ACTA AGROBOTANICA Vol. 64 (4): 23–34 2011 MICROMORPHOLOGY OF FLOWERS, ANATOMY AND ULTRASTRUCTURE OF Chamomilla recutita (L.) Rausch. (ASTERACEAE) NECTARY Aneta Sulborska Department of Botany, University of Life Science in Lublin, ul. Akademicka 15, 20-950 Lublin, Poland e-mail: [email protected] Received: 10.09.2011 Abstract INTRODUCTION Investigations of the micromorphology of flowers and Chamomilla recutita (Asteraceae) is a wild spe- the structure of nectaries in Chamomilla recutita L. (Rausch.) cies growing throughout Europe; in Poland, it is a se- were carried out with the use of stereoscopic, light, scanning getal and ruderal weed (Szweykowscy, 2003). It and transmission electron microscopy. Biseriate glandular tri- chomes consisting of 5-6 cell layers were found on the surface is one of the oldest medicinal plants known in ancient of the corollas of ray and disc florets. Accumulation of secretion Egypt, Greece, and Rome (R u m i ń ska, 1973). within the subcuticular space was accompanied by degradation Due to their structure and location of nectaries, of trichome cells. Secretion release followed rupture of the cuti- the flowers of Asteraceae representatives have been cle in the apical part of the trichome. The ovary of the ray florets classified as flowers with completely hidden nectaries exhibited characteristic ribs covered with epidermis composed (Szafer and Wojtusiakowa, 1969). Lipiń - of radially elongated palisade cells. ski (2010) classified them as hemitropous flowers, in Nectariferous glands were present only in the disc flo- which nectaries are located not deep and are accessible rets. The ring-like nectary (93×163 μm; height x diameter) was to insects with a mouth apparatus of medium-length. located above the inferior ovary. The gland structure was formed According to Fahn (1952, 1988 a, b, 2000), by single-layer epidermis and 5-8 layers of specialised necta- nectaries in Asteraceae represent the type of the sty- riferous parenchyma. Nectar was released via modified 15-20 μm wide stomata. The guard cells were slightly elevated above le (stylopodium) that is located at the style base. Such the surface of the other epidermal cells or were located slightly location of the nectariferous tissue in this family has below them. The stomatal cells were characterised by small ex- also been found by Kuliev (1959), Esau (1973), ternal and internal cuticular ledges. No vascular bundles were Sulborska and Weryszko-Chmielewska observed in the nectary. The gland was supplied by branches (2006, 2007), Wist and Davis (2006) and L i - of vascular bundles reaching the style and ending at the nectary piń ski (2010). Smets (1986) refers to nectaries base. The nectariferous tissue was formed by isodiametric cells in Asteraceae as nectaria persistentia associated with with a diameter of 11-20 μm. The cell interior was filled with persistent flower parts, while Schmid (1988) and electron dense cytoplasm containing a large nucleus, numerous Bernardello (2007) classify them as gynoecial pleomorphic plastids, mitochondria with a distinct system of nectaries. cristae, Golgi bodies, ER profiles, and ribosomes. The plastid A majority of species from the family Asterace- stroma was characterised by presence of pastoglobuli, intraplas- tid tubules, and lighter zones. Several small vacuoles were found ae investigated have nectaries only in the disc florets in each cell. Plasmodesmata were visible in the walls of some (Sammataro et al. 1985; Sulborska and W e - cells. Lighter periplasmic space in which apoplastic transport of ryszko-Chmielewska, 2006, 2007; Wist nectar might take place was observed between the plasmalemma and Davis, 2006). In Heterothalamus alienus, howe- and the cell wall. The presence of an osmiophilic substance in the ver, the nectariferous glands were also present in the intercellular spaces additionally corroborates this assumption. ray florets (V ogel, 1998). The data obtained by Frei (1955) indicate that Key words: Chamomilla recutita, flowers, glandular trichomes, the nectaries in Asteraceae are uniform in terms of lo- nectary, epidermis, nectariferous parenchyma, cation and shape; Lipiń ski (2010) claims that the ultrastructure. nectariferous gland in this family always has the same 24 Aneta Sulborska structure. In turn, Gulyás and Pesti (1966) repor- SEM. Disc florets with nectaries prepared from ted that ring-shaped, funnel-shaped and cone-shaped the anthodia were fixed in a 4% glutaraldehyde solu- glands were observed in the genus Centaurea. In He- tion in 0,1 M phosphate buffer pH 7,0 at the tempera- lianthus annuus flowers, a collar-like discoid nectary ture of 4oC for 12 hours. Next, the material was washed was found, which in cross section exhibited a round with the same buffer 4 times for 20 minutes and dehy- shape or in the form of a 4- to 8-sided polygonal ring drated in alcohol series: 30, 50, 70, 90 and 95%, which (Sammataro et al. 1985). In Inula helenium, the was followed by application of absolute alcohol three nectariferous gland had a shape of a five-armed star times. When dehydrated, the samples were transfer- with rounded tips (Sulborska and Weryszko- red to acetone, critical point dried in liquid CO2 using Chmielewska, 2007), while the nectaries in Achil- Bal-Tec CPD 030 and coated with gold and platinum lea millefolium (Sulborska and Weryszko- using the Polaron SC 7640 sputter coater. The surface Chmielewska, 2006) and Echinacea purpurea, of the nectaries was examined and imaged under the observed from above, were pentagonal (Wist and BS 301 scanning electron microscope coupled with the Davis, 2006). Tescan attachment for digital processing of microsco- The secreted nectar is accumulated in the nar- pe imaging. row corolla tube, and thus elevated by a few millime- TEM. Fragments of disc florets were fixed in tres and accessible to insects (Lipiń ski, 2010). In 2% paraformaldehyde and 2,5% glutaraldehyde in 0,1 the pistillate phase in the disc florets of Echinacea pur- M cacodylane buffer with pH 7,4 at room temperature purea, the basal part of the corolla, which functions as for 2 hours and next at the temperature of 4oC for 12 a nectar reservoir, was substantially enlarged (Wist hours. The samples were then washed with 1 M caco- and Davis, 2006). dylane buffer at the temperature of 4oC for 24 hours The aim of the present study was to investigate and underwent osmosis in 1% OsO4; next they were some aspects of flower micromorphology and morpho- transferred to redistilled water and stained in a 0,5% logical, anatomical, and ultrastructural features of the water solution of uranyl acetate. In the next step, the nectariferous tissue in Chamomilla recutita, as litera- material was dehydrated in an alcohol series and by in- ture does not provide such data. creasing the concentrations of propylene oxide in alco- hol, reaching pure propylene oxide. The samples were MATERIAL AND METHODS embedded in Spurr Low Viscosity resin for 12 hours at the temperature of 70oC. The material thus prepared The Chamomilla recutita L. (Rausch.) flowers was sliced into 60 nm-thick sections with a Reichert studied (in the full bloom phase) originated from the Ultracut S microtome. Ultrathin sections were treated collection of medicinal plants in the UMCS Botani- with an 8% solution of uranyl acetate in 0,5% acetic cal Garden in Lublin. Observations were performed acid and with lead citrate. The observations and docu- with the use of stereoscopic (SM), light (LM), scan- mentation were made with the use of a BS-500 Tesla ning electron (SEM) and transmission electron (TEM) electron microscope. microscopy. SM. In order to localize the nectaries, fresh flow- ers were examined under a stereoscopic microscope RESULTS coupled with a Nikon Coolpix 4500 digital camera. Morphology and anatomy of the flowers LM. For the analysis of morphological and an- atomical features of the nectaries, semi-thin sections The pseudanthia of Chamomilla recutita imita- (0,5 μm thick) were prepared from longitudinal flower ting a single flower were composed of marginal ray fragments and stained with 1% methylene blue with florets and numerous internal disc florets (Fig. 1A). 1% azure II in a 1% water solution of sodium tetrabo- The corolla of the ray florets was connate and formed rate. The material was fixed and embedded in synthetic a tube at the base and an elongated ligule composed of resin following the method used for the transmission three fused petals in the apical part (Fig. 1C). The ray electron microscope. florets had a pistil, but they were sterile. During the microscopic observations, the height The calyx in the disc florets was strongly re- of epidermal cells and diameter of glandular cells were duced and teeth-shaped above the inferior ovary (Fig. measured (n=30), and the number of layers of gland- 1D). The corolla was composed of five fused petals. In forming cell was determined. The stomatal width and its basal part, the corolla tube (mean length 1.6 mm) the height of stomatal cells were measured (n=10). The was narrower, and above ½ of the length, it was wider observations were made under the Jenaval Contrast and had five apical lobes (Fig. 1B). The disc florets microscope and the images were taken with Eclipse were bisexual, fertile, and they bloomed gradually 400, Nikon. from the margin towards the centre of the anthodium Micromorphology of flowers, anatomy and ultrastructure of Chamomilla recutita (L.) Rausch. (Asteraceae) nectary 25 (Fig. 1A). Stamens matured first and next the pistil, mitochondria, Golgi bodies with detaching vesicles, which has a style with a two-lobbed stigma emerged plastids, osmiophilic structures and numerous riboso- from a tube formed by fused stamen heads (Fig. 1B,F). mes (Fig. 3A,C,D). An empty space containing dark, Adaxial and abaxial elongated epidermal cells (papil- fibrillar structures were observed between the walls of lae) containing dense cytoplasm that stained intensely some cells (Fig.