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ISSN: 0017-3134 (Print) 1651-2049 (Online) Journal homepage: http://www.tandfonline.com/loi/sgra20

Pollen-pistil interactions in Grevillea banksii

J. Clare Herscovitch & Anthony R. H. Martin

To cite this article: J. Clare Herscovitch & Anthony R. H. Martin (1989) Pollen-pistil interactions
in Grevillea banksii , Grana, 28:2, 69-84, DOI: 10.1080/00173138909429958

To link to this article: http://dx.doi.org/10.1080/00173138909429958

Published online: 01 Sep 2009.

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 Grana 28: 69-84, 1989

PoIIen- pist i I interactions i n Grevi//eabanksii

The pollen grain, stigma, transmitting tissue and in vitro pollinations

J. CLARE HERSCOVITCH and ANTHONY K. H. MARTIN

Herscovitch, J. C. &: hlartin, A. R. H. 1988. Pollen-pistil interactions in Grevillea bnnksii.

The pollen grain, stigma, transmitting tissue and in vitro pollinations. - G r a m 28: 69-84,
1989. Uppsala 10 hlay 1989. ISSN 0017-3134.
hlorphology and ultrastructure of stigma, transmitting tissue and pollen were examined in
Greuillea banksii R. Br. (Proteaceae), a bird-pollinated shrub with stylar presentation of
pollen. Stigmatic cells are not cuticularised and secrete copious lipidic exudate. Intercellu-
lar material of the thick primary walls of transmitting tissue cells stains positively for
protein. The pollen exine is cavate and foveolate, but labile protein could be detected only
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in the thickened intine below germ pores. Self and non-self pollen of G. bnnksii, germinat-
ed on the stigma, both show normal protein mobilisation and synthesis of wall material.
Ectexine of germinated grains consistently appears saccate. Intra- and interspecific polli-
nations using five other species of Grevil/ea and one of Persoonin as 'foreign' pollen
sources, showed no pollen rejection at the stigma or in the upper transmitting tissue.
Sporophytic control in compatibility regulation, believed most usual with stylar presenta-
tion, is inferred to be absent.
J . Clare Herscovitch, Roynl Botanic Gardens, Sydney, N . S . W. 2000, Aiistralia. Anthony
R . 11. hfartin, Botany Departtnent. University of Sydney, N.S. W.2006, Australia.
(hfanuscript received IS June 1988, revised version accepted 6 October 1988)

Proteaceae are a phylogenetically isolated family of
woody plants of southern hemisphere distribution
(Johnson & Briggs 1975). Greuillea R. Br. (subfam.
Grevilleoideae) comprises trees and shrubs con-
fined to the Australasian Plate. The follicular ova-
ry, with submarginal placentation of the two
ovules, has been described as modified condupli-
cate, a somewhat primitive condition (Haber 1960).
Stylar presentation (deposition of self pollen
from the anthers onto some part of the style, pe-
ripheral to the stigma, from which it is removed
later by an animal vector) typifies many Proteaceae
and the whole genus Greuillea. This facilitates pol-
len collection and deposition on another flower's
stigma (Carolin 1961). Protandry is usual, and nec-
essary for outbreeding.
Self-incompatibility (S.I.) often goes with such
or gametophytic in genetic origin, usually involves
pollen inhibition on stigma or in style respectively.
Heslop-Harrison & Shivanna (1977) surveyed 250
families, typifying the stigmas of Proteaceae as dry-
papillate, a character strongly correlated with spo-
rophytic S.I. and stigmatic inhibition.
In Proteaceae, however. there have been con-
flicting reports about the occurrence of S.I. Ben-
tham (1873), noting the very low fruit set ofBanksin
spp. (Tribe Banksieae), suggested as cause the
capped condition of many flowers (the stigma re-
maining enclosed by the perianth for the entire
flowering time, precluding cross pollination). This
seems to imply S.I. in these species.
Collins & Spice (1986) believe the West Austra-
lian B . prionotes, a species pollinated by honey-
eaters (Melophagoideae), is self-incompatible and
'

stylar presentation. Otherqwise, for total outbreed-
ing, all self pollen must be removed before the
stigma is receptive or it Eould be pushed onto its
own stigma by late visitors and subvert the adapta-
tion (Fzgri & van der Pijl 1979). S.I., sporophytic
show it has a dry stigma.
However, Brough (1933) noted that the species
Greuillea batiksii and G . robiista appeared to set
seed without cross-pollination. Blake (1971) made
similar observations for three species of Batiksia.

5-898361 Grana 28
 70 J . C . Iierscovitch arid A . R . I i . Martin
Table I. Correlatiotis betiueeti fertility control arid uarioirs characteristics of polleri, stigriia arid style
+ = positive correlation; - = negative correlation
Sporoph ytic
S.I.
Stigmatic inhibition ++
Stylar inhibition - +
Pollen nuclei Trinuc.
Culturability Diflic.
Pollen protein hlobile
nail fraction
Ectexinous ivall spaces +
Stigma Dry papillate
Pollen starch
Stylar presentation + +
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A survey shows other contradictory indications
among the observed correlations (Table I).
No Proteaceae have yet been studied as to com-
patibility control. Proteaceae pollen is recorded as
binucleate (Davis 1966) and can be cultured in su-
crose solutions (Brough 1933; Garside 1946), but
very commonly has tectate-perforate exine struc-
ture with large exine cavities, appearing suitable for
storing labile protein, indicative of sporophytic
S.I., as in other families with chambered exines,
e.g. Asteraceae and Malvaceae (Heslop-Hamson et
al. 1973).
We examined pollen-stigma-style relationships in
Greuillea batiksii to resolve the apparent discrepan-
cy between expectation and the sparse observation-
al records, and perhaps note features peculiar to a
genus of gondwanic shrubs hitherto little studied.
G. banhii, a species of northern New South
Wales and southern Queensland is popularly grown
round Sydney. Flowers, c . 4 cm long, are in ra-
cemes, in bracteate pairs, red, zygomorphic, un-
scented, and with copious nectar. Four adherent
tepals form a tube round the long style, topped by a
flattened presenter disc. Floral opening is preceded
by extension of the style, which emerges laterally,
with the disc still covered by the perianth tips con-
taining the anthers (Fig. 1 Ai). With the peeling
away of the tepals, the pollen is exposed on the disc
surface (Fig. 1 Aii), in the centre of which, the
stigma (dry at this time) lies in a shallow pit. The
flowers are chiefly bird-pollinated (honey-eaters).
A close relationship exists between pollinator size
and floral morphology: on each visit, the stigmatic
disc dips onto the same part of the pollinator’s
Gram 28
Gametophytic Self-
S.I. compat. Source
-
Binuc. Binuc. Brewbaker 1967
Easy Easy
hlobile Heslop-Hamson 1978
intine fraction
Wet Wet Heslop-Ilanison
& Shivanna, 1977
+ Baker 8; Baker, 1983
-
head, aiding exact transfer and localisation of pol-
len (Brough 1933).
MATERIALS AND METHODS
Inflorescences came from. two cultivated bushes at Sum-
mer Hill, Sydney, and from a population (used also for
field self-pollination studies) at Crommelin Biological Re-
search Station, Pearl Beach, N.S.W
Mature and immature stigmas with c. 2 mm of style,
were excised under a drop of 3 % glutaraldehyde/O.OS hl
phosphate buffer, pH 6.8, on a imx sheet, then fixed in
the same fixative. post-fixed in OsO,, critical point dried
and gold-coated for SEM. Ripe undehisced anthers were
prepared similarly. Pollen, broken out of anthers, mas
scattered over the surface of double-sided tape. Speci-
mens were viewed in a JShl U3 SEM at 15 kV. TEhl:
excised stigmas and short lengths of style were fixed as
above, dehydrated in acetone, infiltrated in a 1 : 1 mixture
of 100% acetone superdry and Spurr’s resin (Spurr 1969),
100% Spurr’s, and embedded in a second change of
Spurr’s resin. After hardening, specimens were cut on
a Sorvall-Porter-Blum MT2 ultramicrotome, sections
stained in uranyl acetate and Reynolds lead citrate, and
viewed in a Phillips 300 TEM at 60 kV.
For general LM cell structure and for protein-staining
thick sections, Spurr’s resin-embedded material was cut
at 1-2 pm, on a glass knife. LM material was processed
for embedding in GMA. Sections were stained for lipids
using Sudan Black B and Sudan 1V. Insoluble carbohy-
drates were located by the PAS rection, cellulose by
Calcofluor White, and protein by Coomassie Brilliant
Blue. Semi-thin sections were also stained using Toluidine
Blue 0 as a general light-microscope stain. Resin sections
were also stained for protein, using Coomassie Blue and
Fast Green FCF (pH 2.0).
Labile pollen mall proteins and proteins diffusing from
apertures were sought using the stain-fixing medium Coo-
massie Brilliant Blue in a 25: 73 :2 mixture of methanol/
HIO/acetic acid, either by flooding the grains (direct emis-

sions) or by staining the diffusate on prepared agarose gel
films following the method of Heslop-Hamson et al.
(1973).
Laboratory pollinnlions
Almost-mature flower buds from just below opened flow-
ers of the raceme were planted upright in petri dishes on a
medium of 2% agar, 20% sucrose and 0.01 % boric acid in
f 1 2 0 , and kept in a humid atmosphere. We used only
flowers that opened within 24 h, removed the perianth and
brushed all pollen off the presenter with a fine paintbrush.
Stigmas were considered mature when papillae were up-
right and expanded, and exudate obvious (<24 h more).
Pollen w a s transferred to the stigma surface from present-
ers of newly-opened flowers of the same raceme (or of a
bush from a different locality for cross-pollinations), with
fine forceps points.
At I , 2, 4, 7, 18, 24, 48 or 72 h after pollination: ( I )
whole pistils wcre fixed in FAA for Lhl fluorescence
observations with aniline blue stain; or (2) stigmatic re-
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gions were fixed and processed for GhlA embedding; or
(3) stigmatic regions were fixed and processed for TEhl.
Foreign (inlerspecific) pollinations
Creuillea banksii pollen Ras placed on virgin receptive
stigmas of 5 other Greuillea spp. and one of Persoonia
(subfam. Persoonioideae).
After 6 or 24 h, pollinated styles were fixed 24 h or more
in FAA, rinsed in mxter, transferred to 8 N NaOH for 8 h,
and mashed in water for at least 1 h, when stigma and
transmitting tissue were dissected as far as possible from
surrounding style tissue, stained for 3 h or more in 0.05%
aniline blue (as.) in 0.1 M KtHP04 (pH 8),then mounted
in the stain, pressed to separate and flatten the tissues and
viewed by epifluorescence (Martin 1959).
Self-compatibility of the foreign species mas tested after
7 and 48 h.
RESULTS
bfanrre stigmas
At maturity, the presenter of the G . bariksii style
(Fig. 1Aii) is c. 2 mm in diameter. The stigma, in
the central 0.5 mm, is surrounded by a well-cuticu-
larised dry pollen presenting surface (Fig. 1 B, C).
The stigma consists of thin-walled (0.5 pm) unicel-
lular papillae up to 150 pm long, fringing and ex-
tending from a central cavity (Fig. 1 D). No cuticle
could be detected either by LM or EM, the cuticle
of the presenter seeming to end at the stigma edge.
The highly vacuolate stigmatic cells descend to a
depth of almost 1 mm before giving way to trans-
mitting tissue.
The receptive stigma is, however, fully immersed
in a viscous exudate (Fig. lC), staining strongly
with Sudan Black B and Sudan IV (Fig. 1 E), negli-
gibly with PAS (Fig. 1 D) and Coomassie Blue, with
large Sudan-positive droplets in the cytoplasm.
Greoillea batiksii pollen-pistil interoctioris 71
TEM shows the exudate as very smooth-textured
and highly osmiophilic, and osmiophilic droplets
(Fig. 1 F) are located in the vacuole, the cytoplasm
or outside the convoluted plasmalemma but within
the cell wall. Exudate, accumulated in the vacuoles
or vesicles, evidently crosses the plasmalemma and
cell wall and is released into intercellular spaces,
from which it wells onto the surface.
The peripheral cytoplasm contains mitochondria,
dictyosomes and associated vesicles c . 0. I pm dia-
meter, starch-containing plastids and RER in both
vesiculate form and as narrow cisternal strands.
Transmitting tissue.-The transmitting tissue of G .
bariksii occupies the central axis of the style (Fig.
2A). From stigma to base it narrows from 250 Itm
to <60 pm. A ring-shaped transition zone 2-3 cells
cells thick, thin-walled like stigma cells but lacking
lipids (Fig. 2 B , C) occurs between the central stig-
matic cells and the stylar cortical parenchyma.
Transition cells are richly cytoplasmic and the in-
tercellular spaces stain intensely for protein. The
ring narrows to form the central transmitting core.
Transmitting tissue cells are elongated down the
style axis, c. 30-40 pm long and 8-12 pm wide.
They, too, are richly cytoplasmic, with abundant
vesicular RER and large nuclei. Vacuoles may fill
up to one quarter of the cell area in the section.
Mitochondria occur throughout the cytoplasm.
Plastids up to 2.5 pm long are generally peripheral
near the plasmalemma, variable in shape but al-
ways elongated in the same direction as the trans-
mitting tissue itself. Starch is absent. The trans-
verse and longitudinal walls are thick-up to 1.5 pm
(Fig. 2 D), lacking intercellular spaces. Three layers
could be differentiated by TEM-an electron-dense
layer adjacent to the plasmalemma, a narrower,
more electron-lucent layer, and an outer layer
showing as a dark thin line, the latter probably
corresponding to the pectin-rich layer described by
Sassen (1974). If so, this layer, though much thin-
ner in G. banksii, is fundamentally consistent in
structure.
Walls are strongly PAS-positive, the intercellular
zone having a slightly weaker reaction (Fig.2D).
The outer layers also stain intensely for protein,
confirmed by Fast Green FCF (pH 2). The tissue
does not stain with Sudan dyes.
In stylar TS (Fig. 2E), transmitting tissue cells
are grouped each side of the carpellary suture. The
intercellular space between these appressed sur-
Grana 28
Downloaded by [202.62.16.29] at 23:53 30 September 2015

faces is visibly wider than in the tissue itself and
stains intensely for protein, significant in view of
the preferential assortment of pollen tubes along
the suture line (Fig. 3 F, G ) .
Further down the style, the transmitting tissue
becomes more bilobed in TS and at the base forms
two separate tracts, in which, in styles freeze-sec-
tioned to trace pollen tubes to the ovary, the fluor-
escing tubes are seen to follow separate paths.
hlaturc polleri
The mature pollen of G . bariksii is isopolar, oblate,
triporate, amb convex-triangular, c. 45 Itm on a
side. The prominent pore membranes are c. 20 Itm
in diameter, protruding c. 12 Iim. The exine is tec-
tate-foveolate, undulating, with rounded or narrow
supratectal processes (Fig. 2 F , G ) . Small rounded
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elements, sparsely scattered on the surface, may be
Ubisch bodies (Fig. 2 F , H). Tectal foveae range
from c. 0.4 to 1.0 pm in diameter and may appear
blocked by subtectal elements (Fig. 3A). Serial
semi-thin sections of c. 2 Itm showed that the latter
had no attachment to the tectum or foot layer.
Supratectal projections are always associated
with thick columellae that narrow basally. Some-
times two may fuse apically, especially near aper-
ture margins where they are larger and slanted. The
Fig. 1 . A. Greuillea banksii flower: (i) bud just before
opening; perianth encloses style apex and anthers, style
characteristically bent. (ii) open; pollen mass deposited on
expanded stylar apex (presenter, p p ) . I , 2, 3, 4. 5 =
approximate levels at which stigma and style sampled for
pollen tube growth (Fig. 4). Bar = 1 cm.
B. Immature stigma surrounded by cuticularised pollen
presenter from which pollen has been removed (c. 50%
full diameter shown). Papillae stand free from the base.
Exudate beginning to accumulate. p = cuticularised pre-
senter surface. (B and C: SEhl; bar = 0.1 mm.)
C. hlature stigma as in (B). Papillae are completely
immersed in exudate.
D. Longitudinal section (L.S.) of stigmatic papillae ( s p )
and adjacent cells: PAS reaction. Note starch and thin cell
aalls. Exudate has not stained. c = thick cuticle on
presenter, marginal to stigma (arroued). GhlA embedded.
Bar = 40 pm.
E. Section of same stigma as (D) - Sudan Black-stained:
Exudate ( e ) droplets in stigma cells and cuticle (c, ar-
rouvd) strongly stained. Bar = 0.1 mm.
F. TEhl of stigma cell, cell walls ( w ) appearing thick
due to obliquity of section. Large vacuole (v). and drop-
lets (I) at different stages of secretion. Plastids (PO and
mitochondria (in) are also visible. Exudate is highly os-
miophilic. Bar = 2 pm.
Grcvillea bariksii polleri-pistil iriteractiotis 73
endexine appears homogeneous, c. 1 pm thick in
non-apertural areas, but thicker and lamellated near
apertures before thinning to a pore membrane of
sporopollenin lamellae (Fig. 3 B) with no discern-
ible SEM surface pattern (Fig. 2H).
The exine cavities contain amorphous material
(Fig. 3 A) probably tapetal residues. Pollenkitt was
not preserved by the processing, but is presumed to
have been originally present, from the shining sur-
face appearance of fresh grains and their tendency
to adhere to each other.
The non-apertural intine forms a distinctly PAS-
positive thin line between cytoplasm and endexine,
and fluoresces bright blue with Calcofluor, specifi-
cally indicating cellulose. Possible staining with
Coomassie Blue was hard to differentiate from the
adjacent cytoplasm which also stained strongly. By
TEM the intine often appears 2-layered, an outer
thicker, fibrillar zone and an inner electron-lucent
rather alveolar zone (Fig. 3 A), abutting directly
onto the convoluted plasmalemma and in places
appearing invaded by it, making distinction be-
tween the two unclear.
At the aperture, the intine expands to a complex
three-layered structure c. 2 pm thick (Fig. 3 B, Fig.
7A), (probably equivalent to the thicker oncus
(Hyde 1954) of some other porate pollens). This
stains throughout with PAS, but in GMA sections
weak aniline blue fluorescence shows only a thin
layer, presumably callose, structureless at all mag-
nifications, and probably corresponding to the in-
ner electron-lucent layer, non-staining with tolu-
idine blue (Fig. 2 I). The dark inclusions of the
apertural middle layer are Coomassie Blue positive.
The cytoplasm of all apparently viable grains is
filled with amyloplasts with typically 1 (rarely 2 or
3) granule per plastid, up to 2 Iim in diameter (Fig.
2 I-J; Fig. 3 A-C). Scattered cytoplasmic inclu-
sions, irregular or more-or-less spherical, are in-
tensely protein-staining (Fig. 2 J). They correspond
to the highly electron-dense inclusions of c. 0.5 p n
diameter seen by TEM (Fig. 3 A , B, C).
Numerous small vesicular inclusions c. 0.03 Iim
in diameter occur throughout the cytoplasm, often
clustered. All pollen pellets prepared for TEM gave
negative fixation images of these (Fig. 3 A-C), thus
they appear as single electron-lucent membranes
bounding dark rings and electron-lucent cores.
Rarely, a few were seen associated with small
stacks of 3 or 4 dictyosome cisternae c. 0.4 Iim
length (Fig. 3 B) (Herscovitch & hlartin, in prep.).
Grana 28
 74 J . C. IIerscovitch orid A . R . H . Mortin

A
.. _.,......
. . ,

._.._
I.--
.-
-
...<:
.i
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- ! .
_a .
- ...... J
Fig. 2.

Dictyosomes were small and rare, no more than 3
Or 4 being identified in any one thin section of a
grain, occasionally associated with a few small
vesicles as above. Spherical to oval inclusions up to
0.5 I‘m diam. and of different electron densities
\vere noted (Fig. 3 A-C), apparently single-mem-
brane bound. hlitochondria were not abundant
(Fig. 3 0 .
KER was present throughout. Membrane profiles
and tangential sections showed ribosomes densely
packed on the outer surface. The membranes were
very convoluted and the enclosed cisternae highly
distended, filled with a moderately electron-dense,
finely granular intensely staining proteinaceous ma-
terial. The cytoplasm had no affinity for Sudan dyes
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~~
Fig. 2. A. Transmitting tissue. Transverse section (T. S.)
style of G. bonksii c. 10 mm below stigma surface, show-
ing thin-\mlled vacuolate cortical parenchyma (co), vas-
cular bundles and central thick-walled transmitting tissue
( t i ) ; ( e p = epidermis). PAS, GhlA. Bar = 0.2 mm.
B. T. S . of transition zone between stigmatic and trans-
mitting tissue; stain Coomassie Blue (Spurr’s). Dense cy-
toplasm, thin ualls and intense intercellular staining
(white arrow h e a h ) clearly distinguish zone from the
large vacuolated cortical parenchyma cells. Sudan stain-
ing (not figured) shows absence of lipid. Bar (B and C) =
20 pm.
C. As Fig. 2 B in L.S. Transition cells ( t r ) are richly
cytoplasmic and elongated cf. stigmatic and cortical cells
(si and co).
D. Detail of (A) shows thick walls of transmitting tissue,
PAS-stained. hiiddle \\all layer (orroue4 stains more
lightly for carbohydrate. GMA. Bar (D and E) = 40 Itm.
E. As Fig. 2D, Coomassie Blue (Spurr’s). Walls non-
staining, but intense intercellular and suture-line stain
(311).
F. Pollen grain of Greuillen bmksii. Protruding pores
with relatively smooth apertural membranes ( a ) , triangu-
lar amb, tectal surface projections: SEhi. Bar = 20 pm.
G . (F) detail: tectum with undulating surface, foveae
(some blocked), Ubisch body and tectal projections. Bar
(G and H) = 2 pm.
H. (F) detail, region (0):narrow surface projections,
Ubisch bodies and loiver centre, non-patterned pore
membrane and thickened margin to aperture. Bar = 20
Iim.
1. Semi-thin section, Lhl (Spurr’s). Toluidine Blue 0
stained. Abundant starch, thin line of intine, stained cen-
tral layer of aperture ( a ) , non-stained inner layer; loose
exine and thick eolumellae. Bar = 20 Itm. In LXl, exine
(ex) \\as not visible by other stains in either Spurr’s or
GhlA-embedded material (Fig. 2J).
J. As (I), stain Coomassie Blue: dark nuclei and inclu-
sions (protein, pr). Faint stain in central layer of pore
region; exine and starch non-staining. Background stain is
bsa. Bar = 40 pm.
Grevillea batiksii pollen-pistil iriteractiotzs 75
and no inclusions suggestive of lipid were seen
under TEM.
Pollen-wall proteins: results of irnrnersioti
arid diffiisiotiexperirizerits
Itnniersioti.-With 2 min immersion, protein-stained
material was seen diffusing from apertures as
coarse fibrillar strands, and staining intensity in-
creased for up to 10 min (Fig. 3D). No emission
from intra-apertural areas was ever seen, even
though, since amounts in the exine may have been
small and readily dispersed, immediate and con-
tinuous observation took place.
D$Jrsion itilo agar.-Heavy staining of protein
marked contact areas of grain apertures but not
intra-apertural areas except after later diffusion
along contact lines.
Laboralory self-pollirialioris
Grevillea bnriksii pollen tubes fluoresce strongly
with aniline blue, so finding them in stylar TS is
easy. No germination was apparent after I h but in
4 h tubes were long enough to make counts at
different levels (Fig. 1 Aii, Fig. 4).
Though distribution of pollen tubes at the stig-
matic surface was random, they were always found
between levels 1 and 2, sorted into a ring in the
transitional tissue peripheral to the stigma (Fig.
3E). At levels 3 and 4 pollen tubes were in linear
array (Fig. 3F, G) following the more-or-less
straight suture line bisecting the transmitting tissue
at these levels. In vitro styles suffered turgor loss
by 72 h and tubes were seldom seen below level 4,
probably from this causs.
While variability in the 4 h data might arise from
differences in germination time or growth rate, the
fall in pollen tube number down the style in 48 h
pollinations strongly suggests fewer tubes still
growing. It appears from the numbers counted at
level 3 that, no matter how many grains germinat-
ed, the tube number is reduced to a maximum
carrying capacity of the transmitting tissue. With
only two ovules to be fertilised, narrowing of the
transmitting tissue tvould provide a simple mechan-
ism selecting the fastest growing and most vigorous
tubes to effect fertilisation. Field experiment re-
sults (Herscovitch & Martin, in prep.) consistently
showed only two pollen tubes at the base of the
style.
Grana 28
 76 J. C. IIerscovifch arid A . R . I I . Martin
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. :*-
....

Fig. 3 . A. Greuilleo Bnnksii pollen: TEhl of wall and
cytoplasm. Spherical inclusions and amorphous material
in spaces below tectum ( I ) ; endexine (en); intine (11ap-
pears bi-layered; large starch granules (s), clustered small
vesicles (us) and electron-dense inclusions. Bar = 2 Irm.
R. TEhl pore of grain. 3-layered intine structure: inner
electron-lucent (el), middle alveolate with dark inclusions,
outer fine-textured (0);sporopollenin membrane (sn). Cyto-
plasm includes mitochondria, dictyosomes, small vesi-
cles, rer large (0.5 pm) vesicles (ul),electron-dense inclu-
sions and starch granules. Bar = I pm. .
C. TEhl of cytoplasm of inner region of grain. Organ-

Grana 28

60-
UI
2
0
- 30-
6 -
z small numeral at top of bar). Broken line =
10-
Depth in Style
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Ititerspeci~clititergctiericpollitiatiotis
Various crossings were made to see if control of
interspccific incompatibility occurs at the stigma
surface, but in all, pollen germinated well with ap-
parently healthy tube growth (Table 11).
Six hours post-pollination, G. batiksii tubes had
penetrated the stigmatic region of other species and
judging by tube length had entered the transmitting
tissue (Fig. SA). Other species did not penetrate
foreign stigmas so rapidly-at 6 h, tubes had not
reached transmitting tissue (Fig. SB). However,
tubes did not appear abnormally occluded, distort-
ed or burst. Tube lengths in control cross-pollina-
tions of 6 h were similar, indicating that the slower
growth rates were inherent characters of the pollen
and not due to inhibition on foreign stigmas.
By 24 h, pollen tubes in all crosses were further
elks as in (A). rer = endoplasmic reticulum. Bar = 0.5
Clm.
D. Pollen of G. banksii 6 min after immersion in Coo-
rnassie Blue stain-fixative-stained fibrillar material
around apertures; lack of stain around intra-apertural sites
(arrowheads). Bar = 20 pm.
E. Self-pollination: pollen tubes in circular array in
transitional tissue at top of style (level 2 ) . G. bnnisii
selfed 4 h. Aniline blue fluorescence. Bar in E, F and G =
0.2 mm.
F. Section c. level 3: selfed 48 h - linear airangement of
tubes.
G . As (E) and (F), but with addition of Calcofluor to
show surrounding tissue (rr = transmitting tissue).
H. G. banksii selfed 24 h: pollen tube callose plugs and
tube fluorescence. Faint fluorescence in grain (top left
centre). Aniline Blue. Bar = 40 pm.
Greuilleo batiksii polleti-pistil ititeractiotis 77
Fig. 4 . In vitro pollinations of Greuillea bonksii
styles: no. of tubes present at successive levels
I to 4 as in Fig. I A ii. A = 4 h since pollina-
tion, B = 48 h since pollination. Solid line =
selfed; vertical bar = 1 sd each side of mean
(based on no. of obsemtions indicated by
crossed; 0 = 4 h, A = 48 h, each based on
single observed style. There is no statistical
difference between behaviour of pollen on
selfed and crossed styles.
down foreign styles than could be traced, well into
the transmitting tissue, >2 mm in the short-styled
G . triloba, >I0 mm in the longer-styled species,
showing that no inhibition of pollen tube growth,
self or foreign, occurs in the stigmatic region of any
of the species of Proteacae tested.
Gcrminntcd polleti in semi-thiti atid thin scctiotis
The germination time of G. batiksii pollen is very
variable. However, the sequence of events can be
inferred from seeing grains with an emergent tube
in the section, or in semi-thin serial sections, or
showing characteristics recognised from prior study
of germinated grains.
Pollen after gcrttiitiatioti
Exitie.-In both self- and cross-pollinations, the ec-
texine of germinated grains consistently appeared
expanded and saccate (Fig. 5C); columellae be-
came detached either from the foot layer or the
saccate tectum or lay apparently free in the en-
larged cavities (Fig. 5D). This was not seen in
grains without emergent tubes, and never in mature
ungerminated grains, pelleted and fixed for TEhl.
The more vacuolated grains .sho\ved most effect.
Faster primary fixation--Glut/PO,~ for 30 s and
post-fixation in O s 0 4 for 20 min-did not reduce
the effect. We conclude that this effect is more
likely to be associated with pollen germination than
to be a fixation artefact. Abortive grains (Fig. SC)
typically have a highly saccate ectexine but differ
from germinating grains in their smaller size and
abnormally thick endexine.
G r a m 28
 Downloaded by [202.62.16.29] at 23:53 30 September 2015

Fig. 5.

,ipprtiires.--In three grains that had apparently hy-
drated but not yet produced tubes, the apertural
Stmcture and cytoplasm appear significantly differ-
ent from those of pelleted ungerminated grains. The
inner layer of the intine appears broken up and
dispersed in a fibrillar matrix (Fig. 6A and B, Fig.
7 B). The cytoplasm close-by contains spherical
electron-lucent inclusions c. 0.08-0.17 prn diam.,
very like the electron-lucent material of the aper-
ture, each round a dense core. The Coomassie posi-
tive inclusions, held in the alveolate electron-lucent
layer in ungerminated grains (Fig. 3 B), now form a
layer loose in the fibrillar matrix, peripheral to the
area of electron-lucent inclusions.
Apertures ruptured by emergent tubes show no
evidence of the electron-lucent layer of ungerminat-
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ed grains. The intine appears four-layered-the
three outer a sandwich of two coarsely granular-
fibrillar round a central, containing dark inclusions,
just 3s in the incipiently germinated grains. The
contiguous inner layer is finer-textured and more
homogeneous-probably new tube wall (Fig. 5 E,
Fig. 7C); it is texturally distinct from callose.
Thus as the grain cytoplasm is mobilised for ger-
mination, one (usually) of the apertures is modified
to allow tube emergence, the inner textureless elec-
tron-lucent layer being broken up and dispersed. If,
as staining suggests, this layer is callosic, its rela-
tive impermeability and mechanical strength, which
\vould protect the gametophyte from environmental
stresses, would also delay germination. A callose-
Fig. 5 . A . G. boribii pollen tubes in tissue of Greui//tw
rhelettintininna style 6 h after pollination of stigma: c. 500
Iim of tube in photo. Total length c. 900 pm. Aniline Blue
fluor. Bar in A and B = 100 Icrn.
B. G. rhelerrzntitiinrra pollen tubes 6 h after pollination
of G . bnnkrii stigma: c. 400 Iim of tubes is seen here and
transmitting tissue has not been reached.
C. Lhl of G. barilsii germinated pollen (left). Tube
emerged perpendicular to plane of section. Saccate tec-
turn (most so on left). Abortive grain (nb) shows saccate
character but also excessively thick endexine and absence
of cytoplasm. Bar = 10 pm.
D. TEhI of highly saccate tectum of G. boizksii 2 h after
self pollination. Intratectal elements (coo are visible, at-
tached to tecturn (1). foot layer or apparently suspend-
ed in cavity ( 6 ) . Bar = 2 prn.
E. Aperture of newly-germinated G . boriksii 4 h after
cross pollination. Three wall layers, line of electron dense
inclusions ( I ] , inner homogeneous layer ( / I ) . The inner
electron-lucent layer is absent. Cf. Fig. 3 B. TEhI: Bar =
1 Iirn.
Grevillea bariksii pollen-pistil iritcractioris 79
digesting enzyme system is inferred from its break-
down (Roggen & Stanley 1969) and may be among
the emissions detected by Coomassie staining.
Apertures of germinated grains from which no
tube had emerged, are much thickened, multi-lay-
ered, with masses of alveolate electron-lucent in-
clusions (Fig. 6C). The adjacent cytoplasm con-
tains similar inclusions and often appears necrotic.
24 h after pollination, emergent tubes are often
occluded with newly-formed callose, just beyond
the aperture (Fig. 3H).
Cytop[asm.--In earliest tube growth, starch con-
tent and distribution in the grain appear unchanged,
but in tubes known from semi-thin serial sections to
be equal to, or longer than the grain (c. 50 p ) ,
amyloplasts are grouped towards the germ aper-
ture. Appropriate sectioning shows spatial polarisa-
tion of grain contents (Fig. 6 F ) especially in Lhl
preparations stained for starch. Ensuing vacuola-
tion is first seen in germinated grains after 4 h on
the stigma, seeming to start in the amyloplast-de-
pleted cytoplasm furthest from thc germ aperture.
The vacuolated region contains protein accumula-
tions and tubular inclusions. Apertures near the
vacuolated region, from which no tube will emerge,
are heavily occluded, probably with callose (Fig.
6C).
Budding dictysomes are abundant, with numer-
ous associated large vesicles (Fig. 6G). The
vesicles, 0.1-0.2 Iim in diameter, single-membrane
bound, with a fibrillar content, often appear to be
fusing to form larger bodies. Golgi cisternae are c. 1
Itm long (cf. 0.3 pm in ungerminated grains). Mito-
chondria 0.3-0.4 iim diameter are abundant,
massed chiefly at the apertural region. Abundant
small vesicles seem unassociated with particular
organelles or areas.
Most strikingly, in germinated grains, RER is
much less dilated and more vesiculate than in the
ungerminated state, though still highly folded in
places. KER of grains 2 to 4 h post-pollination, with
tubes >I00 pm long, appears elongated, almost
cisternal. Coomassie Blue in GhlA-embedded ma-
terial stains RER as even longer beaded strands,
elongated towards the germ aperture (Fig. 6D),
indicating direction of cytoplasmic movement. The
general post-germination background Coomassie
staining is lighter, probably through the increased
cell volume causing ‘dilution’ of the cytoplasm, as
well as through opening out of the RER.
Coomas’sie’ also intensely stains the aperture
Grana 28
 80 J . C. Herscooitch and A . R . H . Martin
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Fig. 6 . A. Aperture of G. banXsii grain. 2 h after self-
pollination. Sporopollenin membrane shows signs of dis-
persing and mitochondria ( m ) are visible. Note electron-
lucent spherical inclusions (arrow) and the dispersed in-
ner intine layer of the aperture. Bar = 1 pm.
B. As (A), 4 h after pollination. RER is more open. Bar=
0.5 pm.
E ' i .
C. Non-functioning aperture (tube emerged from a dif-
ferent one): 7 h after self-pollination; pore thickly oc-
cluded, electron-lucent inclusions (?callose) in necrotic
cytoplasm (arrow). Bar = 2 pm.
D. Aperture ( a ) of germinating grain, 2 h after cross-
pollination, stain Coomassie Blue: beaded Coomassie-
positive strands, granular staining in cytoplacm and dense
 Greuillea banksii pollen-pistil itzteraciiuns 81
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Fig. 7. Interpretation of intine changes in germination of
Grevillea bonksii pollen grain: A. ungerminated; B. Pollen
2-4 h on stigma; C . just-germinated pollen grain. u =
apertural membrane; c = electron-lucent layer of intine
with callose; d = dictyosome; Ii = homogeneous granular
layer; ip = intine-held protein in middle layer; lu = large
vesicle; r n = mitochondrion; o = outer intine layer; s =
plastidial starch; su = small vesicle; rer = folded ribosom-
al endoplasmic reticulum. Massive callose-containing lay-
er in A is in process of dispersal in B. In C, a thin fibrillar
layer ( / I ) represents this region. Contiguous with it is
newly-formed homogeneous fine-granular inner layer re-
presenting tube wall.

plasmalemma o f germinated grains (Fig. 6 D ) . e d , clumped, a n d m o r e electron-opaque than in the

Dense-stained irregular structures (Fig. 6 E) are ungerminated state (Fig. 6 F ) .
probably the s a m e as t h o s e in the ungerminated
grain, while areas o f dark mottling represent pro-
DISCUSSION
tein-rich ER opening out. Ribosomes (perhaps
owing their clarity t o the m o r e electron-lucent state Stignza and transmitting tissue
of the ground matrix than in t h e ungerminated H e s l o p - H a m s o n & Shivanna (1977) noted that
state) are visible o n t h e ER u n d e r EM, a n d maybe most families are consistent i n stigma type. Their
occur as polysomes in t h e cytoplasm (Fig. 6 G ) .
Post-germination intine material appears retract- Table 11. Interspecificlitztergerieric pullirzntions
+ = successful pollination; - = cross not made ('success-
staining at apex of emerging tube. Cytoplasm in tube more ful' means only that pollen tubes reached transmitting
lightly stained. Starch has not yet moved from grain to tissue. Subsequent fertilisation is not implied)
tube. GMA. Bar in D and E = 10 pm.
E. Same treatment as (D). Coomassic-positive inclu- Pollen parent
sions and densely-stained mottled regions (mo) in interior
of grain. Stigma parent 1 2 3 4 5 6
F. TEhf of incipiently vacuolating grain, cross-pollinat-
ed 4 h. Polarisation in location of starch, protein bodies 1. G. bonksii + + + + + +
b r ) , disorganised intine (i) and retracting plasmalemma. 2. G . rriloba + + + . + + -
Bar = 4 pm. 3. G. buxi/olia + + + - - -
G . Cytoplasm of germinating grain, selfed 4 h. Highly 4. G. r/ieleniaiiniunn + + - + - -
secretory dictyosomes (6). small vesicles (us), some of 5. G. sericea + + - - + -
which seem to be fusing, large vesicles (vf), mitochondria 6. Persooniu piiii/oliu + + - - - +
( i n ) and opening out of rer. Bar = 0.5 pm.

Grana 28
 82 J . C . Herscovitcli arid A:R. H . hfartiri
typification of Proteaceae as dry rested, though, on
only two species. Shivanna (1982) however, admits
there is no firm division between wet and dry stig-
mas. Wet stigmas, often linked with protandry,
may be dry at anthesis, as noted for Proteaceae by
Bentham (1873) and this is true of G . bariksii.
Ultrastructurally, stigma cells of G . bariksii agree
with epidermal cells in such features as large vacu-
ole, peripheral cytoplasm and thin primary wall,
but lack cuticle. The copious lipidic exudate totally
immerses the stigmatic papillae at maturity, and
probably, like cuticle, gives protection against des-
sication and pathogenic attack (Martin & Ortiz
1967). The stigmatic structure and behaviour is
quite different from that of Amyema (Bernhardt &
Knox 1983), another bird-pollinated species, show-
ing that the unusual structure of the latter is not an
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invariable feature of the bird-pollination syndrome.
Wet stigmas correlate with stylar inhibition and
binucleate pollen (Heslop-Hamson 1978, Heslop-
Hamson & Shivanna 1977). The morphology and
exudate are inconsistent with stigmatic compatibil-
ity control, as a liquid interface between pollen and
stigma cells provides no stable receptor site for
cell-cell recognition and rejection (Heslop-Ham-
son 1975, Heslop-Hamson et al. 1975).
Anatomy and ultrastructure of transmitting tissue
have a general similarity in solid-styled genera, e.g.
cotton (Jensen & Fisher 1970), Petunia (Sassen
1974) and Lycopcrsicrrni (Cresti et al. 1976). Wide
intercellular layers, rich in pectic material are com-
mon. G. banksii is not unusual, though finding in-
tercellular protein is interesting; its concentration
mainly close to the suture line along which pollen
tubes pass, suggests a role either in nutrition or
inhibition of tubes.
Pollen rnorpliology
The relatively large pollen exine cavities of G .
bariksii give much potential for storage of labile
protein, but none is present, indicating absence of
sporophyticlstigmatic control of breeding, and cor-
relating with the stigma type. A gametophytic S.I.
system is not precluded (Heslop-Hamson 1978).
Exine cavities may contain other substances be-
sides those involved in recognitiodrejection re-
sponses. Superficial coatings include oily ‘pollen-
kitt’, which may function in adhesion of pollen to
stigmatic papillae as well as self-adhesion of pollen
in relation to visits of pollinators. Hesse (1981)
showed that in tectate pollen of entomophilous
Grana 28
plants, pollenkitt is mainly above the tectum but
that it may be wholly in subtectal spaces in dry-
pollen (anemophilous) species. Possibly this is also
true in development of some ornithophilous spe-
cies. SEMs of dry fresh pollen of other Grevillea
species clearly show foveae bldcked by pollenkitt.
We would anticipate a triple role: adhesion to the
presenter, to pollination vectors and to the recipi-
ent stigma.
In species with a consolidated continuous endex-
ine, enzymes related to germination, tube growth
and cuticle penetration, are in the diffusible intine-
held fraction (Heslop-Hamson et al. 1973) and in
G. banksii seem to be chiefly in the oncus-like
thickening. Simple substrate-film digestion tests
should confirm that the stainable protein emerging
from apertures of G . bariksii is largely enzymic.
Saccate exirie
At anthesis, pollen germ-apertures of most species
are collapsed inwards, and on hydration up to twice
the original volume is accommodated by their out-
ward expansion. But in Grevillca, pores are already
prominent at anthesis and partly filled with thick-
ened intine, so that volume increases cannot be
accommodated in this way. From Rowley’s (1976)
observations it is conceivable that in hydration the
entire grain is stretched by protoplast expansion; as
the tube emerges and the grain vacuolates, the elas-
tic endexine and foot layer would contract, separat-
ing from the loosely attached tectum (Fig. 21; Fig
3A) except at a few points (e.g. pores), leaving the
latter in saccate form.
No other reports of such behaviour are known to
us; enzymic pregermination degradation of sporo-
pollenin has been reported (Gherardini Br Healey
1969, Dickinson & Lewis 1974); from our observa-
tions, this does not seem to be happening in G .
bariksii. Saccate exine has been noted in other spe-
cies of Grevillea (e.g. Erdtman 1966, Fig. 208 B) but
its occurrence has never been explained.
Storage in polleri cytoplasm
The types of storage product and their packaging,
seem to be very species-characteristic (Sassen
1964, Went 1974, Jensen et al. 1968, Larson 1965).
Grevillea pollen contains much starch (Davis
1966), a feature strongly correlated with bird, lepi-
dopteran, wind, and self-pollination (Baker & Bak-
er 1983). Protein is also stored in G . bariksii (Fig.
2 J; Fig. 3A,B,C), both in vacuoles and ER,

though to a lesser extent. Lipid (typical of many
bee-flowers) is virtually absent.
self arid cross polhiations
Pflysiological u. geographical barriers.-In the spe-
cies studied, neither stigma nor upper style bar
pollen penetration, either self, other G . batiksii ge-
nomes, other Greuillerr spp. or more remotely-relat-
ed genera.
It is interesting that G. triloba's stigma is no
barrier to foreign pollen tube penetration as its
surface is cuticularised and dry. In Crocus, the
stigmatic cuticle was shown to be penetrable by
pollen of quite distant families and tubes cannot
penetrate cuticle when stigma surface secretions
are removed, the cutinase system appearing to be
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activated by factors on the stigma surface (Heslop-
Hamson 1977). Cutinase precursors must be pres-
ent in all the species used in the above crosses,
even in G. batiksii, with no detectable stigmatic
cuticle. Heslop-Hamson (1975) had speculated that
cuticle may be involved in control of interspecific
incompatibility. Our data show that even between
closely related species there may be no specificity
in the cutinase system.
If progamic control occurs, morpholphysiological
disparities must arise deeper in the style, maybc via
the tip region of the tube, as in Rhododetrdroti
(Williams et al. 1982) or through failure in hetero-
trophic nutrition. But it seems most likely that ex-
ternal barriers work in the field for these species;
different flowering time, geographical distribution
and pollination syndrome provide effective inter-
specific barriers, possibly making physiological
mechanisms redundant.
hlorphological barriers.-Whitehouse (1950, cited
in hhrtin & Ortiz 1967) suggested that long narrow
styles may filter incompatible tubes eliminated by
inhibition, from compatible tubes. The latter au-
thors were more inclined to relate long styles to
pollination mechanisms. However, the transmitting
tissue of the species they worked with (sweet pota-
to) is of uniform diameter. The functional signifi-
cance of narrowing of the tissue in G . bntiksii in
selecting the most vigorous male genome cannot be
discounted.
In competitive pollinations with self- and cross-
pollen (which we did not attempt) the latter might
be advantaged. But progressive elimination of
Grevillea 'bntiksii polleti-p isti1 ititeractiotis 83
tubes, both in selfed and crossed styles, suggests
not a specific recognition and rejection but an out-
come of space or nutritive constraint imposed by
the narrowing of the transmitting tissue.
ACKNOWLEDGEMENTS
Acknowledgements are included in IIerscovitch & hlartin
in prep. (the concluding part of this account), to be pub-
lished shortly.
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