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Botanical Journal of the Linnean Society, 2015, 177, 202–213. With 4 figures
The embryo sac of Vanilla imperialis (Orchidaceae) is
six-nucleate, and double fertilization and formation of
endosperm are not observed
NETE KODAHL1*, BO B. JOHANSEN2 and FINN N. RASMUSSEN3
1
Department of Plant and Environmental Sciences, University of Copenhagen, Højbakkegård Allé 13,
DK-2630 Taastrup, Denmark
2
Core Facility for Integrated Microscopy, Department of Biomedical Sciences, University of
Copenhagen, Blegdamsvej 3, DK-2200 København K, Denmark
3
Natural History Museum of Denmark, University of Copenhagen, Gothersgade 130, DK-1123
København K, Denmark
Received 20 July 2014; revised 10 October 2014; accepted for publication 19 October 2014
Double fertilization and subsequent endosperm formation are two of the most important synapomorphies of the
angiosperms. Endosperm is generally lacking in Orchidaceae, but Swamy reported the formation of up to ten
endosperm nuclei in Vanilla planifolia in 1947. The observation was documented only by line drawings and has
not been confirmed; however, assumptions about endosperm formation occurring in Orchidaceae are primarily
founded on Swamy’s study. The current study provides the first detailed description of embryo sac formation and
early embryogeny in Vanilla since Swamy, and is the first using modern imaging techniques. Flowers of Vanilla
imperialis were artificially pollinated at 1-week intervals and the resulting fruits were fixed, embedded and
sectioned for light microscopy of embryo sac formation, fertilization and early development of the embryo.
Three-dimensional reconstructions of embryo sacs were obtained using confocal laser scanning microscopy on fixed
ovules. The mature embryo sac contains only six nuclei as a result of the arrested development of the chalazal
nuclei prior to the formation of three antipodals and a polar nucleus. Fertilization was observed 7 weeks after
pollination, but double fertilization and the formation of endosperm did not occur. © 2014 The Linnean Society
of London, Botanical Journal of the Linnean Society, 2015, 177, 202–213.
ADDITIONAL KEYWORDS: antipodal nuclei – B. G. L. Swamy – chalazal nuclei – embryology – endocarpic
trichomes – striking phenomenon – triple fusion – Vanilla fruit.
INTRODUCTION
In the probably largest of all angiosperm families,
Orchidaceae (c. 25 000 species; Dressler, 2005), double
fertilization and endosperm formation are generally
considered to have been lost (Nawaschin, 1900;
Rasmussen, 1995; Clements, 1999). The loss of
endosperm may be connected to the invasion of habitats which are disturbed, patchy and stressed, circumstances which make small seeds particularly
adaptive. Major shifts in juvenile nutrition, e.g. mycorrhizal associations, obviated the need for the
*Corresponding author. E-mail: [email protected]
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production of endosperm in Orchidaceae (Benzing,
1981), but double fertilization and the formation of
a rudimentary endosperm have been reported in a
few genera of orchids (e.g. Pace, 1907; Afzelius, 1916;
Swamy, 1947; Poddubnaya-Arnoldi, 1967; Yasugi,
1983; Vinogradova & Andronova, 2002). One of the
most frequently cited observations of endosperm formation in Orchidaceae was performed on material
of Vanilla planifolia Andrews (Swamy, 1947). The
current study addresses embryo sac formation and
fertilization in the African species Vanilla imperialis
Kraenzl., utilizing light microscopy and confocal laser
scanning microscopy (CLSM) to produce colour photographs and three-dimensional reconstructions of
entire embryo sacs as documentation.
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177, 202–213
EMBRYO SAC OF VANILLA IMPERIALIS
The phylogenetic position of the vanilloid orchids
has been much disputed and ranges from being
embedded in the lower Epidendroideae to a position
as sister group to all other monandrous orchids, corroborated by molecular data and accepted in Genera
Orchidacearum (Dressler, 1993; Cameron et al., 1999;
Freudenstein & Rasmussen, 1999; Rasmussen, 1999;
Cameron, 2003; Chase et al., 2003). Endosperm formation and the unusual seeds with a sclerotic testa
have been regarded as ‘primitive’ traits in Vanilla
Mill. and related genera (Dressler & Dodson, 1960).
EMBRYOLOGY
Microsporogenesis in Orchidaceae is similar to that of
other angiosperms (Johri, 1984; Johri, Ambegaokor &
Srivastava, 1992; McCormick, 1993), but megasporogenesis generally takes place only after pollination of
the flower (Yeung & Law, 1997). Division of the megasporocyte usually results in the formation of a linear
tetrad of megaspores; however, this row is occasionally
reduced to three by omission of division of the micropylar dyad cell after the first meiosis (e.g.
Maheshwari, 1937; Swamy, 1947; Yeung & Law, 1997).
Normal or ‘Polygonum type’ development ensues by
three mitotic divisions of the chalazal megaspore,
resulting in the eight-nucleate embryo sac of most
angiosperms. The Polygonum type embryo sac is considered by many authors to be the ancestral state in
monocotyledons (Maheshwari, 1948; Davis, 1966;
Dahlgren & Clifford, 1982; Johri, 1984; Haig, 1990;
Johri et al., 1992) and has been reported in most
species studied in Orchidaceae. Accordingly, Sharp
(1912) and Clements (1999) stated that an eightnucleate embryo sac is present in most species in
Orchidaceae. However, several observations have
been made of the chalazal nuclei degenerating prior to
their second division, resulting in a six-nucleate
embryo sac containing two chalazal nuclei, an egg cell,
two synergids and a micropylar polar nucleus (e.g.
Pace, 1907; Sharp, 1912; Afzelius, 1916; Prosina,
1930; Swamy, 1945, 1947, 1949; Law & Yeung, 1989).
Yeung & Law (1997) speculated that six-nucleate
embryo sacs may be more common than eight-nucleate
embryo sacs in Orchidaceae. If this is the case, it may
support the theory put forward by Sharp (1912) that
the complexity of embryology in Orchidaceae is
reduced compared with that of other angiosperms.
Nawaschin (1900) was the first to note the lack of
fusion between the polar nuclei and sperm cell, and
hypothesized that this was a general character of the
family. Nevertheless, a number of authors have overlooked his work and have stated that double fertilization occurs ‘as is normal’ in Orchidaceae, but
without presenting any documentation (e.g. Pace,
1909; Sharp, 1912; Swamy, 1947; Fredrikson, 1990).
203
Even though the formation of endosperm is not
common in Orchidaceae, there are a few well-known
and widely cited observations of double fertilization
leading to the formation of a rudimentary endosperm,
especially Swamy (1947). However, interpretations of
embryological structures can be controversial (Yam
et al., 2002), and most observations of endosperm
have been made without the use of modern imaging
techniques. The documentation is mostly line drawings and there is no indisputable evidence of the
nuclei arising as a consequence of fusion between the
polar nuclei and the second sperm cell. Most often
the product of the alleged second fertilization event
does not proliferate, and comments resembling that of
Sharp (1912), ‘the endosperm nucleus disorganizes
without dividing’, are common (e.g. Huang et al.,
1998). Information about endosperm formation in
orchids is summarized by Veyret (1974), Clements
(1999), Vinogradova & Andronova (2002) and Yam
et al. (2002). Yam et al. (2002) present an exhaustive
table of endosperm observations in Orchidaceae,
including negative observations.
B. G. L. SWAMY
AND
VANILLA
PLANIFOLIA
Swamy (1947) reported the formation of up to ten
endosperm nuclei in V. planifolia. The observation
was documented by meticulous line drawings and a
large number of ovules were sectioned for examination. Swamy observed that only one polar nucleus was
formed in V. planifolia, and that this nucleus fused
with one of the sperm nuclei, resulting in a diploid
primary endosperm nucleus. This nucleus divided
and the resulting nuclei moved to the micropylar and
chalazal ends of the embryo sac, respectively. According to Swamy, the micropylar nucleus then divided to
form two or four nuclei and the chalazal nucleus
divided to form seven or eight nuclei, resulting in
8–12 endosperm nuclei. In a few cases, endosperm
formation was arrested after the first or second division. He also observed that ‘as in other orchids the
endosperm is soon used up by the growing embryo’.
This statement may seem ill-founded as the formation
of an endosperm is not common in Orchidaceae;
however, the fact that the endosperm only persists for
a short time could be part of a possible explanation of
why it has only been found in a limited number of
orchids.
MATERIAL AND METHODS
The orchid used for this study is V. imperialis, collected
by C. L. Leakey in Uganda and cultivated as P1977504 in the Botanical Garden, University of Copenhagen, Denmark, voucher at C. Flowers were artificially
pollinated between 30 May and 5 September 2008,
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177, 202–213
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N. KODAHL ET AL.
and fruits of ages 0, 1, 2, 4, 5, 6, 7, 8, 9, 10, 11 and
12 weeks after pollination were collected on
21 August and 5 September 2008. From each fruit,
1-cm pieces were cut 1 cm from the apex, and each
piece was divided into three by cutting between the
placentae.
The resulting pieces were fixed in a solution of 2%
paraformaldehyde (PFA) and 2.5% glutaraldehyde
(GA) in 0.2 mol L−1 phosphate buffer, pH 7.0, rinsed,
dehydrated through a methyl cellosolve series and
embedded in glycol methacrylate (GMA) according to
standard methods (O’Brien & McCully, 1981). The
ovaries were sectioned in 3-μm sections on an LKB
2218 Historange microtome (LKB-Produkter AB,
Bromma, Sweden) and attached to microscope slides
on drops of distilled water. The sections were stained
with periodic acid-Schiff, preceded by overnight incubation in dimedone, and Aniline Blue Black 1% in 7%
acetic acid (PAS/ABB) or with Toluidine Blue (TB),
pH 4.4 (O’Brien & McCully, 1981). Ovules for CLSM
were fixed in 2% PFA in phosphate-buffered saline
(PBS), pH 7.4, overnight, rinsed in 0.05% Triton
X-100 in PBS and stained in 1 μM propidium iodide
(PI) and 0.01% Calcofluor White M2R (CFW, Fluorescent Brightener 28; Sigma-Aldrich, St. Louis, MO,
USA) in PBS, pH 7.4, for 1 h before mounting in
ProLong® Gold (Invitrogen, Grand Island, NY, USA).
All wide-field microscope images were captured using
a Reichert-Jung Polyvar compound microscope and an
Evolution LC digital camera (Media Cybernetics, Inc.,
Bethesda, MD, USA). Image Pro Plus™ v. 4.5 (Media
Cybernetics, Inc.) was used as host program for the
images. CLSM images were captured on a Zeiss LSM
700 microscope using 405-, 488- and 555-nm lasers.
The 405-nm laser excites CFW, the 488-nm laser
excites autofluorescence from lignin, cutin, polyphenolics and other compounds, and the 555-nm laser
excites PI.
RESULTS
The observations of the current study are compared
with those of Swamy (1947) throughout this section in
order to determine the differences or similarities.
Swamy’s drawings have been scaled to match the
photographs of V. imperialis. The magnifications
given in Swamy (1947) seem erratic; if they were
correct, the structures shown would be more than five
times larger than expected. For example, the mature
seeds depicted in fig. 39 (Swamy, 1947) would be
1.6 mm long, but seeds of V. planifolia are usually
about 0.25 × 0.3 mm2 (N. Kodahl, B. B. Johansen & F.
N. Rasmussen, pers. observ.). The most plausible
explanation for these errors is that the magnification
refers to Swamy’s original camera lucida drawings,
which were reduced for printing.
Preceding the pollination, no differentiation of the
ovules from the placenta has taken place (Fig. 1A).
One to two weeks after pollination, differentiation is
evident and the placental ridges have increased
slightly in size (Fig. 1B, C).
Two weeks after pollination, the first megasporocytes are visible and, during weeks three and four, the
megasporocytes develop fully and the two-layered
inner integument differentiates simultaneously
(Fig. 1D–F). The outer integument is in its early stage
at this point (Fig. 1E, F). The ovule is tenuinucellate
with a one-layered nucellus surrounding the megasporocyte.
The first meiotic divisions of the megasporocytes are
observable 5 weeks after pollination (Figs 2A–C, 4A).
The first division is normal, but only the chalazal dyad
cell undergoes the second meiotic division, resulting in
a row of three megaspores. At the time of the meiotic
divisions, the nucellus surrounding the distal part of
the embryo sac is degenerating and, in accordance with
Swamy’s observations, the hypostase is well developed
(Fig. 2A–C). The outer integument is still under development, but it is now apparent that it is at least three
or four cell layers thick (Fig. 2A), which contrasts with
the one- or two-layered outer integuments observed in
other orchids (Wirth & Withner, 1959).
Two of the three megaspores created by the meiotic
divisions degenerate, whereas the chalazal cell
becomes the functional megaspore (Fig. 2B). The
megaspore divides mitotically (Fig. 4B) and the
resulting two nuclei migrate to the micropylar and
chalazal ends of the cell, respectively. The two nuclei
divide mitotically again, resulting in a four-nucleate
embryo sac (as seen in Fig. 2D).
▶
Figure 1. Sections from developing fruits of Vanilla imperialis at 0–4 weeks after pollination (wk.a.p.), PAS/ABB
(periodic acid-Schiff / Aniline Blue Black) staining. A, Placentae from fruit of Vanilla imperialis at 0 wk.a.p. No
differentiation of the ovules from the placental ridges has taken place. B, Developing ovule from fruit of Vanilla imperialis
at 2 wk.a.p. C, Vanilla planifolia (Swamy BGL. 1947. Botanical Gazette 108, 451, courtesy of Chicago University Press).
‘Nucellar papilla before differentiation of archesporial cell’. D, Placentae from fruit of Vanilla imperialis at 4 wk.a.p. Note
the pollen tubes. E, Vanilla planifolia (Swamy BGL. 1947. Botanical Gazette 108, 451, courtesy of Chicago University
Press). ‘Archesporial cell’ and ‘Megaspore mother cell and origin of integuments’. F, Megasporocyte and developing
integuments in fruit of Vanilla imperialis at 4 wk.a.p. II, inner integument; MSC, megasporocyte; P, placenta; PT, pollen
tubes.
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177, 202–213
EMBRYO SAC OF VANILLA IMPERIALIS
Figure 1. See caption on previous page.
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N. KODAHL ET AL.
Figure 2. See caption on next page.
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EMBRYO SAC OF VANILLA IMPERIALIS
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Figure 2. Sections from developing fruits at 5–7 weeks after pollination (wk.a.p.). A, Ovule with two megaspores in
Vanilla imperialis at 5 wk.a.p., PAS/ABB (periodic acid-Schiff / Aniline Blue Black) staining. B, Ovule with two
degenerating megaspores and a functional megaspore in Vanilla imperialis at 5 wk.a.p., PAS/ABB staining. C, Vanilla
planifolia (Swamy BGL. 1947. Botanical Gazette 108, 451, courtesy of Chicago University Press). ‘Row of three cells; two
megaspores and a micropylar dyad cell’. D, Vanilla planifolia (Swamy BGL. 1947. Botanical Gazette 108, 451, courtesy
of Chicago University Press). ‘Four-nucleate embryo sac’. E, Six-nucleate embryo sac in Vanilla imperialis at 7 wk.a.p.
Five of the six nuclei are visible; the egg cell is situated outside of the section. Toluidine Blue staining. F, Six-nucleate
embryo sac in Vanilla imperialis at 7 wk.a.p.; the synergids, polar nucleus and egg cell are visible. Toluidine Blue
staining. G, Another section of the embryo sac seen in (F); the chalazal nuclei are visible. Toluidine Blue staining. H,
Vanilla planifolia (Swamy BGL. 1947. Botanical Gazette 108, 451, courtesy of Chicago University Press). ‘Mature,
six-nucleate embryo sac’. CN, chalazal nuclei; DMS, degenerating megaspores; EC, egg cell; H, hypostase; II, inner
integument; MS, megaspore; N, nucellus; OI, outer integument; PN, polar nucleus; S, synergid.
◀
The two chalazal nuclei do not undergo any further
divisions, but slowly degenerate during the next
4–5 weeks (Fig. 2E, G). The two micropylar nuclei
divide mitotically again, one producing the two synergids and the other giving rise to the egg cell and the
micopylar polar nucleus (Figs 2E, F, H, 3A, B). The
lack of a second division of the chalazal nuclei means
that the mature embryo sac contains no more than six
nuclei, and that a chalazal polar nucleus does not
form (Figs 2E–H, 4C). At this stage, a cell wall is
present around each nucleus in the embryo sac,
meaning that a chalazal polar nucleus capable of
fusing with the micropylar polar nucleus cannot form.
Swamy observed that the nucellus in the distal end
of the embryo sac of V. planifolia had degenerated
completely by the time of the division of the megasporocyte. In V. imperialis, however, it persists until
5–6 weeks after pollination, corresponding to the division of the megaspore.
Seven weeks after pollination, pollen tubes enter
the embryo sac through the micropyle (Fig. 3A). One
of the synergids is degenerating (Fig. 3A). The chalazal nuclei are still visible, as is the egg cell and both
synergids (Fig. 2E–G). The egg cell is well developed,
which indicates that a sperm cell has fused with the
egg cell nucleus (Fig. 3A). According to Swamy, the
other sperm cell fuses with the polar nucleus
(Fig. 3B), but no such observation could be made in
the material at hand. Swamy reported divisions of the
fusion product between the second sperm cell and the
chalazal polar nucleus, but this was not observed in
any of the sections or three-dimensional reconstructions of embryo sacs of V. imperialis.
Eight to nine weeks after pollination, the outer
integument fully envelops the inner integument, but
a large inter-integumental lumen separates them
(Fig. 3E). The condensed, degenerating chalazal
nuclei are still visible.
Ten weeks after pollination, unicellular papillae
cover the placental valves (Fig. 3C, D). There is a
clear separation between the part of the placenta
covered with papillae and the part of the placenta
covered with pollen tubes.
DISCUSSION
VANILLA
IMPERIALIS COMPARED WITH
V. PLANIFOLIA
Integuments and nucellus
In V. imperialis, the ovules only differentiated from
the placentae after pollination, as is also the case for
V. planifolia (Swamy, 1947). This developmental
pattern is uncommon for most angiosperms (Yeung &
Law, 1997), but is predominant in orchids (Tsai et al.,
2008) and may be an adaptation to a combination of
heavy seed set and rare pollination events.
The inner integument developed simultaneously
with the megasporocyte, roughly as in V. planifolia
(Swamy, 1947). However, the outer integument in
V. imperialis was visible prior to the meiotic divisions
of the megasporocyte, whereas, in V. planifolia, it
appeared simultaneously with the divisions. The
outer integument completely enveloped the inner
integument 8 weeks after pollination, which seems
earlier than in V. planifolia.
The outer integument is made up of three to four
layers of cells, or even five to six at the base, in both
Vanilla spp. In most orchids, the outer integument is
one- to two-layered (Swamy, 1947; Wirth & Withner,
1959). The multilayered outer integument may contribute to the very large seeds and could be connected
with a seed dispersal strategy different from the wind
dispersal of most orchids, e.g. endozoochory.
In V. imperialis, the nucellar epidermis did not
degenerate completely until the time of fertilization,
in contrast with V. planifolia, where it had already
been absorbed at the time of the meiotic divisions of
the megasporocyte. According to Lombardi et al.
(2007), lysis of the nucellus normally occurs immediately after fertilization to provide nutrients to the
growing embryo and endosperm. However, the
gradual degeneration observed for V. imperialis and
the differences in timing between V. imperialis and
V. planifolia seem to indicate that the nutrients available from lysis of the nucellus (Norstog, 1974;
Lombardi et al., 2007) may serve various purposes
between species or even within embryogenesis in a
single species.
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N. KODAHL ET AL.
Figure 3. Sections from developing fruits at 7–8 weeks after pollination (wk.a.p). A, Detail of micropylar end of embryo
sac with synergids, egg cell, polar nucleus and pollen tube in Vanilla imperialis at 7 wk.a.p., Toluidine Blue staining. B,
Vanilla planifolia (Swamy BGL. 1947. Botanical Gazette 108, 453, courtesy of Chicago University Press). ‘Double
fertilization’. C, Unicellular papillae cover the placental valves of Vanilla imperialis at 8 wk.a.p., PAS/ABB (periodic
acid-Schiff, preceded by overnight incubation in dimedone, and Aniline Blue Black 1% in 7% acetic acid) staining. D,
Detail of unicellular papillae in Vanilla imperialis at 6 wk.a.p., PAS/ABB staining. E, Embryo sac in Vanilla imperialis
at 8 wk.a.p. with a large inter-integumental lumen separating the inner and outer integuments, PAS/ABB staining. F,
Vanilla planifolia (Swamy BGL. 1947. Botanical Gazette 108, 453, courtesy of Chicago University Press). “ ‘Plate’ of eight
endosperm nuclei (drawn from embryo sac cut transversely)”. G, Embryo sac cut transversely through the tip of the inner
integument in Vanilla imperialis at 8 wk.a.p., PAS/ABB staining. EC, egg cell; PN, polar nucleus; PT, pollen tubes; S,
synergid; UP, unicellular papillae.
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EMBRYO SAC OF VANILLA IMPERIALIS
209
Figure 4. Maximum projections of confocal images of Vanilla imperialis embryo sacs at different developmental stages,
propidium iodide (PI) and Calcofluor White (CFW) staining. A, Projection of 15 images. First meiotic division of the
megasporocyte. A cellulosic wall is formed between the two haploid daughter cells. Notice that the wall between the
hypostase and developing embryo sac is already formed and the nucellus is still present. B, Projection of 13 images.
Two-nucleate embryo sac. The two degenerated megaspores are clearly visible. The nucellus is surrounded by a thin
autofluorescent cuticle. The wall between the hypostase and the embryo sac is well developed, and the nucellus has
degenerated. C, Projection of 29 images. Mature embryo sac showing the six nuclei present at the time of pollination. The
entire embryo sac is surrounded by a thin autofluorescent cuticle. CN, chalazal nuclei; DMS, degenerating megaspores;
EC, egg cell; H, hypostase; PN, polar nucleus; S, synergid.
Embryo sac
The megasporocyte of V. imperialis developed during
the first 3 weeks after pollination and, in the fourth to
fifth week, the meiotic divisions took place. As in
V. planifolia (Swamy, 1947), no more than three
megaspores were formed. This contrasts with the
general pattern in angiosperms (Maheshwari, 1950),
but is not uncommon in Orchidaceae (e.g. Hagerup,
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1945; Swamy, 1947; Law & Yeung, 1989). Two of the
three megaspores degenerated and the chalazal
megaspore became the functional megaspore, and
divided mitotically, as is normal in angiosperms.
No more than six nuclei were distinguishable in the
embryo sac at any time before fertilization. The deviation from the eight-nucleate embryo sac is caused by
the development of the chalazal nuclei being arrested
before a chalazal polar nucleus and three antipodal
nuclei are formed (‘the striking phenomenon’, see
Harling, 1950; Yeung & Law, 1997). This has been
observed a number of times in Orchidaceae (e.g.
Sharp, 1912; Prosina, 1930; Swamy, 1945, 1949; Law
& Yeung, 1989), including Vanilla spp. The embryo
sac of V. planifolia is six-nucleate (Swamy, 1947), but
Krupko, Israelstaem & Martinovic (1954) saw only
one six-nucleate embryo sac for every 15 eightnucleate ones in V. roscheri Rchb.f. According to
Yeung & Law (1997), there is a tendency for orchids
to have a reduced number of nuclei in the embryo sac.
Abe (1977) commented that a seven-celled, eightnucleate embryo sac is in fact uncommon in Orchidaceae. It is not possible to generalize about the
number of nuclei in the embryo sacs of orchids based
on the few reports available and, as stated by Yeung
& Law (1997), past findings of orchid embryo sac
contents may need to be re-evaluated as observations
based on paraffin or plastic sections alone may be
imprecise.
Fertilization
Fertilization was observed 7 weeks after pollination,
but no double fertilization or formation of endosperm
could be observed in V. imperialis, suggesting that
either the second sperm nucleus had not fused with
the polar nucleus or the endosperm nucleus degenerated immediately after fertilization (Veyret, 1974).
This contrasts with the observations of double fertilization and formation of up to ten endosperm nuclei
in V. planifolia (Swamy, 1947).
The endosperm formation observed by Swamy
(1947) occurred by the fusion of the micropylar polar
nucleus and the second sperm nucleus, resulting in a
‘diploid endosperm’. It is debatable whether the
fusion product between one polar nucleus and a
sperm cell should be considered as an endosperm at
all, or rather a second embryo. However, according to
Sargant (1900), endosperm evolved as a diploid tissue
originating from the production of a second embryo.
Friedman (e.g. 1994, 1995, 1998), who conducted
extensive studies of this hypothesis, argued that kin
selection theory (Hamilton, 1964a, b) may account for
embryo altruism and cooperation between products of
double fertilization, i.e. a supernumerary embryo may
have evolved into supportive tissue because of the
inclusive fitness gained from the sister embryo
(Queller, 1982; Friedman, 1995).
Clements (1999) doubted that double fertilization
actually occurs in Orchidaceae at all, stating that
‘evidence that fusion of the second sperm nucleus and
polar nuclei has taken place is often poorly supported
or non-existent, mostly based on interpretations of
cellular content of the embryo sac and illustrated by
line drawings.’ He pointed out that the possibilities
for misinterpretations are many, for example, the
presumed endosperm cells may be derivatives of
the polar nuclei themselves. In support of this,
Poddubnaya-Arnoldi (1960) observed that the chalazal nuclei of Cypripedium insigne Wall. ex Lindl.
[= Paphiopedilum insigne (Wall. ex Lindl.) Pfitzer]
may undergo additional divisions, and it is possible
that this also occurs in other species of orchids. Furthermore, polyembryony has regularly been observed
in Orchidaceae (Yam et al., 2002) and it is possible
that a second embryo could be misinterpreted as
endosperm. Some ovules of V. imperialis showed
similarity with the drawings of endosperm in
V. planifolia (Swamy, 1947) (see Fig. 3F, G), but
examination of neighbouring sections led to the interpretation that the ‘nuclei’, which could be perceived
as endosperm, were in fact cells of the inner integument cut transversely.
Yasugi (1983) reported double fertilization in
Doritis pulcherrima Lindl., but the accompanying
photograph depicts only two unnamed nuclei and no
observations of endosperm were made. According to
Vinogradova & Andronova (2002), double fertilization
occurs in several orchid species. However, the examples depicted are open to discussion. The embryo sac
in figs 4–6 of Vinogradova & Andronova (2002) is
sectioned diagonally and not all nuclei are visible.
The picture of the alleged triple fusion shows only a
few nuclei, and it can be disputed whether these
actually include a sperm nucleus and a polar nucleus.
The interpretation of cells in the embryo sac is
difficult unless all nuclei can be accounted for by
serial sections or three-dimensional reconstructions
and the cellular compartments of the embryo sac are
visualized using a method that differentially stains
both nuclei and cell walls (e.g. PAS/ABB or PI/CFW).
Evidently, once the embryo sac has become cellular,
further rearrangements of the nuclei cannot occur,
and hence no triple fusion and formation of
endosperm can take place.
Yam et al. (2002) list several observations of the
formation of endosperm or lack thereof in Orchidaceae, but they are difficult to assess. It is often not
stated if double fertilization is actually observed and
only about 40 references mention divisions of an
alleged primary endosperm nucleus. Considering the
limited number of observations, the quality of the
© 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177, 202–213
EMBRYO SAC OF VANILLA IMPERIALIS
images presented (if any) and the caveats mentioned
by Clements (1999), it seems impossible to draw any
solidly founded conclusion about the frequency and
distribution of double fertilization and endosperm formation in Orchidaceae.
211
observations of endosperm formation in Orchidaceae
should be critically re-examined and detailed photographic documentation should be provided.
ACKNOWLEDGEMENTS
Placental papillae/endocarpic trichomes
Unicellular papillae were found lining the placental
valves of the ovaries in V. imperialis (Fig. 3C, D).
Similar structures were also observed in Vanilla by,
for example, Swamy (1947), Krupko et al. (1954) and
Roux (1954). Hair-like structures on the adaxial side
of orchid carpels have been observed in many orchid
genera (e.g. Beer, 1857; Malguth, 1901; Hallé, 1986;
Rasmussen & Johansen, 2006 and references
therein), in which there is little doubt that they serve
as hygroscopic elaters, helping the dispersal of seeds.
Dressler (1981) and Freudenstein & Rasmussen
(1999) attached taxonomic significance to these endocarpic trichomes, observing that their presence or
absence seems to characterize larger clades of Orchidaceae. We suggest that the papillae in V. imperialis
and in other species of Vanilla are homologous to
endocarpic trichomes in other groups of orchids, but
with a quite different function. According to Swamy
(1947), Havkin-Frenkel, Pak & French (2002), Joel
et al. (2003) and Odoux et al. (2003), the unicellular
papillae in V. planifolia secrete glucovanillin, the precursor to vanillin. The fruits of V. imperialis are not
fragrant, but produce a white foamy substance which
oozes out of the fruit as it opens distally at maturity.
The seeds are embedded in the substance which has
a slightly sweet taste, a bitter aftertaste and may
function in the dispersal of the seeds.
Conclusions
The observations of V. planifolia (Swamy, 1947) up to
and including the formation of the six-nucleate
embryo sac were confirmed for V. imperialis, although
the timing appears to be different. The reduction from
eight to six nuclei in the mature embryo sac appears
to be widespread in Orchidaceae (e.g. Pace, 1907;
Sharp, 1912; Afzelius, 1916; Prosina, 1930; Swamy,
1945, 1947, 1949; Law & Yeung, 1989, present study),
and six-nucleate embryo sacs may be more common
than eight-nucleate embryo sacs.
No double fertilization was observed and the observation of endosperm formation in V. planifolia
(Swamy, 1947) could not be confirmed. The lack of
double fertilization and formation of endosperm in
V. imperialis, and similar observations from other
species (Krupko et al., 1954; Clements, 1999), causes
the present authors to doubt that double fertilization
and endosperm formation take place in Vanilla,
although differences between species or even variation within the same species cannot be excluded. The
The authors thank L. M. Frederiksen for technical
assistance and H. N. Rasmussen for helpful discussions and critical reading of the manuscript. We
thank the Core Facility for Integrated Microscopy,
Faculty of Health Sciences, University of Copenhagen
for access to the confocal microscopes, and the Botanical Garden, University of Copenhagen for keeping
some vigorous specimens of V. imperialis and providing material at all stages of flowering. University of
Chicago Press has kindly granted permission to
reproduce figures from Swamy BGL. 1947. On the
life-history of Vanilla planifolia. Botanical Gazette
108: 449–456.
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