J . Linn. SOC.(Bot.), 59, 379, p . 253
With 4 plates and 1 text-figure
Printed in Great Britain
Febrwlry, 1966
253
A new interpretation of the structure of the megaspore
membrane in some gymnospermous ovules
BY JOHN M. PETTITT
Department of Palaeontology, British Museum (Natural History), London
(Acceptedfor publication August, 1965)
Communicated by the President
INTRODUCTION
The megaspore membrane in gymnospermous ovules lies between the haploid
gametophyte tissue of the embryo sac and the diploid tissue of the nucellus. I n mature
ovules of some gymnosperms the membrane is often of considerable thickness and from
its behaviour in Sudan stains and other properties it can be classed as a lipid as broadly
defined by Lovern (1955). The evidence available from histochemistry (De Sloover,
1963a) and from acetolysis (Lurzer, 1956) would strongly suggest that the megaspore
membrane in gymnosperms is a t least chemically allied to, if not identical with, the
pollen wall substance sporopollenin, recently investigated chemically by Shaw & Yeadon
(1964).
Recent investigations into the structure of the megaspore membrane in some Palaeozoic fossil gymnosperms using light and electron microscopy (the results are to be
published elsewhere) has revealed that the structure in these fossil ovules is strikingly
different from anything that has yet been described in the ovules of living gymnosperms.
I n an attempt to establish the possible origin of the structural differences a study of the
morphology of the megaspore membrane in the ovules of some living gymnosperms
was undertaken using both light and electron microscopy.
The formation and structure of the megaspore membrane in living gymnosperms
has been observed by a number of workers more or less incidentally during studies on
ovule ontogeny, and the first fully comparative investigation on the subject was published
by Thomson in 1905. Briefly, Thomson regards the megaspore membrane in gymnosperm
ovules as being composed of 2 layers, a n outer ‘suberized’ exosporium and an inner
endosporium. The endosporium, which in the later stages of development is very much
reduced in thickness, is subdivided into 2 homogeneous layers. The outer layer is
‘suberized’ but contains cellulose towards its inner border and the inner layer is composed
mainly of cellulose but in association with a substance resembling pectin. It is worth
noting that Thomson’s histochemical determinations are based on the appearance of
the membrane after treating sections in chlor-zinc iodine, haematoxylin and saffranin
or sulphuric acid and iodine. Thus, he did not employ stains that are specific for either
suberin or pectin. Nevertheless, his results turn out to be remarkably accurate. Although
Thomson’s main concern is with the megaspore membrane he also gives a n account of
the structure and fate of the tissue immediately surrounding the embryo sac. Thomson
finds that during the course of ovule development the cell walls of this tissue, which he
calls the tapetum, become ‘suberized’ and later, in older ovules and mature seeds, the
tissue becomes completely disorganized and is represented merely by debris. Thomson
also noted that in the seeds of many genera a ‘suberized’ layer was present over the
18
254
JOHN
M. PETTITT
outer walls of the superficial prothallial cells and which is often in close contact with the
megaspore membrane.
More recently, Favre-Duchartre (1956) has described the development of the megaspore
membrane in Ginkgo biloba L. This author, using more refined histochemical techniques
than Thomson, finds that the female prothallus in Ginkgo is a t f i s t surrounded by a
pecto-cellulosic membrane, about 1 p in thickness, which stains with ruthenium red.
This, by analogy with pollen wall terminology, he calls the intine (Text-fig. 1A). Later,
as the coenocytic prothallus enlarges, the surface of this membrane is covered by a
series of perpendicular rod-like elements of uniform length which are sudanophilic and
which Fane-Duchartre calls the exine, again by analogy with pollen wall terminology
(Text-fig. 1 A). At this stage in development a mass of particles having the same staining
properties as the exine is detectable lying between it and the cells of the nucellus. These
particles, which are deposited on the surface of the exine, are interpreted as originating
from the degenerating tapehl cells. As cellular differentiation becomes detectable in
the embryo sac of Ginkgo the prothallial tissue is seen to separate from the megaspore
membrane and the outer cellsof the gametophyte are covered by a sudanophilic component
which Faae-Duchartre terms the cuticle (Text-fig. 1 A).
It may be mentioned here that recent work on pollen wall formation (see for example,
Rowley, 1959,1964-4;Larson & Lewis, 1962; Heslop-Harrison, 1963a, b, 1964) shows quite
clearly that, in contradistinction to the events in the Ginkgo ovule, the main part of the
pollen intine is deposited after the formation of the exine.
The most detailed account of megaspore membrane development and structure is
that published by De Sloover in 3 papers (1963a, b, 1964) on Encephlartos poggei Asch.
Using Sudan black and a variety of other staining procedures on sections, De Sloover
finds that in the early stages the embryo sac is surrounded by a 2-layered membrane
neither of which is coloured by the Sudan stain. The double membrane increases in
thickness and a t the coenocytic stage of prothallial development an outer sudanophilic
layer is detectable which can be resolved as a series of radially orientated rods subtended
by a homogeneous layer which is unaffected by Sudan black but which colours pink-red
with ruthenium red (Text-fig. 1 B).
When the gametophyte of E. p g g e i is a t the cellular-alveolar stage the megaspore
membrane is composed of 5 layers. De Sloover designates the layers Cl-C5, C1 being
the outermost, sudanophilic layer and C5 the inner cellulosic layer which is in continuity
with the radial walls of the prothallial cells. Layers C3 and C4 are regarded as having
differentiated from the homogeneous inner layer present a t the coenocytic stage. From
histochemical evidence and the results of acetolysis De Sloover concludes that layers
Cl-C3 are composed of sporopollenin, C4 is pectic and C5 cellulosic. The stratzcation
of the megaspore membrane detected in E. p g g e i is shown on the right of Text-fig. 1 B,
although the diagram represents a somewhat later stage in development when layer C4
is absent.
As the embryo sac increases in volume and cell walls begin to differentiate in the
prothallus, the inner cellulosic layer (C5) becomes detached from the remainder of the
membrane by the total disappearance of the pectic layer ( 0 4 ) . At about this stage there
is differentiation of the radial elements into clavate structures with divided bases which
are joined by a continuous membrane represented by layer C2.
I n the seed the megaspore wall is crushed against the surrounding tissue as the embryo
sac expands, so that the radial rods become completely disorganized. The final formative
process of the megaspore wall is reached by the deposition of a new Sudan black-staining
layer on the outside of the inner layer (C5). De Sloover, like Favre-Duchartre (1956),
calls this membrane the cuticle (Text-fig. 1).
n- G I - - - - l l n c 9 - z.1 f ~ l l r \ - ; l ~ - o - n u ~ h a r t r p nanTItR
j,@,.rninalaavhased aa more
The megaspore membrane in some g y m n o s p e r m ovules
255
Layers C1 and C2 would correspond to the ectexine of Faegri’s classilkation (1956);
C2 being the ‘foot layer’. C3 would be the endexine of Faegri whilst the pectic layer C4,
which does not survive in the seed, is regarded as equivalent to the medine detected by
Saad (1961) in the pollen walls of angiosperms.
De Sloover records that in ovules of E . poggei, when the prothallus is a t the cellularalveolar stage of development, a very clearly defined jacket of cells is differentiated
immediately outside the embryo sac. The cells comprising the jacket stretch and
EARLY
MATURE
A
B
C
Text-fig. 1. Diagrammatic representation of sections through the megaspore membranes in
gymnosperm ovules. A. Ginkgo biloba (after Favre-Duchartre, 1956). B. Encephalartos poggei
(after De Sloover, 1963a, b ) . C. Zamkfioridana, Pinus ezcelsa etc. c, cuticle; e , exine; i,
intine; m, megaspore membrane; n, nexine; 8, sexhe; t, tapetum; tm, tapetal membrane. For
full explanationsee text.
disintegrate following the expansion of the megaspore. During this phase, staining and
acetolysis reveal that the walls of the jacket cells as well as small granules that have
become apparent in their cytoplasm and in the spaces between the cells have the same
properties as the exine of the megaspore wall. I n more mature ovules and in the seed
the jacket is no longer detectable but the acetolysis-proof granules are seen attached
t o the megaspore exine.
Both Favre-Duchartre (1956) and De Sloover (19636, 1964) suggest that the exine
material in ovules originates in the tapetum or jacket cells surrounding the megaspore.
I n one of his publications De Sloover (19636) summarizes the evidence that development
of the megaspore membrane in E . poggei is centripetal. This process he maintains, departs
256
JOHN
M.P
E
~
sharply from the centrifugal differentiation of the pollen wall in angiosperms described
by Heslop-Harrison (1963a, b). D e Sloover accepts that the acetolysis-proof granule8
which remain after the degeneration of the jacket cells are secretory products of this
tissue and do in fact contribute towards the structure of the exine; but he argues that
their deposition on to the already structurally distinct outer layer, whilst centrifugal,
is essentially a casual process and should not, therefore, be regarded as exine formation
s e w stricto.
MATERIALS AND METHODS
The ovules and seeds used in this study were principally from ZamiaJEoridanaA. DC.
and Pinus excelsa Wall. This material was supplemented with ovules and seeds of a
number of other genera including Encephlartos frtdericiguilielmi Lehm. PUN.,
Cyeas
revoluta Thunb., -urn
u h Brongn., Pinus sylvestris L., T a x w bamta L. and Juniperus
excelsa Bieb. Since none of this material wm specifically fixed for electron microscopy,
cytoplasmic detail was not preserved. However, the h e structure of the megaspore exine,
which was of immediate interest, was not noticeably altered by the use of conventional
cytological fixatives. For comparison, male fructilications of Encephalartos willosus Lem.,
Ginkgo biloba L. and T a x w baccata L. were examined.
Conifer male and female reproductive structures intended for light microscopy were
fixed in either Carnoy I (ethanol-acetic)or in 10% neutral formalin as soon after collection
as possible. All the cycad and cinkgo material was selected from museum collections
stored in either alcohol or formalin. The original fixative in the latter case was presumably
formalin, and it is known that a t least some of the alcohol-stored collections were fixed
in formalin-acetic-alcohol.
After dehydration through an ethanol series the o d e s and pollen sacs were embedded
in paraffin wax (m.p. 56-58°C) using either methyl benzoate-celloidin, chloroform or
benzene as a clearing agent. Sections were cut a t 4-8 p, dewaxed, carried to water and
subjected to one of the following histochemical procedures: (1) A saturated solution
of Sudan black B in 70 % ethanol or its more specific acetylated derivative in 70 yo
ethanol (Casselman, 1954) and carmalum. (2) Nile blue sulphate or Cain’s modification
of this test (see Casselman, 1962). (3) The periodic acid/SchifE (PA/S) test according to
Hotcbkiss (see Pearse, 1953). Control preparations, omitting the periodic acid treatment,
were carried through a t the same time. (4)0.02 yoruthenium red is distilledwater. Sections
stained in Sudan black B and carmalum, Nile blue sulphate and in ruthenium red were
mounted in Farrant’s medium and those treated by the PA/S procedure in Depex after
dehydration.
Conifer pollen sacs for electron microscopy were fixed in 1 % osmium tetroxide,
buffered with Verona1acetate to pH 7.3, for 14 hr. a t room temperature, in 6 yophosphate
buffered glutaraldehyde (pH 7.1) a t about 4°C for 17 hr. followed by 14 hr. in buffered
osmium tetroxide, or in 2 yo unbuffered potassium permanganate a t room temperature
for 2 hr. (Mollenhauer, 1958) immediately following collection. After dehydration in a
graded ethanol series (5 min. changes) the tissue was embedded in a 3 :1 mixture of
n-butyl :methyl methacrylate containing 1yo benzoyl peroxide or in a resin mixture of
Araldite M-Epon 812 (Larson, 1964) after impregnation a t reduced pressure.
Ultra-thin sections were cut using glass knives, mounted on copper grids with carbon
reinforcement and examined with a Siemens’ ‘Elmiskop la’ microscope operating a t
60 kV. Thicker sections were cut a t intervals for examination by direct light microscopy
and by phase contrast.
To supplement the histochemical detection of sporopollenin in wax sections the
acetolysis procedure of Erdtman (1954)was employed. After the removal of the wax the
preparations were carried to water and immersed in glacial acetic acid for a few minutes
before being transferred to acetolysis solution kept a t about 60°C in a water bath. When
acetolysis was completed, the sections were re-immersed in glacial acetic acid and carried
The megaspe menzbrane in some gymnospermous ovules
257
through decreasing concentrations of the acid to distilled water (several changes) and
stained with Sudan black B.
Megaspore membranes were isolated from entire or half ovules and seeds by the usual
acetolysis procedure (see Erdtman, 1954) and mounted in glycerine jelly. Those intended
for electron microscopy were embedded in methacrylate or Araldite M-Epon 812 as
described above.
OBSERVATIONS
1. The ovule
I n ovules of Zamia Jloridana when the embryo sac is in the free nuclear phase and the
nuclei are in a parietal position a very thin (less than 0.5 p) sudanophilic membrane is
detectable surrounding the megaspore (Text-fig. 1 C, m). Acetolysis of the sections leaves
the membrane unaffected and clearly discernible (Pl. 1, fig. 1 ) . The tapetal cells a t this
time show signs of degeneration and in sections stained with acetylated Sudan black B
the tapetal cytoplasm in some cells, particularly those towards the inside of the tissue,
is strongly sudanophilic. This Sudan colouration of the tapetal cytoplasm seems to be
caused by finely dispersed inclusions which cannot readily be resolved as discrete lipoidal
bodies. The tapetal cell walls are also noticeably sudanophilic and after section acetolysis
they survive as thin, resistant, perforate membranes which colour with acetylated
Sudan black B (Pl. 1, figs. 1, 2). The walls of the innermost tapetal cells are somewhat
different in appearance after section acetolysis. They are represented by either thin
sheets of resistant material, the inner surfaces of which are covered with small sudanophilic
droplets, or merely by aggregations of droplets which are usually less than 0.5 p in
diameter. The droplets are clearly seen under phase contrast illumination (Pl. 1, fig. 3)
and there is some evidence to suggest that they coalesce to form clusters which arrange
themselves against the tapetal cell walls. Similar aggregations can be detected on the
outer surface of the megaspore membrane.
The blue colouration of the megaspore membrane and tapetal droplets after treatment
of non-acetolyzed sections with Nile blue sulphate can be taken t o indicate that these
components contain a high proportion of acidic lipids (Pearse, 1953; Casselman, 1962)
and rather unexpectedly, both give a faintly positive PAjS reaction before, but not
after acetolysis.
,4s ovule development proceeds the sudanophilic megaspore membrane in Z. Jloriduna
increases in thickness. A comprehensive ontogenetic series was not available but a t a
time when the sudanophilic membrane is about 2.4 p thick, completely homogeneous
and without ornamentation, and the embryo sac contains a thin-walled tissue, an inner
non-lipoidal membrane is detectable. This layer stains intensely with ruthenium red
(Pl. 1, fig. 8). The total thickness of the megaspore membrane a t this stage is 3.6 p and
there is no measurable variation in different regions. The tapetum has become reduced
in thickness and its cells clearly more disorganized (Pl. 1, fig. 7 ) , no doubt partly as a
result of the increase in volume of the embryo sac.
If complete ovules of Z. Jloridana a t the developmental stage described above are
acetolyzed a resistant cellular sac representing the tapetum and containing a thin homogeneous megaspore membrane is released (Pl. 1, figs. 6 6 ) . Ultra-thin sections (Pl. 2,
fig, 6) show the somewhat granular composition of the megaspore membrane after
acetolysis.
The events leading towards the maturation of the embryo sac are accompanied by
the rapid disorganization of the tapetum. The tapetal cell walls and cytoplasm become
strongly sudanophilic.
I n unacetolyzed sections of Pinus excelsa ovules with mature archegonia, large lipoidal
droplets are revealed in the degenerating tapetal cytoplasm after Sudan black B staining.
Some of the droplets are apparently coalescing t o form aggregations against a thick
membrane which is present on the inner side of the tapetal tissue (Pl. 2, figs. 4, 5 ) .
258
JOHN M. PETTITT
Examination of this inner membrane with the electron microscope after acetolysis shows
a high concentration of droplets arranged along the surface (PI. 3, fig. 4 ) and from the
ultrastructural evidence i t would appear that the membrane itself originates by first
the partial and then the complete coalescence of the acetolysis-resistant droplets.
I n many sections the membrane is found disassociated from the collapsed tapetal
cells and lying between these and the gametophyte. But the occasional connexion between
it, the tapetal cells and the periphery of the embryo sac suggests that such separation is
an artifact caused during preparation.
I n ovules of Z.$widana with well-formed archegonia, the behaviour of the sudanophilic
tapetal cells can be readily followed in a single longitudinal section as their degeneration
and collapse a t the micropylar and chalaza1 ends of the ovule is less advanced than a t
the sides. Examination of Sudan black B preparations shows the tapetum as a well-defmed,
cellular tissue, colouring strongly with the Sudan stain and undoubtedly undergoing
some compression from the expanding gametophyte. As the tissue is followed laterally
it is seen to become progressively more compressed until it is represented merely by a
thin sudanophilic membrane which under low magmfkations looks more or less homogeneous (Pl. 2, fig. 1). Lipoidal droplets which are detectable as inclusions in the tapetal
cells would seem to become incorporated into the membrane, and in some locations they
can be detected adhering to the outer surface (Pl.2, fig. 2).
It would appear that this phenomenon of tapetal cell degeneration, compaction and
coalescence of the walls and contents to form an acetolysis-proof membrane occurs in
a diversity of gymnosperm ovules. P1. 3, figs. 1, 2 are electron micrographs of nonacetolyzed tapetal membranes from mature ovules of Cycas revoluta and Encephalartos
fredericiguilielmi. I n both these figures irregular sporopollenin droplets, many of which
in Cycas are hollow, are evidently coalescing to form a compact structure. I n surface
view under the light microscope the membranes appear as in P1. 2, fig. 3.
At the time the tapetum begins to degenerate the double-layered megaspore membrane
in Zamia is present as a morphologically distinct structure in close contact with the cells
of the prothallus. It can be stated here that a t no time during its development is there
any noticeable ornamentation in the form of radially arranged elements on the outer
surface of this membrane. As the gametophyte enlarges the megaspore membrane is
stretched and the inner, pectinous layer is no longer detectable as a distinct inner zone
in sections stained with ruthenium red.
I n all the genera studied the increase in volume of the gametophyte is accompanied
by a reduction in thickness in the outer, sudanophilic layer of the megaspore coat, so
that in sections of mature ovules i t can be seen as a blue-staining membrane usually less
than 1p i n thickness after treatment with acetylated Sudan black B (PI.2, fig. 4; Text-fig.
1C). Electron micrographs of sections cut through the peripheral prothallial cells show
that the membrane has developed strong anticlinal walls (Pl. 3, fig. 6) which can be
clearly seen under phase contrast as a cellular reticulum on the surface of the membrane
after acetolysis (PI. 3, fig. 3).
The continued blue colouration obtained after the Nile blue sulphate test indicates
that the tapetal material and the sudanophilic component of the megaspore membrane
still contain acidic lipids and in addition, both continue to give a weakly positive reaction
after the PA/S test.
I n certain ovules of 8. $oridam the abortive condition described by Smith (1910)
was encountered. I n her publication Smith illustrates a proliferation of what she calls
the ‘ spongy tissue’ in ovules where the megaspore had failed to develop. Longitudinal
sections of such ovules, acetolyzed and stained with acetylated Sudan black B show
that the walls of the ‘spongy tissue’ are acetolysis-resistant and sudanophilic. These
observations suggest that the tissue in question is formed by the proliferation of the
tapetum, and although the megaspore has failed to develop beyond a very rudimentary
stage, the secretory function of the tissue continued normally.
The m e g a s p e membrane i n some gymnospermous ovules
259
I n the seeds of the genera investigated the tapetal membrane is present as a thin,
granular sudanophilic structure situated between the embryo sac and the remnants of
the nuclleus. The megaspore membrane i6represented by a somewhat thinner sudanophilic
covering closely associated with the outermost cells of the prothallus (Pl.3, fig. 5).
2 . The pollen sac
According to Goebel’s (1905) classification the tapeta in the pollen sacs of Ginkgo
biloba, Encephalartos villosus and Taxus baccata are of the secretory type, in which the
cells remain intact a t the borders of the locules during the greater part of pollen develoment. If the tapetal cells of these genera are examined a t a stage when the pollen wall
is fully formed, sudanophilic droplets can be detected dispersed in the cytoplasm and
particularly large accumulations can be seen lining the inside of the cell walls (Pl. 4,
figs. 2 , 5 ) . That these droplets are chemically similar to sporopollenin is suggested not
only by their affinity for Sudan black but also by their resistance to acetolysis. I n at
least one genus, Ginkgo, the deposition takes the final form of a thin sheet of material
present as a continuous covering on the tapetal cell walls. After acetolysis, the covering
remains as a thin sac bearing a clear cellular reticulum, scattered with small droplets
and enclosing a mass of pollen grains (Pl. 4, fig. 4).
Prior to the dissolution of the tapetal cells in the pollen sacs of Taxus baccata lipoidal
material is commonly present as discrete droplets arranged along the cell walls. The
droplets stain intensely with acetylated Sudan black B (Pl. 4, fig. 2). Electron micrographs of glutaraldehyde-fixed osmium treated material give additional evidence of
their arrangement and suggest that in some cells there is in fact coalescence of the smaller
droplets to form large aggregations (Pl. 4, figs. 1,3).
DISCUSSION
The developmental process of the membrane surrounding the megaspore in gymnospermous ovules described above differs from those published by Favre-Duchartre (1956)
in Ginkgo and De Sloover ( 1 9 6 3 ~b), 1964) in Encephakrtos poggei in one major respect.
Whereas these authors find the ontogenetic increase in thickness and complexity as
progressive development affecting the original membrane present in the early stages,
my observations would suggest that in the mature ovules of Zamia Jloridana and Pinus
exceba a t least, the true megaspore membrane is represented as a thin acetolysis-proof
membrane closely associated with the peripheral cells of the embryo sac.
This membrane is evidently what both Fame-Duchartre and De Sloover call the cuticle
in their material (Text-fig. 1)and to which Thomson (1905)and Liirzer (1956) also draw
attention. The membrane that these authors (and others) have called the megaspore
membrane in the mature ovule and seeds would appear to be what I interpret as the
remains of the tapetal tissue and which cannot be regarded as part of the megaspore
membrane sensu stricto. Admittedly, the tapetal membrane surrounds the megaspore
and if by definition, the megaspore membrane is merely the membrane that surrounds
the megaspore, the term could justifiably be applied. However, this would invalidate the
assumed homology between the megaspore membrane in the ovule and the pollen exine,
which recent studies (e.g., Rowley, 1959; Larson & Lewis, 1962; Heslop-Harrison,
1963a, b ) clearly show is not formed from collapsed tapetal cells.
There are definite indications that very similar events take place in the tapetal cells
of the gymnosperm ovule and the tapetal cells of the pollen sac, the net result in both
cases being the accumulation of acetolysis-resistant, sudanophilic droplets which in the
ovule and in some cases also in the pollen sac coalesce t o form a membrane. The products
are basically the same and a single name should be applied to both. That this name should
be the ‘megaspore membrane’ is, to say the least, undesirable, for two reasons. Firstly,
the megaspore membrane in a t least some gymnosperm ovules would be what is called
the tapetum in the pollen sac, and clearly, the term megaspore membrane could not be
260
JOHN
M. P E ~ T . ~
applied to the latter. Secondly, attention would be directed away from the fact that a
membrane is present in close contact with the gametophyte which in all probability is
the true megaapore membrane and the homologue of the pollen exine.
The disappearance of the pectinow layer (04)and the formation of the cuticle recorded
by De Sloover (1963a,3) may be interpreted as the loss of the inner layer during expansion
of the gametophyte, resulting in the closer association of the sudanophilic layer and the
outer cells of the gametophyte, rather than the deposition of a new layer. An alternative
suggestion is that the layer (a)
becomes modified during the growth of the prothallus
so that it is not easily detected with the optical microscope. Electron micrographs indicate
that this in fact may be the case. It is of interest that Van Der Pluijm (1964) reports
the presence of a cuticle surrounding the embryo sac in an angiosperm.
There is nothing new in the discovery of products resembling sporopollenin in the
tapetal cells of pollen sacs; the fact has been known, as Heslop-Harrison (1962) points
out, for a hundred years.
Heslop-Harrison (1962, 19633) has published electron micrographs which show that
there is an accumulation of a substance resembling sporopollenin that is deposited as
plaques on the walls of the secretion tapeta in Cannabis sativa and Silene pendula prior
to the dissolution of the cells. The author presents evidence which suggests that the
sporopolleninplaques may originate in cytoplasmic organellesthat resemble mitochondria,
in size and shape, and points out that the presence of the plaques could be taken to indicate
that the sporopollenin is synthesized in the tapetal cells.
Although the origin of the acetolysis-proof lipoidal material (sporopollenin) of the
megaspore membrane in gymnosperm ovules has yet to be investigated ultrastructurally
it would be surprisingif the mechanism was found to be very different from that involved
in the formation of the pollen exine. The presence of a tegillate membrane surrounding
the embryo sac of Juniperus excelsa (Pl. 2, fig. 7 ; see also Liirzer, 1956) offers evidence
that the structural complexity of the megaspore membranein some ovules closelyparallels
that of some pollen exines.
The positive PA/S reaction of the sudanophilic material in the tapetal cells and the
megaspore membrane is open to two interpretations. Firstly, that the tapetal secretion
and the membrane substance are composed of a mixture of lipoidal material and carbohydrate. The fact that a positive reaction was not obtained after acetolysis would lend
support to this interpretation. It is interesting to note that Rowley (1964) has suggested
that the pollen exine may be functionally comparable with the mammalian placenta,
acting as a distribution manifold and filter and being concerned with substrate transport
to the protoplast, rather than just a product of the transport. If this suggestion is correct,
then the possibility that the PA/S test is demonstrating that a substance or substances
other than lipids is being transported from the tapetum to the megaspore exine cannot
be overlooked.
However, an alternative explanation is available. Gomori (1958) draws attention to
the fact that unsaturated fatty acids can be oxidized by periodic acid to aldehyde and
the latter would react with Schiff’s reagent to give a red colour characteristic of a positive
Schiff reaction. The blue colouration obtained after the Nile blue sulphate test would
indicate that both the tapetal secretion and the sudanophilic megaspore exine contain
acidic lipids which could include unsaturated fatty acids, and presumably these could
be oxidized in the manner suggested by Gomori.
Returning to the morphology of the megaspore membrane, there is evidently considerable structural variation in the membranes surrounding the megaspores in the
ovules of living gymnosperms. Liirzer (1956)illustrates acetolyzed megaspore membranes
from 8 conifer species under phase contrast and establishes that they are divisible into
2 distinct types; the Pinaceae-type which has an ornament (sexine) of baculate rods and
the Cupressaceae-typewhich is tegillate. There is close structural correspondencebetween
Liirzer’s illustration of the membrane from Pinus mugo and the tapetal membrane of
c
The megaspore membrane in some gymnosperms ovules
261
Pinus excelsa published here (Pl. 3, fig. a), and support is added to her interpretation
that, for example, Juniperus sabina has a tegihte membrane by the electron micrographs of the same structural complexity in Juniperus excelsa (Pl. 2 , fig. 7).
I n the ovules of certain Carboniferous pteridospems and cordaites, however, the
megaspore ,membranesare much simpler in construction and ultrastructurally resemble
the exines of some pteridophyte spores. The evidence for this will be presented elsewhere.
SUMMARY
A light and electron microscope investigation of the tapetum during stages in the
ontogeny of a variety of gymnospermous ovules reveals that eventually this tissue
degenerates and forms a thick acetolysis-resistant membrane (the tapetal membrane)
between the embryo sac and the nucellus. This membrane is not considered part of the
megaspore membrane in the sense that that term is used in free-sporing heterosporous
plants.
In the mature ovules of the genera investigated the true megaspore membrane is
present as a thin, acetolysis-proof membrane closely associated with the peripheral
prothallial cells and internal l a the thicker tapetal membrane. This megaspore membrane
can be related to a somewhat more elaborate structure that is present at the earlier stages
of development.
There is strong evidence to suggest that the events in the tapetum of the ovule are to
some extent paralleled in the tapetum of the pollen sac.
ACKNOWLEDGEMENTS
I am most grateful to the Director of the Royal Botanic Gardens, Kew and to Professor
T. Delevoryas of Yale University for supplying material. I should also like to express
my very sincere thanks to Professor Dennis Lacy of the Department of Zoology, St.
Bartholomew’s Medical College, London for allowing me free use of the electron microscope and technical facilities in his department. Iam also very grateful to Professor Lacy,
Dr Brian Lofts and Dr W. G. Chaloner for much helpful advice and discussion.
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W. G. B., 1962. HiStochemiical Techniqm. London.
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DE SLOOVER,
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J. L., 1963b. Jaquette endospermique et diff6rentiation de l’exine m6gasporale chez
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DE SLOOVER,
chez Encephalartos poggei Aach. Cellule, 64: 149-200.
ERDTMAN,
G., 1952. Pollen morphology and plant tuxononzy. Angiosperms. Stockholm.
ERDTMAN,
G., 1954. An. introduction to pollen analysis. Waltham, Mass.
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K., 1956. Recent trends in pdynology. Bot. Rev., 22: 639-64.
FAVRE-DUCHARTRE,
M., 1956. Contribution B 1’Qtudede la reproduction chez le Ginkgo bilboa. Revue
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N 1962.
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J., 1963a. An ultrastructural study of pollen wall ontogeny in Silene penduza.
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HESLOP-HARRISON,
J., 1963 b. Ultrastructural aspects of differentiation in sporogenous tissue. 17th
Symposium of the Society f o r Experimental Biology, 3 1 6 4 0 .
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J., 1964. Cell walls, cell membranes and protoplasmic connections during meiosis
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D. A., 1964. Processing pollen and spore exines for electron microscopy. Stain Tech., 39:
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JOHN M. PETTITT
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D. A. & LEWIS,C. W., 1962. Pollen wall development i n P a r k i n s o n b aculeuta. Qranapalynol.,
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J. R., 1959. The h e structure of the pollen wall in the Commelinaceae. C r a m palynol., 2 :
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J . R., 1964. Formation of the pore in pollen of Poa annua. Pollen Physiology and Fertilization
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EXPLANATION OF PLATES
&ATE
1
Z a m i a floridam
Fig. 1. Acetolyzed section from a young ovule showing the resistant tapetal cell walls and megaspore
membrane (m).Acetylated Sudan black B preparation. x 90.
Fig. 2. Details of the tapetal cell walls from the section in Fig. 1. x 480.
Fig. 3. Walls of the innermost tapetal cells after section acetolysis. Thin sheets of resistant material
are covered with sudanophilic droplets. Acetylated Sudan balck B preparation under anoptral contrast.
x 1200.
Fig. 4. Part of an acetolyzed megaspore membrane isolated from a complete ovule in which the embryo
sac contained a thin-walled tissue (see Fig. 8). x 100.
Fig. 5. Cellular 8&0 representing the tapetum, containing the megaspore membrane, released from an
acetolyzed ovule at the same stage of development as that in Fig. 8. x 8.5.
Fig. 6. Details of the tapetal B&C shown in Fig. 5. Small droplets can be seen adhering to the cell walls.
x 320.
Fig. 7. Acetolyzed section of the tapetum from a n ovule at the same stage of development as that in
Fig. 5 . Compare with Fig. 1. Acetylatd Sudan black B preparation. x 100.
Fig. 8. Section through a n unacetolyzed megaspore membrane showing the stratification after
acetylated Sudan black B staining. The outer (upper) layer stains strongly, the inner (lower) layer not
at all. x 400.
PLATE2
Zamia jlcwidam, P i n w excelsa, Encephalartos fredericiguilielmi, Juniperus excelsa
Fig. 1. Part of a longitudinal section of Zamiajlcwidana ovule with well-formed archegonia, showing
the behaviour of the tapetum. For explanation see text. Sudan black B and camalum preparation.
x 20.
Fig. 2 . Detail of the membrane formed by the collapse of the tapetal tissue. The section is the same
as Fig. 1. The gametophyte tissue is t o the right of the figure. Note that there are droplets adhering
to the outside (left) of the membrane. x 600.
Fig. 3. Surfme view of the tapetal membrane isolated by acetolysis from a mature ovule of Encephalmtos fredericiguilielmi. Sudan black B preparation. x 100.
Fig. 4. Acetylated Sudan black B-stained section of a Pinw excelsa ovule with mature archegonia.
The gametophyte tissue is towards the lower part of the figure. tm, tapetal membrane; m, megaspore
membrane. Note that there are sudanophilic droplets in the tapetal cytoplasm. x 350.
Fig. 5. Tapetal membrane of a Pin- ezcelsa ovule at the same stage of development as that in Fig. 4.
The arrows indicate coalescent droplets. Anoptral contrast. x 600.
Fig. 6. Electron micrograph of a section through the acetolyzed megaspore of Zamiafloridana a t the
same stage of development as that shown in PI. 1, fig. 4. x 10,000.
Fig. 7. Electron micrograph of part of the megaspore membrane in the mature ovule of Juniperus
ezcelsa. x 10,000.
Juuru . Linn. Sur . flu/ . l·ot . .-J\1 . Xu. ;nH
.IOHN
:\1. PETTITT
Plate L
{ Fuc lu y f l .
~H2)
Juurn. Li11 n . ,')'oc. Hut. T'o/. ;)\). So. :370
J O H~
~f.
PETTiTT
Plate :2
Jou rn . Li1111. Sw. /ful. l"uf. ;)!1 . .\"u . :l/!1
JOHN
~1.
l'I·: 'J'TITT
Plak il
.Journ. Linn. 8uc . B ot . Fol . 5!l, So. 379
,JOHN 1\f. PETTITT
Plate 4
The megaspore memhrane in same gymnospermous ovules
PLATE
263
3
Cycas revoluta, Encephalartos fredericiguilielmi, Pinus excelsa, Pinus sylvestris
Fig. l. Electron micrograph of a section through the tapetal membrane from a mature ovule of Cycas
revoluta. x 15,000.
Fig. 2. Electron micrograph of a section through the tapetal membrane from a mature ovule of
Encephalartos fredericiguilielmi. Collapsed nucellar tissue occupies the upper part of the figure. A
surface view of the membrane is shown in Pl. 2, fig. 3. x 10,000.
Fig. 3. Acetolyzed megaspore membrane from a mature ovule of Cycas revoluta. Anoptral contrast.
x375.
Fig. 4. Electron micrograph of a section through acetolyzed tapetal and megaspore membranes of
Pinus excelsa. The developmental stage is the same as that in Pl. 2, figs. 4, 5. tm, tapetal membrane;
m, megaspore membrane. For full explanation see text. x 10,000.
Fig. 5. The tapetal membrane (tm) and megaspore membrane (m) in the seed of Pinus sylvestris.
Prothallial tissue occupies the lower part of the figure. Acetylated Sudan black B and carmalum
preparation. x 350.
Fig. 6. Electron micrograph of a section through the peripheral prothallial cells of Encephalartos
fredericiguilielmi. The megaspore membrane is the outer electron-dense layer; the arrow indicates an
anticlinal wall in the membrane. Compare with Fig. 3. x 2,500.
PLATE 4
Taxus baccata, Ginkgo biloba, Encephalartos villosus
Fig. l. Electron micrograph of a section through a Taxus baccata pollen sac tapetal cell containing
electron-dense lipid droplets. (Fixed in glutaraldehyde, post-fixed in osmium tetroxide.) x 15,000.
Fig. 2. Light micrograph of a section of the same tissue as Fig. 1. Sudan black B staining reveals the
presence of droplets arranged along the cell walls. x 800.
Fig. 3. Electron micrograph of a section through a pollen sac tapetal cell of Taxus baccata. The upper
part of the figure is occupied by a large lipoidal body formed by the coalescence of small droplets.
(Fixed in glutaraldehyde, post-fixed in osmium tetroxide.) x 10,000.
Fig. 4. Tapetal membrane from Ginkgo biloba pollen sac. The membrane is acetolysis-resistant and
encloses a mass of pollen grains. x 95.
Fig. 5. Sudanophilic droplets in the cytoplasm and aggregated against the walls of the pollen sac
tapetum in Encephalartos villosus. Sudan blackBand carmalum preparation. x 560.