Flora 255 (2019) 86–97

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Flora
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Structure of the pistil and pollen tube growth in Adansonia L. species: T

Implications for fertilization efficiency
Elysée N. Rasoamananaa, , Onja Razanamaroa, Jean-Luc Verdeilb, Fabienne Lapeyre Montesb,
⁎

Perle Ramavovololonaa, Pascal Danthuc,d

a
Université d’Antananarivo, Mention Biologie et Ecologie végétales, BP 906, 101 Antananarivo, Madagascar
b
CIRAD, UMR AGAP, F-34398 Montpellier Cedex 5, France
c
CIRAD, UR HortSys, Campus de Baillarguet, 34398 Montpellier Cedex 5, France
d
UR HortSys, Université de Montpellier, CIRAD, 34998 Montpellier Cedex, France

ARTICLE INFO ABSTRACT

Edited by Alessio Papini The success of fertilization depends on an array of complex physical and physiological interactions between the
Keywords: pollen and the pistil. To better understand the fertilization process in Malagasy baobabs, pistil structure and
Adansonia pollen tube growth were investigated in three species of Adansonia: A. grandidieri (section Brevitubae), A. ma-
Anatomy dagascariensis and A. rubrostipa (Longitubae section). The pistil consists of an ovary with 6–9 locules, a very long
Pollen pistil interaction hollow type style and six to nine lobed wet, papillate stigma. The pollen tubes grow in intercellular secretions in
Hollow style the stigma and join the stylar canal to reach the locule of the ovary and then the micropyle of the ovules. The size
Gametophytic selection of the central stylar canal shrinks as it approaches the ovary. This restriction leads to gametophytic competition
by reducing the number of pollen tubes that reach the ovules. The pollen tubes reach the ovules four to six days
after pollination. The pollen tubes of the two Longitubae species grow faster than those of A. grandidieri. This
information on the progamic phase of baobabs is essential to understand the mode of reproduction of baobabs.

1. Introduction phenomenon is interspecific pollination. While the floral biology, floral

Baobabs (Adansonia, Malvaceae) are tropical trees that grow in
Madagascar, Africa and Australia. There are eight or nine species
worldwide, as debate continues concerning the recently discovered
ninth species. Six of the species are endemic to Madagascar (Baum,
1995a; Cron et al., 2016; Pettigrew et al., 2012). The genus Adansonia is
subdivided into three sections: Brevitubae, Longitubae and Adansonia
sections. All Malagasy species are grouped in Brevitubae (A. grandidieri
Baill. and A. suarezensis H. Perr.) and Longitubae (A. za Baill., A. ru-
brostipa Jumm. and H. Perr., A. madagascariensis Baill. and A. perrieri
Capuron) sections. Despite the fact baobabs are one of the emblematic
species of Madagascar, knowledge of their biology, ecology and evo-
lution is still incomplete. The introgression and hybridization phe-
nomenon, a form of evolution of long lived species, was identified by
Leong Pock Tsy et al. (2013) in Longitubae species. The origin of this
scents and comparative pollination biology of baobabs have already
been studied (Andriafidison et al., 2006; Baum, 1995b; Razanamaro
et al., 2015; Ryckewaert et al., 2011), little is known about the phe-
nomena involved in the successful fertilization of baobabs.
The success of pollen grains in ovule fertilization depends on the
physical and physiological interactions between the pollen and the
pistil. These interactions begin with recognition of pollen by the stigma
and continue with the progression of the pollen tube to the embryo sac
(Cheung, 1996, 1995; Cisneros-López et al., 2010; Gawel and Robacker,
1986; Shivanna, 1982). In this way, the stigmatic papillae supply the
right environment for pollen germination (Edlund, 2004) and the style
provides mechanical facilitation and the nutrients required for the de-
velopment of the pollen tube (Gawel and Robacker, 1986). Up to now,
there has been no detailed description of the anatomy of the pistil of the
genus Adansonia. To our knowledge, baobabs have one of the longest

⁎
Corresponding author.
E-mail address: elyseenoro@yahoo.fr (E.N. Rasoamanana).

https://doi.org/10.1016/j.flora.2019.04.005
Received 13 November 2018; Received in revised form 27 March 2019; Accepted 17 April 2019
Available online 19 April 2019
0367-2530/ © 2019 Published by Elsevier GmbH.
E.N. Rasoamanana, et al.
Table 1
Locations of the species investigated.
Species Sections Sampling site Geographical coordinates
A. grandidieri Brevitubae Morondava 20°10'12’’S, 44°31'04’’E
A. madagascariensis Longitubae Tsaramandroso 16°22’4.46’’S, 47°2’56.94’’E
A. rubrostipa Longitubae Ifaty 23°4′39.98′′ S 43°37′2.23′′ E
styles in Angiosperms (up to 20 cm long), so the encounter between the
male gamete contained in the pollen grain and the female gamete
contained in the ovule involves a long journey for the pollen tube. The
ultrastructure of pollen grains of the genus Adansonia and the possible
role of the apertural system on the pollen hydration and germination
were described in a previous paper (Rasoamanana et al., 2014), but no
information has been published about pollen tube growth and its
pathway. To estimate how much time is required for pollen to reach the
ovules and the mode of progression of the pollen tube inside the pistil,
here we describe the structure of the pistil, pollen germination and the
pathway the pollen tube takes to reach the ovule in three species of
baobab: A. grandidieri (Brevitubae), A. madagascariensis and A. ru-
brostipa (Longitubae).
2. Materials and methods
2.1. Study species and sites
Baobabs grow in the dry forests of western Madagascar. All mate-
rials were collected from their natural habitats (Table 1). A. grandidieri
(Brevitubae) flower in the cool, dry season (June to August) while A.
madagascariensis and A. rubrostipa (Longitubae) flower during the hot,
wet season in February-March. Brevitubae flowers open at dusk
whereas Longitubae flowers open at nightfall (Baum, 1995b;
Ryckewaert et al., 2011). A. grandidieri has white flowers with short
floral parts, a short staminal tube (about 1.5 cm long); and 700–800
stamens (Fig. 1A). The two species of Longitubae have delicate, elon-
gated flowers with brightly colored petals ranging from orange to red in
A. madagascariensis and from yellow to orange in A. rubrostipa (Fig. 1B;
C). They have a long staminal tube (6–10 cm long), fewer stamens (A.
madagascariensis has 90–100 and A. rubrostipa 100–150). In all species,
there is only one long style, and the ovary contains numerous ovules.
Baobab pollination requires a pollen vector. The main pollinators
are Sphingidae but in Brevitubae, nocturnal mammals (lemurs and
bats) and bees also visit the flowers (Andriafidison et al., 2006; Baum,
1995b; Ryckewaert et al., 2011).
Fig. 1. Comparison of the flowers in the three species of baobab. (A) A. grandidieri. Brevitubae section (B) A. madagascariensis. Longitubae section (C) A. rubrostipa.
Longitubae section. Petals (pe), sepals (sp), stamens (sm), stigmata (arrow), style (st).
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Flora 255 (2019) 86–97
2.2. Observation of pollen nuclei
A total of 15 individuals per species were for pollen. Sufficient
mature pollen was obtained by placing 3–4 anthers directly on a mi-
croscope slide. The pollen grains were stained with 0.005% DAPI so-
lution (0.1 M sodium phosphate (pH 7), 1 mM EDTA, 0.1% Triton X-
100, 0.4 μg/ml DAPI) (Coleman and Goff, 1985). The nuclei were
counted under a Leica DM4500B microscope, with UV epi-illumination
X 20 magnification (filter A4).
2.3. Pistil histology
Unpollinated pistils were collected from three distinct individuals
per species. Immediately after sampling, the pistils and ovaries were
placed in a tube containing the fixation solution prepared as follows:
50 ml of 0.2 M sodium phosphate buffer, pH 7, 20 ml of 10% paraf-
ormaldehyde, 2 ml of 50% glutaraldehyde, 28 ml distilled water and 1 g
of caffeine. One drop of tween-80 was added and vacuum was applied
for 15 min. Pistil samples were incubated for 48 h at 4 °C, then placed in
50% ethanol for 1 h and preserved in 70% ethanol at 4 °C until analysis.
Samples were then dehydrated by successively immersing them in 70%
ethanol for 30 min, 95% ethanol (2 × 30 min) and ethanol for 8 h
(Buffard-Morel et al., 1992). As the pistils are long, fragments of the
samples were collected at the top (near the stigma), in the middle and at
the end of the style (near the ovary) (Fig. 2). The fragmented samples
were then impregnated for 24 h at 4 °C in a medium containing Tech-
novit 7100 resin (100 ml; Kulzer, Wehrheim, Germany) following the
manufacturer’s instructions and finally embedded in resin at 37 °C.
Longitudinal and transversal sections (5–7 μm) of each fragment were
cut using a Leica RM2255 microtome (Germany).
The collected sections were double stained according to Buffard-
Morel et al. (1992). Briefly, polysaccharides were stained dark pink
with periodic acid-Schiff (PAS) (Sigma, ref. 3952016), and soluble
proteins were stained blue with naphthol blue-black (Fisher et al.,
1968) (NBB, Sigma, ref. 195243). Sections were then mounted in Iso-
mount (Labonord ref. 05547535).
Observations were made under a microscope under a bright field
(Leica DM4500B, Leica, Germany), connected to a Retiga 2000R
camera (Q Imaging Co.). The pictures were processed using Volocity
4.0.1 software (Improvision, Lexington, MA, USA).
2.4. Pollen tube pathway
For each species studied, pollination was carried out by hand on
emasculated flowers at the anthesis, using fresh pollen from a dehiscing
anther. Ten individuals per species were sampled for the treatment.
E.N. Rasoamanana, et al. Flora 255 (2019) 86–97

Since preliminary data showed no pollen activity before 36 h post

pollination; pollinated pistils from three individuals were collected at
36 h, two at 96 h, two at 120 h and three at 144 h after pollination. All
the samples were preserved in 70% ethanol.
After rinsing with distilled water, longitudinal sections (80 μm) of
the style and the ovary were cut with a vibratome (Microm, HM50 V,
Germany) and cleared in a 8NNaOH solution at room temperature for
16 h until the tissues became transparent (translucent). They were then
rinsed in distilled water and stained with decolorized aniline blue 0.1%
K2NPO4 pH 10 (Kho and Baër, 1968). On each sampling occasion,
pollen tube growth was examined on the stigma and on the style (at
levels 1, 2, 3 and 4 in Fig. 2) and on the ovary, using a Leica DM 4500
epifluorescence microscope equipped with an A4 filter.

2.5. Data measurement and processing

For the morphometry, a total of 13 flowers belonging 5–6 in-

dividuals per species were studied. The length of the style, the depth of
the stigma, and the size of the ovaries were measured using graph
paper. The number of stigmatic lobes was counted under a binocular
microscope.
To describe pistil anatomy, the stylar canal was measured directly
on the photos using Image J software. Measurements were made at
three locations on the style: the top, the middle and the end (Fig. 2). For
each species, one measurement per point was made on five pistils from
different individual.
The statistical calculation comprised the mean and standard de-
viation.
For the pollen tube, the pollen growth rate was calculated by the
average length of the pistil (mm) divided by the average time (in hours)
required for the tubes to reach the ovules. The quantity of pollen tubes
was evaluated by the total thickness of all the tubes.

3. Results

3.1. Style and stigma morphology and structure

The stigma is composed of six to nine short stigmatic branches with

Fig. 2. Location of the cross sections of the pistil: on the stigma (1), at the top of
the style (2), in the middle of the style (3), at the end of the style (4) and on the
ovary (5).
regular contours in A. grandidieri, and with irregular contours in the two
species of Longitubae (Fig. 3A, B, C). The stigmatic surface is rough.
The depth of the stigma is on average 1.09 ± 0.30 mm in A. grandidieri,
2.13 ± 0.35 mm and 3.43 ± 1.4 mm in A. madagascariensis in A. ru-
brostipa. The single style is straight, tapered and thinner at the top,
densely villous below and glabrous above, white, 72.92 ± 7.48 mm in
length in A. grandidieri; red and longer in A. madagascariensis and A.
rubrostipa (respectively 148.37 ± 16.15 mm and 242 ± 20.27 mm).
No marked differences were observed in the structure of either the
pistil or the ovary in the species examined (Fig. 4A). The stigmas of
Adansonia are of the wet type; secretory papillae and polysaccharide
compounds are located outside the papillae. The stigmatic zone is made
up of elongated and densely arranged glandular unicellular papillae
cells with large intercellular spaces (Fig. 4B, C). The papillar cells have
thin cuticles and dense cytoplasm. PAS was positive for polysaccharide
in the cell walls of the papillae, while NBB staining was positive for the
cytoplasm (Fig. 4B, C). Carbohydrates were detected in intercellular
exudates (Fig. 4C).
The style is hollow with a canal running through its center (Fig. 6A,
B). The central stylar canal is lined with papillary cells rich in protein,
an elongated longitudinal cell with intercellular spaces (Fig. 5D–F).
Near the stigma, the stylar canal is still multi-radiated form but gra-
dually merges into a single channel a quarter in length of the style
(Fig. 5A, C). The vascular bundles in this region are arranged in a single
circle (Fig. 6C). Both the diameter of the stylar canal and the quantity of

88
E.N. Rasoamanana, et al.
Fig. 3. Comparison of the stigma morphology of three Adansonia species under
a binocular microscope. (A) A. grandieri, (B) A. madagascariensis and (C) A.
rubrostipa. lobe (lb), stigma papillae (p), stylar canal (sc).
stylar papillae and exudates decrease towards the ovary whereas the
diameter of the style increases (Table 2, Fig. 5A, B). At the base of the
style, the stylar canal is very narrow and the papillae are fewer in
number (Fig. 5A, B).
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Flora 255 (2019) 86–97
Fig. 4. Structure of Adansonia stigma under a light microscopy. (A) General
view of the stigma in A. rubrostipa in longitudinal section. (B) Zoom on the
stigmatic papillae in longitudinal section of A. grandidieri. (C) Cross section at
the end of the stigma in A. madagascariensis. Exudates (ex), stigma papillae (p);
parenchyma tissue (pt).
The structure of the style consists of a layer of epidermis with cu-
ticle, a parenchymal cortex and a central stylar canal (Fig. 5). The
epidermal cell is sub-rectangular and covered with a thick cuticle
(Fig. 5C). The cortical cells of the style are circular, large but variable in
size in cross section and sub-rectangular in longitudinal section
(Fig. 5B, C, E). The vascular region is formed of two vascular bundles
circles alternating with mucilage (Figs. 5C, 6 D). In the vascular regions,
E.N. Rasoamanana, et al.
the cortical cells are smaller (Fig. 5C, D). Their wall is thin and contains
some polyphenols and many PAS-positive granules, which are possibly
reserve starches (Fig. 5F).
3.2. Ovary morphology and structure
The baobab ovary is rounded conical and hairy (Figs. 7 and 8A);
ovaries range in size from 1 to 1.5 cm. A. rubrostipa and A. mada-
gascariensis (Longitubae) have semi-inferior ovaries and A. grandidieri
has superior ovaries. The ovary is plurilocular with six to nine carpels
similar to the number of the stigmatic lobes (varies among species but
also in individuals of the same species) (Figs. 7 and 8A, B). The carpels
contain numerous ovules (227 ± 9 in A. grandidieri, 166 ± 29 in A.
madagascariensis and 171 ± 18 in A. rubrostipa) on axile placentation
(Figs. 7 and 8B).
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Fig. 5. Anatomical structure of Adansonia style
(A, B, C, D) A. madagascariensis. (E, F) A.
grandidieri. (A) Cross section at the top of the
style showing the multilobed stylar canal and
the stigmatic papillae (B) Zoom on the section
showing the style papillae with the exudates,
the vascular bundles and the cortex. (C) Cross-
section of the mid-style with unilobed stylar
canal. (D) Zoom on the section showing the
vascular bundles, a stylar canal lined with pa-
pilla, mucilage and an accumulation of starch
(arrow) (E) Longitudinal section of the style.
(F) Longitudinal view of the papillae lining the
stylar canal. Epidermis (ep), cortex (co), mu-
cilage (mu), stigma papillae (p), polyphenol
(pp), stylar canal (sc), stylar papillae (sp),
vascular bundles (vb).
The ovary wall shows zonal differentiation comprising 1) the dorsal
epidermis, 2) the cortex, 3) the vascular zone, 4) the circumlocular
zone, and 5) the ventral epidermis (Figs. 7C, and 8 C). The epidermis is
composed of small circular cells elongated by pluricellular trichomes
(Figs. 7D, 8 C). The cortex is formed by larger parenchyma cells that
gradually increase in size near the vascular zone (Fig. 7D). In the
longitudinal section, the vascular zone is formed of parenchyma tissue
mixed with longitudinal vascular bundles. In the cross section, the
vascular bundles are arranged in one circle (Fig. 7C) showing the
normal orientation of xylem and phloem (Fig. 7E). Plentiful mucilage
was observed in this zone. The circumlocular zone comprises ground
tissue of parenchyma cells embedded with two circles of vascular
bundles that are smaller than those in the main vascular zone (Fig. 7F).
The ventral epidermis is the inner ovary epithelium which after fe-
cundation, possibly contributes to the formation of the endocarp. The
E.N. Rasoamanana, et al. Flora 255 (2019) 86–97

Fig. 6. Schematic drawing of stigma and style. (A) Longitudinal view of the pistil showing (1) the stigma, (2) the top of the style and (3) the middle of the style. (B)
Schematic representation of the surface of the stigma. (C) Cross section at the top of the style; note the vascular bundle arranged in a single circle. (D) Cross section in
the middle of the style showing two vascular bundle circles. epidermis (ep), cortex (co), mucilage (mu), stigma papillae (p), stylar canal (sc), stylar papillae (sp), lobe
(lb), vascular bundle (vb).

Table 2
Diameter of the style and of the stylar canal at different locations along the style (N = 5).
A. madagascariensis A. rubrostipa A. grandidieri

Style (μm) Stylar canal (μm) Style (μm) Stylar canal (μm) Style (μm) Stylar canal (μm)

Top of style 1177 ± 80 528 ± 62 912 ± 79 592 ± 48 1347 ± 87 455 ± 68

Mid-style 1366 ± 93 290 ± 45 1564 ± 134 284 ± 43 1890 ± 112 336 ± 49
Base of style 1604 ± 102 163 ± 34 1863 ± 152 234 ± 55 1883 ± 145 87 ± 22

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E.N. Rasoamanana, et al.
ventral epidermis is formed by one sub-rectangular cell layer with cu-
ticle (Fig. 7F).
The stylar canal is located toward the central axis of the insertion of
the ovary in the septal tissue (Fig. 7A). It is directly linked just below
the insertion of the style (Fig. 9A). The stylar canal passes through the
locular region and extends into several channels in the ovarian cavity
(Fig. 9B). The stylar canal is lined with papillae that produce PAS po-
sitive exudates (Fig. 9C).
The ovule is anatropous, bitegmic, crassinucellar and has a long
funicle (Fig. 9E). The outer integument is thicker than the inner. The
inner integument forms the micropyle; cells of the outer integument
grow outward to form an exostome (Fig. 9F). The vascular bundles pass
through the outer integument. The ovule is rich in protein. The outer
wall of the nucellus and those of the adjacent cells of the inner
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Flora 255 (2019) 86–97
Fig. 7. (A) A. grandidieri. Longitudinal section
of the whole ovary. B) A. madagascariensis.
Cross section of the whole ovary. (C) A.
grandidieri Cross section of the different zones
of the ovarian wall: cortex, vascular zone, cir-
cumlocular zone. (C) Zoom on the cortex. (D,
E) A. grandidieri. Zoom on the vascular zone (F)
A. grandidieri Zoom on the circumlocular zone,
note the vascular bundles arranged in two
circles. Circumlocular zone (cz), cortex (co),
dorsal epidermis with trichomes (de), locule
(lo), mucilage (mu), ovule (ov), ovary wall
(ow), phloem (phl), septum (se), stylar canal
(sc), vascular bundle (vb), vascular zone (vz),
ventral epidermis, xylem (xyl).
integument have a thick coating of cutin along the micropyle. The
micropyle is zig-zag in shape (Fig. 9F).
3.3. Pollen tube growth and pathway at the stigma and the style
The pollen grains of Adansonia are 3-colporate, rarely 4-colporate.
The ectexine is formed by a perforate tectum with isolated spinules. The
pollen is binucleate, composed of one generator cell and one vegetative
cell (Fig. 10A). The sperm cells form during pollen tube germination.
No pollen was observed to germinate less than 36 h after pollina-
tion; eight hours after pollination, pollen grains are only hydrated. A
large number (about 200) of pollen grains have to be deposited on the
stigma to enable germination of the pollen tube (unpublished data).
Pollen grains germinated along the entire stigmatic area (Fig. 10B). At
E.N. Rasoamanana, et al.
Fig. 8. Schematic drawing of the ovary. (A) Longitudinal section of the ovary
(B) Cross section of the ovary (C) Cross section of the ovary wall. circumlocular
zone (cz), cortex (co), locule (lo), mucilage (mu), ovary wall (ow), ovule (ov),
phloem (phl), septum (se), stylar canal (sc), stylar papillae (arrow), trichome
(tr), vascular bundle (vb), vascular zone (vz), ventral epidermis (ve), xylem
(xyl).
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Flora 255 (2019) 86–97
36 h after pollination, the pollen grains start germinating on two or
three pores of the pollen (Fig. 10C), then continue to grow on only one
of the pores (Fig. 10D). The pollen tubes pass through the stigmatic
papillae then grow all along the style inside the stylar canal (Fig. 11A).
We hypothesize that the pollen tubes feed on the carbohydrate secre-
tions produced by the papillary cells lining the stylar canal. A pro-
gressive reduction in the number of pollen tubes growing in the style
was observed at the middle of the style (Fig. 11B, C) and even fewer
near the ovary (Fig. 11D).
3.4. Pollen tube growth and pathway to the ovary
At the ovary, the pollen tubes pass through the exudates present in
the placenta. Pollen tubes grow between the two rows of ovules, and
one by one, bend 90° to grow between two adjacent ovules toward the
micropylar side (Fig. 12A). After arriving at the micropylar side, the
pollen tube again bends about 90° to reach the inner integument
(Fig. 12B).
The growing pollen tube reaches the ovule three to six days after
pollination. The pollen tube growth rate is estimated at
1.13 ± 0.26 mm h−1 in A. grandidieri; 8.22 ± 4.74 mm h−1 in A.
madagascariensis, and 5.38 ± 1.9 mm h−1 in A. rubrostipa. Not all the
ovules are reached by pollen tubes.
4. Discussion
To our knowledge, this study is the first to reveal the anatomy of the
pistil in the genus Adansonia. The style of Adansonia is longer than that
of other known species of Malvaceae (Snow and Spira, 1991). Mucilage
secreting structures are common features in most families belonging to
the Malvales order (Marzinek and Mourão, 2003). The stigma is of the
wet type and the style has a hollow stylar canal that is also found in
other unrelated species like Lilium sp. (Liliaceae) (Janson et al., 1994).
4.1. Pollen tube development along the pistil
In the present study, we demonstrate that several factors are in-
volved in the development of the pollen tube along the pistil of
Adansonia. The first interaction occurs at the stigmatic papillae where
exudates trigger the germination of the pollen grains. Concerning the
pollen, in a previous paper (Rasoamanana et al., 2014), we suggested
that the ultrastructure of the sporoderm around the germinal pores of
baobab pollen may be related to the function of hydration and germi-
nation of the pollen tubes in baobabs. On the other hand, on the pistil a
considerable amount of exudates was observed on the surface of the
pollinated stigma, a frequent phenomenon in compatible pollination,
and our histochemical data confirm the results reported by Onus
(2000). Like in other wet stigma species, proteins and carbohydrates are
detected in the stigma exudates of baobab (Onus, 2000). As shown in
Capsicum eximium (Solanaceae), the stigmatic proteins found in baobab
species could be involved in the recognition of the pollen and in-
compatibility response (Onus, 2000).
After passing through the stigmatic papillae, the pollen tube de-
velops inside the stylar canal. In baobab species, a delay of about 36 h
has been identified. Delays have also been reported in other species
E.N. Rasoamanana, et al.
including some Talinum mengesii (Portulacaceae) populations, in which
germination was delayed for up to two hours after pollination (Murdy
and Carter, 1987). According to these authors, this delay may be a
strategy to promote the accumulation of pollen on the stigma and in-
duce simultaneous germination. The presence of exudates rich in car-
bohydrate lining the baobab stylar canal suggests the exudates can be
used as nutrients for pollen tube growth, as observed in other species
with a hollow style (Kroh et al., 1970; Labarca and Loewus, 1973).
In the ovary of baobabs - like in many multiovulated species - the
whole placenta is covered by secretory cells (Gonzàlez et al., 1996).
Furthermore, pollen tube guidance strategies have been demonstrated,
for example, in peach, only ovules in secretory phase are approached by
the pollen tube (Herrero, 2000). As this secretion does not appear in all
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Flora 255 (2019) 86–97
Fig. 9. Anatomy of the style and ovule. A.
grandidieri. (A) Longitudinal section at the en-
trance of the ovary. Note the narrow stylar
canal. (B) Longitudinal section of the septum.
(C) Zoom on the stylar canal at the top of the
ovary lined with papillae. (D) Cross section of
part of the ovary with a central stylar canal and
ovules in axile placentation. (E) Longitudinal
section of an entire ovule. (F) Zoom on the zig-
zag entrance of the embryo sac (dashed arrow).
Chalaza (ch), cortex (co), embryo sac (es), fu-
nicle (fu), inner integument (it), nucellus (nu),
outer integument (ot), septum (se), stylar canal
(sc), stylar papillae (sp), vascular bundle (vb),
micropyle (arrow).
ovules, all ovules are not reached by pollen tube. It is possible that in
baobabs, there is also a strategy for pollen tube guidance that can select
the ovules to be fertilized.
4.2. Gametophytic selection
In the genus Adansonia, physical constraints limit further develop-
ment of the pollen tube because of the reduction in the diameter of the
stylar canal along the style. There are also fewer papillary cell and
exudates after the middle of the style, which may limit the resource for
pollen tube development. One effect of this event is that the number of
pollen tubes decreases from the stigma to the end of the style. The re-
duction in the diameter of the stylar canal as well as the decrease of the
E.N. Rasoamanana, et al.
stylar papillae are doubtless anatomical and physiological adaptations
that favor the selection of pollen tubes. This phenomenon is observed in
number of species with the goal of promoting pollen competition
(Cruzan, 1986; Herrero and Hormaza, 1996; Matthews et al., 1999).
Gamete selection is a form of sexual selection known to genetically
improve plant populations (Charlesworth et al., 1987; Sari-Gorla et al.,
1992). In the present study, gametophytic selection affects sporophyte
generation, as the physical restriction and limitation of resource allows
only the strongest gametes to cross the barrier. We suggest that in
baobabs, pollen selection is one form of gene selection to increase the
quality of the generation.
4.3. Implications for pollination
As baobabs are animal pollinated species (Andriafidison et al., 2006;
Baum, 1995b; Ryckewaert et al., 2011), the time required for male
gametes to reach the ovules needs to be known to study the effective-
ness of a pollinator. Compared to other species, the average growth rate
of the pollen tubes in baobabs is relatively fast (about 118 mm/day).
For example, it ranges from less than 1 mm/day in Chamelaucium
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Flora 255 (2019) 86–97
Fig. 10. (A) Adansonia binucleate pollen grains
stained with DAPI (A. madagascariensis). Nuclei
were pushed out of the pollen by osmotic
pressure. (B) Adhesion of pollen to the stigma.
(C) Zoom on the beginning of pollen tube
germination on the two germinative pores
(arrow) 36 h after pollination. (D) Pollen tube
growing from a single pore (36 h after polli-
nation). Exudates (ex), nuclei (n), stigma pa-
pillae (p), pollen (pl), pollen tube (plt).
uncinatum (O’Brien, 1994) to about 240 mm/day in Zea mays (Barnabas
and Fridvalszky, 1984). The pollen tubes of A. grandidieri have a slower
growth rate (1.14 mm.h−1) than those of the two species of Longitubae
A. madagascariensis and A. rubrostipa (respectively 8.22 mm h−1 in A.
madagascariensis, and 5.38 mm h−1). The rate of pollen tube growth
may be genetically determined (Tilton, 1984), but is influenced by
other factors such as temperature (Adaniya and Higa, 1988), pollen
competitiveness (Niesenbaum, 1999; Snow and Spira, 1991) and self-
incompatibility (Lush and Clarke, 1997; Visser and Marcucci, 1983).
The long journey traveled by the pollen tubes and the selection of male
gametes along the pistil may be a limiting factor the fertilization of
ovules. This situation may limit pollination in baobabs, in which low
natural regeneration has been reported (Razanameharizaka, 2009).
Details of pistil structure and composition enabled us to characterize
the state of the pistil at anthesis as a prelude to determining the changes
that occur with successful pollination, and the factors and processes
involved. This study adds new information that will be useful for
breeding schemes and provides a basis for understanding pollen-pistil
interactions in baobabs.
E.N. Rasoamanana, et al. Flora 255 (2019) 86–97

Fig. 11. A. rubrostipa. Progression of the pollen tube (A) in the stigma, (B) at the top of the style, (C) in the middle of the style, (D) near the ovary. Note the decrease
in the density of the pollen tubes of the stigma at the entrance of the ovary. Pollen (pl), pollen tube (plt).

Fig. 12. Progression of the pollen tubes into

the ovary. (A) A. rubrostipa. Progression of the
pollen tubes through the stylar canal present in
the placenta. Note the ramification of the
pollen tubes to reach the ovary. (B) A. grand-
idieri. Pollen tube penetration at the micro-
pylar side of the ovule. Funicle (fu), ovule (ov),
pollen tubes (plt), septum (se).

96
E.N. Rasoamanana, et al.
Declarations of interest
On behalf of all authors, I declare that there are no interests of
conflict in this paper.
Acknowledgements
The fieldwork of this work was supported by International
Foundation for Sciences [grant numbers: 5013-1]; the laboratory work
was carried out thanks to DESI-CIRAD's travel grant. We also thank
Daphne Goodfellow for her careful revision of our English.
References
Adaniya, S., Higa, T., 1988. Effects of temperature and relative humidity on pollen ger-
minability and pollen tube growth in the style of Zingiber mioga Roscoe. J. Jpn. Soc.
Hortic. Sci. 57, 43–51.
Andriafidison, D., Andrianaivoarivelo, R.A., Ramilijaona, O.R., Razanahoera, M.R.,
MacKinnon, J., Jenkins, R.K.B., Racey, P.A., 2006. Nectarivory by endemic Malagasy
fruit bats during the dry season. Biotropica 38, 85–90.
Barnabas, B., Fridvalszky, L., 1984. Adhesion and germination of differently treated
maize pollen grains on the stigma. Acta Bot. Hung. 30, 329–332.
Baum, D.A., 1995a. A systematic revision of Adansonia (Bombacaceae). Ann. Mo. Bot.
Gard. 82, 440–470.
Baum, D.A., 1995b. The comparative pollination and floral biology of baobabs
(Adansonia-Bombacaceae). Ann. Mo. Bot. Gard. 82, 322–348.
Buffard-Morel, J., Verdeil, J.L., Pannetier, C., 1992. Embryogenèse somatique du cocotier
(Cocos nucifera L.) à partir de tissus foliaires: étude histologique. Can. J. Bot. 70,
735–741.
Charlesworth, D., Schemske, D.W., Sork, V.L., 1987. The evolution of plant reproductive
characters; Sexual versus natural selection. In: Stearns, S.C. (Ed.), Evolution of Sex,
pp. 317–335.
Cheung, A.Y., 1995. Pollen-pistil interactions in compatible pollination. Proc. Natl. Acad.
Sci. U. S. A. 92, 3077.
Cheung, A.Y., 1996. Pollen—pistil interactions during pollen-tube growth. Trends Plant
Sci. 1, 45–51.
Cisneros-López, M.E., Mendoza-Onofre, L.E., Zavaleta-Mancera, H.A., González-
Hernández, V.A., Mora-Aguilera, G., Córdova-Téllez, L., Hernández-Martínez, M.,
2010. Pollen–pistil interaction, pistil histology and seed production in A×B grain
sorghum crosses under chilling field temperatures. J. Agric. Sci. 148, 73.
Coleman, A.W., Goff, L.J., 1985. Applications of fluorochromes to pollen biology. I.
Mithramycin and 4′,6-Diamidino-2-Phenylindole (Dapi) as vital stains and for
quantitation of nuclear DNA. Biotech. Histochem. 60, 145–154.
Cron, G.V., Karimi, N., Glennon, K.L., Udeh, C., Witkowski, E.T.F., Venter, S.M.,
Assogbadjo, A.E., Baum, D.A., 2016. One African baobab species or two? A re-eva-
luation of Adansonia kilima. S. Afr. J. Bot. 103, 312.
Cruzan, M.B., 1986. Pollen tube distributions in Nicotiana glauca: evidence for density
dependent growth. Am. J. Bot. 73, 902–907.
Edlund, A.F., 2004. Pollen and stigma structure and function: the role of diversity in
pollination. Plant Cell 16, S84–S97. https://doi.org/10.1105/tpc.015800.
Fisher, D.B., 1968. Protein staining of ribboned epon sections for light microscopy.
Histochemie 16, 92–96.
Gawel, N.J., Robacker, C.D., 1986. Effect of pollen-style interaction on the pollen tube
growth of Gossypium hirsutum. Theor. Appl. Genet. 72, 84–87.
Gonzàlez, M.V., Coque, M., Herrero, M., 1996. Pollen-pistl interaction in kiwifruit
(Actinidia deliciosa; Actinidiaceae). Am. J. Bot. 83, 148–154.
Herrero, M., 2000. Changes in the ovary related to pollen tube guidance. Ann. Bot. 85,
79–85.
97
Flora 255 (2019) 86–97
Herrero, M., Hormaza, J.I., 1996. Pistil strategies controlling pollen tube growth. Sex.
Plant Reprod. 9, 343–347.
Janson, J., Reinders, M.C., Valkering, A.G.M., Tuyl, J.M.V., Keijzer, C.J., 1994. Pistil
exudate production and pollen tube growth in Lilium longiflorum Thunb. Ann. Bot. 73,
437–446.
Kho, Y.O., Baër, J., 1968. Observing pollen tubes by means of fluorescence. Euphytica 17,
298–302.
Kroh, M., Miki-Hirosige, H., Rosen, W., Loewus, F., 1970. Incorporation of label into
pollen tube walls from myoinositol-labeled Lilium longiflorum pistils. Plant Physiol.
45, 92–94.
Labarca, C., Loewus, F., 1973. The nutritional role of pistil exudate in pollen tube wall
formation in Lilium longiflorum II. Production and utilization of exudate from stigma
and stylar canal. Plant Physiol. 52, 87–92.
Leong Pock Tsy, J.-M., Lumaret, R., Flaven-Noguier, E., Sauve, M., Dubois, M.-P., Danthu,
P., 2013. Nuclear microsatellite variation in Malagasy baobabs (Adansonia,
Bombacoideae, Malvaceae) reveals past hybridization and introgression. Ann. Bot.
112, 1759–1773.
Lush, W.M., Clarke, A.E., 1997. Observations of pollen tube growth in Nicotiana alata and
their implications for the mechanism of self-incompatibility. Sex. Plant Reprod. 10,
27–35.
Marzinek, J., Mourão, K.S., 2003. Morphology and anatomy of the fruit and seed in de-
velopment of Chorisia speciosa A. St.-Hil.-Bombacaceae. Braz. J. Bot. 26, 23–34.
Matthews, M.L., Gardner, J., Sedgley, M., 1999. The Relationship between transmitting
tissue, pollen tube, and ovule number: a study across 10 Angiosperm families. Int. J.
Plant Sci. 160, 673–681.
Murdy, W.H., Carter, M.E.B., 1987. Regulation of the timing of pollen germination by the
pistil in Talinum mengesii (Portulacaceae). Am. J. Bot. 74, 1888.
Niesenbaum, R.A., 1999. The effects of pollen load size and donor diversity on pollen
performance, selective abortion, and progeny vigor in Mirabilis jalapa
(Nyctaginaceae). Am. J. Bot. 86, 261–268.
O’Brien, S.P., 1994. Pistil structure and pollen tube pathways in Leptospermum myrsinoides
and L. continentale (Myrtaceae). Ann. Bot. 73, 225–230.
Onus, A.N., 2000. Structure of the stigma and style in Capsicum eximium and the effects of
pollination. Turk J Bot 24, 337–346.
Pettigrew, F.R.S., Jack, D., Bell, K.L., Bhagwandin, A., Grinan, E., Jillani, N., Meyer, J.,
Wabuyele, E., Vickers, C.E., 2012. Morphology, ploidy and molecular phylogenetics
reveal a new diploid species from Africa in the baobab genus Adansonia (Malvaceae:
Bombacoideae). Taxon 61, 1240–1250.
Rasoamanana, E.N., Razanamaro, O., Ramavovololona, P., Ramamonjisoa, R.Z., Verdeil,
J.L., Danthu, P., Suárez-Cervera, M., 2014. Pollen wall ultrastructure of the genus
Adansonia L. species. Plant Syst. Evol. 301, 541–554.
Razanamaro, O., Rasoamanana, E., Rakouth, B., Randriamalala, J.R., Rabakonadrianina,
E., Clément-Vidal, A., Pock, Leong, Tsy, J.-M., Menut, C., Danthu, P., 2015. Chemical
characterization of floral scents in the six endemic baobab species (Adansonia sp.) of
Madagascar. Biochem. Syst. Ecol. 60, 238–248.
Razanameharizaka, J.H.H., 2009. Régénération, démographie, physiologie de la graine et
des plantules du genre Adansonia à Madagascar. Antananarivo.
Ryckewaert, P., Razanamaro, O., Rasoamanana, E., Rakotoarimihaja, T.,
Ramavovololona, P., Danthu, P., 2011. Les Sphingidae, probables pollinisateurs des
baobabs malgaches. Bois For. Trop. 307, 57–68.
Sari-Gorla, M., Pe, M.E., Mulcahy, D.L., Ottaviano, E., 1992. Genetic dissection of pollen
competitive ability in maize. Heredity 69, 423.
Shivanna, K.R., 1982. Pollen-pistil interaction and control of fertilization. In: Johri,
P.B.M. (Ed.), Experimental Embryology of Vascular Plants. Springer, Berlin
Heidelberg, pp. 131–174.
Snow, A.A., Spira, T.P., 1991. Differential pollen-tube growth rates and nonrandom fer-
tilization in Hibiscus moscheutos (Malvaceae). Am. J. Bot. 78, 1419–1426.
Tilton, V.R., 1984. Stigma, style, and pollen recognition in Galinsoga quabriradiata Ruiz &
Pav. (Compositae). Caryologia 37, 423–433.
Visser, T., Marcucci, M.C., 1983. Pollen and pollination experiments. IX. The pioneer
pollen effect in apple and pear related to the interval between pollinations and the
temperature. Euphytica 32, 703–709.