J. Cell Sd.

47, 365-383 (1981) 365

Printed in Great Britain © Company of Biologists Limited ig8i

POLLEN TUBE DEVELOPMENT IN PETUNIA

HYBRIDA FOLLOWING COMPATIBLE AND
INCOMPATIBLE INTRASPECIFIC MATINGS

MARIA HERRERO* AND H. G. DICKINSON

Department of Botany, Plant Science Laboratories, University of Reading,
Whiteknights, Reading RG6 2AS, U.K.

SUMMARY
Pollen tubes formed following compatible and incompatible intraspecific matings in
Petunia have been examined with light and electron microscopes. Compatible and incom-
patible tubes develop in an identical fashion on the stigma but, on entry into the top 1 mm of the
stylar transmitting tissue changes occur both to the cytology of the tubes and their rates of
growth. The early cytological changes are common to tubes of both compatibilities but, although
both types of tube accelerate on entry into the style, incompatible tubes grow more slowly than
compatible. Cytological differences became apparent between compatible and incompatible
tubes following a short period of growth in the style, the latter possessing thicker cell walls
and a cytoplasm packed with both organelles and reserves. Incompatible tubes subsequently
burst or simply cease growth and die. The characteristic image afforded by this cytoplasm
resembles that of burst or dead compatible tubes, except in that proportions of the cell com-
ponents may differ. These data are discussed in terms of current models proposed to explain
pollen tube growth and the operation of the self-incompatibility response in Petunia.

INTRODUCTION
As in many plants with gametophytic control of pollen compatibility with respect
to the style, pollen tubes of Petunia formed following a self-mating fail to reach the
ovary. Growing more slowly than tubes from a cross-pollination they eventually cease
development in a region some two-thirds of the way down the style. Since much is
known of the physiology of and structures involved in pollen tube growth, it is not
unreasonable to expect that the differences in development between tubes of different
compatibilities might easily be explained by changes in the ultrastructure and physio-
logy of the tube cytoplasm. This has not proved to be the case.
Striking ultrastructural differences between compatible and incompatible tubes
were reported as early as 1966 by van der Pluijm & Linskens who described the walls
of incompatible tubes as being far thicker than those derived from compatible crosses.
In a comprehensive investigation into the cytology of pollen tube growth in Lyco-
persicum (de Nettancourt et al. 1973), the arrest of incompatible pollen tubes was
reported to result partly from the cessation of protein synthesis, itself caused by
ribosome-coated endoplasmic reticulum forming into concentric whorls (de Nettan-
court et al. 1974), and from the binding of an 'incompatibility protein' to
wall-precursor containing bodies in the tube cytoplasm. Such results were not fully
• Present address: INIA, Apartado 202, Zaragoza, Spain.
366 M. Herrero and H. G. Dickinson
confirmed by work on Oenothera (Dickinson & Lawson, 19756) where growth of
incompatible tubes appeared to be accompanied in the first instance by changes in
the carbohydrate metabolism of the tube, with resultant changes in the cell wall.
Data from this investigation did, however, reveal that considerable modification of
the cytoplasmic components involved in cellulose biosynthesis is required to permit
the very rapid rate of growth characteristic of these tube cells.
Because the elongating pollen tubes are embedded in stylar tissue, physiological
investigations have not been easy. Work by van der Donk (1974) has, however, shown
differences between tubes of differing compatibilities to become evident very soon
after pollination. So much so that Linskens (1975) has proposed the 'recognition'
stage of the self-incompatibility response to operate in the stigma.
Despite the considerable effort expended in these and other investigations, we are
still in ignorance both as to the point in tube metabolism at which the self-incompa-
tibility mechanism has its primary effect and, indeed, of the means by which pollen
tube elongation is arrested. We report here an investigation into these events in
Petunia, the results of which are discussed in terms of the accompanying changes that
occur in the tissue of the pistil (Herrero & Dickinson, 1979).

MATERIALS AND METHODS

Details of the plant material used in this study, together with the methods of preparation of
tissue for light and electron microscopy are set out in Herrero & Dickinson (1979). The methods
for staining resin-embedded material with Coomassie Brilliant Blue (CBB) are described by
Fisher (1968).

RESULTS
Pollen germination and tube growth at the stigma surface
Within 30 min of pollination most grains germinate (Fig. 2) and develop a tube at
least 30 jim in length. Conspicuous changes overcome the pollen during early stages
in germination, firstly a fibrogranular matrix (Figs. 1, 3) that encased the mature grain

Fig. 1. Colpal region of uninucleate microspore (p), still retained in the anther.
Fibrils (/) are visible in the sculptured exine, facing the loculus (/). x 8620.
Fig. 2. Scanning electron micrograph of mature pollen grain (g) on the stigma (s). Note
the absence of any deposition on the exine surface. The globules (/) are presumably
derived from the stigmatic fluid. A germinal pore (p) is also visible, x 5640.
Fig. 3. Low-power transmission micrograph of material shown in Fig. 6. The exine (e)
is free from fibrils. The vegetative nucleus (n) and a germinal pore (/>) are also shown,
x 2050.
Fig. 4. Light-microscopic preparation of pollen grain germinating on the stigma,
reacted to reveal acid phosphatase. Higher levels of the enzyme, which appears to be
in packets, are contained in the tube (f) than the grain (g). x 730.
Pig. 5. Tip of pollen tube growing on the stigmatic surface. Note the spherical
vesicles (arrows). The cell wall (w) is fibrous and disorganized, and the ground cyto-
plasm very electron-opaque, x 16300.
Pollen tube development in Petunia 367
368 M. Herrero and H. G. Dickinson
in the anther becomes no longer detectable and, secondly, marked changes occur in
the pollen cytoplasm. Here, instead of the electron-opaque protoplast characteristic
of the grain prior to dispersal, a more electron-lucent cytoplasm is seen, containing
increased amounts of rough endoplasmic reticulum, dictyosomes, and associated
vesicles. The plasma membrane, previously smooth and featureless, now appears
active, and is formed into numerous small projections associated with vesicles. Other
components of this germinating protoplast are droplets of unsaturated lipid, diffuse
fibrous masses, mitochondria, the vegetative nucleus and the generative cell. Although
all the microbodies present in the cytoplasm appear identical under the electron
microscope, light-microscopic histochemical tests indicate 2 classes of microbody to
be present, one containing acid phosphatase (Fig. 4) and the other a peroxidase. The
peroxidase-containing bodies survive for only some 60 min after germination, whereas
those containing acid phosphatase may be found throughout development of the pollen
tube. Clearly the cytoplasm of the young pollen tube on the stigma surface is contin-
uous with that of the pollen grain, but since differences occur in the composition of
the cytoplasm between the various regions of the tube, these areas are probably more
helpfully described individually.
At the tube tip, the cytoplasm is comparatively electron-opaque and the protoplast
surface very irregular, such that the plasma membrane is undetectable (Fig. 5). At this
surface are numerous vesicles, similar to those seen near dictyosomes deeper in the
cytoplasm. The wall of the pollen tube tip consists solely of loosely woven fibrils
showing no particular organization (Fig. 5).
Back from the tip, the plasma membrane of the tube becomes better defined and the
fibrils of the wall organized in particular directions (Fig. 6). Even at this point, the
presence of callose is neither indicated by electron microscopy nor by cytochemical
tests. While only small mitochondria may occasionally be discerned in the cytoplasm
of the tube tip, here these organelles are far more frequent. Conspicuous also are large
aggregates of fibrous material, similar to that of the tube wall, and masses of folded
membrane reminiscent of the myelin figures of animal cells. Numerous vesicles still
populate this cytoplasm, many of which appear associated either with the plasma
membrane or with the fibrous masses (Fig. 6).
Examination of the pollen tube wall close to the grain reveals it to consist of an
outer, well organized fibrillar layer and an inner, electron-lucent layer adjacent to the
plasma membrane (Fig. 7). Conspicuous in this cytoplasm are large numbers of

Fig. 6. Transverse section of pollen tube, as depicted in Fig. 5, but further back from
the tip. Large fibrous masses are present (/), as are vesicles (arrows) and mitochondria
(w). «>, wall, x 21400.
Fig. 7. As Fig. 5, further back along the tube towards the grain, showing the presence
of a thin electron-lucent layer (p) between the plasma membrane (arrows) and the
fibrous wall (w). x 15200.
Fig. 8. Transverse section of pollen tube, as depicted in Figs. 6, 7. Here the cytoplasm
is more electron-lucent, containing elements of rough endoplasmic reticulum (e),
mitochondria (m), and a paramural body (p). The lipidic stigmatic fluid (s) is also
visible, x 14170.
Pollen tube development in Petunia 369

. 8
370 M. Herrero and H. G. Dickinson
membranous cisternae, normally identified with paramural bodies (Fig. 8). This
cytoplasm is also rich in rough endoplasmic reticulum, mitochondria, plastids and
microbodies (Figs. 7, 8). Light-microscopic tests indicate callose to be present in this
wall (Fig. 10) and bodies containing acid phosphatase to populate the protoplast. By
the time the tube has grown to a length of some 200 /tm the vegetative nucleus and the
generative cell are also to be found in this region (Fig. 9). A large vacuole has developed
in the pollen grain by this stage, sometimes extending into the tube itself. The cyto-
plasm remaining in the grain is rich in rough endoplasmic reticulum.
It is striking that no differences exist in germination, cytoplasmic content and wall
development between compatible and incompatible grains growing on the stigmatic
surface.

Pollen tube growth in the transmitting tissue

Between 2 and 3 h after pollination the first compatible and incompatible pollen
tubes pass through the 'neck' of the style and into the transmitting tissue. Details of
the organization of these cells are given elsewhere (Herrero & Dickinson, 1979).
Coincident with the arrival of the pollen tubes a proportion of transmitting tissue cells
burst, and the tubes continue their growth in a matrix formed partly from the residue
of these ruptured cells (Fig. 11). Pollen tube growth in the style differs significantly
from that on the stigma. This difference is reflected in the mode of wall synthesis, the
composition of the tube cytoplasm and, of course, the further alterations to tube
development that result from operation of the self-incompatibility system.

The cytology of compatible pollen tubes

The changes to the compatible pollen tube as it enters the transmitting tissue are
few. The small dictyosome vesicles characteristic of growth on the stigma appear to
be replaced by larger vesicles, some 0-25 /tm in diameter. Despite the fact that
dictyosomes are less frequent in the cytoplasm of the tube tip, these organelles seem

Fig. 9. Transverse section through a pollen tube growing in the stigmatic fluid ( / ) .
Both the vegetative nucleus (y) and the generative cell (g) are visible, x 10400.
Fig. 10. Pollen grains developing in the stigma stained to reveal the presence of callose
in the tube walls (arrows). Light micrograph, x 320.
Fig. 11. Pollen tube (t) growing through a stylar matrix formed of cytoplasm (c)
derived from ruptured cells. Note adjacent intact stylar cells (J). x 26000.
Fig. 12. Transverse section through a pollen tube growing through the style, taken
near to the tip. Vesicles (arrows) containing organized fibrillar content may be seen
merging with the plasma membrane (p) and discharging their content into the wall (tv).
x 18500.
Fig. 13. Detail of pollen tube growing in the stylar tissue showing the protoplast
periphery some distance from the tube tip. Here fibrillar ( / ) and electron-lucent (/)
layers of the wall may be discerned, as may paramural membranous bodies (j>).
x 38600.
Fig. 14. Low-power light micrograph of a compatible cross-pollination stained to show
the presence of callose (c) in the 'plugs' formed in the pollen tubes following the
passage of the generative cell, x 90.
Pollen tube development in Petunia

14
372 M. Herrero and H. G. Dickinson
the most likely source of the larger vesicles, which, like the small vesicles, may be
seen merging with the plasma membrane of the tube tip (Fig. 12). Unlike the smaller
vesicles, however, these vesicles observed during growth in the style regularly contain
an organized fibrillar content. The general electron opacity of the tube cytoplasm is also
greater when in the style, and much of this electron-opacity can be shown, using
enzymic digestion, to be sensitive to protease. Polyribosomes are also present in far
greater numbers in these cells. In the tube cytoplasm further away from the tip, more
paramural bodies may be seen (Fig. 13), and light-microscopic histochemistry reveals
bodies containing esterase to be present in this region. Further back in the tube,
callose commences to be formed at precisely the same distance from the tip as when
the tube was growing on the stigma. The organelles associated with this synthesis
are also similar to those found in earlier development of the tube.
Once the generative cell has passed down the tube, some 6 h after pollination
(Fig. 14), callosic plugs are formed. On the stigma, transverse sections of these' empty'
tubes retain their original rounded appearance, while in the style they present an
infolded or collapsed aspect (Fig. 15). The depth of the callosic wall varies during
growth of the pollen tube, for, as it enters the lower style it becomes very thin, such
that it is detectable in only few preparations. However, as the tube approaches the
ovary (Fig. 16) callose once more becomes evident. In addition, profiles indicating the
presence of burst pollen tubes are frequently conspicuous in this lower region of the
style. The necrotic cytoplasm of these ruptured tubes regularly contains spherical
bodies, apparently composed of radiating fibrils, and measuring about 0-25 /im in
diameter. Enzymic digestion reveals the electron-opaque matrix of these spheroids to
be insensitive to protease while the surrounding cytoplasm is digested by this enzyme.
In many ways this discharged cytoplasm resembles that of some incompatible pollen
tubes when examined either in the stigma or style 24 h after pollination (Fig. 17).
Over the course of tube growth down the style, the generative cell continues to
follow closely behind the tip. The generative cell (Fig. 18) which measures about
3 /tm long and 2 /im wide, is bounded by an electron-lucent space, contains mito-
chondria and a complex assembly of microtubules. No plastids appear to be present.
As the generative cell passes through a particular length of tube, a callosic plug is
formed behind it.

Fig. 15. Transverse section through a pollen tube (/) growing in the style, but follow-
ing passage of the generative cell. Note how the wall is infolded (arrows), x 14400.
Fig. 16. Light-microscope preparation of a compatible pollen tube entering the
ovary (0), stained to reveal callose. Note the callosic plug (p) and the pollen tube (t)
itself, x 460.
Fig. 17. Necrotic cytoplasm from burst compatible pollen tubes at the base of the
stylar transmitting tissue. Note the presence of the spherical fibrillar bodies (arrows)
characteristic of degenerate pollen tube cytoplasm, x 6700.
Fig. 18. Transverse section through a pollen tube showing the generative cell (g).
The nucleus (n) is clearly visible, and small wefts of microtubules (arrows) may be
discerned in the tube cytoplasm. The electron-lucent wall (w) between generative
and vegetative cells is also evident, x 40050.
Pollen tube development in Petunia

18
374 M. Herrero and H. G. Dickinson

Differences between the growth in the style of compatible and incompatible pollen tubes
There appear to be no totally novel structures in the developing incompatible pollen
tube, although the balance between cytoplasmic components may sometimes be
altered considerably. The first indications of the incompatibility response may be
seen soon after the pollen tube penetrates the transmitting tissue. These include an
increased rate of apposition of the outer fibrillar wall at the tube tip (Fig. 19) and a
densely ' packed' aspect of the cytoplasm, especially in the region of the generative
cell (Fig. 20). Examination of incompatible tubes growing deep in the transmitting
tissue reveals them to possess much thicker walls (Fig. 21). Both the fibrillar and the
callosic electron-lucent walls are far more heavily deposited, and in regions distant
from the tip, small crescent-shaped fibrillar inclusions may frequently be seen
apparently immobilized inside the electron-lucent component of the wall (Fig. 21).
The general cytoplasm of the tubes, both at the tips and elsewhere, is strikingly
electron-opaque, but this electron opacity may be considerably reduced by treatment
with protease (Fig. 22). The packing of cytoplasmic components is also increased, with
greater numbers of almost all the inclusions previously described. Staining with
Coomassie Brilliant Blue and treatment with PAS shows increased levels of protein
and carbohydrate in these tubes (Figs. 23, 24).
Within 24 h of growth in the transmitting tissue, a conspicuous change overcomes
the cytoplasm of the majority of incompatible tubes. A marked increase in electron
opacity occurs followed by a loss of identifiable form by most cytoplasmic inclusions
(Fig. 25). Coincident with these events is the appearance of inclusions, between
0-2 and 0-3/tm in diameter, apparently composed of radiating fibrils (Fig. 26). As with
the similar bodies seen in burst compatible tubes these inclusions are insensitive to
protease, in sharp contrast to the cytoplasm investing them which is rapidly digested
by this enzyme (Fig. 27). Also evident in the electron micrographs of this material are
profiles indicating that a proportion of these incompatible tubes rupture (Fig. 28).

Fig. 19. Tangential section through the tip of an incompatible pollen tube (t) growing
in the style. Note the increased deposition of fibrillar precursors (arrows) into the
tube wall (w). The increased electron-opacity of the cytoplasm is also evident, x 17 500.
Fig. 20. Sectioned incompatible pollen tube showing thickened fibrillar ( / ) and
electron-lucent (/) walls, the generative cell (g), and electron-opaque cytoplasm,
x 15340.
Fig. 21. Detail of incompatible pollen tube deep in the transmitting tissue, showing
fibrillar wall precursor elements (arrows) apparently embedded in the electron-
lucent wall (/)• X I I 580.
Fig. 22. Material as shown in Fig. 21, but following treatment with protease. The
cytoplasm (c) appears to be particularly sensitive to this enzyme, x 7370.
Pollen tube development in Petunia 375
376 M. Herrero and H. G. Dickinson

28
 Pollen tube development in Petunia 377

DISCUSSION
Germination of the pollen grain and early tube growth on the stigma surface
The first event to take place following the arrival of the pollen on the stigma is the
disappearance of the fibrillar coating of the exine. Although tapetally-synthesized
pollen grain coatings have been implicated in mechanisms of self-incompatibility
(Dickinson & Lewis, 1973 a, b), this has only been shown in plants where pollen
compatibility is sporophytically controlled. In Petunia where compatibility is con-
trolled by the pollen genotype such an involvement would not be expected, a conclu-
sion supported by the work of Fett, Paxton & Dickinson (1976) in Lilium, and Gilissen
& Brantjes (1978) on Petunia, where washing of pollen was shown not to affect the
self-incompatibility response.
Activation of the pollen cytoplasm follows at approximately the same time as the
disappearance of the fibrillar coating, with the anticipated increase in numbers of
dictyosome vesicles, first reported by Larson (1965), and the increase in rough
endoplasmic reticulum and polysome frequency described by Linskens, Schrauwen &
Koning (1970). Once it has emerged from the grain, the manner of growth of the
pollen tube differs little until it enters the transmitting tissue, and resembles closely
the style of growth observed in vitro (Kroh, 1967). The disorganized fibrils at the tip
of the tube presumably consist of polymerized and part-polymerized cellulose
(O'Kelley & Carr, 1954), deposited from the vesicles present in the subjacent cyto-
plasm (Sassen, 1964). These vesicles have been shown to contain both cellulose and the
enzymes necessary for its synthesis (Engels, 1974). Both the work here reported, and
previous studies (Van de Woude, Morre & Bracker, 1971) indicate dictyosomes to be
the source of these vesicles. How they are transferred to the growing tip is not clear.
Work with inhibitors (Mascarenhas & Lafountain, 1972) has indicated that micro-
tubules are unlikely to be involved in this process, but that cytochalasin-sensitive

Fig. 23 A. Incompatible pollen tubes (arrows) growing in the style reacted with PAS
to reveal carbohydrate. Note the dense staining of the tube cytoplasm. Light micro-
graph, x 1800. B. As Fig. 23 A, but a compatible tube. The lack of staining of the
tube cytoplasm (arrows) is evident. Light micrograph, x 1900.
Fig. 24A. As Fig. 23 A, but stained with Coomassie Brilliant Blue. The tube contents
(arrows) stain strikingly, x 1440. B. As Fig. 23 B, but stained with Coomassie Brilliant
Blue. The tubes (arrows) appear to contain little protein, x 1600.
Fig. 25. Necrosis in an incompatible pollen tube (t) some 24 h after entering the
transmitting tissue. Note the thickened walls (iv), the presence of spherical fibrillar
bodies (arrows), and the lack of identifiable organelles in the cytoplasm, x 4670.
Fig. 26. Detail of cytoplasm of a tube such as that shown in Fig. 25. The spherical,
fibrillar bodies (s) are clearly evident, together with some membrane (arrows), x 40910.
Fig. 27. Ruptured incompatible pollen tube (i) in the transmitting tissue following
digestion with protease. Note the sensitivity of this necrotic cytoplasm to the enzyme,
x 337°-
Fig. 28. As Fig. 27, but without enzymic digestion. Most of the features shown in
Figs. 25 and 26 are also evident in this cytoplasm (c). x 2710.
378 M. Herrero and H. G. Dickinson
elements, presumably microfilaments, organize the movement of these vesicles via
cytoplasmic streaming.
Since the material deposited at the tube-tip is eventually destined to form part of
the cylindrical wall of the tube it must be flexible, a property no doubt conferrred
upon it by the loose packing of the cellulosic microfibrils. Once finally deposited in
the tube wall, these fibrils are then reinforced by a layer of callose. Little is known of
the cytoplasmic structures involved in the synthesis of this glucan; Jensen & Fisher
(1970) suggested that dictyosome vesicles were responsible for its deposition, whereas
Cresti & van Went (1976) described callose synthesis in elements of' rough' endoplas-
mic reticulum. Most recently, however, both Cresti, Pacini, Ciampolini & Sarfatti
(1977) and Ciampolini & Cresti (1977) have again indicated dictyosome vesicles as
being the source of this polymer. Cell fractionation experiments by Helsper, Veerkamp
& Sassen (1977) have revealed vesicles from the tube cytoplasm to contain /?-glucan
synthetase, and to be capable of synthesizing alkali-insoluble 1-3 glucans. Elsewhere
in plants where callose synthesis can be stimulated, for example in the stigmatic
papillia of Raphanus (Dickinson & Lewis, 1973 a), the structures involved appear to be
irregular-shaped vesicles, sometimes tube-like, emanating from elements of the
endoplasmic reticulum. While nothing from the present investigation points directly
to the identity of structures involved in callose synthesis, it is interesting that para-
mural bodies (Marchant & Robards, 1968), composed of irregular tubules and
vesicles, are present without exception at the sites of callose deposition.
The mechanism by which the generative cell moves into the tube is not evident from
results obtained so far. However, since cytoplasmic streaming must of necessity be
recirculatory in nature, it could hardly move this cell steadily down the tube. The
assembly of microtubules around the generative cell is most striking, and it is not
impossible that it is concerned with its mobility (Hoefert, 1971). Sanger & Jackson
(1971), however, consider these microtubules to be responsible for maintaining the
ellipsoid shape of the cell, rather than its movement, and more recently Cresti, van
Went, Willemse & Pacini (1976) have identified fibrous masses in the pollen tube
cytoplasm with the motility of the generative cell.
The cytology of the stigma appears in no way to be disturbed by the passage of the
pollen tube (Herrero & Dickinson, 1979), confirming the conclusion by Konar &
Linskens (1966) that this tissue plays a passive role at this stage. What is taken up by the
tube tip during this phase of growth is unknown, but it has tacitly been assumed that
much of this development is supported by the reserves in the pollen grain (Linskens,
1964; Dickinson & Lawson 1975 a). The significance of the identical development of
compatible and incompatible pollen tubes on this tissue is not easily evaluated. This
similarity also extends to growth rate, tubes of both compatibilities growing at a rate
of 150/im h"1 (Herrero & Dickinson, 1980). Certainly it may be that no factors are
present in the stigma to affect the tube according to its compatibility, but it may
equally be that the tube, growing with the aid of pollen-held reserves, may not yet be
sensitive to the molecules responsible for the arrest of incompatible pollen tubes.
 Pollen tube development in Petunia 379

The changes in tube growth that occur on entry into the transmitting tissue
The alterations to the cytology of the tube-tip that take place in the neck of the
style may, in the main, be explained in the difference between the growth rates of the
tubes; compatible tubes, for example, increase to a rate of some 520/im h"1 (Herrero
& Dickinson, 1980). Certainly the cellulosic tube wall continues to be formed at the
tube tip from vesicle-held precursors, but their source now appears to be cisternae of
the endoplasmic reticulum. Fibrils often become apparent in these vesicles as they
near the walls, and the vesicles themselves often fuse together before reaching the cell
wall. In Oenothera there is evidence that this process becomes even further modified,
with the phosphorylation stages necessary for the formation of the cellulose being
carried out in the vesicles while appressed closely to mitochondria (Dickinson & Law-
son, 1975 a). The increased electron-opacity of the cytoplasm presumably results from
increased levels of protein in this region of the tube.
It is in the neck of the style that a considerable difference in growth rate becomes
evident between tubes of differing compatibilities, for incompatible tubes only increase
in growth rate by 190 /im h"1 compared with the 370 /im h"1 of compatible tubes
(Herrero & Dickinson, 1980). Ascher (1966) has suggested growth on the stigma and
in the style to be controlled by 2 different operons, and that incompatible tubes fail to
activate a high-velocity operon controlling stylar growth which enables it to utilize
stylar materials for its subsequent development. If this is true, incompatible growth
may thus be regarded as autotrophic. Such an inference has been supported by work
of Rosen & Gawlick (1966) on the structure of tube tips, and also van der Donk (1975),
who explained lack of growth of incompatible tubes in terms of an inability to activate
the stylar genome, condemning the tube to grow only as long as its reserves lasted.
Results from Herrero & Dickinson (1980), and those presented here do not wholly
confirm these conclusions. Certainly, incompatible tubes do grow more slowly in the
style than compatible ones, but both types accelerate on entry into this tissue. Incom-
patible tubes, however, cease growth apparently while still containing plentiful
reserves which, of course, may either remain from the pollen grain or be derived from
the pistil. Equally, when growing in the transmitting tissue they resemble far more
closely in structure and growth rate compatible tubes in the same region of the style,
rather than tubes on the stigma. The incompatible tube, therefore, appears to undergo
many of the changes experienced by compatible tubes on entry into the transmitting
tissue, but suffers in addition some other effect resulting in the reduced growth rate.
Incompatible tubes are capable of accumulating labelled precursors from the stigma
to levels equivalent to those found in compatible tubes (Kroh, Miki-Hirosige, Rosen &
Loewus, 1970), and appear to contain higher levels of detectable reserves, de Nettan-
court et al. (1974) propose that protein synthesis is inhibited in incompatible tubes, a
conclusion which receives support from Cresti et al. (1977) who showed, that gamma-
irradiated compatible pollen tubes resembled in many ways incompatible tubes. On
the other hand, our cytochemical data indicate that incompatible tubes contain high
levels of proteins but, of course, we know nothing of the character of these polypep-
tides.
380 JVf. Herrero and H. G. Dickinson
Summarizing the evidence available from this present study, it seems most likely
that both compatible and incompatible tubes undergo identical initial changes on
entry into the transmitting tissue. These changes must involve an increase in uptake of
materials from the pistil'and, in incompatible tubes, one or more of these materials
must induce a metabolic imbalance that results in a decrease in tube growth rate.
The bursting of many of the compatible tubes at the base of the transmitting tissue
is perplexing. The microscopic data indicate that the tube walls are lacking the
electron-lucent callosic layers here and it is not impossible that the absence of this
rigid wall-layer is responsible for the rupture of the tubes. Why callose deposition
should cease in this region is not clear. It is known that the oxygen tension is very low
in this part of the style (Linskens& Schrauwen, 1966), and Tiipy (1959) has proposed
that callose production is closely linked to respiration. This may be the case, but since
cellulose and callose share much the same biosynthetic pathway, it is not easy to see
how one carbohydrate should be formed, in the absence of, or at the expense of the
other.

The differences in pollen tube growth following compatible and incompatible matings
Since the compatibility of pollen tubes appears to become established as soon as
they enter the top of the transmitting tissue, this neck region of the style probably
plays the most important role in the self-incompatibility system in Petunia. Examina-
tion of incompatible tubes in the stylar neck presents a confusing picture, but perhaps
the most conspicuous features of such material 24 h after pollination are the small,
spherical fibrillar vesicles. These bodies have been considered in most detail by de
Nettancourt et al. (1973) who interpret them as resulting from the admixture of
'incompatibility protein' with pollen tube wall precursors, and point out the sirnilarity
between these inclusions and the bodies liberated when pollen tubes encounter the
synergid cells prior to fertilization (Jensen & Fischer, 1968; Diboll, 1968; van Went,
1970; Vazart, 1971). Dickinson & Lawson (1975&) concluded somewhat differently
from work on Oenothera. They pointed out that these structures were.characteristic
solely of burst or dead pollen tubes and did not constitute a primary product-of the
self-incompatibility response. The chemical constitution of these bodies has yet to be
fully elucidated, but Cresti & van Went (1976) conclude that they are fich in Callose,
and form the basis of the callosic plugs characteristic of growing pollen tubes'.
Since we here present evidence that these fibrillar bodies are also present in burst
compatible tubes, the inference must be reinforced that they represent precursors of
the cellulosic portion of the pollen tube wall which have, either as a result of tube
bursting or necrosis, autopolymerized in situ.
Although the fibrillar bodies seem not to be involved in the first events of the
incompatibility response, the construction of the pollen tube wall becomes evidently
different between tubes of different compatibilities only some 7 h after pollination.
That increased amounts of callose were present in incompatible tubes was established
as early as 1957 (Linskens & Esser, 1957), and shortly afterwards Tupy (1959)
suggested that this did not necessarily represent an increased pace of callose synthesis,
but could reflect a decreased rate of tube extension. When structural investigations of
 Pollen tube development in Petunia 381
these events began, it became clear that the components of the pollen tube wall
differed not only in their quantity, but also in their disposition. Schlosser (1961) for
example, reported thickening of the tube wall at the tip, while van der Pluijm &
Linskens (1966) described thickening of the tube walls themselves, de Nettancourt
et al. (1973) suggested that modification of the tube tip resulted in the demise of
incompatible tubes, proposing that rupture of these cells followed a disappearance of
the callosic wall and an expansion of the outer fibrillar wall into the intercellular spaces
of the conducting tissue.
Although many of the features described by de Nettancourt et al. (1973) are present
in our material, we interpret them differently and suggest, as did Tupy (1959), that
all the events observed may be explained in terms of a decreased rate of tube growth.
Thus, we believe the thickened walls of the incompatible pollen tube to result from
deposition of precursors at a rate equal to that in the compatible tubes which are, of
course, growing faster. This continuity of 'normal' metabolism in tubes which are
elongating slowly would not only explain the thick walls, but also the apparent
continuing synthesis of cellulose once formation of the callose wall has commenced,
for the 'change-over point' of wall precursor deposition would be 'focussed' in a far
shorter length of tube. Further, most other cell products would also be accumulated
at a rate appropriate to a compatible tube-elongation rate, thus explaining the high
levels of reserves and other cytoplasmic constituents of the incompatible tubes.
No unequivocal data point to the reason behind this slow growth. Rosen (1961)
suggested that pollen tubes extend by hydrostatic pressure acting upon a 'weakened'
tip. Extending this hypothesis to the situation in Petunia, the slow growth of incom-
patible tubes must result from increased wall pressure, or a decrease in turgor
pressure. Heslop-Harrison (1978) explored the possibility that the tube wall becomes
modified by a change in activity of cell-wall synthesizing enzymes, but it is equally pos-
sible that incompatible tubes are unable to break down stylar materials into moieties
which provide the necessary osmotic pressure to drive the tubes as fast as those
resulting from compatible matings.

One of us (M.H.) would thank Reading University and the OECD for financial support.
Thanks are also due to the Royal Society for the provision of photomicrographic apparatus,
and to the Electron Microscope Unit of the Plant Science Laboratories, University of Reading.

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(Received 17 May 1979 - Revised 12 August 1980)