Turkish Journal of Botany Turk J Bot (2013) 37: 306-315 http://journals.tubitak.gov.tr/botany/ © TÜBİTAK Research Article doi:10.3906/bot-1202-20

Anther development and cytochemistry in aestivus ()

Filiz VARDAR*, Işıl İSMAİLOĞLU, Meral ÜNAL Department of Biology, Faculty of Arts and Sciences, Marmara University, Göztepe, 34722, İstanbul, Turkey

Received: 10.02.2012 Accepted: 18.09.2012 Published Online: 15.03.2013 Printed: 15.04.2013

Abstract: Asphodelus aestivus Brot. (Asphodelaceae) anthers were analysed to provide a detailed understanding of the events that lead to pollen grain development, accompanied by cytochemical observations at different ontogenic stages. An anther locule of A. aestivus was bordered by 4 different layers: the tapetum, the middle layer, the endothecium, and the epidermis. At the tetrad stage, secretory tapetal cells enlarged maximally, while they showed degeneration at the young microspore stage. During the course of the degeneration, vacuolisation and reduction in the volume were conspicuous. Cytochemical analysis revealed that tapetal cells presented an intense reaction with regard to protein, insoluble polysaccharides, and lipids, although the rest of the wall cells reacted weakly throughout development. The entire wall layer cells were rich in starch grains as well. The ultrastructural outcomes confirmed that orbicules were embedded within the dissolving tapetal walls. Young pollen cytoplasm was rich in mitochondria and short ER cisterns, and encircled by the intine and exine. The exine of mature pollen was of the tectate-columellate type. The intine became thicker and consisted of 2 sublayers at the sulcus. The mature pollen had the following morphological characteristics: monosulcate, heteropolar, oblate-spheroidal, widely elliptical in polar view, rhombic, and large. The cytoplasm of a mature pollen grain was filled with starch grains, insoluble polysaccharides, and proteins.

Key words: Asphodelus aestivus, Asphodelaceae, anther development, tapetum, cytochemistry

1. Introduction pollen structure and development, and the accompanying The anther is a morphologically simple organ of the flower changes within the anther yield an array of characters concerned with microsporogenesis and production of potentially useful for assessing phylogenetic relationships. pollen grains, which undergo a series of morphological Our data will allow a new look at this aspect of the and physiological changes until reaching maturity. The development and cytochemistry of anthers in Asphodelus diversity of the cells and tissues, developmental events, and aestivus Brot. (Asphodelaceae), which is a common spring- critical stages during anther ontogenesis have been at the flowering geophyte encountered on the Mediterranean centre of interest since generative reproduction in coast. A. aestivus formations represent the last degradation depends on proper pollen structure and function. The stage of Mediterranean type ecosystems and are often referred to as “asphodel deserts” resulting from frequent chance of effective pollination and fertilisation of maternal fires and overgrazing (Pantis & Margaris, 1988). plants decreases due to defects in the development of the Besides being the dominant life form in many degraded microspore or the surrounding nutritive layer, the tapetum Mediterranean ecosystems, root tubers of A. aestivus are (Bohdanowicz et al., 2005). During microsporogenesis, the used as food for humans and animals (Sawidis et al., 2005). tapetum plays a secretory role, providing essential nutrients The genus Asphodelus Reichb. is a circum- to the developing sporogenous tissue (Pacini, 2000). In Mediterranean genus that includes 5 sections and is addition, pollen coat components are secreted from active represented by 16 species (Lifante, 1996). The perianth of tapetal cells, which undergo structural and biochemical the actinomorphic flower ofA. aestivus has a distinct calyx changes during the final phase of cell differentiation and and corolla and the white petals have a dark stripe through death (Leśniewska & Charzyńska, 2000). the centre. Furthermore, the entomophilous flower of A. Grant (1981) emphasised the importance of studying aestivus secretes a considerable amount of nectar, which is the sexual process and reproduction for obtaining an involved in pollination (Sawidis et al., 2008). understanding of the evolution of certain taxa. Similarly, Although A. aestivus is of economic and ecological Taylor and Osborn (2006) stated that investigations of importance, little attention has been given to the * Correspondence: [email protected] 306 VARDAR et al. / Turk J Bot structural and cytological aspects of the male and female polysaccharides, Coomassie Brilliant Blue for proteins, gametophyte. Schuster et al. (1993) carried out a detailed Sudan Black B for lipids, and Auramine O for sporopollenin experiment on pollination-dependent female reproductive and exine, as previously described (Vardar & Ünal, 2011; success in A. aestivus, a self-compatible outcrosser. The Vardar et al., 2012). The sections were photographed with reproductive biology of A. aestivus was analysed by Lifante ProgRes Capture Pro 2.6 software, assisted by a Jenoptik (1996), including phenology of the flowers, pollen and 122CU colour camera and an Olympus BX-51 microscope. nectar production, pollinators, and reproductive efficiency. Moreover, pollen morphology was examined by Kosenko 3. Results and Sventorzhetskaya (1999) in the family Asphodelaceae Anther development, microsporogenesis, and pollen to elucidate the phylogenetic relationship. Attention development were analysed from the premeiotic stage to has also been paid to the anatomy and ultrastructure of the mature pollen stage. This period was divided into 4 floral nectaries of A. aestivus (Weryszko-Chmielewska et stages: al., 2006). Sawidis et al. (2008) studied the morphology, 1. Premeiotic stage, anatomy, and fine structure of the flower glands (nectary, 2. Tetrad stage, osmophores, and mucilage gland) of A. aestivus. The 3. Young pollen stage (after release of microspores antimicrobial activity of different concentrations of A. from tetrad), aestivus extracts against bacteria and yeasts was also 4. Mature pollen grain stage (just before anther tested (Oskay et al., 2007). To the best of our knowledge, dehiscence). no report has been published on anther development and 3.1. Anther development cytochemistry in A. aestivus, although it seems to be very The undifferentiated anthers of Asphodelus aestivus were significant, not only for systematic comparisons, but also ovoid and consisted of meristematic cells encircled by for knowledge of pollen development and fertilisation. an epidermal layer. Concurrent with development, the The present paper is the first research on anther anther turned out tetrasporangiate. In each anther lobe development in A. aestivus, as well in the genus hypodermal cells differentiated into archesporial cells and Asphodelus, by the application of light, fluorescence, became more remarkable. These archesporial cells divided and electron microscopy (TEM-SEM) to provide a in a plane parallel to the outer wall of the anther (periclinal detailed understanding of the events that lead to pollen divisions), cutting off parietal cells toward the epidermis grain development. Information on the development and primary sporogenous cells toward the interior of the of the male reproductive structures in A. aestivus will anther. The outer parietal layer formed only endothecium, help advance our understanding of its reproductive whereas the inner one divided and formed the middle behaviour, and contribute to our understanding of its layer as well as the tapetum (monocotyledonous type). taxonomic relationship with closely related taxa within the Meanwhile, the sporogenous cells enlarged and underwent Asphodelus/Asphodelaceae. mitotic divisions once, generating pollen mother cells (PMCs). The enlargement of anthers was in progress 2. Materials and methods during the meiotic division of PMCs. Flower buds of Asphodelus aestivus (Asphodelaceae) In Asphodelus aestivus an anther locule was bordered growing in natural habitats in the vicinity of Beykoz, by 4 different layers: the tapetum, the middle layer, the İstanbul (Turkey), were collected in March and April. endothecium, and the epidermis at the early developmental One anther from each flower bud was gently dissected stages. The secretory tapetal cells, with emphatic nuclei and squashed in 0.2% aceto-orcein for estimation of the and large volume, resembled PMCs. At the beginning development stage. of meiosis of the PMCs, tapetal cells underwent mitotic Flower buds were fixed in 2.5% glutaraldehyde and 2% divisions. At the end of tapetal mitosis, no cell plate was paraformaldehyde in 0.05 M cacodylate buffer at pH 7 for formed; therefore, 2 diploid nuclei remained inside the 4 h at room temperature and post-fixed in 1% osmium cell. In some cells primary divisions were followed by tetroxide in the same buffer for 2 h at room temperature. secondary divisions. In a single cell 1-, 2-, 3-, and rarely The samples were dehydrated in ethanol series, and 4-nuclei were observed. Additionally, large polyploid embedded in epoxy resin using propylene oxide. Ultrathin nuclei were created by the nuclear fusion (Figure 1). sections (~70 nm) were contrasted with uranyl acetate After callose dissolution the tapetum started to undergo and lead citrate, and examined with a JEOL JEM 1011 substantial changes in cell organisation, including nucleus transmission electron microscope (TEM). morphology. The nuclear degeneration was characterised For cytochemical observations, the osmication step by loss of spherical shape, distinct shrinkage in volume, was omitted from the fixation. To semithin sections (1 nuclear deformation, formation of an irregular mass, and µm) were applied periodic acid-Schiff (PAS) for insoluble absence of nucleolus prior to the degradation of chromatin.

307 VARDAR et al. / Turk J Bot

a b c d

e f g h

Figure 1. Mitosis in the tapetum of Asphodelus aestivus. a- tapetal cell with single nucleus. b- metaphase. c- telophase. d- binucleated cell. e- telophase of secondary mitosis. f- trinucleated cell. g- 4-nucleated cell. h- polyploid nucleus resulted from nuclear fusion. The scale bar 1 µm in (h) applies also to (a)–(g).

By observation of PAS-stained sections, we defined during the entire developmental stages. Tapetal cells starch in anther wall layers at different development stages. accumulated protein from the premeiotic stage to the At the premeiotic stage, the epidermis and endothecium mature pollen stage. The amount of protein displayed a contained large starch grains, although the middle layer peak at the mature pollen stage during the course of tapetal was low in starch. In comparison to the other wall cells, degeneration (Figure 2). the cytoplasm of tapetal cells was filled with PAS-positive Lipid staining results obtained with Sudan Black B material and small starch grains. At the tetrad stage, the indicated that the epidermis, endothecium, and middle expanded tapetal cells became denser, and contained layer were poor in lipoidal substances during the entire many larger starch grains and intensive PAS-positive developmental stages. However, the tapetal cytoplasm material. It was obvious that tapetal cells enlarged contained a small amount of lipoidal substances at the premeiotic stage. Concurrent with development, the maximally at the tetrad stage and the volume of the cells tapetal cytoplasm started to accumulate more lipids. underwent reduction at the young pollen stage. Moreover, After pollen release from the tetrad, at the locular side vacuolisation was conspicuous at this stage. Although of the tapetal cytoplasm, more densely stained lipoidal large starch grains were still visible in tapetal cells, they substances, which were in contact with the exine, attracted started to regress in the epidermal and endothecial cells. At attention (Figure 2). the mature pollen stage, the epidermis and endothecium To clarify the lipoidal substances released from tapetum were devoid of starch grains. Similarly, as reduction more detailed analysis was required. Sporopollenin- of tapetal cells progressed starch grains disappeared, specific fluorochrome Auramine O observations but degenerating tapetum was still full of PAS-positive confirmed that these lipoidal substances were orbicules material (Figure 2). (Ubisch bodies) with sporopolleninous sheathes (Figure Protein analysis, performed with Coomassie Brilliant 3). The ultrastructural outcomes also confirmed that Blue, revealed that tapetal cells were rich in protein in these orbicules were rounded and embedded within the comparison with the other wall layers. The epidermis, dissolving radial and inner tangential tapetal walls (Figure endothecium, and middle layer were poor in protein 4).

308 VARDAR et al. / Turk J Bot

Ep

En ML T

a b c d

e f g h

j k l

Figure 2. Semithin sections of Asphodelus aestivus anthers at different developmental stages stained with PAS (a–d), Coomassie Brilliant Blue (e-h) and Sudan Black B (i-l). a, e, i- Anther wall cells at premeiotic stage. b, f, j- Enlarged tapetal cells at tetrad stage with large starch grains (arrow). c, g- Young microspore stage, vacuolisation (v) in tapetal cells. d, h- Mature pollen stage, degenerating tapetum. k- Mature pollen stage, lipoidal substances (arrow) at the locular face of tapetum. l- Lipoidal substances (arrow) are in contact with exine (double arrow). Ep: Epidermis; En: Endothecium; ML: Middle layer; T: Tapetum. The scale bar 10 µm in (l) applies also to (a)–(k).

Along with the pollen maturation and anther dehiscence the middle layer and tapetum degenerated completely. After degeneration of the tapetal cells the epidermis and endothecium existed in the mature anther wall, and pollen grains remained in the loculus. 3.2. Microsporogenesis and pollen development a Microspore mother cells of A. aestivus showed regular meiotic division, and the processes of pollen development progressed normally within each individual anther as well as in all anthers of the same flower. Meiotic division was followed by simultaneous cytokinesis. Callose accumulation started at the corners of PMCs in the early stages of prophase I. Microspore tetrads mostly showed a tetrahedral arrangement, and 4 microspores b were separated from each other by a callose wall. At the end of meiosis the callose wall surrounding tetrads Figure 3. Semithin sections of Asphodelus aestivus anthers at underwent progressive lysis, and the young microspores different developmental stages stained with Auramine O. a- were liberated into the locule cavity. The free microspores, Orbicules at the locular face of tapetum (arrow) at young pollen which had spherical and centrally located nuclei, started stage. b- Orbicules (arrow) and exine (arrow head) at mature to become round. The mature pollen had the following pollen stage. The scale bar 10 µm in (b) applies also to (a). morphological characteristics: monosulcate, heteropolar,

309 VARDAR et al. / Turk J Bot

a b

Figure 4. Ultrastructure of tapetum. a- Rounded orbicules at the locular face of tapetum (arrow). b- Orbicules in contact with exine (arrow). The scale bar 0.5 µm in (b) applies also to (a). oblate-spheroidal, widely-elliptical in polar view, rhombic, and of tectate-columellate type. Although the intine was and large. The sulcus was long, wide, and extending to the considerably thinner than the exine, in the sulcus region ends of the pollen grain; hence, the pollen content was it became thicker and consisted of 2 sublayers: an outer capable of protruding (Figure 5). intine (exintine) and an inner intine (endintine) (Figure Ultrastructural studies revealed that the pollen wall 6). The bicellular state of the pollen grains ofA. aestivus exine (primexine) started to form in the callose wall at the persisted to anthesis. tetrad stage. After callose dissolution, the exine developed The surface of the exine was perforate. It had rough and got thicker. Young pollen cytoplasm was rich in sculpturing that looked like separate tubercules on a distal mitochondria and short ER cisterns, and encircled by the face along the sulcus margins (perforate-rugulate) (Figure intine and exine. The exine of mature pollen was very thick 7).

ab

cd

Figure 5. Pollen grains of Asphodelus aestivus at different stages. a- Young microspore. b- Proximal face of mature pollen. c- Distal face of mature pollen. d- Protruding content of pollen. The scale bar 10 µm in (d) applies also to (a)–(c).

310 VARDAR et al. / Turk J Bot

a b

c d

Figure 6. Exine formation in Asphodelus aestivus. a- Primexine (arrow) in tetrads. b- Exine (arrow) and intine (double arrow) walls of a young pollen grain. c- Thinner intine (double arrow) and tectate-columellate type exine of a mature pollen grain. d- Thick and double layered intine (double arrow) at sulcus region. C = callose, gn = generative nucleus. Scale bar = 0.5 µm.

Cytochemical analysis indicated that the cytoplasm development was ignored in the genus Ashodelus. This of a mature pollen grain was filled with starch grains, paper reports observations on the developmental and insoluble polysaccharides, and proteins but lacked lipid. cytochemical characteristics of the male reproductive It was detected that a well-defined exine was made up organ in Asphodelus aestivus Brot., incorporated in of lipoidal substances and protein but the intine was Asphodelaceae. composed of insoluble polysaccharides and protein. The The anther wall development in Liliaceae was reported thicker intine around the sulcus represented strong PAS- as monocotyledonous type (Davis, 1966), including Gasteria positivity (Figure 8). verrucosa (Keijzer, 1987), Anemarrhena asphodeloides After the anthers attained their maximum size the (Chen et al., 1988), Ophiopogon xylorrhizus (He et al., 1998), pollen sacs combined through tissue fusion and mature and Ornithogalum virens (Leśniewska & Charzyńska, 2000). pollen grains were scattered into the environment Furthermore, it was indicated that the cells of the glandular concurrent with dehiscence of the anther. tapetum became multinucleate but that nuclear fusions may occur (Davis, 1966), as was presented in A. aestivus. 4. Discussion Additionally, Oksala and Therman (1977) described The family Liliaceae was formerly a paraphyletic group endomitosis in the anther tapetum of Eremurus. that included a great number of genera now contained The anther development events occur in a precise in other families, including Asphodelaceae. Although chronological order correlated with microspore/pollen the antimicrobial activity (Oskay et al., 2007) and floral grain development (Platt et al., 1998). Those criteria for nectary (Weryszko-Chmielewska et al., 2006; Sawidis determination and selection of the developmental stages et al., 2008) were most widely analysed, reproductive of anthers are more precise than morphological criteria

311 VARDAR et al. / Turk J Bot

a b

c d

Figure 7. SEM micrographs of mature pollen. a- Entire pollen grain in distal view. b- Perforate-rugulate exine in distal view. c- Entire pollen grain in proximal view. d- Perforate exine in proximal view. based only on the size of flower buds or anthers (Koltunow Keijzer (1987) reported that in Gasteria verrucosa et al., 1990). tapetal cells were covered with sporopollenin containing The tapetum has attracted much attention because of tapetal membranes and orbicules after the disappearance its apparently nutritive physiological role during pollen of tapetal cell walls. They also indicated that the exine was development by secreting numerous substances into the made up of sporopollenin, which was derived from the loculus (Clément & Audran, 1995), and by regulating the tapetum. chemical composition of the locular fluid (Souvré et al., Several researchers confirmed that accumulation and 1987). As described by Pacini (1994), tapetal metabolites mobilisation of starch is a common character of the anther released into the loculus are in the form of insoluble enveloping layers in the angiosperms (Bhandari, 1984). It polysaccharides, proteins, and enzymes (such as callase), has been also stated that the products of starch mobilisation and pollenkitt, tryphine, and recognition substances. in the anther wall are transported to the loculus and used Moreover, orbicules originate in the cytoplasm of the for pollen metabolism (Reznickova, 1983). tapetal cells as lipoidal pro-orbicular bodies, accumulating Clément et al. (1994) carried out a PAS reaction and below the membrane and eventually extruding to the indicated that the tapetal cytoplasm of Lilium was lacking locular face where they provide sporopollenin precursors in starch but the tapetal vacuoles accumulated soluble for exine formation (Shivanna, 2003). polysaccharides during the growth phase. Therewith

312 VARDAR et al. / Turk J Bot

a b c Figure 8. Cytochemistry of Asphodelus aestivus pollen grains. a- Insoluble polysaccharide depositions and starch grains (arrows) stained with PAS. b- Protein depositions stained with Coomassie Brilliant Blue. c- Exine stained with Sudan Black B. The scale bar 10 µm in (c) applies also to (a) and (b). they suggested that these polysaccharides may be used 2012), the time of the degeneration varies greatly from by the tapetal cells for 2 kinds of functions: providing species to species. As we described previously, in Latyhrus energy (De Block & Debrouwer, 1993) and being secreted undulatus the tapetal cells started to degenerate at the into the loculus (Bhandari, 1984). Consequently, starch vacuolated pollen stage and degenerated completely at the accumulation in the outer cell layers during the early stages mature pollen stage (Vardar & Ünal, 2011). The present of pollen development represents a temporary storage paper provides the first information on the timing of tapetal for anther sugars. The mobilisation of these reserves degeneration in A. aestivus. The multinucleated tapetal may be partly used by sporophytic cells for growth and cells developed maximally at the tetrad stage; underwent differentiation (Keijzer & Willemse, 1988) and partly some alterations such as vacuolisation, reduction in transmitted to the microspores via the locular fluid (Pacini volume, and degeneration at the young pollen stage; and & Franci, 1983; Bhandari, 1984). degenerated completely just before anther dehiscence. Cytochemical results from A. aestivus anthers showed A. aestivus underwent simultaneous cytokinesis in that the cells of the epidermis, endothecium, and middle the PMCs and isobilateral microspore tetrads, compatible lamella gave a weak reaction for protein and lipids. with the family Liliaceae (Davis, 1966). The morphological However, during development the anther wall cells were characters of pollen obtained from TEM and SEM studies rich in starch grains. At the young pollen stage starch grains were consistent with those of other Asphodelus species started to regress. According to our results, the tapetal (Kosenko & Sventorzhetskaya, 1999); however, the intine cells of A. aestivus accumulate protein, polysaccharides, structure was indicated for the first time in the genus and lipoidal substances throughout development. The Asphodelus. results confirmed that lipoidal derivatives were in In conclusion, during anther wall development in contact with pollen grains, and had a possible role in regard to structural development and accumulation of pollen wall formation. Several researchers reported that organic compounds (protein, insoluble polysaccharide, polysaccharides, proteins, and lipids in pollen cytoplasm and lipid), tapetal cells of A. aestivus came to the fore. had important metabolic roles in pollen germination and The tapetal cells, which were rich in polysaccharides, pollen tube formation (Hess, 1993; Li et al., 1995). proteins, and lipoidal substances, degenerated at the Chen et al. (1988) revealed that, as the tapetal walls mature pollen stage. Our data provide a new look at the of Anemarrhena asphodeloides (Liliaceae) began to be aspect of male reproductive potential of A. aestivus and the disorganised, the plasmolemmas of tapetal cells gradually genus Asphodelus. On the other hand, the timing of tapetal moved inwards. During the later stages of tapetum development, degeneration, and pollen maturation will be development, the nuclei disappeared and cytoplasmic the focus of future programmed cell death investigations. vacuolation of tapetal cells was evident. In the meantime, Although there are some molecular phylogenetic studies a number of pro-orbicules were seen in the tapetal on the family Liliaceae (İkinci, 2011), ultrastructural and cytoplasm. cytochemical features of pollen will also provide useful The cells of secretory tapetum maintain their position, characters for assessing relationships within this genus and eventually undergo degeneration in situ towards the and family. end of pollen development (Pacini et al., 1985). Although, in recent years, it has been reported that tapetal cells Acknowledgement undergo programmed cell death throughout degeneration This work was supported by the Research Foundation of (Papini et al., 1999; Wu & Cheung, 2000; Vardar & Ünal, Marmara University (BAPKO no. FEN-A-110908-0224).

313 VARDAR et al. / Turk J Bot

References

Bhandari NN (1984). The microsporangium. In: Johri BM (ed.). The Lifante DZ (1996). Reproductive biology of Asphodelus albus Embryology of Angiosperms, pp. 53–157. Berlin, Heidelberg, (Asphodelaceae). Systematics and Evolution 200: 177– New York, Tokyo: Springer. 191. Bohdanowicz J, Szczuka E, Świerczyńska J, Sobieska J & Kościńska- Oksala T & Therman E (1977). Endomitosis in tapetal cells of Pajak M (2005). Distribution of microtubules during Eremurus (Liliaceae). American Journal of Botany 64: 866–872. regular and disturbed microsporogenesis and pollen grain Oskay M, Aktaş K, Sarı D & Azeri C (2007). Asphodelus aestivus development in Gagea lutea (L.) Ker.-Gaw. Acta Biologica (Liliaceae)’un antimikrobiyal etkisinin çukur ve disk diffüzyon Cracoviensia 47: 89–96. yöntemiyle karşılaştırmalı olarak belirlenmesi. Ekoloji 62: 62– Chen ZK, Wang FH & Zhou F (1988). On the origin, development 65 (in Turkish). and ultrastructure of the orbicules and pollenkit in the tapetum of Anemarrhena asphodeloides (Liliaceae). Grana 27: 273–282. Pacini E (1994). Cell biology of anther development. In: William EG, Clarke AE & Knox RB (eds.) Genetic Control of Self- Clément C & Audran JC (1995). Anther wall layers control pollen incompatibility and Reproductive Development in Flowering sugar nutrition in Lilium. Protoplasma 187: 172–181. Plants, pp. 289–308. Dordrecht: Kluwer. Clément C, Chavant L, Burrus M & Audran JC (1994). Anther starch Pacini E (2000). From anther and pollen ripening to pollen variations in Lilium during pollen development. Sexual Plant presentation. Plant Systematics and Evolution 222: 19–43. Reproduction 7: 347–356. Pacini E & Franchi GG (1983). Pollen grain development in Smilax Davis OL (1966). Systematic Embryology of the Angiosperms. New aspersa L. and possible function of the loculus. In: Mulcahy DL York: John Wiley & Sons. & Ottaviano E (eds.) Pollen Biology and Implications for Plant De Block M & Debrouwer D (1993). Engineered fertility control in Breeding, pp. 183–190. Amsterdam: Elsevier. transgenic Brassica napus space L.: Histochemical analysis of Pacini E, Franchi GG & Hesse M (1985). The tapetum: its form, anther development. Planta 189: 218–225. function and possible phylogeny in Embryophyta. Plant Grant V (1981). Plant Speciation. New York: Columbia University Systematics and Evolution 149: 155–185. Press. Pantis J & Margaris NS (1988). Can systems dominated by asphodels He TH, Rao GY, You RL & Zhang DM (1998). Embryological studies be considered as semi-deserts? International Journal of on endangered Ophiopogon xylorrhizus (Liliaceae). Acta Biometeorology 32: 87–91. Phytotaxonomica Sinica 36: 305–309. Papini A, Mosti S & Brighigna L (1999). Programmed cell death Hess MW (1993). Membrane coatings on the generative cell surface events during tapetum development of angiosperms. of freeze-substituted pollen. Protoplasma 176: Protoplasma 207: 213–221. 84–88. Platt KA, Huang AHC & Thomson WW (1998). Ultrastructural İkinci N (2011). Molecular phylogeny and divergence times estimates study of lipid accumulation in tapetal cells of Brassica napus L. of Lilium section Liriotypus (Liliaceae) based on plastid and cv. westar during microsporogenesis. International Journal of nuclear ribosomal ITS DNA sequence data. Turkish Journal of Plant Science 159: 724–737. Botany 35: 319–330. Reznickova SA (1983). Metabolism of reserve substances in the Keijzer CJ & Willemse MTM (1988). Tissue interactions in developing anther. In: Erdelska O (ed.) Fertilization and the developing locule of Gasteria verrucosa during Embryogenesis in Ovulated Plants, pp. 57–62. Bratislava, Veda. microsporogenesis. Acta Botanica Neerlandica 37: 475–492. Sawidis T, Weryszko-Chmielewska E, Anastasiou V & Bosabalidis Keijzer CJ (1987). The process of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally AM (2008). The secretory glands of Asphodelus aestivus flower. dehiscing anthers. New Phytologist 105: 487–498. Biologia 63: 1118–1123. Koltunow AM, Truettner J, Cox KH, Wallroth M & Goldberg RB Sawidis T, Kalyba S & Delivopoulos S (2005). The root–tuber (1990). Different temporal and spatial gene expression patterns anatomy of Aspodelus aestivus. Flora 200: 332–338. occur during anther development. Plant Cell 2: 1201–1224. Schuster A, Noy-Meir I, Heyn CC & Dafni A (1993). Pollination Kosenko VN & Sventorzhetskaya OY (1999). Pollen morphology in dependent female reproductive success in a self-compatible the family Asphodelaceae (Asphodeleae, Kniphofieae). Grana outcrosser, Asphodelus aestivus Brot. New Phytologist 123: 38: 218–227. 165–174. Leśniewska J & Charzyńska M (2000). Tapetal plastids in Shivanna KR (2003). Pollen Biology and Biotechnology. New Ornithogalum virens: from meristematis stage to pollen coat. Hampshire: Science Publishers Inc. Acta Biologica Cracoviensia 42: 141–149. Souvré A, Albertini L & Audran JC (1987). Le grain de pollen des Li YQ, Southworth D, Linskens HF, Mulcahy DL & Cresti M (1995). Angiospermes. Apports de la biopalynologie et perspectives Localization of ubiquitin in anthers and pistils of Nicotiana. biotechnologiques. Bulletin de la Société Botanique de France Sexual Plant Reproduction 8: 123–128. 134: 87–112.

314 VARDAR et al. / Turk J Bot

Taylor ML & Osborn JM (2006). Pollen ontogeny in Brasenia Vardar F, İsmailoğlu I & Ünal M (2012). Embryological and (Cabombaceae, Nymphaeales). American Journal of Botany 93: cytological features of Gagea bohemica (Liliaceae). Turkish 344–356. Journal of Botany 36: 462–472. Vardar F & Ünal M (2011). Cytochemical and ultrastructural Weryszko-Chmielewska E, Sawidis T & Piotrowska K (2006). observations of anthers and pollen grains in Lathyrus undulatus Anatomy and ultrastructure of floral nectaries of Asphodelus Boiss. Acta Botanica Croatica 70: 53–64. aestivus Brot. (Asphodelaceae). Acta Agrobotanica 59: 29–42. Vardar F & Ünal M (2012). Ultrastructural aspects and programmed Wu HM & Cheung AY (2000). Programmed cell death in plant cell death in the tapetal cells of Lathyrus undulatus Boiss. Acta reproduction. Development 44: 267–281. Biologica Hungarica 63: 52–66.

315