Pollen size varies extensively among angiosperm species and partially reflects evolutionary adaptation of each species to the pollination and fertilization environment. Size of uninuclear microspores in Aesculus parviflora was analyzed and compared with the size of microspores in Aesculus hippocastanum, Aesculus carnea, and Aesculus flava. The microspores came from closed flower buds of different size (3, 4, and 5 mm) isolated from lower (female flowers), middle (bisexual flowers), and upper (male flowers) segments of inflorescences. Aesculus parviflora had smaller microspores than Aesculus carnea and Aesculus flava, but larger microspores than Aesculus hippocastanum. All analyzed microspores showed bimodal distribution in all investigated species of the genus Aesculus.Projekat ministarstva br. 14302

Miljković, Danijela

Radojević, Ljiljana

Zdravković-Korać, Snežana R.

Ćalić-Dragosavac, Dušica D.

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

INTRODUCTION
Pollen size varies extensively among angiosperm 
species. This variation partially reflects evolution-
ary adaptation of each species to the pollination 
and fertilization environment. Thus, the competi-
tive advantages of large pollen may be outweighed 
by the numerous advantages of small pollen, given 
the specific reproductive environment of the species 
(Sarkissian and Harder, 2001). Pollen dimorphism 
has been detected in anthers of some woody spe-
cies, including Aesculus hippocastanum (Radojević 
1989, 1991; Ćalić et al., 2003-2004), Aesculus carnea 
(Marinković and Radojević, 1992), and Aesculus 
flava (Ćalić-Dragosavac et al., 2008). Also, scanning 
and transmission electron microscopy of the genus 
Aesculus confirmed significant differences in pol-
len size and shape (Pozhidaev, 1995). In statistics, 
a bimodal distribution is a continuous probability 
distribution with two different modes. These appear 
as distinct peaks (local maxima) in the probability 
density function.
The buckeye genus Aesculus is one of orna-
mental trees, notably the horse chestnut (Aesculus 
hippocastanum), which is grown for its winter buds, 
large leaves, and striking inflorescence. The genus 
Aesculus has 13-19 species and is one of the most 
remarkable examples of intercontinental disjunc-
tion of plants in the Northern Hemisphere. Aesculus 
hippocastanum (horse chestnut) can rarely be found 
in woods as a cultured species, althrough it grows 
under different ecological conditions. Because of 
its beautiful and dense crown, horse chestnut is 
frequently planted either as a solitary tree or in 
avenues. Aesculus carnea is an artificial hybrid of 
A. hippocastanum L. and A. pavia L.; because of 
its beautiful red inflorescence, it is widely used as 
an ornamental tree. Aesculus species have different 
medicinal or cosmetic uses. The bark of the horse 
chestnut contains small amounts of gallic and tannic 
acids, which are used in industry. 
Bottlebrush buckeye (Aesculus parviflora Walt.) 
is one of the most beautiful flowering shrubs in 
North America. Aesculus parviflora flowers, as well 
as the flowers of Aesculus hippocastanum, Aesculus 
carnea, and Aesculus flava, located in the basal part 
of panicle are female and fertile, while flowers in 
COMPARATIVE ANALYSIS OF MICROSPORE SIZE VARIABILITY
IN THE GENUS AESCULUS (HIPPOCASTANACEAE)
DUŠICA ĆALIĆ-DRAGOSAVAC1, SNEŽANA ZDRAVKOVIĆ-KORAĆ1,
DANIJELA MILJKOVIĆ2, and LJILJANA RADOJEVIĆ1
1Department of Plant Physiology, Siniša Stanković Institute for Biological Research, 11060 Belgrade, Serbia
2Department of Evolutionary Biology, Siniša Stanković Institute for Biological Research, 11060 Belgrade, Serbia
Abstract — Pollen size varies extensively among angiosperm species and partially reflects evolutionary adaptation of 
each species to the pollination and fertilization environment. Size of uninuclear microspores in Aesculus parviflora was 
analyzed and compared with the size of microspores in Aesculus hippocastanum, Aesculus carnea, and Aesculus flava. The 
microspores came from closed flower buds of different size (3, 4, and 5 mm) isolated from lower (female flowers), middle 
(bisexual flowers), and upper (male flowers) segments of inflorescences. Aesculus parviflora had smaller microspores 
than Aesculus carnea and Aesculus flava, but larger microspores than Aesculus hippocastanum. All analyzed microspores 
showed bimodal distribution in all investigated species of the genus Aesculus.
Key words: Aesculus, Hippocastanaceae, pollen dimorphism, microspore diameter
UDC 582.746.56:581.33:575
795
Arch. Biol. Sci., Belgrade, 61 (4), 795-800, 2009                  DOI:10.2298/ABS0904795C
D. ĆALIĆ-DRAGOSAVAC ET AL.796
the middle are bisexual and those on top are male 
(Heywood, 1978). As for inflorescences, we can 
define three different segments independent of 
flower sexuality. Female flowers (segment A) have 
pistils and stamens, but if the anthers do not open 
stamens will peak prematurely. On the other hand, 
bisexual flowers (segment B) have normally devel-
oped and functional pistils and stamens. Male flow-
ers (segment C) have undeveloped pistils and never 
form fruits (Heywood, 1978). 
MATERIALS AND METHODS
The influence of the position of closed flower buds 
in relation to the axis of the inflorescence on micro-
spore size in four species of Aesculus is examined 
in this paper. Anthers were collected from elite A. 
parviflora, A. hippocastanum, and A. flava trees, 
growing in the Botanical Garden of Belgrade, as well 
as from an A. carnea Hayne tree growing in a local 
park. The flowers of all four species are bisexual and 
zygomorphic. Uninuclear microspores were isolated 
from closed flower buds of different size (3, 4, and 5 
mm) orginating from inflorescence segments A, B, 
and C of A. parviflora, A. hippocastanum, A. carnea, 
and A. flava. 
Anthers were longitudinally resected and free 
microspores were stained with a 1% orcein solution 
prepared in 45% acetic acid (Fig. 1). Three hundred 
microspores were analyzed from each closed flower 
bud. Aceto-orcein-treated microspores were viewed 
Fig. 1. 1) Inflorescence with closed flower buds of Aesculus parviflora. 2) Microspores of A. parviflora stained with aceto-orcein 
(small microspore – arrow; x 40). 3) Microspores of A. parviflora stained with fluorescein-diacetate (x 10). 4) DAPI staining uni-
nucleate (N-nucleus) microspores (ultraviolet exciter BP340-380 and Y50 barrier filter; x 100).
COMPARATIVE ANALYSIS OF MICROSPORE SIZE VARIABILITY IN THE GENUS AESCULUS 797
with a Leica DMRB microscope (Wetzlar, Germany) 
and analyzed using the UTHSCSA Image Tool ver-
sion 3.0 (San Antonio, USA) software program. The 
results were analyzed using a completely random-
ized design and tested according to the least signifi-
cant difference (LSD) test.
A rapid method with fluorescein diacetate (FDA) 
(Heslop-Harrison and Heslop-Harrison, 1970) and 
4’, 6-diamidino-2-phenylindole (DAPI) (Colleman 
and Goff, 1985) was used to determine pollen 
viability, as well as pollen dimorphism in all types 
of flowers. These reagents were also used to observe 
cell divisions in the microspores. Fluorescein diac-
etate (2 mg l-1) dissolved in acetone was diluted (1: 
1) with a 0.5 M sucrose solution. The contents of 
anthers were squeezed out and stained with a DAPI 
(1 μg ml-1) solution prepared in distilled water and 
examined as previously described. The FDA and 
DAPI-treated microspores were viewed with an 
Olympus BX 51 microscope fitted with an ultraviolet 
exciter filter of the B 12 or BP 340-380 type for DAPI 
in combination with a Y 50 barrier filter. 
RESULTS AND DISCUSSION
Uninuclear microspores of A. parviflora, as well 
as uninuclear microspores of Aesculus hippocasta-
num, A. carnea, and A. flava, showed differences 
in size, shape, staining intensity, fluorescence, and 
viability after treatment with fluorescein diacetate. 
Microspores were isolated from closed flower buds 
measuring 3, 4, and 5 mm in length and different 
segments of inflorescences (A, B, and C) (Fig. 1, 
part 1).
Microspores of A. parviflora (14.04-18.56 μm) 
were smaller than microspores of A. hippocastanum 
(16.90-19.29 μm), A. carnea (21.85-26.18 μm), and 
A. flava (23.85-27.18 μm) (Table 1). Also, micro-
spores of all studied species can be divided into two 
groups: small and lightly staining (with acetoorceine 
and acetocarmine); and large and densely staining 
(Fig. 1, parts 2 and 3). Small uninuclear microspores 
(Fig. 1, part 4) were embryogenic, while large ones 
were nonembryogenic. Variability in microspore 
size was confirmed by the presence of bimodal dis-
tribution (Fig. 2).
Table 1. Means values () and standard errors (SE) of microspores size (in µm), were isolated from closed flower buds of different 
length size (3, 4, and 5 mm) from three segments of inflorescences: A (female flowers), B (bisexual flowers), and C (male flowers).
*The values of different letters are significantly different at the 0.05 probability level according to protected LSD test.
Species Length of flower buds (mm)
Diameter of microspores ( ± SE)(µm)
A B C
A. parviflora 14.04 ± 0.19d 16.09 ± 0.19d 18.58 ± 0.31c
A. hippocastanum 3 16.90 ± 0.18c 18.98 ± 0.16c 19.29 ± 0.16c
A. carnea 21.85 ± 0.22b 24.05 ± 0.26b 26.18 ± 0.27b
A. flava 23.85 ± 0.23a 25.05 ± 0.28a 27.18 ± 0.29a
A. parviflora 18.97 ± 0.17d 20.44 ± 0.24d 20.84 ± 0.22d
A. hippocastanum 4 20.46 ± 0.20c 22.14 ± 0.17c 22.31 ± 0.16c
A. carnea 23.87 ± 0.21b 26.02 ± 0.23b 28.16 ± 0.20b
A. flava 26.87 ± 0.22a 28.02 ± 0.27a 30.16 ± 0.26a
A. parviflora 21.19 ± 0.23c 22.63 ± 0.22c 23.02 ± 0.26c
A. hippocastanum 5 22.34 ± 0.17c 22.88 ± 0.14c 23.43 ± 0.20c
A. carnea 25.34 ± 0.25b 27.55 ± 0.29b 30.32 ± 0.31b
A. flava 28.34 ± 0.21a 30.25 ± 0.23a 33.32 ± 0.25a
D. ĆALIĆ-DRAGOSAVAC ET AL.798
Fig. 2. Bimodal distribution of uninuclear microspore size (in 3, 4, and 5 mm closed flower buds) from inflorescence segments A, B, 
and C of A. parviflora, A. hippocastanum, A. carnea, and A. flava.
COMPARATIVE ANALYSIS OF MICROSPORE SIZE VARIABILITY IN THE GENUS AESCULUS 799
The 3-mm-long flower buds contained signifi-
cantly smaller microspores compared to the 4- and 
5-mm-long flower buds in all (A-C) segments of the 
inflorescence. Depending on bud size, the smallest 
microspores were in segment A (from 23.85 to 28.34 
μm), the largest in the segment C (from 27.18 to 
33.32 μm). In addition, average size of microspores 
varied depending on bud size and segment, by about 
10 μm. The two characteristic peaks had different 
values for microspores derived from flower buds of 
the same length, but from different segments.
The presence of variability and bimodal dis-
tribution of size in uninuclear microspores of four 
investigated species of the genus Aesculus (Fig. 2) 
was in correlation with the results of Nägeli (1998) 
and Ćalić et al. (2003-2004). Our findings that uni-
nuclear microspores isolated from closed flower 
buds showed differences in size, shape, staining 
intensity, fluorescence, and viability in four species 
of Aesculus are in agreement with results on Aesculus 
hippocastanum (Radojević, 1989, 1991; Radojević et 
al., 2000; Ćalić et al., 2003-2004), Aesculus carnea 
(Marinković and Radojević, 1992), and Aesculus 
flava (Ćalić-Dragosavac et al., 2008). Our results 
confirm differences of microspore size in 13 species 
of the genus Aesculus (Pozhidaev, 1995).
Acknowledgment — This study was financially supported by the 
Ministry of Science and Environmental Protection of Serbia 
(Grant No. 143026).
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D. ĆALIĆ-DRAGOSAVAC ET AL.800
REZIME
АЛЕКСАНДРА НИКОЛИЋ1, С. КНЕЖЕВИЋ2, МИЛА КРСМАНОВИЋ3, М. МИЦЕВ3,
М. РИСТАНОВИЋ4 и ДРАГИЦА РАДОЈКОВИЋ1
1Институт за молекуларну генетику и генетичко инжењерство, 11010 Београд, Србија
2Прва хируршка клиника, Клинички центар Србије, 11000 Београд, Србија
3Одељење за хистопатологију, Институт за болести дигестивног система, Клинички центар Србије,
11000 Београд, Србија
4Институт за хуману генетику, Медицински факултет, Универзитет у Београду, 11000 Београд, Србија
Ово истраживање је имало за циљ да се 
анализира присуство мутације у кодону 12 
гена K-ras у панкреасном ткиву пацијената 
са карциномом панкреаса и да се процени да 
ли ова мутација представља потенцијални 
молекуларни маркер за карцином панкреаса у 
српској популацији. Истраживање је обухватило 
анализу 40 узорака ткива панкреаса пацијената 
са клиничком дијагнозом карцинома панкреаса. 
Присуство мутације у кодону 12 гена K-ras 
анализирано је методом PCR-RFLP (polymerase 
chain reaction - restriction fragment length poly-
morphism). Резултати истраживања указују да 
је мутација у кодону 12 гена K-ras у ткиву 
панкреаса присутна са високом учесталошћу (66 
%) код пацијената са карциномом панкреаса.


Comparative analysis of microspore size variability in the genus Aesculus (Hippocastanaceae)

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Comparative analysis of microspore size variability in the genus Aesculus (Hippocastanaceae)

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