ACTA AGROBOTANICA Vol. 60 (2): 3–8 2007 DEVELOPMENT OF THE POLLEN IN THE ANTARCTIC FLOWERING PLANT COLOBANTHUS QUITENSIS (KUNTH) BARTL. Irena Giełwanowska 1, 2, Anna Bochenek 1, Ewa Szczuka 3 1Department of Plant Physiology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-719 Olsztyn, Poland e-mail:[email protected] 2Department of Antarctic Biology, Polish Academy of Sciences, Ustrzycka 10/12, 02-141 Warsaw, Poland 3Department of Plant Anatomy and Cytology, UMCS University, Akademicka 19, 20-033 Lublin, Poland Received: 20.10.2007 Summary The summer season is considered to last for abo- Colobanthus quitensis (Kunth) Bartl. produced two ty- ut 5 months, more or less from November to March, ta- pes very small bisexual fl owers. In the Antarctic natural con- king especially day and night length into account. Con- ditions chasmogamic and cleistogamic fl owers most often form sidering the length of the snow-cover period in the areas fi ve stamina with short fi laments. Two microsporangia with uncovered by ice, the growth season of seminiferous a three-layer wall form in the anther. Microspore mother cells, plants occurring here may be shortened by 1-2 months. which develop into microspores after meiosis, form inside the Irrespective of other factors affecting the plants, this microsporangium. Microsporocytes of Colobanthus quitensis time is critical for the formation of generative cells and are surrounded with a thick callose layer, the special wall. After seeds (Edwards, 1974; G i e ł wanowska et al. meiosis, the callose wall is dissolved and microspores are rele- 2006). ased from the tetrad. The production of proorbicules, orbicules Macroscopic observations of fl ower and seed and peritapetal membrane, and the construction of a complex development in Antarctic pearlwort and Antarctic hair sporoderm with numerous apertural sites were observed. When grass were conducted in various areas of the Maritime microspore and pollen protoplasts underwent necrosis, probably Antarctic and in different growth seasons. Greene and as a result of temperature and osmotic stress, sporoderm lay- ers formed around microspores, and the cell tapetum did not Holtom (1971) described the fl ower formation in Co- disintegrate. However, woody wall layers did not accumulate in lobanthus quitensis and Deschampsia antarctica on the endothecium cells. South Orkney Islands. Brown (1912) and H oldga- t e (1964) observed specimens of Antarctic pearlwort and hair grass which fl owered and dispersed seeds in Key words: Colobanthus quitensis, microsporangia, microsporo- the communities of South Georgia, while Holtom and genesis, male gametophytes, tapetal cells Greene (1967) on some of the South Shetland Is- lands. INTRODUCTION As only few descriptions of generative cell deve- lopment in Antarctic fl owering plants have been publi- Colobanthus quitensis (Kunth) Bartl. is the only shed (G i e ł wanowska, 2005; G i e ł wanowska fl owering dicot of the Antarctica. It has grown together et al. 2005; G i e ł wanowska et al. 2006), in this with Deschampsia antarctica Desv. (Poaceae) for a few work we present some stages of microsporogenesis and thousand years. It effectively colonises surfaces of the pollen development in Colobanthus quitensis. Antarctic geobotanical zone uncovered by melting ice (Edwards, 1974; A lberdi et al. 2002). In the MATERIALS AND METHODS extremely hostile Antarctic conditions, Colobanthus quitensis (Caryophyllaceae) fl owers profusely almost The experimental materials were fl ower buds of every year. After pollination and fertilization it produc- Colobanthus quitensis in different development stages. es seeds. Antarctic pearlwort seeds are often dead due The materials were collected during the 26th expedition to unfavourable microhabitat conditions and the short to the Antarctic, organized by the Department of An- cold summer (Convey, 1996; G i e ł wanowska et tarctic Biology, Polish Academy of Sciences in Warsaw. al. 2006). Flower buds of Colobanthus quitensis were collected 4 Irena Giełwanowska, Anna Bochenek, Ewa Szczuka during the Antarctic summer, from December 2001 moved from various areas of the tapetum towards pollen to March 2002, in the vicinity of the Arctowski Polar grains, and in the form of bigger structures, orbicules, Station (62º09.8´ S and 58º28.5´W). joined the developing sporoderm (Fig. 5 ). A dozen or so Light and electron microscopy of apertural sites devoid of electron-dense sporopollenin The material for light and electron microsco- material (Fig. 5, ap) differentiated in the forming sporo- py was prepared by a standard method, i.e. fi xed in an derm. In the semi-thin sections, stained with toluidine osmic acid-glutaraldehyde solution. The material was blue, proorbicules and orbicules were visible in the form dehydrated in an alcohol-acetone series, and embedded of blue-green drops (Fig. 5 and Fig. 6). Orbicules settled in Poly Bed 812 epoxy resin. Semi-thin and ultra-thin on the surface of pollen grains until the fi nal stage of sections were prepared in a Leica (Ultracut R) ultrami- tapetal disintegration and atrophy. At the same time, en- crotome, using glass and diamond knives, stained with dothecium with conspicuous ridge-like wall thickenings toluidine blue, and toluidine blue and azure B, following was already fully developed in the microsporangium Pearse (1962), and then examined and photographed wall (Fig. 5). under a light microscope, models Olympus and Nikon It is worth noting that, in the case of microspo- Optiphot II. All preparations to be photographed un- re or pollen protoplast necrosis, as a result of tempe- der a light microscope were mounted in glycerin. Ultra- rature or osmotic stress, tapetum did not disintegrate, -thin sections (60 – 90 nm) were placed onto a copper and endothecium with woody ridges did not develop, wire gauze (300 meshes) and contrasted with a saturated although layers were almost normally deposited on the aqueous solution of uranyl acetate and lead citrate, fol- sporoderm (Fig. 3). lowing Reynolds (1973). Observations and electro- When pollen was mature and contained a bicel- nograms were made in a JEOL JEM 100S transmission lular and tricellular male gametophyte, Colobanthus electron microscope. quitensis microsporangia burst open at a specifi c site, stomium, on the inner (adaxial) side of the anther (Fig. RESULTS 4 and Fig. 7). After anther dehiscence some pollen re- ached the surface of the receptive cells on the fi ve-part Colobanthus quitensis fl owers usually produced feathery stigma, while some remained inside the micro- 5 stamina with short fi laments. sporangium and germinated (Fig. 4). In the anther there were two microsporangia, which had a regular circular shape in cross section. Four DISCUSSION cell layers were visible in the microsporangium wall. The most external layer was epidermis under which, A small number of microspore mother cells diffe- almost on its entire length, there was a layer of starch- rentiated in each of the two microsporangia of the Colo- -fi lled cells which transformed into endothecium. Only banthus quitensis anther. However, already in the early at some places there was a middle layer of parenchyma. fi rst meiotic prophase these cells were surrounded with A nutritive tissue, tapetum (Fig. 1), developed inside. a thick callose wall and were isolated in this way until The central part of the microsporangium was occupied the tetrad stage. Callose began to hydrolise when the by archesporial tissue from which, as a result of meiosis, construction of sporoderm started, just as in other plants microspores were formed. Before meiosis began, micro- (Heslop-Harrison et al. 1986 ). spore mother cells constructed special callose walls. Cal- Microsporgenesis in Deschampsia antarctica was lose surrounded microsporocytes until the tetrad stage. completely different. In the microsporocyte cell walls After meiosis, when callose disintegrated, microspores of the Antarctic hair grass, deposition of a thick callose were released from the tetrads, and a complex cell wall, layer was not observed. From the stage of sporogenic sporoderm, formed on the surface of their protoplasts. tissue cells, through all the meiotic stages, to tetrads, The developing mother cells of the male game- microsporocyte cell walls of the Antarctic hair grass tophyte, from the premeiotic stage, through meiosis, remained thin. This feature distinguishes Deschampsia to the stage of double-cell pollen grains (Fig. 2), were antarctica from other grasses, in which callose special surrounded and nourished by the cell tapetum. Cells walls of meiotic cells are common (McQuade and of the secretory tapetum contained one or two nuclei. Young-Rottler, 1984; Rodkiewicz et al. They retained their individuality until the gametophy- 1996), as in other angiosperms (P a cini et al. 1985). te stage. In the thick cytoplasm of tapetal cells, before In the microspore of Colobanthus quitensis, af- microspores were released from the tetrads, osmophillic ter the fi rst mitosis, the formed generative nucleus does droplets appeared and moved inside the pollen sac (Fig. not break off the sporoderm, but remains fi rmly attached 3). Apart from small lipid droplets, vast areas containing to the inner surface of the pollen wall. After mitosis, the such material visible within the cytoplasm and on the generative cell stays close to the sporoderm, but it is se- borderline with the pollen sac, could be observed in the parated from the vegetative cell by a very delicate wall tapetal cells. This material, in the form of proorbicules, with a little amount of fi brous material. The typical wall Development of the pollen in the Antarctic fl owering plant Colobanthus quitensis (Kunth) Bartl. 5 Fig. 1. Fragment of a microsporangium in Colobanthus quitensis. Layers of the wall; tapetum (ta), parenchymal middle layer (ml) and epidermis (ep). Very thick callose walls (arrows) of the microspore mother cells are visible. 1200 x. Fig. 2. Binucleate pollen grains are visible in the microsporangium. Tapetum is disintegrated (ta). 2000 x. Fig. 3. Microsporangium with disturbed pollen development. There is no developed endotecium in the microsporangium wall.