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The elasticity of the exine
John Rowley & John Skvarla
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Grana 39: 1±7, 2000
The elasticity of the exine
JOHN R. ROWLEY and JOHN J. SKVARLA
Rowley, J. R. & Skvarla, J. J. 2000. The elasticity of the exine. ± Grana 39: 1±7. ISSN 0017-3134.
We pressed pollen grain exines of ten genera with sizes ranging from about 20 to over 100 mm in
diameter past a piston in a close ®tting cylinder. The clearance between piston and cylinder was about
20 mm. Except for exines of Betula all the other pollen types were at least twice the clearance diameter
and could be expected to be greatly deformed, crushed or fractured. Cracks were evident with the light
microscope in some grains and a few were clearly deformed but most appeared intact, even exines of
Zea mays at a diameter of 100 ± 110 mm. With scanning electron microscopy cracks were apparent in
most of the large grains (Zea, Lilium, Pinus, Crinum and Epilobium) but not in the smaller grains
(Betula, Ephedra, Tulipa, Fagus and Typha). We also found many exines within exines. In some cases,
e.g., Lilium, the exines entered through apertures but in other grains such as Zea and Pinus, exines
came in through cracks which had opened during acetolysis or centrifugation, then closed so tightly
that the cracks were dif®cult to see with light microscopy. This opening and closing of cracks in exines
means that the pollen grain exine is very ¯exible and resilient and capable of withstanding shock
without permanent deformation. To regain their original form the exine components that were severely
cracked, ruptured or partly separated must spring back together like the partly separated halves of a
tennis ball.
John R. Rowley, Botany Department, Stockholm University, SE-106 91 Stockholm, Sweden; John J.
Skvarla, Oklahoma Biological Survey and Department of Botany-Microbiology, University of Oklahoma,
Norman, OK 73019-0245, USA.
(Manuscript accepted 1 November 1999)
Traverse (1986: in Palynos) reported ®nding sapotaceous MATERIAL AND METHODS
tricolporate pollen grains inside the large monosulcate pollen
of a palm. He suggested that as many as eight of the smaller
pollen grains had invaded the large grain during centrifu-
gation following acetolysis. He requested comments from
readers. Salgado-Labouriau (1987) sent a letter to Palynos in
reply. She had seen many large grass grains ®lled with tetrads
of Mimosa pollen. When she turned the grains over she
found that they appeared to be entire and without cracks.
The small Mimosa grains were thought to have entered the
large grass grains through their pores during acetolysis.
Our approach to these observations of pollen inside other
pollen grains came about as a result of efforts by Belin and
Rowley to release pollen grain-held antigens as a source of
proteins in pollen of Betula (Belin & Rowley 1971). A so
called ``X-press'' was one of the procedures tested. The X-
press is a freeze press designed by Edebo (1960). In its use
pollen in buffer was frozen at ca 270³C in a cylinder of the
press. Then with a hydraulic press the frozen mixture was
forced through a small aperture in the cylinder. As seen by
light microscopy most exines appeared to be intact following
such treatment. Belin & Rowley (1971) found, however,
using transmission electron microscopy (TEM), that most
exines had been fractured and many exine fractures ®tted
together rather closely.
Pollen grains used in the experiments are listed below along with
their size data. Pollen of Lilium, Typha, Betula, Zea, Epilobium and
Tulipa were obtained from Allergon AB (Allergon AB, Sweden).
Fagus and Crinum came from the Stockholm University campus and
greenhouses. Ephedra pollen grains came from Qatar and Pinus
from the University of Oklahoma campus (Table I).
The pollen grains were dry, except for those of P. resinosa, which
had just been shed from living microsporangia. The pollen was put
into phosphate buffer. After one hour under vacuum in the buffer
the mixture was divided into two centrifuge tubes.
The contents of one of these tubes were:
± pressed by a piston down into a Dounce homogenizer tube
and up out of the tube three times,
± dehydrated in 100% ethanol, covered by acetolysis acid
mixture and heated to 100³C for 10 min,
Table I. Pollen used in our experiment.
The size indicated was after acetolysis.
Taxa
Average size (mm)
Lilium regale
35695
42
42
21 ± 25
100 ± 110
85
Fagus sylvatica
Typha latifolia (tetrad)
Betula pendula
Our aim in this experiment was to see if pollen grains of a Zea mays
Epilobium angustifolium
Tulipa gesneriana
Ephedra foliata
Crinum purpurascens
Pinus resinosa
variety of sizes, like Betula in the experiment above, as well
as sapotaceous and Mimosa pollen grains as previously
mentioned, still appear more-or-less intact after great
distortion and fracturing. Such information will be useful
for explaining pollen exine elasticity.
35
18 ± 28635 ± 54
55675
50675
# 2000 Taylor & Francis. ISSN 0017-3134
Grana 39 (2000)

2
J. R. Rowley and J. J. Skvarla
Grana 39 (2000)

Elasticity of the exine
3
± washed, centrifuged and examined by light microscopy and
scanning electron microscopy (SEM).
Those exines which contained exines were mostly the larger
ones, i.e., Zea, Crinum and Lilium. The smaller (ca
20 ± 40 mm) exines were mainly those of Betula although
the larger (ca 40 ± 80 mm) exines of Fagus, Pinus, Epilobium
and tetrads of Typha were seen in Lilium. The aperture of
Lilium is large enough to permit entry of these exines, and
some exines of Lilium were clearly cracked (Figs. 11, 12).
Probably all pollen grains of reasonable size with sulcate
apertures would permit entry of other smaller exines, e.g.,
Crinum in Figs. 13 ± 16. The pore of Zea is much too small
for any of the grains in our mixture and grains within exines
of Zea must have entered by a fracture in the exine. Many
exines of Zea were ¯attened and showed concavities; exines
of other species were frequently nested in such concavities
(Fig. 3 and probably also one of the Betula exines in Fig. 5).
The through-focus Figs. 13 ± 16 of a Crinum exine show
that the Betula exine is inside the Crinum one. The through-
focus series of Zea (Figs. 17 ± 19) shows that a Betula exine
and portion of a broken Pinus exine are within the Zea
grain. The crack in the Zea grain that allowed entry of
Betula and Pinus is essentially closed but shows as two
bright parallel lines (Fig. 19).
The contents of the other tube were:
acetolyzed then centrifuged and washed in water, then pressed by
the piston six times as above and then examined as above.
Photomicrographs were taken under oil at X40/Na 1.0 or X100/
Na 1.3 with planapochromatic objectives. In preparation for
scanning electron microscopy (SEM) the exines were dried using
a critical point drying alternative, hexamethyldisilazane (Chissoe
et al. 1994), sputter-coated with gold/palladium (60/40) and
examined with a JEOL 880 SEM equipped with a lanthanum
hexaboride gun.
RESULTS
Except for Epilobium very few of the large exines, Zea,
Lilium, Crinum, Pinus, Fagus, Typha and Tulipa, showed
obvious distortion or cracks by LM after piston/cylinder
treatment either before or after acetolysis. The large and
gaping crack in the Zea exine (Fig. 1) was exceptional in
LM observations. There were rare examples of exines
broken of Zea and Pinus with the ``halves'' detached; part of
a Pinus exine is in the Zea exine in Fig. 18. Exines of Betula,
Fagus, Typha and Ephedra showed no obvious distortion,
except for one Fagus grain of the several hundred Fagus
exines seen with LM, which had an exine of Betula nested in
DISCUSSION
it (Fig. 5). Most exines of Epilobium, however, were Only when apertures are large do exines get into other exines
deformed to some extent (Figs. 3, 8 & 10). without the inner or outer exines being fractured or cracked.
Except for Ephedra (if passed between piston and cylinder This is certainly the case for Lilium and probably also for
lengthwise) and Betula, all the other exines could be Crinum. Entry of exines into a grain like Zea with a very
expected to be compressed to at least half their diameter small aperture must be through a fracture or crack. We
on being pressed between the piston and cylinder. Since supposed that the exine on each side of an extensive crack
relatively few exines were distorted these results show that could, on being pressed through a narrow space, separate
exines are extremely ¯exible and elastic which allows them like the halves of a broken tennis ball. Like the broken
to recover their original form after compression and even tennis ball the still connected halves of a broken exine could
after extensive fracturing.
return to a nearly complete form although, of course, still
In samples prepared for SEM, frequent cracks (fractures) cracked. Other exines might enter the cracked grain when
occurred in the largest exines Zea (Figs. 6, 9), Lilium the crack is open as during acetolysis, centrifugation or
(Figs. 11, 12), Pinus (Fig. 7), and Epilobium (Figs. 8, 10) but immediately following being pressed by the piston through
rarely in the other exines. Exines of Typha (Figs. 7 & 9) were the cylinder.
not distorted and the Fagus exine (Fig. 9) was not crumpled.
Recognition that exines ¯ex between hydrated and dry
The sacci were separated to some extent from the body of conditions is apparent in the works of von Mohl (1835),
the Pinus exine in Fig. 7 but the exine was not distorted. The Mirbel (1833), Fritzsche (1834) and Meyen (1828) between
exines were severely crushed but sprang back to their the years 1830 and 1839 (see text and references in
original form.
Wodehouse (1935). The concept of harmomegathy (Wode-
Many exines were seen inside exines that had been pressed house 1935) involves exine ¯exibility in its accommodation
between piston and cylinder. The frequency of exines within to volume changes. Payne (1972) found that the entire
exines was similar in acetolyzed and unacetolyzed exines. pollen wall, not only the apertures, functions in volume
Figs. 1 ± 5. Light micrographs of acetolyzed or unacetolysed exines pressed between a piston and tight ®tting (ca 20-mm clearance) cylin-
der. Bars~10 mm (All photomicrographs are at the same magni®cation). (1) An extensive crack in Zea. (2) The exine of Zea is clearly
cracked and has a large opening that partly shelters an exine of Fagus (arrow). Two Betula exines (arrowheads) lie within the broken
Zea. On passing the piston and cylinder the Zea exine must have been distorted to a diameter about that of the Betula exines. The
exines of Pinus (asterisks) appear to be intact. (3) Three exines of Betula within a Zea exine (note pore, circled) and a tetrad of Typha
(T) outside nested in an exine concavity. The exine of Epilobium (star) is greatly damaged. The exine of Fagus (arrow) and several of
Pinus (one marked by an asterisk) and Betula (arrowhead) do not appear damaged. (4) Exine of Pinus within an exine of Lilium. Gaps
in the exine of Lilium at either side of the grain are ends of the large aperture (arrowheads). (5) An exine of Zea enclosing three
Betula exines. The arrow at the upper right points to a crack seen as a bright line on the surface of the Zea exine. This crack could
be traced part way under the exine at lower focal levels. The greatly damaged exine at the lower left is Fagus overlaid by or partly
enveloping an exine of Betula (arrowhead). The Pinus exine appears to be intact.
Grana 39 (2000)

4
J. R. Rowley and J. J. Skvarla
Grana 39 (2000)

Elasticity of the exine
5
regulation. Bolick (1981) measured forces involved in
The exine of Epilobium angustifolium seems to be an
changes in exine form, such as bending. Heslop-Harrison exception to the above situation since its exine was seen in
(1979), in his work on the hydrodynamics of pollen, many cases to remain deformed following compression
discussed the dilation of the cytoplasm and thus substantial between piston and cylinder. This result might be explained
or even expected, perhaps, by the exceptional structural
complexity of the onagraceous pollen exine as reported by
Skvarla et al.1975, 1976, Keri & Zetter 1992, Praglowski
et al. 1994, and Rowley & Claugher 1991.
expansion of grains, during their rehydration in the
pregermination interval on stigmatic surfaces.
During microspore development to pollen grain maturity
it is clear that in most species there is a certain degree of
enlargement of the circumference of the exine. In Zea the
volume increases ca 1,200 times from microspores in the
tetrad at ca 10 mm in diameter to mature pollen at ca
120 mm. How this increase is accomplished with regard to
the exine is not clearly de®ned, as far as we know.
Stretching, which is an indication of plasticity, is mentioned
in reports of exine development. Banerjee et al. (1965)
showed that columellae were arranged in ®les of twos and
threes under muri early in development of the reticulate
exine of Sparganium longifolia. Later in development, as the
microspores/pollen grains enlarged, the muri of the greatly
enlarged reticulations were ``supported'' by a single ®le of
columellae. Not only were the columellae uniserate rather
than multiserate under the muri but they were also more
widely separated than in younger stages (Banerjee et al.
1965: Figs. 12 ± 17). Banerjee et al. show that exines can be
stretched. The extent of stretching can be appreciated in
their camera lucida drawings and measurements of separa-
tion of columellae throughout development. Columellar
separation increases from 0.14 mm during tetrad time to
0.35 mm at maturity. Takahashi (1980) in work on
development of the reticulate structure of Hemerocallis
pollen agreed with the above interpretation.
Pollen exines may enter other pollen grains through large
apertures. If the aperture is small, grains probably enter
through breaks or cracks that are open during acetolysis,
centrifugation or, in our experiment, when they are pressed
by a piston past the close ®tting cylinder.
Fractured pollen grain exines are like broken tennis balls.
They can be almost completely separated by a fracture but
the remaining ``wall'' will be enough to ``snap'' the
components back to a very nearly intact-appearing form.
The clearance of our piston/cylinder device is ca 20 mm, thus
only Betula could be expected to be undamaged. Most of
the exines, even the large ones (Zea and Lilium),
appeared undamaged although they must have been
greatly compressed (¯attened). Thus our foremost conclusion
is that the exine structure is very ¯exible, elastic and strong.
ACKNOWLEDGEMENTS
We thank W. F. Chissoe, University of Oklahoma and Samuel
Roberts, Noble Microscopy Laboratory, for his assistance with the
scanning electron microscopy. We wish to thank Dr Nina I.
Gabarayeva for many helpful suggestions on an early draft of our
manuscript.
A remarkable aspect of our experiment is the condition of
the muri of the Lilium reticulum. Muri are supported by
relatively slender and tall columellae about 0.5 mm in height
(Dickinson 1970, Southworth 1985, and Takahashi 1995).
The large exines of Lilium were greatly compressed by our
methods. In our SEMs we have selected a damaged aperture
in Fig. 6 and a broken exine in Fig. 7 because exines of
other grains were evident within them, but most exines of
Lilium were without obvious damage especially with respect
to the muri (Fig. 5). Even in Fig. 7 where the exine is broken
the muri appear without modi®cation up to the edge of the
fractures. The exine structure that allows such damaging
treatment without deformation is strong but plastic and
¯exible and certainly not brittle.
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been large enough to allow entry of any of the exines in our mixture. (7) Exines of Betula (arrowhead), Pinus (asterisk) and Typha
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Grana 39 (2000)

6
J. R. Rowley and J. J. Skvarla
Figs. 13 ± 16. Through focus LM series of a Betula pollen grain inside a Crinum grain. The Crinum grain is 1-sulcate and large
(Table I). The grain of Betula may have entered the Crinum exine via the aperture but the Crinum exine may have been split open and
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high focal level. Surface spines of Crinum and the two Betula exines at the top of the ®gure are in focus. (14) Optical section that is
nearly medial. Spinules of Crinum are seen in pro®le. The Betula grain is in better focus, and the two Betula grains above are going
out of focus. (15) Below the medial section of Crinum. The Betula exine is better focused inside the Crinum exine. The two Betula
exines at the top of the ®gure are greatly out of focus. (16) A low level of focus on the Crinum exine. The spinules on the lower side
of the Crinum exine are in focus. The two Betula grains above are now out of focus. These ®gures show that the Betula exine is within
the exine of Crinum.
Grana 39 (2000)

Elasticity of the exine
7
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