternal plastid inheritance. In spite of the overall strong maternal bias, there exists a spectrum of species in which plastid inheritance ranges from purely maternal to predominantly paternal. 2. Factors that influence the pattern of plastid inheritance operate both before (often long before) and after fertilization. For example, several different mechanisms for exclusion of plastids from particular cells, none of which is completely effective on its own, may operate sequentially during both gametogenesis and embryo-genesis. There appears to exist a general trend such that the more highly evolved the organism, the more numerous the mechanisms employed and the earlier they first come into operation. The pattern of plastid inheritance shown by a species represents the efficiency or lack of efficiency of these combined mechanisms. 3. In the newly-formed zygote of many unicellular algae, the plastids from both gametes are present and there is direct competition between them. Often the plastid from one mating type (usually the ‘invading’ male gamete, where this can be identified) quickly degenerates. Species such as Chlamydomonas are unusual in that the plastids from the two gametes fuse. In spite of this, inheritance of plastid DNA is normally uniparental. How this is accomplished remains unclear. In oogamous algae, the paternal plastids which enter the egg cell are frequently fewer in number and smaller in size than those contributed by the female gamete. The reduced contribution of paternal plastids can result from asymmetrical cell division or from differential timing of cell and plastid division during spermatogenesis. 4. In species ranging from unicellular algae to angiosperms, plastids may be partially or completely debarred from particular cells at critical stages during the reproductive cycle. An important factor in this form of plastid elimination is their postioning with respect to the nucleus prior to a cell division. When plastids closely encircle the nucleus, they are usually incorporated equally into the two daughter cells; when the plastids are concentrated at some distance from the nucleus, they are frequently excluded from one daughter cell. 5. Elimination of plastids from a gamete prior to plasmogamy prevents direct competition between the two types of plastid in the zygote or embryo. Perhaps the most effective method of excluding paternal plastids from the egg cell has been achieved by some lower land plants; the plastids migrate to the posterior part of the spermatozoid, and are discarded from there in a discrete vesicle before the egg is reached. 6. Plastid inheritance in conifers appears to be unique. In those species in which the derivation of plastids in the pro-embryo can be determined, it has been found that they come only from the male gamete. Maternal plastids are positively excluded from the pro-embryo and later degenerate. 7. In most angiosperm species plastid inheritance is maternal; in only a few species is it regularly biparental. The first step towards exclusion of paternal plastids often takes place in the uninucleate pollen grain where the plastids may be concentrated at the pole of the cell farthest from the site of the future generative cell. Any plastids that succeed in entering the generative cell may degenerate before the gametes are released from the pollen tube. Even if paternal plastids reach the egg, they are at a disadvantage because they are (a) entering an environment that is essentially alien, and (b) normally present in much smaller numbers than maternal plastids. Later, when the zygote divides, the few paternal plastids may fail to become incorporated in the small terminal cell which gives rise to the embryo proper. 8. There appears to be no consistent evolutionary progression in the use of more efficient mechanisms to influence plastid inheritance; most of the mechanisms associated with exclusion of paternal plastids in angiosperms, for example, can also be found in one or other species of green alga. The primary factors that influence plastid inheritance appear to be (I) direct competition in the zygote between plastids of the two parental types – the principal mechanism operating in isogamous algae, but also operating in some angiosperms; and (2) the divergent evolution of the two types of gamete - on the one hand a small male gamete with a minimum of cytoplasm which is capable of moving (spermatozoid) or being moved (pollen) efficiently, and, on the other hand, a large egg cell with numerous organelles, which is well able to act as ‘host’ for the future zygote. Many of the additional mechanisms that influence the pattern of plastid inheritance seem to be the more or less ‘accidental’ result of other evolutionary events.     

Why are plastids maternally inherited in epilobium? Ultrastructural observations during microgametogenesis

We have investigated the sequential stages of microgametogenesis by electron microscopy, to determine the basis of maternal inheritance of plastids in Epilobium. The development of both the vegetative and generative cells has been followed using a semi-artificial growth system for pollen tubes. The generative cells inside the pollen grain contains numerous mitochondria, 5–8 proplastids, and, in contrast to the vegetative cytoplasm, only a few vacuoles. When the generative cell has divided into the two sperm cells inside the pollen tube, small vesicles deriving from dicytosome cisternae become abundant. These vesicles appear to form vacuoles by fusion which then contain remnants of fibrillar, globular or membranaceous material. It is suggested that this material derives from proplastids as the proplastids disappear either before or shortly after the generative cell has divided, concurrently with the appearance of the ‘remnants’ in the vacuoles. The mitochondria of the sperm cells remain intact.     

Intergeneric pollen – megagametophyte relationships of conifers in vitro

 Germinating pollen from larch (Larix occidentalis), Sitka spruce (Picea sitchensis) and white pine (Pinus monticola) were co-cultured with megagametophytes dissected from cones of other genera (Pseudotsuga menziesii, Larix×eurolepis and Pinus monticola). Pollen was presented to megagametophytes possessing archegonia which were either alive, degenerating or dead. In addition, pollen was presented to fertilized megagametophytes and to megagametophytes that had been cut in half. Megagametophyte penetration by pollen tubes and male gamete release into archegonia were verified by serial sections of glycomethacrylate-embedded specimens. Pollen tubes penetrated through any part of the apex of the megagametophyte. Division of the body cell into the two gametes was regularly observed. Delivery of gametes was confirmed between spruce and larch. Pollen tubes also penetrated fertilized megagametophytes, dead or degenerating archegonia as well as wounded and/or cut surfaces. This demonstrates the inability of the male gametophyte to optimize its mating efforts, since it is unable to differentiate between healthy and unhealthy archegonia. The megagametophyte cells are unable to optimize male selection. They may produce secretions of a generally attractive nature, as pollen is attracted to the apex of the megagametophyte, but archegonia themselves do not produce pollen-specific signals of either a promotive or inhibitory nature. These results open new avenues for the development of novel breeding strategies where natural breeding barriers may be bypassed. Received: 19 March 1998 / Accepted: 29 April 1998     

Cytological Studies of the Cytoplasmic Inheritance in Lilium regale and L. davidii

The distribution and characteristics of plastids and mitochondria in the generative and sperm cells of Lilium regale Wils. and L. davidii Duch. were described. In L. regale there were few plastids and abundant mitochondria in the newly formed generative cell. When the generative cell became free in the vegetative cytoplasm, the plastids degenerated completely within the generative cell. It was further proved by DAPI fluorescent technique that there was no organell DNA in the generative cell within the mature pollen grain or the pollen tube. However, distribution of the plastids was strictly polarizable during the division of the micmspore in L. davidii, resulting the lack of plastids in the newly formed generative cell. Data of RFLP analysis comparable between L. davidii, L. longifiorum and their interspecific hybrid have also proved the plastid inheritance in L. davidii to be of uniparental maternal transmission. Although the mitoehondria were observed both in the generative and sperm cells of L. regale and L. davidii but their DNA was decomposed in the male gametophyte stage. Therefore the mitochondda in the sperm cell could not be transmitted into the offspring. The results provided the detail, cytological evidence that organelles in the microgametophyte are incapable of genetic transmission in the two species of Lilium.     

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.     

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.