Dragićević, Ivana

Pemac, Danijela

Radojević, Ljiljana

Ćalić-Dragosavac, Dušica D.

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

Variability of size in uninucleate microspores was observed in 
yellow buckeye (Aesculus flava Marshall). Microspores were iso-
lated from closed flower buds (3, 4 and 5 mm) and different seg-
ments (A, B and C) of inflorescences. Investigation of bimodal 
distribution confirmed the presence of pollen dimorphism in 
all types of flowers.
Aesculus flava (syn. A. octandra) is a species of buckeye 
native to eastern North America, from Pennsylvania, west to 
eastern Illinois, and south to the northernmost parts of Alabama 
and Georgia. Pollen dimorphism has been detected in anthers 
of some woody species. Uninuclear microspores isolated from 
closed flower buds in horse chestnut (R a d o j e v i ć , 1989, 1991; 
Ć a l i ć  et al., 2003/4) and red chestnut (M a r i n k o v i ć  and 
R a d o j e v i ć , 1992) showed differences in size, shape, stain-
ing intensity, fluorescence, viability, and embryogenic potential 
(R a d o j e v i ć  et al., 2000). Moreover, pollen orginating from 
13 species of the genus Aesculus showed differences in size and 
shape, pore position, sculpturing of the colpal membrane, and 
sculpturing of the mesocolpia (P o z h i d a e v, 1995). The small 
yellow-green inflorescence of Aesculus flava is about 17 cm 
long and 7 cm wide. Composed of an upright panicle of many 
solitary flowers, it appears in mid-May, the inflorescence clearly 
rising above the expanded foliage.
As in flowers of Aesculus hippocastanum, A. flava flowers 
located in the basal part of the panicle are female and fertile, 
while flowers in the middle are bisexual and those on the top 
are male (H e y w o o d , 1978). 
The female flowers (segment A) have pistils and stamens, 
but the stamens peak prematurely and the anthers do not open, 
while bisexual flowers (segment B) have normally developed 
and functional pistils and stamens.
The male flowers (segment C) have undeveloped pistils and 
never form fruits (H e y w o o d , 1978). 
Uninuclear microspores were isolated from closed flower 
buds of different size (3, 4, and 5 mm) orginating from inflo-
rescence segments A, B, and C. Anthers were resected and free 
microspores were stained with 1% orcein solutions prepared 
in 45% acetic acid (Fig. 1). Three hundred microspores were 
analyzed from each closed flower bud. Aceto-orcein-treated 
microspores were a DMRB microscope from Leica (Wetzlar, 
Germany) and analyzed with the Image Tool software program 
(version 3.0) from UTHSCSA (San Antonio, USA). The results 
were analyzed using a completely randomized design.
VARIABILITY AND BIMODAL DISTRIBUTION OF SIZE IN UNINUCLEAR MICROSPORES OF 
AESCULUS FLAVA MARSHALL. Dušica Ćalić-Dragosavac1, Danijela Pemac2, Ivana Dragićević3, and 
Ljiljana Radojević1.  1Department of Plant Physiology and 2Department of Evolutionary Biology, Siniša 
Stanković Institute for Biological Research, 11060 Belgrade, Serbia; 3Institute of Botany, Faculty of Biology, 
University of Belgrade, 11000 Belgrade, Serbia.
UDC  581.33:582.766.5:575
3P
Arch. Biol. Sci., Belgrade, 60 (1), 3P-4P, 2008		 	 	 	 	 																	              DOI:10.2298/ABS080103PC
Keywords: Yellow buckeye, pollen dimorphism, microspore diameter
Fig. 1-4. Microspores of Aesculus flava stained with aceto-orcein (smaller microspore – arrow; x40) and fluorescein-diacetate (Fig. 2, x20; 
and Fig. 3, x40). Fig. 4. DAPI-staining uninucleate (N-nucleus) microspores (ultraviolet exciter BP340-380 and Y50 barrier filter; x40).
Flower buds 3 mm in length contained significantly smaller 
microspores than 4- and 5-mm-long flower buds in all (A-C) 
segments of the inflorescence. Independent of bud size, the 
smallest microspores (from 23.85 to 28.34 µm) were in segment 
A, the largest (from 27.18 to 33.32 µm) in segment C. In addi-
tion, average microspore size varied by about 10 µm, depending 
on bud size and segment. Microspores of A. flava have shorter 
and longer diameters in all the investigated flower buds. It has 
been suggested that uninuclear microspores of A. flava, like 
microspores of A. hippocastanum (R a d o j e v i ć , 1989, 1991; 
Ć a l i ć  et al., 2003/4), can be grouped in two classes: small, 
lightly staining (with aceto-orceine and fluorescein-diacetate); 
and large, densely staining (Figs. 1, 2, and 3). Small uninuclear 
microspores (Fig. 4) are androgenic, while large ones are 
non-androgenic (R a d o j e v i ć , 1989, 1991). Variability of 
microspore size was confirmed by the presence of a bimodal 
distribution, with lower and higher peaks (Fig. 5). The two 
characacteristic peaks had different values for microspores 
derived from flower buds of the same length, but originating 
from different segments. The presence variability and a bimodal 
distribution of size in uninuclear microspores of A. flava (Fig. 5) 
is in agreement with the results of N ä g e l i  (1998) and Ć a l i ć 
et al. (2003/4). 
Acknowledgment - This study was financially supported by 
the Serbian Ministry of Science and Environmental Protection 
(Grant 143026).
References: - Ćalić, D., Zdravković-Korać, S., Pemac, D., and 
Lj. Radojević (2003/4). Biol. Plant. 47, 457-458. - Nageli, M. 
(1998): Ph. D. Thesis No.12808. ETH, Zürich. - Heywood, V. H. 
(1978). In: Heywood,V. H. (Ed.), Flowering plants in the World, 
194-196. Oxford – London - Melbourne. - Marinković, N., Lj. 
Radojević (1992). Plant Cell Tissue Organ Cult. 31, 51-59. - 
Pozhidaev, A. E. (1995). Grana 4, 10-20. - Radojević, Lj. (1989). 
Arch. Biol. Sci. (Belgrade) 41, 137-143. - Radojević, Lj. (1991). In: 
Bajaj, Y. P. S. (Ed.), Biotechnology in Agriculture and Forestry, 
111-141. Springer – Verlag, Berlin – Heidelberg. - Radojević, Lj., 
Marinković, N., and S. Jevremović (2000). In Vitro Cell Dev. Biol. 
Plant. 36, 464-469.
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Fig. 5. Bimodal distribution of microspore size (in 3, 4, and 5 
mm closed flower buds) from segments A, B and C of Aesculus 
flava inflorescences.


Variability and bimodal distribution of size in uninuclear microspores of Aesculus flava Marshall

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Variability and bimodal distribution of size in uninuclear microspores of Aesculus flava Marshall

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