Annals of Botany 87: 469±475, 2001
doi:10.1006/anbo.2000.1359, available online at http://www.idealibrary.com on
Successful Fertilization and Seed Set from Pollination on Immature Non-dehisced Flowers
of Eucalyptus globulus
H . T R I N D A D E {, L . C . B OAVI D A {} , N. B O R R A L H O{ and J. A . F E I JOÂ *{}
{RaõÂz, Instituto de InvestigacËaÄo de Floresta e Papel, Apartado 15, 2065-Alcoentre, Portugal, {Instituto Gulbenkian
de CieÃncia, PT-2780-156 Oeiras, Portugal and }Centro de Biotecnologia Vegetal, Faculdade de CieÃncias,
Universidade de Lisboa, Campo Grande, Ed.C2.PT-1749-016 Lisboa, Portugal
Received: 2 June 2000 Returned for revision: 30 August 2000 Accepted: 12 December 2000 Published electronically: 7 February 2001
To minimize the number of visits required to achieve seed-set from hand pollination whilst maintaining full control of
the reproductive process in Eucalyptus globulus, we investigated the importance of style maturation on the progamic
phase of reproduction. Controlled pollinations were performed in: (1) fully receptive styles; (2) immature cut styles at
or after ¯ower dehiscence; and (3) immature cut styles before ¯ower dehiscence. We show here that pollen germination
and tube growth to the ovule occurred in all three treatments, and all produced viable seeds. Remarkably, tubes
growing on immature sliced styles of non-dehisced ¯owers reached the ovules by day 7, 1±2 d faster than tubes
growing on normal receptive stigmas. Moreover, more tubes entered the ovary in this kind of pollination and,
consequently, more viable seeds were produced. We established histochemically that tube growth in immature sliced
styles is concomitant with the presence of lipid secretion. These results fully support the eciency of the so-called
`one-step pollination' method in Eucalyptus globulus, which we now prove can be applied more successfully to
immature non-dehisced ¯owers. General application of this procedure is likely to have bene®cial impacts on the
# 2001 Annals of Botany Company
planning and eciency of large-scale genetic improvement programs.
Key words: Eucalyptus globulus, one-step pollination, lipids, pollen, fertilization.
I N T RO D U C T I O N
In recent decades Eucalyptus globulus has become one of
the most planted trees for paper pulp production. Its
economic importance implies a demand for genetic
improvement. Vegetative propagation is unsuccessful, so
improvement and production are based mainly on seeds,
implying successful sexual reproduction. Detailed descriptions of ¯ower anatomy and pollination in the genus have
been made (Grin, 1982; Sedgley and Smith, 1989; Sedgley
et al., 1989; Gore et al., 1990; Hingston and Potts, 1998). In
short, receptivity of the stigma is associated with the
presence of exudate, which promotes pollen tube growth
(Knox, 1984; Sedgley and Smith, 1989). As a protandrous
species, dehiscence (operculum fall) is soon followed by
pollen release from anthers (anthesis), but peak receptivity
only occurs 5±10 d later. Thus, to some extent pollination
of a ¯ower by its own pollen (autogamous self-pollination)
is prevented, but crossing with other ¯owers at di€erent
developmental stages on the same tree (geitonogamous selfpollination) is not (Tibbits, 1989; Hardner et al., 1996).
Early studies in Eucalyptus (E. regnans) were unsuccessful
in terms of seed set when pollination was carried out before
peak receptivity (Grin and Hand, 1979). The prereceptive stigma is dry, preventing adherence and pollen
grain germination. This may be overcome by wounding the
stigma or style, usually by a transverse cut or longitudinal
* For correspondence. Fax ‡351 214407970, e-mail jose.feijo@fc.
ul.pt
0305-7364/01/040469+07 $35.00/00
sectioning. The exposed tissue readily exudes a drop of
secretion, sucient to support pollination. This approach
led to the development of the so-called `one-stop' or `onestep pollination' techniques (OSP): in short, the prereceptive bud is emasculated, the stigma is severed or cuto€ (allowing the intracellular secretion to be released) and
the desired pollen is applied, all in a single visit. Recent
studies using this approach in Eucalyptus globulus have
shown that fertilization and seed-set are successful, if not
improved (Harbard et al., 1999; Williams et al., 1999). This
procedure is much more ecient than the traditional
method, reducing the number of visits from three or more
to one, with less labour and no bagging required. A possible
improvement would be to extend OSP to ¯owers in an
earlier stage of development, before ¯ower dehiscence
(operculum fall). This would be more ecient in terms of
the use of ¯owers and would decrease seed costs. This
approach was attempted by Harbard et al. (1999), but the
preliminary results were disappointing with losses of 50 %
seeds compared to post-dehiscence sliced styles.
It should be emphasized that these methods were
developed from a strict seed-set perspective, without attention to the reproductive process, such as pollen germination
and tube growth. Accordingly, we decided to investigate the
feasibility of pollinating ¯owers prior to dehiscence, and
anthesis. A simple factorial design, using well-known
E. globulus parents, was used to generate the di€erent
crosses. To ensure that the cytological basis for failure
would be understood, we followed the style secretion closely
# 2001 Annals of Botany Company
470
Trindade et al.ÐSeed-set after Pollination on Immature Flowers in Eucalyptus globulus
T A B L E 1. Experimental design used for pollination and seed set experiments: three mother parents were used and these were
crossed with a mixture of the three pollen lots
Flower
Style
Mother tree
Pollen
Before dehiscence
At dehiscence
At receptivity (*)
Transversely sliced
Transversely sliced
Intact
MN14, MN15, MN22
MN14, MN15, MN22
MN14, MN15, MN22
PL143, MN35, RE25
PL143, MN35, RE25
PL143, MN35, RE25
* 4/7 d after dehiscence/anthesis.
and characterized its content. Pollen germination and tube
growth up until ovule penetration ( progamic phase) were
fully characterized. We conclude that fertilization is
possible as soon as the style starts to accumulate lipids
along the transmitting tissue. In these conditions, cut styles
of ¯owers pre-dehiscence (and pre-anthesis) supported
pollen germination, tube growth and better seed production. This increases the eciency of OSP procedures
considerably, and is likely to have a positive impact on
large-scale pollination programmes.
M AT E R I A L S A N D M E T H O D S
Plant material
®rst treatment, intact styles were also used as controls to
examine spatio-temporal processes in pollen tube germination and growth. For every type of cross, capsules were
collected and ®xed after 1, 2, 5, 8, 24 h and 2, 5 and 7 d after
pollination. The material was ®xed for 3 d at 4 8C in a
solution containing 4 % paraformaldehyde (w/v), and 1 %
CaCl2 (w/v) in distilled water, saturated with CaCO3 (Baker,
1946) and autoclaved in 10 % sodium sul®te for approx. 1 h
to soften the tissues. Squashed ¯owers were used to assess
pollen tube germination and growth following incubation
with 0.1 % de-colourized aniline blue (Martin, 1959) for
24 h. Slide preparations were observed under ¯uorescence
microscopy (Nikon TE300), with blue (500 nm) or UV
(400 nm) excitation.
Experiments were conducted using selected clones in a seed
orchard. For this study six clones were selected from
previous data on capsule retention, ease of accessibility of
¯owers for hand pollination, and seed production. Clones
MN14, MN15 and MN22 were used as female ¯ower
parents and clones PL143, MN35 and RE25 as pollen
donors. These clones were crossed in a factorial design as
detailed in Table 1. For seed-set experiments, each mother
was fertilized with a single pollen donor. Each cross was
divided in two subsets: at least nine ¯owers were used to
visualize pollen tube growth, while the others were collected
and scored for seed set. For the early pollination stages,
branches were collected from each clone, the ¯owers were
tagged and emasculated, and brought into the laboratory.
Pollination was performed on the same day for ¯owers in a
given branch at di€erent stages of development, using a
mixture of pollen from the three donor clones. Later
pollination stages were performed in the ®eld, using
previously emasculated and isolated ¯owers. Data were
tested for randomness and homogeneity (Levene) and
analysed by two-way analysis of variance (ANOVA), with
Bonferroni post-hoc analysis for line, column and pair
comparison using the package SYSTAT (v. 7.0 for
Windows, SPSS Co.).
Histochemical staining was performed on fresh, un®xed
material, using longitudinally sliced styles collected from
¯owers before anthesis, at anthesis and at receptivity. Total
lipids were assessed using Sudan Black B (Pearse, 1980).
The sample was immersed in a saturated solution of 0.3 %
Sudan Black in 70 % ethanol for 15 min, followed by rapid
wash in 70 % ethanol and water. Polysaccharides were
detected using the PAS assay as described in McManus
(1948). Incubation for 30 min with fresh 1 % tetrahydrate
sodium borate was followed by 10 min in 1 % periodic acid,
a rapid wash in distilled water, immersion in Schi€ reagent
for 30 min and a ®nal wash for 3 2 min in 0.5 % sodium
metabisul®te. Starch was visualized by Lugol (IKI) staining
for 15 min. Protein content was assessed with the Ninhydrin-Schi€ reaction (Yasuma and Ichikawa, 1953). Sliced
styles were incubated overnight at 37 8C in a 0.5 %
ninhydrin solution prepared in ethanol, washed for 5 min
in distilled water and reacted with Schi€ reagent for 30 min
at room temperature. Styles were visualized with a stereo
microscope (Leica IM4) and photographed with Kodak
Elite II 400 ASA ®lm.
Pollen tube growth
Ovule development
Three stages of pistil development were tested: (1) ¯owers
previously emasculated and bagged at dehiscence/anthesis
were allowed to reach receptivity; (2) ¯owers at dehiscence/
anthesis (operculum about to fall and emasculated); or (3)
¯owers prior to dehiscence/anthesis (operculum was
removed and ¯owers emasculated). In all treatments, styles
were transversely cut approximately half-way down their
length, generating a drop of exudate. Additionally to the
Using the same ¯ower developmental stages, capsules
were ®xed to follow ovule development using light
microscopy (LM) and scanning electron microscopy
(SEM). The material was ®xed in 2.5 % glutaraldehyde in
0.05 M sodium cacodylate bu€er, pH 7.2 for 3 d at 4 8C,
dehydrated on a graded ethanol series, embedded in a
graded mixture of ethanol : LR-White resin and polymerized
for 24 h at 60 8C. For SEM, using the same ®xation
Stigma and style characterization
Trindade et al.ÐSeed-set after Pollination on Immature Flowers in Eucalyptus globulus
procedure used for LM, the material was dehydrated in a
graded acetone series, critical-point dried in liquid CO2 ,
mounted on aluminium stubs and sputter coated with gold.
Samples were viewed with a Jeol JSM T-220 SEM. Semithin sections of about 2±5 mm were made with a LKBUltrotome ultramicrotome. Finally, fertilized capsules
previously ®xed in the same ®xing solution were frozen by
rapid immersion in liquid nitrogen and stored at ÿ180 8C.
Sections (20±30 mm thick) were made with a Leica cryomicrotome, dehydrated in a graded ethanol series, stained
with toluidine blue and examined using transmitted light
microscopy.
Seed set
Pollinations were carried out as described above in March
1999. After 6 months, capsules were collected and viability
rate and seed production were scored per capsule. Seed
germination tests were carried out in vitro in semi-solid
medium containing 3 % sucrose. Germination was evaluated on day 10.
R E S U LT S
Cut styles of non-dehisced ¯owers support better pollen
germination and tube growth
Receptive stigmas of intact styles (control) are shown in
Figs 1 and 2. Most pollen grains had adhered and hydrated
8h after pollination. Pollen tube growth was clearly
detectable 24 h after pollination (Fig. 1). After 48 h, pollen
tubes reached 0.5±1 mm into the style, and 7 d after
pollination a few pollen tubes had reached the base of the
style (Fig. 2). Observations of capsules after pollination on
sliced styles at peak stigmatic receptivity are shown in
Figs 3 and 4. They show a similar pattern of pollen tube
growth, with germination starting on day 1 and pollen
tubes reaching the base of the style after 7 d (Fig. 4). These
observations provide direct evidence that pollen grains are
able to adhere to, hydrate and ®nd their way through the
transmitting tissue in the drop of secretion that emerges
from a newly-wounded surface, immediately after cutting.
However, hydration and germination may take longer, since
pollen tubes reach the ovary at the same time as in non-cut
receptive styles but only have to travel about half the
distance. The most relevant issue we wanted to address was
the possibility that the same treatment could be equally
successful in cut styles of clearly closed ( pre-dehiscence/
anthesis) capsules. Wounding the unreceptive stigma/style
is also sucient to promote pollen grain adhesion,
hydration and germination (Fig. 5) and successful seed
set. In these ¯owers, pollen tube growth was faster than in
mature styles with wounded or intact styles, and at day 7
tubes formed organized bundles at the base of the style
(Fig. 6). Moreover, the number of pollen tubes reaching the
ovary was substantially larger and, using the SEM, pollen
tubes were seen entering the ovule loci as well as in the
micropyle of some ovules at day 7 (data not shown).
471
Histochemical characterization of stylar exudates shows an
important role for lipids
Histochemical characterization of the style tissue before
and during anthesis, and at peak stigmatic receptivity is
presented in Table 2. As shown, lipids are the main
component of the stigmatic exudate and the only component that showed a signi®cant variation during pistil
maturation. Figures 7±9 show the sequence of its accumulation as the pistil matures. Lipids are already present in
immature styles of ¯owers before dehiscence, accumulating
®rst in the upper part which will develop into the stigma
(Fig. 7). From there, they are secreted along the transmitting
tissue cells, even before dehiscence (Fig. 8). While these
types of images from in toto preparations do not give the
best spatial accuracy, the patchy and irregular structure of
the Sudan reaction provides an indication that lipids may
already have accumulated extracellularly along the transmitting tissue walls in this stage (Fig. 8). They are then gradually accumulated as the ¯ower matures, and eventually when
the pistil becomes receptive they are secreted to the stigma
surface where they accumulate within a sticky drop of
exudate (Fig. 9). The other components investigated ( proteins and polysaccharides) showed a much more irregular
and weak reaction. Proteins, detected by the NinhydrinSchi€ reaction, were weakly present along the transmitting
tissue, and showed no major variations in the stigma before
or after dehiscence (Figs 10 and 11). Polysaccharides,
detected by the PAS reaction, were weakly present on the
stigma cells and did not show major variations in the
transmitting tissue of styles sectioned before or after dehiscence (Figs 12 and 13). Neither protein nor polysaccharide
patterns could be correlated with the ability to set seed and
thus may not be essential for successful pollen-pistil
interactions to occur. This was con®rmed by the fact that
in stages in which lipids were not observable, pollen tubes
could not grow into the style. Therefore, the presence of
lipids along the extracellular spaces of the transmitting
tissue seems to be a diagnostic character for the stage of
maturity of the style required for OSP to be successful.
Ovules are functionally mature before ¯ower dehiscence and
produce viable seeds
Ovule structure and morphology were compared between
the selected ¯ower developmental stages (immature and
mature capsules). Observations performed with optical and
scanning electron microscopes (Figs 14 and 15) show that
most fertile ovules in a mature ovary seem to be fully
receptive with a developed inner and outer integument and a
complete embryo sac. However, in immature capsules,
ovules are smaller and there was some heterogeneity in
ovular development within the same ovary. Full maturation
after OSP takes about 5±6 d, but in this case this does not
seem to constitute a functional barrier for pollen tubes to
enter the ovules when these become receptive to fertilization.
Con®rmation for this ®nding came from seed-setting
results. Pollination performed in selected trees using ¯owers
at and prior to dehiscence/anthesis allowed viable seed
production, with good germination rates (Table 3). All traits
472
Trindade et al.ÐSeed-set after Pollination on Immature Flowers in Eucalyptus globulus
T A B L E 2. Histochemical characterization of the style and stigmatic exudate formed during pistil maturation
Style and exudate
Polysaccharides
Starch
Proteins
Lipids
Pre-dehiscence
Dehiscence
At receptivity (*)
‡/ÿ
‡/ÿ
‡/ÿ
‡/ÿ
‡/ÿ
‡
‡/ÿ
‡/ÿ
‡/ÿ
‡‡
‡‡
‡‡‡
* 4/7 d after dehiscence/anthesis.
Results are shown on an arbitrary qualitative scale of detection, de®ned as absence (ÿ), variable (‡/ÿ), presence (‡) and accumulation (‡‡/
‡‡‡). See Figs 7±9 for detailed images of lipid characterization.
T A B L E 3. Pollination success, seed-set and germination following pollination of E. globulus ¯owers at di€erent stages of
maturity
MN14
MN22
MN15
Average
Pollination success ( % capsules)
Receptive ¯ower
At dehiscence
Before dehiscence
17.1 + 2.2a
67 + 0.0d
16.6 + 6.8d,e
12.6 + 3.1b
44.4 + 10.5d
8.3 + 3.4e
12.7 + 2.1b
22.2 + 5.2
50 + 10.2
14.1 + 2.5c (n ˆ 164)
44.6 + 5.2 (n ˆ 27)
25 + 6.8c (n ˆ 36)
Seeds/capsule
Receptive ¯ower
At dehiscence
Before dehiscence
5.1 + 1.0
22.5 + 3.9g
25 + 1.0g
19.0 + 2.9f
8.8 + 0.4
28 + 1.0h
20.8 + 4.0f
20.0 + 5.7f
33 + 7.6h
15.0 + 2.6i (n ˆ 35)
17.1 + 3.3i (n ˆ 18)
28.7 + 3.2 (n ˆ 18)
Seed germination rate
Receptive ¯ower
At dehiscence
Before dehiscence
48.6 + 5.6j
67.3 + 11.5
84.0 + 5.5m
53.1 + 4.4j
48.8 + 0.1
71.4 + 4.3
77.2 + 5.3k
75.6 + 3.1k
84.1 + 0.4m
59.6 + 5.1l (n ˆ 29)
63.9 + 4.9l (n ˆ 12)
80.0 + 3.4 (n ˆ 12)
Pollination success is the ratio of the number of collected vs. pollinated capsules; seed/capsule is the average number of seeds collected per
capsule; seed germination rate is the number of seeds that germinated (compared with the total number plated) showing distinctive cotyledons and
root growth. Data are means + s.e. The three categories were analysed by two-way analysis of variance (ANOVA), and searched by indexed
column, line or, if needed, pair correlation by the Bonferroni test for post-hoc analysis. Values with the same superscripts did not di€er
signi®cantly (P 4 0.05).
assessed (crossing success, number of seeds per capsule, and
germination rate) were generally better for ¯owers pollinated at or prior to dehiscence. Particularly signi®cant was
the trait seed number per capsule: the number of seeds in
capsules pollinated at ¯ower dehiscence/anthesis (17.6) or
receptive ¯owers (14.9) was clearly surpassed by the number
produced following pollination before dehiscence (28.7)
(P 5 0.0001; Table 3). This is also consistent with the
previous observation that in ¯owers before dehiscence, a
higher number of pollen tubes reached the ovules. Also very
signi®cant was the improved viability of the seeds, rising
from 60 and 64 % in the receptive and dehisced fertilizations
to 80 % in the pre-dehisced capsules (P 5 0.001; Table 3).
DISCUSSION
One-step pollination on immature pre-dehiscence ¯owers
improves seed production and germination
Although one-step pollination (OSP) has already been
described for E. globulus, to the best of our knowledge
this is the ®rst report in which capsules were successfully
fertilized before dehiscence/anthesis, resulting in increased
pollen tube growth, seed-set and viability rates. Underpinning this result was the monitoring of the cytological
steps during the progamic phase, for which this paper
constitutes the ®rst account in E. globulus. Furthermore, a
putative functional relationship between the success of
pollination and the presence of lipids in the transmitting
tissue is proposed, allowing us to establish the earliest
developmental stage at which OSP procedures can be
applied. With regard to controlled pollination techniques
in E. globulus, our work represents an obvious advantage
not only in reducing the cost per ¯ower but also in increasing
the number and quality of seeds produced per cross.
The practice of style slicing/severing in ¯owers at anthesis
in Eucalyptus (anthesis is often used synonymously with
dehiscence in the Eucalyptus literature since both occur
simultaneously) was ®rst reported by Potts and Cauvin
(1988; also Cauvin, 1988). This treatment was used to overcome inter-speci®c barriers and it was successful for hybrid
seed production. Extension of the concept to E. globulus was
later reported by Harbard et al. (1999). While these authors
gave attention to capsule production, they did not present
data on the cytological success of the crosses tested.
Therefore, while it was shown that OSP was an improvement
compared to controls in receptive ¯owers, pre-dehiscence
crosses were concluded to be less e€ective. This conclusion
is, however, somewhat ¯awed by the fact that these authors
focused their analyses on the number of capsules formed,
overlooking the number of seeds per capsule. Thus, while
their success in terms of numbers of capsules formed is
Trindade et al.ÐSeed-set after Pollination on Immature Flowers in Eucalyptus globulus
F I G S 1±15. Figure captions over page.
473
474
Trindade et al.ÐSeed-set after Pollination on Immature Flowers in Eucalyptus globulus
comparable to ours, an obvious net gain is obtained when
the number of seeds per capsule is taken into account (see
Table 3). This result becomes even more important when
seed germination rates are taken into account.
Immature pre-dehisced styles support better pollen tube
growth
We ®rst became aware of the above conclusion when few
pollen tubes were seen entering the ovary of receptive
pollinated and intact styles, given the number of pollen
grains observed in the upper part of the style. This dramatic
reduction suggests that control mechanisms in the pistil
select which pollen tubes develop (Cruzan, 1986; Herrero,
1992; Herrero and Hormanza, 1996). The reduction in the
number of pollen tubes progressing down the style has also
been noted in E. woodwardii (Sedgley and Smith, 1989). In
contrast, after pollination of pre-dehisced sliced styles, a
large number of pollen tubes followed the transmitting
tissue path and entered the ovary and ovular loci in pollen
tube bundles; this was not usually seen after pollination of
intact receptive ¯owers or cut styles after dehiscence. This
may be explained by di€erent conditions that operate in the
pistil of ¯owers at di€erent developmental stages, since a
variety of mechanisms may act to restrict pollen tube
growth. In an immature style the molecular mechanisms by
which the pistil acts as a selective barrier to fertilization are
not completely operational before ¯ower receptivity
(De Nettancourt, 1997). The morphological di€erences
between young and mature transmitting tissue cells were,
in this case, observable in the accumulation of secretion, but
this feature accounts only for the sustainability of growth.
Other molecules which act as selective barriers to pollen
tube growth are among the last components formed during
maturation. Therefore, in immature buds, the transmitting
tissue cells would be expected to contain a range of inactive
precursors (Herrero and Dickinson, 1980). In this respect,
we propose a mechanistic description of the reported success
of immature pollinations based on two sequential factors:
(1) cutting the upper part of the style provides enough
secretion on the cut surface, in terms of lipids, for the pollen
to adhere and germinate; and (2) absence of selective
mechanisms of pollen tube development, which evolve
rapidly between dehiscence and style receptivity, allows
more tubes to reach the ovary. Successful fertilization is
possible since our observations of pollen development, ovule
morphology and seed set strongly suggest that the female
part is functional several days before dehiscence.
Lipids in the transmitting tissue are diagnostic features of
style competence
The presence of lipids in stigmatic exudates has long been
reported (Konar and Linskens, 1966), but the issue gained
F I G . 1. Fluorescence micrograph of an aniline blue stained receptive intact style showing pollen tube growth 1 d after pollination. Bar ˆ 60 mm.
F I G . 2. 7 d after pollination, pollen tubes have grown to the bottom of intact receptive styles. Bar ˆ 25 mm.
F I G . 3. Fluorescence micrograph of the upper portion of a receptive sliced style (ST) showing pollen tube penetration and deposition of callose
plugs, 1 d after pollination. Bar ˆ 50 mm.
F I G . 4. Pollen tube progression in the bottom of receptive cut styles, 7 d after pollination. Bar ˆ 70 mm.
F I G . 5. Fluorescence micrograph of cut styles obtained from ¯owers before dehiscence (operculum fall), 1 d after pollination. Many pollen tubes
are seen and callose plugs are also visible. Bar ˆ 100 mm.
F I G . 6. Pollen tube growth in the lower part of the style in pollinated immature styles 7 d after pollination. Bar ˆ 50 mm.
F I G . 7. Lipid staining by Sudan Black in a section of a style obtained from very immature ¯owers. Although lipids are already visible in some
places, this style is still unable to promote good pollen tube growth. Bar ˆ 500 mm.
F I G . 8. Sudan Black staining in styles a few days before ¯ower dehiscence. Lipid accumulation along the pistil, notably in the extracellular space,
is already very apparent. This kind of style is able to promote pollen tube growth if tubes are allowed to adhere directly to the presumably lipidrich component of this extracellular secretion, either by slicing or wounding the style. Bar ˆ 500 mm. TT, Transmitting tissue.
F I G . 9. Receptive styles showing dark colouration with Sudan staining, revealing much lipid accumulation, conspicuously in the stigma exudate.
Bar ˆ 500 mm.
F I G . 10. Protein staining using the Ninhydrin-Schi€ reaction in a style before dehiscence (stage equivalent to Fig. 7). A weak reaction is detected
in the stigma layer (arrow) but no signi®cant reaction distinguishes the transmitting tissue. Bar ˆ 500 mm.
F I G . 11. Receptive style. The pattern of protein staining is similar and restricted to the stigma (arrow), although the inner structure of the style is
clearly developed. Bar ˆ 500 mm.
F I G . 12. Polysaccharide, as detected by the PAS reaction, in a sectioned style prior to dehiscence (stage equivalent to Fig. 7). A weak reaction
occurs on the transmitting (arrow) and the continuing tracts into the ovary. Bar ˆ 500 mm.
F I G . 13. Receptive style. Again there is no signi®cant modi®cation on the staining pattern, and the intra-ovarian tracts show the same pattern of
staining. Bar ˆ 500 mm.
F I G . 14. Light microscopy image of semi-thin cross-sections of ovules (OV) on pre-dehisced capsules. Ovules are almost fully developed, showing
the complete formation of both integument layers. Bar ˆ 300 mm.
F I G . 15. Scanning electron microscope image of ovules of pre-dehisced capsules. Ovules (OV) show the ®nal campilotropic con®guration, with
well de®ned funiculus (arrow). Although extracted during the preparation procedures, the embryo sac (ES) is almost full size. Bar ˆ 300 mm.
Trindade et al.ÐSeed-set after Pollination on Immature Flowers in Eucalyptus globulus
functional relevance with the recent ®nding that lipids may
have an important role in de®ning water availability
interfaces and consequent guidance (Wolters-Arts et al.,
1998). In E. globulus, we detected them extracellularly along
the transmitting tissue and in the stigmatic secretion,
generally with the same intensity and pattern, even after
style cutting. This is in accordance with the observations of
Wang et al. (1996), who found chemical similarities between
the exudate formed when the style is cut near the tip and the
receptive stigma exudate. Unfortunately, the chromophore
used, Sudan Black, is not speci®c enough to provide data
other than the presence of total lipids and lipophilic
substances. However, correlation between histochemical
detection of lipids and style competence for tube growth
seems to indicate an important role for lipids in the
progamic phase. Otherwise lipids may constitute a marker
of development that correlates with pistilar functionality.
AC K N OW L E D GE M E N T S
J.A.F. acknowledges partial ®nancial support from
FundacËaÄo CieÃncia e Tecnologia (FCT-PRAXIS 3/3.2/
2100/FLOR/96) for laboratory support. L.C.B. acknowledges a research fellowship (FCT-PRAXIS, GGP/XXI/
BIC/3746/96).
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