COMPARATIVE ANALYSIS OF MICROSPORE SIZE VARIABILITY IN THE GENUS AESCULUS (HIPPOCASTANACEAE)

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.


INTRODUCTION
Pollen size varies extensively among angiosperm species.This variation partially reflects evolutionary adaptation of each species to the pollination and fertilization environment.Thus, the competitive 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 species, including Aesculus hippocastanum (Radojević 1989(Radojević , 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 pollen 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 ornamental 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 disjunction 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)
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 developed and functional pistils and stamens.Male flowers (segment C) have undeveloped pistils and never form fruits (Heywood, 1978).

MATERIALS AND METHODS
The 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 with a Leica DMRB microscope (Wetzlar, Germany) and analyzed using the UTHSCSA Image Tool version 3.0 (San Antonio, USA) software program.The results were analyzed using a completely randomized design and tested according to the least significant 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 diacetate (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.

Microspores of
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)  The 3-mm-long flower buds contained significantly 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.
influence of the position of closed flower buds in relation to the axis of the inflorescence on microspore 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.