Download synchronous pollen mitosis and the formation of the generative cell

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
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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).)
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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
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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-
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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.
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J. (19666). Cytoplasmic connections between angiosperm meiocytes.
Ann. Bot. 30, 221-230.
LARSON, D. (1963). Cytoplasmic dimorphism within pollen grains. Nature, Land, zoo, 911-
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MOHLETHALER,
K. (1967). Ultrastructure and formation of plant cell walls. A. Rev. PL Physiol.
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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,
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{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 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.
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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.
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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.
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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.
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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.
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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.
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