2986 Advances in Environmental Biology, 6(11): 2986-2991, 2012 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

A study of microsporogenesis and pollen morphology in babakhanloui ()

Rahmani Hamideh, Majd Ahmad, Arbabian Sedigheh, Sharfnia Fariba, Mehrabian Sedigheh

Department of Biology, North Tehran Branch, Islamic Azad University, Tehran, Iran

Rahmani Hamideh, Majd Ahmad, Arbabian Sedigheh, Sharfnia Fariba, Mehrabian Sedigheh; A study of microsporogenesis and pollen morphology in Crataegus babakhanloui (Rosaceae)

ABSTRACT

In this study, microsporogenesis and pollen morphology of Crataegus babakhanloui were studied. The , in different developmental stages, were removed, fixed in Formalin -glacial acetic acid- alcohol (FAA), stored in 70% ethanol, embedded in paraffin and then sliced at 8-10 μm by rotary microtome. Staining was carried out by periodic Acid Schiff (PAS) and contrasted with hematoxylin. Scanning electron microscope (SEM) was used to analyze the mature pollen grains. The results indicated that anthers wall development followed the dicotyledonous type and were tetrasporangiate witch composed of epidermal layer, endothecium layer, two rows of middle layers and then tapetum layer. Tapetum was dimorphic in early and late process. Microspore tetrads are tetrahedral and Pollen grains are shed at bicellular stage. Pollen grains are tricolporate, medium size and prolate. Exine sculpturing is striate with perforations on grain surface.

Key words: Crataegus babakhanloui, Microsporogenesis, Pollen grain.

Introduction in diameter, almost spherical, purplish-black and dusty with 3-4 stones. Hawthorn (Crataegus spp.) ornamentally and Hawthorns provide food and shelter for many medically has a big name in science history. The species of birds and mammals, and the flowers are genus Crataegus belongs to the subfamily Maloideae important for many nectar-feeding [3,17]. in the Rosaceae family and it is estimated that they Hawthorn are consumed fresh or processed into include 150 to 1200 species [8]. It is grows in jams, jellies, soft drinks, candies and canned fruits. northern temperate regions such as East Asia, Hawthorns have been used in traditional Europe, and eastern North America [36] and has 17 medicine, and there is considerable interest in testing endemic species in Iran, One of indigenous species hawthorn products for evidence-based medicine [18]. which is Crataegus babakhanloui [24]. Clinical investigations and other research suggest Crataegus babakhanloui specie is or that extracts of hawthorn fruits, flowers and small , mostly growing to 2-3 meters, with have multiple health effects including small pome and (rarely) thorny branches. hypolipidaemic, anti-atherosclerotic, antioxidant, Hairy leaves are 3-6 cm long and 2-5 cm broad, with ant-diabetic, hypotensive, Immunomodulatory, 3-7 shallow, forward-pointing lobes on each side of cardioprotective, antiatherogenic, anti-inflammatory, the , obovate and deeply lobed, sometimes almost Hepatoprotective, antihypercholesterolaemia and to the midrib, with the lobes spreading at a wide blood vessel relaxing activities [38,27,16]. angle. Hairy Petiole is length of about 2 cm diameter. Active ingredients found in hawthorn include The hermaphrodite flowers are produced in corymbs tannins, flavonoids (such as vitexin, rutin, quercetin, of 9-15, each about 12 to 15 mm diameter and hyperoside), oligomeric proanthocyanidins with five sepals which are three angle and flat, five (OPCs, such as epicatechin, procyanidin, and white petals, 20 stamens and three or four (rarely particularly procyanidin B-2), flavone-C, triterpene two) styles. Generally flower abundantly during late acids (such ursolic acid, oleanolic acid, and spring and early summer. Their open and crataegolic acid), and phenolic acids (such as caffeic unspecialized flowers are pollinated by flies, beetles, acid, chlorogenic acid, and related phenolcarboxylic bees and occasionally by ants (Dickson [13], acids) [40,38]. Notably, nothing is known about the Christensen pers. obs., cited in Christensen [8]). basic anther structure and development of pollen the They usually produce large numbers of fruits during specie Crataegus babakhanloui. late summer and autumn [8]. The fruit is 10-12 mm

Corresponding Author Rahmani Hamideh, Department of Biology, North Tehran Branch, Islamic Azad University, Tehran, Iran. E-mail: [email protected] Tel: +989124269497 2987 Adv. Environ. Biol., 6(11): 2986-2991, 2012

Present research is the first study on samples were dehydrated during alcohol series, and microsporogenesis and pollen morphology of Iranian embedded in paraffin after the process of paraffin indigenous specie of Crataegus. This paper offers a saturation in toluene. With rotary microtome, partial remedy to this situation by providing an sections of 8-10 μm thickness were taken from the account of anther wall, tapetum, microspore and materials embedded in paraffin. Staining was carried male gametophyte development and pollen out with PAS (periodic Acid Schiff) according to morphology in Crataegus babakhanloui. protocol suggested by Yeung [37] and contrasted The main aim of this paper is to present a Meyer’s Hematoxylin. They were mounted in detailed study on microsporogenesis and Pollen Canada balsam, examined with a Zeiss Axiostar plus morphology of Crataegus babakhanloui and than to light microscope. Many sample studied fore each use the result of pollen feature for evaluating of stage and photomicrographs. taxonomic relationships among Crataegus species. C: palynological studies: Material and Methods Using a SEM model XL30 (Philips), Flowers A. material: were opened under a binocular microscope using a needle. Pollen grains were mounted on 12.5 mm In this study flowers buds were collected at diameter stubs and than coated in a sputter coater different developmental stage between 9:00 - 10:00 with 25nm of gold palladium at an accelerating AM, during the period 9th may - 5 November in years voltage of 15-25 kV. SEM photographs from the 2011-2012 from Abharak in north of Iran (Fig.1). equatorial and polar view and exine of pollen grains were captured at 500 x, 2500x, 2500x and 7500x B. morphological studies: magnifications. The terminology follows that of Punt et al., [30] and Halbritter et al., [19]. Flowers were fixed in FAA (Formalin -glacial acetic acid- alcohol). After the fixation process; the

Fig. 1: Photo of Crataegus babakhanloui.

Result: nuclei are evident in the tapetal cells at this point (Fig. 2, 1 and 2). Two basic type of tapetum Male gametophyte development: hasamoeboidal (periplasmoidal) type and secretory type is visible in this species, as at the start of The anther is tetrasporangiate and dehisces microsporogenesis see secretory tapetum and final longitudinally. From the cross section we see that the stage is (periplasmoidal) type. In early stage of anther wall consists of five layers from outer to meiosis, Microsporocytes separate with callose inner: an epidermis, endothecium, two rows of walls; tapetal cells more or less enlarge (Fig. 2, 3 and middle layer and a layer of tapetum (Fig. 2). 4) following meiosis I, macrospore dyads have Microspores and pollen grains are produced resulted. After meiosis II numerous tetrads of from pollen mother cells (Microsporocytes), PMCs microspores appear, tetrad shape is tetrahedral and within loculus anthers (microsporangia or pollen the wall formation taking place after each stage of sacs) of the flower. Microsporocytes are recognizable meiosis. Soon, the wall surrounding the 4 microspore by their large volume, dense cytoplasm, and cells dissolve and the microspores in the pollen sac conspicuous nuclei. The single cell layer of tapetum are released. As a result, the mitotic division of the surrounding the anther loculus is first recognizable at microspore is unequal and gives origin to two cells: the early Microsporocyte stage (Fig. 2, 1 and 2). the cells of a darker and smaller generative nucleus Large volume, dense cytoplasm and one or two large and a lighter and larger vegetative nucleus appear. So

2988 Adv. Environ. Biol., 6(11): 2986-2991, 2012 pollen grains have two-celled at the time of shedding 3-10). Exin (Ex) and Intin (In) are formed in this stage (Fig.

Fig. 2: (1): Section of single loculuse showing early microspore mother cell (MmC). The loculuse is lined by epiderm (Epi), Endothecium (End), middle layer (m.l.) and tapetum (T). (2): tetrasporangiate anther showing early microspore mother cell (MmC), epiderm (Epi), Endothecium (End), middle layer (m.l.) and tapetum (T). (3, 4): Section of single loculuse showing porophase meiosis I, callosic wall are visible that surrounding early meiosis stages. (5): Pollen grain in dyad stage (arrow show cell wall). (6): tetrahedral tetrads in the anther loculus.

2989 Adv. Environ. Biol., 6(11): 2986-2991, 2012

Fig. 3: ( 7): Section of single loculuse showing later tetrad stage. Not those extensions of tapetal cells are visible (Tp). (8): cross section of adult anther (9): endothecium(End) layer are tangentially elongated and have spiral lignin thickenings (10): mature pollen grain with Exin (Ex) and Intine (In), nucleus (Nu).

Fig. 4: (1,2): SEM micrographs of pollen grain of Crataegus babakhanloui (3)The exin pattern of pollen of Crataegus babakhanloui.

By the time the pollen grains are mature, the During meiosis, the cells of endothecium layer tapetal cells have completely degenerated, are tangentially elongated and have spiral meanwhile the cells of middle layers grow radially thickenings which are made of lignin. At the time of and tangentially as the anther matures. In meiotic dehiscence, the endothecium is still intact the anther division, the middle layers cells start a slow connective varies from round to flat. Crystals of degeneration process. After the tetrads are formed, Calcium oxalate are common in the anther walls. the inner most of the middle layers begins to degenerate and all the middle layers become flatted General pollen morphological featuers: during the free microspore stage and have degenerated completely at the mature pollen grain Pollen grains tricolporate. Polar axis (P) (37- ) stage. The epidermis in C. babakhanloui preserves its 38 (-40) and equatorial axis (E) (18) 20 (-21) μm. vitality until the period of anther splitting. Shape (P/E) is medium size and prolate. Pollen side walls in polar view are convex or circular-angular.

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