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 Crataegus babakhanloui (Rosaceae) 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 flowers, 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 Hawthorn (Crataegus spp.) ornamentally and medically has a big name in science history. The genus Crataegus belongs to the subfamily Maloideae in the Rosaceae family and it is estimated that they include 150 to 1200 species [8]. It is grows in northern temperate regions such as East Asia, Europe, and eastern North America [36] and has 17 endemic species in Iran, One of indigenous species which is Crataegus babakhanloui [24]. Crataegus babakhanloui specie is shrubs or small trees, mostly growing to 2-3 meters, with small pome fruit and (rarely) thorny branches. Hairy leaves are 3-6 cm long and 2-5 cm broad, with 3-7 shallow, forward-pointing lobes on each side of the leaf, obovate and deeply lobed, sometimes almost to the midrib, with the lobes spreading at a wide angle. Hairy Petiole is length of about 2 cm diameter. The hermaphrodite flowers are produced in corymbs of 9-15, each flower about 12 to 15 mm diameter with five sepals which are three angle and flat, five white petals, 20 stamens and three or four (rarely two) styles. Generally flower abundantly during late spring and early summer. Their open and unspecialized flowers are pollinated by flies, beetles, bees and occasionally by ants (Dickson [13], Christensen pers. obs., cited in Christensen [8]). They usually produce large numbers of fruits during late summer and autumn [8]. The fruit is 10-12 mm

in diameter, almost spherical, purplish-black and dusty with 3-4 stones. Hawthorns provide food and shelter for many species of birds and mammals, and the flowers are important for many nectar-feeding insects [3,17]. Hawthorn fruits are consumed fresh or processed into jams, jellies, soft drinks, candies and canned fruits. Hawthorns have been used in traditional medicine, and there is considerable interest in testing hawthorn products for evidence-based medicine [18]. Clinical investigations and other research suggest that extracts of hawthorn fruits, flowers and leaves have multiple health effects including hypolipidaemic, anti-atherosclerotic, antioxidant, ant-diabetic, hypotensive, Immunomodulatory, cardioprotective, antiatherogenic, anti-inflammatory, Hepatoprotective, antihypercholesterolaemia and blood vessel relaxing activities [38,27,16]. Active ingredients found in hawthorn include tannins, flavonoids (such as vitexin, rutin, quercetin, and hyperoside), oligomeric proanthocyanidins (OPCs, such as epicatechin, procyanidin, and particularly procyanidin B-2), flavone-C, triterpene acids (such ursolic acid, oleanolic acid, and crataegolic acid), and phenolic acids (such as caffeic acid, chlorogenic acid, and related phenolcarboxylic acids) [40,38]. Notably, nothing is known about the basic anther structure and development of pollen the specie Crataegus babakhanloui.

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 microsporogenesis and pollen morphology of Iranian indigenous specie of Crataegus. This paper offers a partial remedy to this situation by providing an account of anther wall, tapetum, microspore and male gametophyte development and pollen morphology in Crataegus babakhanloui. The main aim of this paper is to present a detailed study on microsporogenesis and Pollen morphology of Crataegus babakhanloui and than to use the result of pollen feature for evaluating of taxonomic relationships among Crataegus species.

samples were dehydrated during alcohol series, and embedded in paraffin after the process of paraffin saturation in toluene. With rotary microtome, sections of 8-10 μm thickness were taken from the materials embedded in paraffin. Staining was carried out with PAS (periodic Acid Schiff) according to protocol suggested by Yeung [37] and contrasted Meyer’s Hematoxylin. They were mounted in Canada balsam, examined with a Zeiss Axiostar plus light microscope. Many sample studied fore each stage and photomicrographs. C: palynological studies:

Material and Methods A. plant material: In this study flowers buds were collected at different developmental stage between 9:00 - 10:00 AM, during the period 9th may - 5 November in years 2011-2012 from Abharak in north of Iran (Fig.1). B. morphological studies:

Using a SEM model XL30 (Philips), Flowers were opened under a binocular microscope using a needle. Pollen grains were mounted on 12.5 mm diameter stubs and than coated in a sputter coater with 25nm of gold palladium at an accelerating voltage of 15-25 kV. SEM photographs from the equatorial and polar view and exine of pollen grains were captured at 500 x, 2500x, 2500x and 7500x 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: Male gametophyte development: The anther is tetrasporangiate and dehisces longitudinally. From the cross section we see that the anther wall consists of five layers from outer to inner: an epidermis, endothecium, two rows of middle layer and a layer of tapetum (Fig. 2). Microspores and pollen grains are produced from pollen mother cells (Microsporocytes), PMCs within loculus anthers (microsporangia or pollen sacs) of the flower. Microsporocytes are recognizable by their large volume, dense cytoplasm, and conspicuous nuclei. The single cell layer of tapetum surrounding the anther loculus is first recognizable at the early Microsporocyte stage (Fig. 2, 1 and 2). Large volume, dense cytoplasm and one or two large

 

nuclei are evident in the tapetal cells at this point (Fig. 2, 1 and 2). Two basic type of tapetum hasamoeboidal (periplasmoidal) type and secretory type is visible in this species, as at the start of microsporogenesis see secretory tapetum and final stage is (periplasmoidal) type. In early stage of meiosis, Microsporocytes separate with callose walls; tapetal cells more or less enlarge (Fig. 2, 3 and 4) following meiosis I, macrospore dyads have resulted. After meiosis II numerous tetrads of microspores appear, tetrad shape is tetrahedral and the wall formation taking place after each stage of meiosis. Soon, the wall surrounding the 4 microspore cells dissolve and the microspores in the pollen sac are released. As a result, the mitotic division of the microspore is unequal and gives origin to two cells: the cells of a darker and smaller generative nucleus 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 Exin (Ex) and Intin (In) are formed in this stage (Fig.

3-10).

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 tapetal cells have completely degenerated, meanwhile the cells of middle layers grow radially and tangentially as the anther matures. In meiotic division, the middle layers cells start a slow degeneration process. After the tetrads are formed, the inner most of the middle layers begins to degenerate and all the middle layers become flatted during the free microspore stage and have degenerated completely at the mature pollen grain stage. The epidermis in C. babakhanloui preserves its vitality until the period of anther splitting.

 

During meiosis, the cells of endothecium layer are tangentially elongated and have spiral thickenings which are made of lignin. At the time of dehiscence, the endothecium is still intact the anther connective varies from round to flat. Crystals of Calcium oxalate are common in the anther walls. General pollen morphological featuers: Pollen grains tricolporate. Polar axis (P) (37- ) 38 (-40) and equatorial axis (E) (18) 20 (-21) μm. Shape (P/E) is medium size and prolate. Pollen side walls in polar view are convex or circular-angular.

2990 Adv. Environ. Biol., 6(11): 2986-2991, 2012

Colpus width is 29-31 μm. Endoapertures (Pori) Circular or almost square in outline, Exine striateperforate; striate (ridge –and – valley – pattern) crowded, usually parallels together or colpus, 0/270/33 μm in width and perforations 0/07 – 0/1 μm in length (fig 4)

1.

2.

Discussion: 3. In this study, we describe the detailed developmental and morphological characters of pollen grains of the one specie Crataegus in Iran. According to Khatamsaz [24] howthron has 17 indigenous species in Iran .one of the species which is Crataegus babakhanloui. The anther wall development coincides with the dicotyledonous type [10]. In family Rosaceae, anthers are monosporangiate, the method of microsporogenesis in Crataegus babakhanloui is the unparallel those reports for other species of Rosaceae, Such as having tetrasporangiate, two type of tapetum [26,5]. Microspore mitosis is an asymmetric division. This division can also be viewed as a determinative division in that the resulting two cells have very different cell fates [22]. Because of a lake of sufficient data about embryology from other Crataegus, we have not compared the microsporogenesis characters of C. babakhanloui with those of many other species. In the general evaluation of pollen surface, the pollen grain shows striate type of exine sculpture with perforations in the grooves confirming the general characteristics for the pollen of Rosaceae [32]. The most frequent exine sculpturing in family Rosaceae is striate or striate- perforate [14,39] our result also show striate- perforate and confirming the general characteristics for the pollen of Rosaceae [15]. In current study, pollen size is smaller than the samples analyses by Domnez [14] Medium size and prolate pollen grain and very small diameter of perforations. Numerous studies performed about Pollen morphology of Crataegus in the world. European species have been examined several workers [15,6,31], while that some species from north America was studied by Dickinson and Phipps [13] and Hebda et al. [20] Zhou et al [39] from Turkish Crataegus species, Aytug et al [1], Pehlivan [28] and Donmez [14] have been studied. But pollen morphology of Iranian species such as Crataegus babakhanloui has not been examined. The consequences of our study are consistent with the results of pollen morphological investigation of other Crataegus species. References

 

4.

5.

6.

7. 8.

9.

10. 11.

12.

13.

Aytuğ, B., 1967. Studies on pollen morphology and important gymnosperms of Turkey. Istanbul University Faculty of forest publications. No:1261/114]. Barnes, J., L.A. Anderson and J.D. Phillipson, 1996. Herbal Medicines. A guide for health care professionals. First Ed. Pharmaceutical Press Publication, London, pp: 284287. Bass, D.A., N.D. Crossman, L.S. Lawrie and M.R. Lethbridge, 2006. The importance of population growth, seed dispersal and habitat suitability in determining plant invasiveness, Euphytica, Special Issue: Plant Breeding and Crop Domestication as Sources of New Invasive Species, 148(1-2): 97-109, DOI: 10.1007/s10681-006-5944-6. Belz, G.G., R. Butzer, W. Gaus and D. Loew, 2002. Camphor-Crataegus berry extract combination dose-ependently reduces tilt induced fall in blood pressure in orthostatic hypotension. Phytomedicine, 9: 581-588. Bhojvani, S.S. and W.Y. Soh, 2001.Current trends in the embryology of Angiosperms. Kluver Acad. Publishers, Dordrecht, Netherlands. Bombosi, P., 2000. Crataegus monogyna; Crataegus laevigata. – In: Buchner, R. & Weber, M.. 2000 onwards, PalDat - a palynological database: Descriptions, illustrations, identification , and information retrieval. http://www.paldat.org/ Brown, D., 1995. Encyclopedia of herbs and their uses. Dorling Kindersley Publishers. Christensen, K.I., 1992. Revision of Crataegus sect.Crataegus and Nothosect. Crataeguineae (Rosaceae-Maloideae) in the old World. Syst Bot Monogr, 35: 1-199. Chu, C.Y., M.J. Lee, C.L. Liao, W.L. Lin, Y.F. Yin and T.H. Tseng, 2003. Inhibitory effect of hot-water extract from dried fruit of Crataegus pinnatifida on low-density lipoprotein (LDL) oxidation in cell and cell-free systems. J. Agric. Food Chem., 51: 7583-7588. Davis, G.L., 1966. Systematic embryology of the angiosperms. John Wiley & Sons, New York. Dickinson, T.A. and C.S. Campbell, 1991. Population structure and reproductive ecology in the Maloideae (Rosaceae). Syst. Bot., 16: 350362. Dickinson, T.A., 1983. Crataegus crus-galli L. sensu lato in Southern Ontario: Phenotypic variation and variability in relation to reproductive behavior. PhD Thesis. London, ON, Canada: University of Western Ontario. Dickinson, T.A. and J.B. Phipps, 1986. Studies in Crataegus (Rosaceae: Maloideae) XIV. The breeding system of Crataegus crus-galli sensu lato in Ontario. Am. J. Bot., 73: 116-130.

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

14. Donmez, E.O., 2008. Pollen morphology in TurkishCrataegus (Rosaceae), Bot. Helv., 118: 59-70. 15. Eide, F., 1981. Key for Northwest European Rosaceae pollen. Grana., 20: 101-118. 16. Erl-Shyh, K., W. Chau-Jong and L. Wea-Lung, 2007. Effects of polyphenols derived from fruit of Crataegus pinnatifida on cell transformation, dermal edema and skin tumor formation by phorbol ester application. Food and Chemical Toxicology, 45: 1795-1804. 17. Ferrazzini, D., I. Monteleone and P. Belletti, 2008. Small-scale genetic diversity in oneseed hawthorn (Crataegus monogyn acq.), European Journal of Forest Research, 127(5): 407-414, DOI: 10.1007/s10342-008-0224-a J8. 18. Fong, H. and J. Bauman, 2002. Alternative Medicines for Cardiovascular Diseases-Hawthorn, Journal of Cardiovascular Nursing,16(4): 1-8. 19. Halbritter, H., M. Weber, R. Zetter, A. FroschRadivo, R. Buchner and M. Hesse, 2007. PalDat – illustrated handbook on pollen terminology. Vienna: Society of the Promotion of Palynological Research in Austria; 20. Hebda, R.J., C.C. Chinnappa and B.M. Smith, 1988. Pollen morphology of the Rosaceae of Western Canada I. Agrimonia to Crataegus. Grana., 27: 93-113. 21. Heywood, Vernon H. ed., 1993. Flowering Plants of the World. New York: Oxford University Press. 22. Horvitz, H.R. and L. Herskowitz, 1992. Mechanisms of asymmet- ric cell division: Two Bs or not two Bs, that is the question. Cell, 68: 237-255. 23. Kao, E.S., C.J. Wang, W.L. Lin, Y.F. Yin, C.P. Wang and T.H. Tseng, 2005. Antiinflammatory potential of fl avonoid contents from dried fruit of Crataegus pinnatifi da in vitro and in vivo. J Agr Food Chem., 53: 430-6. 24. Khatamsaz, M., 1991. Flora of Iran, No:6,241267, Research institute of forests and rangelands. 25. Kocyildiz, Z.C., H. Birman, V. Olgac, K. Akgun-Dar, G. Melikoglu and A.H. Mericli, 2006. Crataegus tanacetifolia leaf extract prevents L-NAMEinduced hypertension in rats: a morphological study. Phytother. Res., 20: 6670. 26. Lersten, N.R., 2004. Flowering plant embryology, Blackvell Publishing, Iowa, USA. 27. Ljubuncic, P., I. Portnaya, U. Cogan, H. Azaizeh and A. Bomzon, 2005. Antioxidant activity of Crataegus aronia aqueous extract used in traditional Arab medicine in Israel. Journal of Ethnopharmacology, 101(1-3): 153-161. 28. Pehlivan, S., 1995. Atlas of Allergic Pollens in Turkey .Ed. 1rd. Ünal Ofset, Ankara.

 

29. Phipps, J.B., R.J. O’Kennon and R.W. Lance, 2003. Hawthorns and medlars. Royal Horticultural Society, Cambridge, UK. 30. Punt, W.P., P. Hoen, S. Nilsson and L. Thomas, 2007. Glossary of Pollen and Spore Terminology. Rev. Paleobot. Palyno., 143: 1-81. 31. Reitsma, T.J., 1966. Pollen morphology of some European Rosaceae. Acta Bot. Neerl., 15: 290307. 32. Richard, P.J.H., 1981. Paléophytogéographie postglaciaire en Ungava par l’analyse pollinique. Collection Paléo-Québec, 13: 153. 33. The American Horticultural Society, 2002. The American Horticultural Society Encyclopedia of Plants and Flowers. New York: DK Publishing. 34. The American Horticultural Society, 2002. The American Horticultural Society Encyclopedia of Plants and Flowers. New York: DK Publishing. 35. Wichtl, M., 1996. Herbal drugs and phytopharmaceuticals. In N. Bisset Ed., A handbook for practice on a scientific basis, CRC: Medpharm Scientific Publishers, pp: 161-166. 36. Yao, M., H.E. Ritchie and P.D. BrownWoodman, 2008. A reproductive screening test of hawthorn. J Ethnopharmacol., 118: 127-32. 37. Yeung, E.C., 1983. Histological and histochemical staining procedures. In: Cell culture and somatic cell genetics of plants, Orlando. Vasil, I. K. (Ed). Florida: Academics Press, pp: 689-697. 38. Zhang, P. and S. Xu, 2001a. Chemical constituents from the leaves of Crataegus pinnatifida Bge. var. major N.E.Br. Acta Pharmaceutica Sinica, 36(10): 4-4. 39. Zhou, L.H., Z.X. Wei and Z.Y. Wu, 1999. Pollen morphology of Prunoideae of China (Rosaceae). Acta Bot. Yunnan, 21: 207-211. 40. Zou, S.Q. and W. Chen, 2006. Determination of two components in Crataegus cuneata Sieb. et Zucc. by HPLC-PAD. Food Science (in Chinese), 27(11): 438-440.