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-HARRISONInstitute of Plant Development and Department of Botany,University of Wisconsin, Madison, Wisconsin, U.S.A.

SUMMARYIn 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 synchroneitycan be traced to the persistence of cytoplasmic connexions between the cells, from the meioticprophiase 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 amor-phuos polar structure lying against the spore wall. The cell plate formed after pollen mitosis ishemispherical, and its curved growth is related to a radial spread of the microtubules of thephragmoplast after the telophase of the division. The plate itself, and the wall derived from it,is identifiable as callose by its fluorescence properties. In the later development of the gameto-phyte, growth of the callose wall continues until the originally hemispherical generative cellbecomes separated from the spore wall. The cell then assumes a spherical shape and moves tothe vicinity of the vegetative nucleus, where it remains freely suspended, bathed in the cyto-plasm 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 eachanther loculus, the synchroneity can be traced to the existence of a system of cellularinterconnexions establishing complete cytoplasmic continuity throughout (Heslop-Harrison, 1966 a, b). Links between neighbouring meiocytes are usually broken beforemetaphase II, and where this occurs synchroneity is progressively lost thereafter.Within a single meiocyte, nuclei may become asynchronous if a dividing wall isformed at the dyad stage, or remain synchronous through meiosis II if the tetradcleavage is delayed until the completion of both divisions.

In species forming loose pollen, there are no interconnexions after the completionof meiosi9 and the release of the spores from the tetrads, and accordingly there is noclose synchronization in the later pollen mitosis. However, in some of the massulateorchids where the pollen is formed in aggregates of several hundred cells, the nucleiof all cells in a single massula are closely synchronized, not only during the meioticdivisions themselves but, subsequently, throughout pollen mitosis (Barber, 1942;Heslop-Harrison, 1953). If it be indeed true that strict synchroneity in nuclear be-haviour depends upon the sharing of a common cytoplasmic environment, it is to beexpected that in these orchids there should be a system of intercellular connexionspersistent after meiosis until the ripening of the pollen. The existence of these channels

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458 J. Heslop-Harrison

is demonstrated in this paper, and some observations on the pollen mitosis and theformation 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 duringmaturation, halved, and one half transferred immediately to 1*5% glutaraldehydebuffered in o-i M phosphate buffer at pH 7-0. The other hah0 was prefixed 15-20 minin acetic-alcohol (1:3), and stained in acetic orcein before squashing for microscopicobservation; from this material the developmental stage was established. Half-pollinia selected for electron microscopic study were retained in glutaraldehyde for48 h at room temperature, and then thoroughly washed in tap water and impregnatedwith 1 % osmium tetroxide buffered in o-1 M phosphate buffer at pH 7-0 for 2h at4 °C. The tissue was then washed once more, and dehydrated through an alcoholseries before embedding in Araldite. Sections were cut with glass knives, mounted onFormvar-coated grids, and post-stained with uranyl acetate in saturated solution in90 % ethanol. Observations were made with an AEI EM 6 microscope.

Pollinia for optical microscopy were fixed in Langlet's fixative, wax embedded andsectioned at 10-15 /*• Phase-contrast studies of the spindle were made with unstainedmaterial, and for fluorescence microscopy of callose, sections were treated withaqueous 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 thepollen mitosis together; the degree of synchroneity may be judged from the appearanceat metaphase, seen in Fig. 2. The overall sequence of events from the prophase of thedivision is illustrated in Figs. 3-9, and the behaviour of the spindle is summarized inFig. 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 plateslie parallel to the outer wall, and the spindle is truncated on this side (Figs. 2, 4). Theanaphasic movement carries the chromosomes destined to be incorporated into thevegetative cell (tube) nucleus towards the centre of each cell, while those forming thegenerative nucleus move up to the wall, with further truncation of the spindle (Fig. 5).The telophase nuclei differ in shape, the generative one remaining conspicuouslyflattened (Figs. 5, 6). The subsequent changes principally concern the spindle. Thephragmoplast expands laterally, and as this happens the generative nucleus rounds up.With further lateral expansion, the relationship of the spindle fibres with the originalplane of the metaphase plate disappears, and the phragmoplast becomes an assemblageof 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 wallbehind the generative nucleus, and in favourable sections tangential to the wall theycan 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 out-spreading 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 polarstructure observable in electron micrographs lies in the region P. (c) Telophase, withearly initiation of the cell plate, (D), (E) and (F), Lateral fanning out of the spindlemicrotubules, with continued growth of the cell plate to give a complete hemisphericalwall. In (E), A marks the region shown in the electron micrograph, Fig. 26, and the verti-cal line the plane of the section shown in the micrograph of Fig. 25. (G), Continuedgrowth of the generative cell wall in the plane of the spore wall, (H), Generative cellseparated from the spore wall and lying against the indented vegetative nucleus.(1), Condition at the time of pollen maturation, (g, generative nucleus; v, vegetativenucleus; w, cellulose cell wall (equivalent of the intine in the mature spore).)

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its fluorescence properties after aniline blue staining, the cell plate and the wallarising from it is of callose at all stages. The sequence of fluorescence photomicro-graphs in Figs. 15-17 can be related to the phase-contrast series (Figs. 5—11). Fig. 15shows the earliest appearance of the cell plate at a time when the equatorial plane ofthe phragmoplast is still flat. Subsequently, the cell plate grows laterally, curving as itdoes so, and giving more and more intense fluorescence, as evident in the two massulaeof Fig. 16. At the close of cytokinesis the generative cell is seen to be enclosed in abrilliantly 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 re-action, shows little fluorescence, and is presumably primarily cellulosic.

Continued growth of callose occurs subsequently in the plane of the parent cell walluntil the generative nucleus and associated cytoplasm are wholly enclosed in acallose sheath. The cell so formed then becomes detached from the parent cell walland 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 ofD. fuchsii have been published in an earlier paper (Heslop-Harrison, 19666). It hasbeen shown that with the passage into the meiotic prophase, the original plasmodes-mata are replaced by massive protoplasmic strands, the cytomictic channels of Gates(1911). Most of the strands formed in D. fuchsii are multiple, although no singlestrand exceeds 1 /i in diameter. These connecting filaments of protoplasm correspondto those Unking the meiocytes in non-massulate angiosperms and they are of com-parable dimensions (Heslop-Harrison, 1964, 19666). In non-massulate species, thelinks between the meiocytes are normally eliminated before meiosis II, and none isformed at all between the sister spores of a tetrad, but in the massulate orchids studiedsome at least of the connexions persist, and the walls formed between the spores arealso perforated and accommodate cytoplasmic strands of dimensions similar to thoseof 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 ofthis kind can be traced in all parts of any particular massula, suggesting that cyto-plasmic continuity exists throughout. The fact offers an explanation for the syn-chronized behaviour of the cells during pollen mitosis and the subsequent cytokinesisseen, for example, in Figs. 2 and 15-17.

As described above, the pollen mitosis is asymmetrical, the spindle becoming trun-cated at the pole facing the wall (Fig. 4). In early anaphase, however, the spindle micro-tubules curve inwards from the margin of the chromosome plate, focusing towards acentral region. A distinctive body was always observed close to the cell wall at thepoint of convergence, in a position, that is, corresponding to that of the centriole in ananimal 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 ofribosomes. 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 entangle-ment 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 abouto-6 /i, lying closely apposed to the plasmalemma, with a rather dense granularinterior and some suggestion of a lighter peripheral zone.

During meiosis, each massula is invested in a thick callose sheath, and thinnercallose walls exist between the meiocytes themselves (Fig. 14). The callose invest-ment is drastically reduced by the onset of the pollen mitosis (Fig. 15). As alreadyindicated, by this time the principal material of the internal spore walls is cellulose,although slight residual fluorescence does suggest the persistence of some callosecomponent. When mature, each massula is invested in the equivalent of an exine(Fig. 18). Judged from optical staining properties and electron density followingKMnO4 and OsO4 fixation, the exine material is closely similar chemically to thesporopollenin of species with pulverulent pollen. The coating is thickest on the outerfaces adjoining the tapetum, but it extends between the massulae, and particles areoccasionally visible even between the pollen cells of a single massula. The electron-transparent layer within the exine in Fig. 18 corresponds to the intine, and from itsstaining properties it is evidently cellulose, as in species with free pollen. As may beseen 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 beregarded as having an intine but no exine.

The formation of the cell plate after the pollen mitosis follows the general patternnow well established for normal somatic cell divisions (Miihlethaler, 1967), andrecently described in detail by Pickett-Heaps & Northcote (1966 a, 6) for wheatmeristems and the stomatal complex of young wheat leaves. There are novel features,however, and since the outcome of the division is a hemispherical wall, comparisonwith the process described by Pickett-Heaps & Northcote (19660) for the formationof the curved wall of the subsidiary cells of the wheat stomatal complex is of someinterest.

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 subsidiarycell. The corresponding region in the orchid microspore would be that marked A inFig. IE. Were the preliminaries of the division as described by Pickett-Heaps &Northcote, microtubules should be seen in transverse section in these positions. How-ever, these regions show no special features in the spore cell, and the cytoplasm in thevicinity is not invaded by microtubules until the extending phragmoplast approachesthe 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 theusual form of a flattened aggregate of unit-membrane bounded vesicles (Fig. 21).From the earliest appearance of these vesicles the equatorial zone reveals callosefluorescence (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 equatorof the phragmoplast, but as the aggregation of vesicles begins, it seems that mostterminate in the equatorial plane (Fig. 22). As the cell plate becomes better defined,the microtubules become constricted more and more, focusing towards the remainingapertures (Fig. 23). With the consolidation of the central region the main concentra-tion of microtubules extends marginally, curving to produce the aspect seen in phase-contrast 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 wallwith that of the parent cell. The microtubules of the expanded phragmoplast can beseen converging from the cytoplasm of the tube cell, and there is some indication thatnear the parent cell wall they pass without interruption into the cytoplasm of theprospective 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 runalong it towards the polar structure lying behind the telophase generative nucleus.

As lateral growth of the generative cell wall progresses, a junction with the parentspore cell wall is established. This is seen in Fig. 27. The callose of the generative cellwall has spread out slightly along the surface of the cellulose of the spore wall, forminga little foot within the plasmalemma; the contrast in electron density between the twopolysaccharides and the relative homogeneity of the callose is clearly seen.

Dictyosomes are present throughout the period of formation of the generative cellwall (Fig. 26), but only on the vegetative cell face. Dictyosome-derived vesicles arepresent in the cytoplasm, but the material so far examined does not offer convincingevidence 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 cellbecomes isolated, since plasmodesmata are not formed. The cytoplasm enclosed withinthe new wall bears a normal ribosome population, and a few profiles of endoplasmicreticulum are usually apparent (Fig. 28). The only organelles present are mito-chondria, and it is noteworthy that plastids are seemingly entirely excluded.

DISCUSSION

The observations recorded above suggest that the synchronization of mitoses in thespores of the massulate orchids is, like the earlier synchronization of meiosis, a con-sequence of the sharing of a common cytoplasmic matrix. In effect, the massula forms asyncytium from mid-leptotene to the time of pollen germination. The fact accountsnot only for the synchroneity in division, but for the complementation observed be-tween the aneuploid nuclei arising from meiosis in triploid dactylorchid hybrids. Allsuch nuclei enter pollen mitosis, whatever their chromosome numbers, presumablybecause within a single massula there are, in aggregate, integral numbers of balancedgenomes (Heslop-Harrison, 1953). However, the confused situation resulting from dis-oriented mitosis in numerous aneuploid nuclei prevents the normal differentiation ofvegetative 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 normallyfollows meiosis in species with free pollen is essential to permit the haploid genomes tofunction 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 themassulate orchids must naturally be to prevent the assertion of independence amongthe numerous nuclei within the massula. All will differ in consequence of gene segre-gation, but again because of compensation the differences cannot be expressed infunctions located in the cytoplasm. This implies that haploid incompatibility systemswould be unworkable; and indeed no self-incompatibility is known in the genusDactylorchis (Heslop-Harrison, 1954), which is well enough equipped with otherdevices to enforce outbreeding.

Detailed fine-structural studies of pollen formation in five other orchidaceousgenera have been made by Chardard (1958,1962). In the genera examined by Chardardmassulae are not formed, but the pollen grains remain coherent in the tetrads. Pollenmitosis in each of the four spores of the tetrad is synchronized, and here again theeffect is related to the persistence of massive cytoplasmic interconnexions between thespores after the completion of meiosis, strikingly well illustrated in Chardard's electronmicrographs. Different tetrads within each loculus of the anther behave asynchro-nously, since the cytoplasmic interconnexions present between the mother cells aresevered after the meiosis, as in species with pulverulent pollen.

In the dactylorchids described here, the cells of each massula are interdependentthroughout meiosis and the initial development of the gametophytes, but the massulaein each loculus are isolated from each other from preleptotene onwards by the forma-tion of the investing callose walls (Fig. 14). This isolation is reflected in the asynchronyobservable between massulae, through meiosis and subsequently. The obviousimplication of this, namely that so far as the control of meiosis is concerned there canbe no pervasive system operating throughout the loculus, will be discussed in afurther paper.

The observations on the formation of the generative cell wall demonstrate again thebasic morphogenetic role played by the spindle microtubules in cytokinesis even wherethe product is a hemispherical wall. The departures from the scheme described byPickett-Heaps & Northcote (19666) for wall formation in the stomatal cell complex ofwheat have already been noted. Essentially in the present instance we see a phragmo-plast acting to form an enclave within another cell by lateral expansion and cuivatureafter a mitosis, rather than a demarcation by microtubules of wall 'contact points' inadvance of nuclear division. Nevertheless the conclusion that the microtubules fulfiltheir morphogenetic function by directing the initial disposition of vesicles that latercoalesce to form the cell plate seems inescapable. A novelty here is that the vesiclescontain 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 freesuspension within the cytoplasm of the vegetative cell of the gametophyte is broughtabout by what is seemingly a unique process, namely the growth of a callose wall overthe surface of a cellulose wall followed by separation of the two. Evidently a processof 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 generativecell, nor the participation of callose as a structural material.

The cytoplasm of the generative cells of the orchid species studied contains most ofthe expected organelles, but it appears to be devoid of plastids, or at least of bodiesidentifiable as plastids by the criteria applicable to somatic cells. Chardard (1958,1962) similarly found plastids to be entirely absent from the generative cells of theeight orchid species studied by him, and recorded further that mitochondria could notbe distinguished within the thin pellicle of cytoplasm between the nucleus and thegenerative cell wall. On the other hand, Bopp-Hassenkamp (i960) reported thepresence of plastids and mitochondria in the generative and sperm cells of LiUum, whileDiers (1963) showed that all the expected organelles, including plastids, were presentin the generative cell cytoplasm of Oenothera hookeri. Larson (1963), in a fine-structuralstudy of the generative cells of several species of other families, noted that plastidswere 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 theflowering plants, one with, and one without, plastids.

In all accounts of the generative cell there is agreement that its protoplast becomesentirely isolated from the cytoplasm of the vegetative cell by the formation of a wallimpenetrated by plasmodesmata or wider protoplasmic interconnexions. The genera-tive cell moves through the pollen tube to effect fertilization, and such free migrationwould naturally itself preclude the persistence of plasmodesmata. However, it maybe surmised that it is in any event essential for the gamete genomes to be insulatedfrom activating factors in the vegetative cell cytoplasm during the period of vigorousmetabolism following upon pollen germination.

REFERENCES

ARENS, K. (1949). Provo de calose par meio da microscopia a luz fluorescente e aplicacoes dometodo. 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, 539-549-HESLOP-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 floweringplants. Endeavour 35, 65-72.

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HESLOP-HAHRISON, J. (19666). Cytoplasmic connections between angiosperm meiocytes.Ann. Bot. 30, 221-230.

LARSON, D. (1963). Cytoplasmic dimorphism within pollen grains. Nature, Land, zoo, 911-912.

MOHLETHALER, K. (1967). Ultrastructure and formation of plant cell walls. A. Rev. PL Physiol.18, 1-24.

PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (1966a). Organisation of microtubules and endo-plasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1,109—120.

PICKETT-HEAPS, J. D. & NORTHCOTE, D. H. (19666). Cell division in the formation of thestomatal complex of young leaves of wheat. J. Cell Sci. i, 121-128.

{Received 22 November 1967—Revised 17 December 1967)

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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 thedivision, x ca. 800.Fig. 3. Prophase in the outer cells of the massula. The outer wall already bears asporopollenin coat (sp), overlying an inner cellulose layer corresponding to theintine (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 thatbelow. The lateral spread of the spindle and the formation of the curved cell plate canbe traced in these two massulae. x ca. 1400.

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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 theflattened generative nucleus to show the regular radial orientation of the spindlefibres, which are to be interpreted as the optical image produced by aggregates of themicrotubules observed electron microscopically, x ca. 1850.

Fig. 9. Central area of the cell plate now consolidated; fibres visible only in theperipheral 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 indentedvegetative nucleus, x ca. 1600.

Fig. 13. Mature pollen, showing the generative cell lying suspended in the vegetativecell cytoplasm, x ca. 1700.

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Figs. 14-17 are fluorescence photomicrographs of aniline blue-stained massulae.Fig. 14. Massulae in late meiotic prophase. Each is surrounded completely by a heavycallose wall, and callose extends between the individual meiocytes, although these arelinked by cytomictic channels, x ca. 600.Fig. 15. Pollen mitosis, telophase state corresponding to that in the upper massula ofFig. 6. x ca. 620.Fig. 16. Two adjacent massulae showing the progressive curvature of the cell plateand 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 acomplete hemisphere enclosing the generative cell, x ca. 620.

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Figs. 18-31 are electron micrographs of glutaraldehyde-OsO4 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 thecorner of a massula judged to be daughters of the same meiosis. ' t', marks the site ofanother 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 theequatorial region, but it is evident that most terminate in the darker zones between thefirst callose-containing vesicles. Generative nucleus off tc the left, vegetative off to theright, x ca. 55000.

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Fig. 22. Later formation of the cell plate, showing the unit membrane-bounded callosemasses derived from aggregation of the earlier vesicles, x ca. 90000.

Fig. 23. Final phase in generative cell-wall formation. The remaining microtubulesconverge towards the last perforations in the callose wall in the peripheral region, asseen in Fig. 9. x ca. 60000.

Fig. 24. Section in the region A of Fig. 1 E, showing the microtubules of the expandedphragmoplast converging into the cytoplasm of the generative cell from the vegetativecell, x ca. 72000.

Fig. 25. Section in the plane of the vertical line of Fig. 1 E. The microtubules areseen curving towards the plasmalemma and others running along it. x ca. 130000.

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Fig. 26. Dictyosome with peripheral vesicles lying on the vegetative cell side of thenewly forming cell plate, x ca. 85000.Fig. 27. Junction of the callose generative cell wall with cellulose spore wall. The twowall materials can be distinguished by their different electron opacity, and the callose isseen already to be spreading out to form an island behind the plasmalemma. Even-tually this extends centripetally until the generative cell is wholly ensheathed withcallose. x ca. 50000.Fig. 28. Generative cell and a portion of the surrounding vegetative cell cytoplasmshortly after the completion of the cell plate, but before the subsequent thickeningand the ingrowth of callose along the spore wall, x ca. 35000.

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