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J. Celt Sci. 3, 457-466 (1968) Printed in Great Britain SYNCHRONOUS POLLEN MITOSIS AND THE FORMATION OF THE GENERATIVE CELL IN MASSULATE ORCHIDS J. HESLOP-HARRISON Institute of Plant Development and Department of Botany, University of Wisconsin, Madison, Wisconsin, U.S.A. SUMMARY In orchd species forming microspores in aggregates, the pollen mitotic division occurs synchronously in all cells of each massula, as do the earlier meiotdc divisions. The synchroneity can be traced to the persistence of cytoplasmic connexions between the cells, from the meiotic prophiase until pollen maturation. The mitosis giving the generative nucleus is asymmetrical, and the spindle is truncated at one side, where the microtubules converge towards an amorphuos polar structure lying against the spore wall. The cell plate formed after pollen mitosis is hemispherical, and its curved growth is related to a radial spread of the microtubules of the phragmoplast after the telophase of the division. The plate itself, and the wall derived from it, is identifiable as callose by itsfluorescenceproperties. In the later development of the gametophyte, growth of the callose wall continues until the originally hemispherical generative cell becomes separated from the spore wall. The cell then assumes a spherical shape and moves to the vicinity of the vegetative nucleus, where it remains freely suspended, bathed in the cytoplasm of the vegetative cell but insulated from it by the completely ensheathing callose wall. INTRODUCTION In those flowering plants with simultaneous meiosis in all mother cells of each anther loculus, the synchroneity can be traced to the existence of a system of cellular interconnexions establishing complete cytoplasmic continuity throughout (HeslopHarrison, 1966 a, b). Links between neighbouring meiocytes are usually broken before metaphase II, and where this occurs synchroneity is progressively lost thereafter. Within a single meiocyte, nuclei may become asynchronous if a dividing wall is formed at the dyad stage, or remain synchronous through meiosis II if the tetrad cleavage is delayed until the completion of both divisions. In species forming loose pollen, there are no interconnexions after the completion of meiosi9 and the release of the spores from the tetrads, and accordingly there is no close synchronization in the later pollen mitosis. However, in some of the massulate orchids where the pollen is formed in aggregates of several hundred cells, the nuclei of all cells in a single massula are closely synchronized, not only during the meiotic divisions themselves but, subsequently, throughout pollen mitosis (Barber, 1942; Heslop-Harrison, 1953). If it be indeed true that strict synchroneity in nuclear behaviour depends upon the sharing of a common cytoplasmic environment, it is to be expected that in these orchids there should be a system of intercellular connexions persistent after meiosis until the ripening of the pollen. The existence of these channels 29-2 458 J. Heslop-Harrison is demonstrated in this paper, and some observations on the pollen mitosis and the formation of the generative cell wall are put on record. MATERIALS AND METHODS Observations were made on two species, Dactylorchis fuchsii and D. purpureUa, grown in cultivation. Complete pollinia were dissected out at various times during maturation, halved, and one half transferred immediately to 1*5% glutaraldehyde buffered in o-i M phosphate buffer at pH 7-0. The other hah0 was prefixed 15-20 min in acetic-alcohol (1:3), and stained in acetic orcein before squashing for microscopic observation; from this material the developmental stage was established. Halfpollinia selected for electron microscopic study were retained in glutaraldehyde for 48 h at room temperature, and then thoroughly washed in tap water and impregnated with 1 % osmium tetroxide buffered in o-1 M phosphate buffer at pH 7-0 for 2h at 4 °C. The tissue was then washed once more, and dehydrated through an alcohol series before embedding in Araldite. Sections were cut with glass knives, mounted on Formvar-coated grids, and post-stained with uranyl acetate in saturated solution in 90 % ethanol. Observations were made with an AEI EM 6 microscope. Pollinia for optical microscopy were fixed in Langlet's fixative, wax embedded and sectioned at 10-15 /*• Phase-contrast studies of the spindle were made with unstained material, and for fluorescence microscopy of callose, sections were treated with aqueous aniline blue at o-oi %, following Arens (1949). OBSERVATIONS The pollen mitosis At the end of meiosis, each massula contains 300-400 spore nuclei. These enter the pollen mitosis together; the degree of synchroneity may be judged from the appearance at metaphase, seen in Fig. 2. The overall sequence of events from the prophase of the division is illustrated in Figs. 3-9, and the behaviour of the spindle is summarized in Fig. 1. The prophase nuclei come to lie close to the periclinal walls within the massulae, a disposition particularly well seen in the outer cells (Fig. 3). The metaphase plates lie parallel to the outer wall, and the spindle is truncated on this side (Figs. 2, 4). The anaphasic movement carries the chromosomes destined to be incorporated into the vegetative cell (tube) nucleus towards the centre of each cell, while those forming the generative nucleus move up to the wall, with further truncation of the spindle (Fig. 5). The telophase nuclei differ in shape, the generative one remaining conspicuously flattened (Figs. 5, 6). The subsequent changes principally concern the spindle. The phragmoplast expands laterally, and as this happens the generative nucleus rounds up. With further lateral expansion, the relationship of the spindle fibres with the original plane of the metaphase plate disappears, and the phragmoplast becomes an assemblage of fibres radiating from a centre between the generative nucleus and the adjacent wall. As may be seen from Figs. 6 and 7, a zone of fibres comes to lie close to the wall behind the generative nucleus, and in favourable sections tangential to the wall they can be seen as a complete ring radiating from the position of the nucleus (Fig. 8). Pollen mitosis and generative cell formation 459 Formation and characteristics of the generative cell wall The first evidence of the deposition of a cell plate appears soon after telophase (Fig. 6), and the plate continues thereafter to spread marginally, following the outspreading phragmoplast, until it forms a complete hemisphere (Fig. 7). Judged from Fig. 1. Diagrammatic summary of the events of the pollen mitosis, (A) Metaphase, and (B) anaphase, showing the asymmetrical spindle (cf. Hagerup, 1938). The polar structure observable in electron micrographs lies in the region P. (c) Telophase, with early initiation of the cell plate, (D), (E) and (F), Lateral fanning out of the spindle microtubules, with continued growth of the cell plate to give a complete hemispherical wall. In (E), A marks the region shown in the electron micrograph, Fig. 26, and the vertical line the plane of the section shown in the micrograph of Fig. 25. (G), Continued growth of the generative cell wall in the plane of the spore wall, (H), Generative cell separated from the spore wall and lying against the indented vegetative nucleus. (1), Condition at the time of pollen maturation, (g, generative nucleus; v, vegetative nucleus; w, cellulose cell wall (equivalent of the intine in the mature spore).) 460 J. Heslop-Harrison its fluorescence properties after aniline blue staining, the cell plate and the wall arising from it is of callose at all stages. The sequence of fluorescence photomicrographs in Figs. 15-17 can be related to the phase-contrast series (Figs. 5—11). Fig. 15 shows the earliest appearance of the cell plate at a time when the equatorial plane of the phragmoplast is still flat. Subsequently, the cell plate grows laterally, curving as it does so, and giving more and more intensefluorescence,as evident in the two massulae of Fig. 16. At the close of cytokinesis the generative cell is seen to be enclosed in a brilliantly fluorescing hemispherical cap on the side facing the vegetative cell (Fig. 17). At this stage its outer wall is that of the parent cell, which gives a strong PAS reaction, shows little fluorescence, and is presumably primarily cellulosic. Continued growth of callose occurs subsequently in the plane of the parent cell wall until the generative nucleus and associated cytoplasm are wholly enclosed in a callose sheath. The cell so formed then becomes detached from the parent cell wall and moves into the vicinity of the vegetative nucleus, which assumes a crescent shape (Fig. 12). As the pollen matures, the enlarged tube nucleus rounds up once more, although still remaining in close association with the generative cell (Fig. 13). Electron microscopy Electron micrographs of the pre-meiotic archesporium and leptotene meiocytes of D. fuchsii have been published in an earlier paper (Heslop-Harrison, 19666). It has been shown that with the passage into the meiotic prophase, the original plasmodesmata are replaced by massive protoplasmic strands, the cytomictic channels of Gates (1911). Most of the strands formed in D. fuchsii are multiple, although no single strand exceeds 1 /i in diameter. These connecting filaments of protoplasm correspond to those Unking the meiocytes in non-massulate angiosperms and they are of comparable dimensions (Heslop-Harrison, 1964, 19666). In non-massulate species, the links between the meiocytes are normally eliminated before meiosis II, and none is formed at all between the sister spores of a tetrad, but in the massulate orchids studied some at least of the connexions persist, and the walls formed between the spores are also perforated and accommodate cytoplasmic strands of dimensions similar to those of the strands connecting the meiocytes. A link between spores is illustrated in Fig. 19. The location of the cells shown, at the •corner of a massula, indicates that they are the products of the same meiosis. Links of this kind can be traced in all parts of any particular massula, suggesting that cytoplasmic continuity exists throughout. The fact offers an explanation for the synchronized behaviour of the cells during pollen mitosis and the subsequent cytokinesis seen, for example, in Figs. 2 and 15-17. As described above, the pollen mitosis is asymmetrical, the spindle becoming truncated at the pole facing the wall (Fig. 4). In early anaphase, however, the spindle microtubules curve inwards from the margin of the chromosome plate, focusing towards a central region. A distinctive body was always observed close to the cell wall at the point of convergence, in a position, that is, corresponding to that of the centriole in an animal mitosis. Marginally, this body is approached by numerous microtubules, mostly oriented radially, but with many distributed more randomly. In section the Pollen mitosis and generative cell formation 461 converging microtubules are seen to lie each in a clear zone, forming a halo free of ribosomes. At the edge of the polar structure the microtubules form a loose meshwork; a few may enter it, but it is evident that the body itself is not simply a dense entanglement of microtubules, as is evident from the electron micrograph of the central region, (Fig. 20). The polar structure is seemingly a hemisphere, with a diameter of about o-6 /i, lying closely apposed to the plasmalemma, with a rather dense granular interior and some suggestion of a lighter peripheral zone. During meiosis, each massula is invested in a thick callose sheath, and thinner callose walls exist between the meiocytes themselves (Fig. 14). The callose investment is drastically reduced by the onset of the pollen mitosis (Fig. 15). As already indicated, by this time the principal material of the internal spore walls is cellulose, although slight residual fluorescence does suggest the persistence of some callose component. When mature, each massula is invested in the equivalent of an exine (Fig. 18). Judged from optical staining properties and electron density following KMnO4 and OsO4 fixation, the exine material is closely similar chemically to the sporopollenin of species with pulverulent pollen. The coating is thickest on the outer faces adjoining the tapetum, but it extends between the massulae, and particles are occasionally visible even between the pollen cells of a single massula. The electrontransparent layer within the exine in Fig. 18 corresponds to the intine, and from its staining properties it is evidently cellulose, as in species with free pollen. As may be seen from Fig. 3, this layer is continuous with the internal walls of the outer spores. Since the spores within the massula have similar cellulose walls, they may thus be regarded as having an intine but no exine. The formation of the cell plate after the pollen mitosis follows the general pattern now well established for normal somatic cell divisions (Miihlethaler, 1967), and recently described in detail by Pickett-Heaps & Northcote (1966 a, 6) for wheat meristems and the stomatal complex of young wheat leaves. There are novel features, however, and since the outcome of the division is a hemispherical wall, comparison with the process described by Pickett-Heaps & Northcote (19660) for the formation of the curved wall of the subsidiary cells of the wheat stomatal complex is of some interest. According to these authors, prior to the division giving the subsidiary cell nucleus, bands of microtubules consistently form, apposed to the lateral wall of the parent cell, marking in advance the junction zones of the future curved wall of the subsidiary cell. The corresponding region in the orchid microspore would be that marked A in Fig. IE. Were the preliminaries of the division as described by Pickett-Heaps & Northcote, microtubules should be seen in transverse section in these positions. However, these regions show no special features in the spore cell, and the cytoplasm in the vicinity is not invaded by microtubules until the extending phragmoplast approaches the wall, and their orientation is then radial, as evident in Fig. 8. The cell plate is initiated in the equatorial region of the phragmoplast, taking the usual form of a flattened aggregate of unit-membrane bounded vesicles (Fig. 21). From the earliest appearance of these vesicles the equatorial zone reveals callose fluorescence (Fig. 15, which corresponds approximately in respect to mitotic stage to 462 J. Heslop-Harrison Fig. 21). The microtubules of the spindle initially show continuity across the equator of the phragmoplast, but as the aggregation of vesicles begins, it seems that most terminate in the equatorial plane (Fig. 22). As the cell plate becomes better defined, the microtubules become constricted more and more, focusing towards the remaining apertures (Fig. 23). With the consolidation of the central region the main concentration of microtubules extends marginally, curving to produce the aspect seen in phasecontrast images such as Fig. 6. Ultimately the margins of the hemispherical plate approach the parent cell wall. Fig. 24 is of the area A in the diagram, Fig. 1 E, before fusion of the generative wall with that of the parent cell. The microtubules of the expanded phragmoplast can be seen converging from the cytoplasm of the tube cell, and there is some indication that near the parent cell wall they pass without interruption into the cytoplasm of the prospective generative cell. Fig. 25 is of a section along the vertical line in Fig. IE. Evidently the microtubules curve down towards the plasmalemma, and then run along it towards the polar structure lying behind the telophase generative nucleus. As lateral growth of the generative cell wall progresses, a junction with the parent spore cell wall is established. This is seen in Fig. 27. The callose of the generative cell wall has spread out slightly along the surface of the cellulose of the spore wall, forming a little foot within the plasmalemma; the contrast in electron density between the two polysaccharides and the relative homogeneity of the callose is clearly seen. Dictyosomes are present throughout the period of formation of the generative cell wall (Fig. 26), but only on the vegetative cell face. Dictyosome-derived vesicles are present in the cytoplasm, but the material so far examined does not offer convincing evidence to suggest that they contribute to the growing callose wall. With the linking of the new callose wall with that of the spore, the generative cell becomes isolated, since plasmodesmata are not formed. The cytoplasm enclosed within the new wall bears a normal ribosome population, and a few profiles of endoplasmic reticulum are usually apparent (Fig. 28). The only organelles present are mitochondria, and it is noteworthy that plastids are seemingly entirely excluded. DISCUSSION The observations recorded above suggest that the synchronization of mitoses in the spores of the massulate orchids is, like the earlier synchronization of meiosis, a consequence of the sharing of a common cytoplasmic matrix. In effect, the massula forms a syncytium from mid-leptotene to the time of pollen germination. The fact accounts not only for the synchroneity in division, but for the complementation observed between the aneuploid nuclei arising from meiosis in triploid dactylorchid hybrids. All such nuclei enter pollen mitosis, whatever their chromosome numbers, presumably because within a single massula there are, in aggregate, integral numbers of balanced genomes (Heslop-Harrison, 1953). However, the confused situation resulting from disoriented mitosis in numerous aneuploid nuclei prevents the normal differentiation of vegetative and generative cells, and although the triploid massulae survive for a period, they are infertile. Pollen mitosis and generative cell formation 463 It has been argued that the isolation of the spores in the tetrad which normally follows meiosis in species with free pollen is essential to permit the haploid genomes to function independently of the diploid parent and of each other (Heslop-Harrison, 1964, 1966 a, b). The effect of the persistent sharing of a common cytoplasm in the massulate orchids must naturally be to prevent the assertion of independence among the numerous nuclei within the massula. All will differ in consequence of gene segregation, but again because of compensation the differences cannot be expressed in functions located in the cytoplasm. This implies that haploid incompatibility systems would be unworkable; and indeed no self-incompatibility is known in the genus Dactylorchis (Heslop-Harrison, 1954), which is well enough equipped with other devices to enforce outbreeding. Detailed fine-structural studies of pollen formation in five other orchidaceous genera have been made by Chardard (1958,1962). In the genera examined by Chardard massulae are not formed, but the pollen grains remain coherent in the tetrads. Pollen mitosis in each of the four spores of the tetrad is synchronized, and here again the effect is related to the persistence of massive cytoplasmic interconnexions between the spores after the completion of meiosis, strikingly well illustrated in Chardard's electron micrographs. Different tetrads within each loculus of the anther behave asynchronously, since the cytoplasmic interconnexions present between the mother cells are severed after the meiosis, as in species with pulverulent pollen. In the dactylorchids described here, the cells of each massula are interdependent throughout meiosis and the initial development of the gametophytes, but the massulae in each loculus are isolated from each other from preleptotene onwards by the formation of the investing callose walls (Fig. 14). This isolation is reflected in the asynchrony observable between massulae, through meiosis and subsequently. The obvious implication of this, namely that so far as the control of meiosis is concerned there can be no pervasive system operating throughout the loculus, will be discussed in a further paper. The observations on the formation of the generative cell wall demonstrate again the basic morphogenetic role played by the spindle microtubules in cytokinesis even where the product is a hemispherical wall. The departures from the scheme described by Pickett-Heaps & Northcote (19666) for wall formation in the stomatal cell complex of wheat have already been noted. Essentially in the present instance we see a phragmoplast acting to form an enclave within another cell by lateral expansion and cuivature after a mitosis, rather than a demarcation by microtubules of wall 'contact points' in advance of nuclear division. Nevertheless the conclusion that the microtubules fulfil their morphogenetic function by directing the initial disposition of vesicles that later coalesce to form the cell plate seems inescapable. A novelty here is that the vesicles contain callose or callose precursors, and no cellulosic wall is ever formed. The transition of the generative cell from a lateral position against one wall to free suspension within the cytoplasm of the vegetative cell of the gametophyte is brought about by what is seemingly a unique process, namely the growth of a callose wall over the surface of a cellulose wall followed by separation of the two. Evidently a process of this kind must occur widely in the development of male flowering-plant gameto- 464 J. Heslop-Harrison phytes, not necessarily involving a wall so well-defined as that of the orchid generative cell, nor the participation of callose as a structural material. The cytoplasm of the generative cells of the orchid species studied contains most of the expected organelles, but it appears to be devoid of plastids, or at least of bodies identifiable as plastids by the criteria applicable to somatic cells. Chardard (1958, 1962) similarly found plastids to be entirely absent from the generative cells of the eight orchid species studied by him, and recorded further that mitochondria could not be distinguished within the thin pellicle of cytoplasm between the nucleus and the generative cell wall. On the other hand, Bopp-Hassenkamp (i960) reported the presence of plastids and mitochondria in the generative and sperm cells of LiUum, while Diers (1963) showed that all the expected organelles, including plastids, were present in the generative cell cytoplasm of Oenothera hookeri. Larson (1963), in a fine-structural study of the generative cells of several species of other families, noted that plastids were identifiable in most, but that they were rare and structurally much simplified. Evidently the possibility is open that two types of generative cell may exist in the flowering plants, one with, and one without, plastids. In all accounts of the generative cell there is agreement that its protoplast becomes entirely isolated from the cytoplasm of the vegetative cell by the formation of a wall impenetrated by plasmodesmata or wider protoplasmic interconnexions. The generative cell moves through the pollen tube to effect fertilization, and such free migration would naturally itself preclude the persistence of plasmodesmata. However, it may be surmised that it is in any event essential for the gamete genomes to be insulated from activating factors in the vegetative cell cytoplasm during the period of vigorous metabolism following upon pollen germination. REFERENCES K. (1949). Provo de calose par meio da microscopia a luz fluorescente e aplicacoes do metodo. Lilloa 18, 71-75. BARBER, H. N. (1942). The pollen-grain division in the Orchidaceae. J. Genet. 43, 97-103. BOPP-HASSENKAMP, G. (i960). Elektronmikroskopische Untersuchungen an Pollenschlauchen zweier Liliaceen. Z. Naturf. 15, 91—94. CHARDARD, R. (1958). L'ultrastructure des grains de pollen d'Orchidees. Revue Cytol. Biol. vig. 19, 223-235. CHARDARD, R. (1962). Recherches sur les cellules-meres des microspores des Orchidees. fitude au microscope electronique. Revue Cytol. Biol. vig. 24, 1-148. DIKRS, L. (1963). Elektronmikroskopische Beobachtungen an der generativen Zelle von Oenothera hookeri Torr. et Gray. Z. Naturf. iSb, 562-566. GATES, R. R. (1911). Pollen formation in Oenothera gigas. Ann. Bot. 25, 909—940. HAGERUP, O. (1938). A peculiar asymmetrical mitosis in the microspore of Orchis. Hereditas 34. 94-96. HESLOP-HARRISON, J. (1953). Microsporogenesis in some triploid Dactylorchid hybrids. Ann. Bot. 17, 539-549HESLOP-HARRISON, J. (1954). A synopsis of the Dactylorchids of the British Isles. Ber. geobot. Forsch. Inst. RUbelfiir 1953, pp. 53-82. HESLOP-HARRISON, J. (1964). Cell walls, cell membranes and protoplasmic connections during meiosis and pollen development. In Pollen Physiology and Fertilisation (ed. H. F. Linskens), pp. 39-47. Amsterdam: North Holland Publishing Co. HESLOP-HARRISON, J. (1966a). Cytoplasmic continuities during spore formation in flowering plants. Endeavour 35, 65-72. ARENS, Pollen mitosis and generative cell formation 465 J. (19666). Cytoplasmic connections between angiosperm meiocytes. Ann. Bot. 30, 221-230. LARSON, D. (1963). Cytoplasmic dimorphism within pollen grains. Nature, Land, zoo, 911- HESLOP-HAHRISON, 912. MOHLETHALER, K. (1967). Ultrastructure and formation of plant cell walls. A. Rev. PL Physiol. 18, 1-24. J. D. & NORTHCOTE, D. H. (1966a). Organisation of microtubules and endoplasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1, PICKETT-HEAPS, 109—120. PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (19666). Cell division in the formation of the stomatal complex of young leaves of wheat. J. Cell Sci. i, 121-128. {Received 22 November 1967—Revised 17 December 1967) 466 J. Heslop-Harrison Figs. 2-13 show pollen mitosis and generative cell formation, phase contrast. Fig. 2. Metaphase in one massula, showing the high level of synchroneity in the division, x ca. 800. Fig. 3. Prophase in the outer cells of the massula. The outer wall already bears a sporopollenin coat (sp), overlying an inner cellulose layer corresponding to the intine (w). x ca. 1600. Fig. 4. Metaphase, showing the truncated spindle, x ca. 1400. Fig. 5. Late anaphase; vegetative nuclei to the left, generative to the right, x ca. 1400. Fig. 6. Telophase in the upper massula, nuclei at the close of the division in that below. The lateral spread of the spindle and the formation of the curved cell plate can be traced in these two massulae. x ca. 1400. Journal of Cell Science, Vol. 3, No. 3 J. HESLOP-HARRISON {Facing p. 466) Fig. 7. Continuous curved cell plate in a pollen grain at the corner of a massula. x ca. 1400. Fig. 8. Section of a cell from the same massula as Fig. 7, but cut in the plane of the flattened generative nucleus to show the regular radial orientation of the spindle fibres, which are to be interpreted as the optical image produced by aggregates of the microtubules observed electron microscopically, x ca. 1850. Fig. 9. Central area of the cell plate now consolidated; fibres visible only in the peripheral position. The stage corresponds to that seen in the electron micrograph, Fig. 23. x ca. 1400. Figs. 10, 11. Completion of the callose wall and rounding off of the generative cell, x ca. 1400. Fig. 12. Generative cell detached from the spore wall, and lying against the indented vegetative nucleus, x ca. 1600. Fig. 13. Mature pollen, showing the generative cell lying suspended in the vegetative cell cytoplasm, x ca. 1700. Journal of Cell Science, Vol. 3, No. 3 J. HESLOP-HARRISON Figs. 14-17 are fluorescence photomicrographs of aniline blue-stained massulae. Fig. 14. Massulae in late meiotic prophase. Each is surrounded completely by a heavy callose wall, and callose extends between the individual meiocytes, although these are linked by cytomictic channels, x ca. 600. Fig. 15. Pollen mitosis, telophase state corresponding to that in the upper massula of Fig. 6. x ca. 620. Fig. 16. Two adjacent massulae showing the progressive curvature of the cell plate and the increase in thickness of callose. x ca. 620. Fig. 17. Massula at a stage corresponding to that of Fig. 10. The callose wall forms a complete hemisphere enclosing the generative cell, x ca. 620. Journal of Cell Science, Vol. 3, No. 3 J. HESLOP-HARRISON Figs. 18-31 are electron micrographs of glutaraldehyde-OsO 4 fixed material: c, callose;£«, generative nucleus; m, mitochondrion; p, plasmalemma; s, lipid droplets; sp, sporopollenin exine; w, cellulose cell wall (equivalent of the intine). Fig. 18. Corners of adjacent massulae, ensheathed in the equivalent of the exine. x ca. 7300. Fig. 19. Cytoplasmic channel, ' / ' , between two spores, from their position in the corner of a massula judged to be daughters of the same meiosis. ' t', marks the site of another link in a different plane, x ca. 16000. Fig. 20. Central region of the polar structure, late anaphase. x ca. 80000. Fig. 21. Early deposition of the cell plate. Some microtubules may still traverse the equatorial region, but it is evident that most terminate in the darker zones between the first callose-containing vesicles. Generative nucleus off tc the left, vegetative off to the right, x ca. 55000. Journal of Cell Science, Vol. 3, No. 3 J. HESLOP-HARRISON Fig. 22. Later formation of the cell plate, showing the unit membrane-bounded callose masses derived from aggregation of the earlier vesicles, x ca. 90000. Fig. 23. Final phase in generative cell-wall formation. The remaining microtubules converge towards the last perforations in the callose wall in the peripheral region, as seen in Fig. 9. x ca. 60000. Fig. 24. Section in the region A of Fig. 1 E, showing the microtubules of the expanded phragmoplast converging into the cytoplasm of the generative cell from the vegetative cell, x ca. 72000. Fig. 25. Section in the plane of the vertical line of Fig. 1 E. The microtubules are seen curving towards the plasmalemma and others running along it. x ca. 130000. Journal of Cell Science, Vol. 3, No. 3 W J. HESLOP-HARRISON Fig. 26. Dictyosome with peripheral vesicles lying on the vegetative cell side of the newly forming cell plate, x ca. 85000. Fig. 27. Junction of the callose generative cell wall with cellulose spore wall. The two wall materials can be distinguished by their different electron opacity, and the callose is seen already to be spreading out to form an island behind the plasmalemma. Eventually this extends centripetally until the generative cell is wholly ensheathed with callose. x ca. 50000. Fig. 28. Generative cell and a portion of the surrounding vegetative cell cytoplasm shortly after the completion of the cell plate, but before the subsequent thickening and the ingrowth of callose along the spore wall, x ca. 35000. Journal of Cell Science, Vol. 3, No. 3 27 tr> W 28' J. HESLOP-HARRISON