ACTA AGROBOTANICA Vol. 60 (1): 1 12 2007 DISTRIBUTION AND STRUCTURE OF INTERNAL SECRETORY RESERVOIRS ON THE VEGETATIVE ORGANS OF INULA HELENIUM L. (ASTERACEAE) Aneta Sulborska Departament of Botany, Agricultural University of Lublin ul. Akademicka 15, 20 950 Lublin e mail: [email protected] Received: 16.04.2007 Summary land it grows most frequently in thickets, forest fringes The aim of the study was to investigate the structure and and near rivers, as well as it is grown in herb gardens topography of endogenous secretory tissues of Inula helenium L. (Strzelecka and Kowalski, 2000; L e w k owicz- By using light and electron microscopy, morphological and ana M o s i e j , 2003; S z weykowscy, 2003). tomical observations of stems, leaves and rhizomes were made. Inula helenium, like many other plants from It was shown that in the stems secretory cavities were situated the Asteraceae family, has multiple applications in in the vicinity of phloem and xylem bundles. The number of therapeutics. It is attributable to the presence of the reservoirs reached its maximum value (34) at shoot flowerig biologically active compounds contained in excre- termination, whereas the cavities with the largest diameter were tions of secretory tissues situated in different parts observed at full flowering stage (44.6 μm). In the leaf petioles of plants. and midribs, the reservoirs also accompanied the vascular bun The medicinal raw material of elecampane com- dles, and their number and size increased along with the growth prises its roots and rhizomes containing 1-3% of essential of the assimilation organs. Observations of the cross sections of the rhizomes revealed the presence of several rings of secre oil, polyacetylene compounds, a bitter substance alan- tory reservoirs. The measurements of the cavities showed that topicrin, mineral salts and 44% of inulin (O ż arowski as a rule the reservoirs with a larger dimension were located in and J aroniewski, 1987; S t r z elecka and Kowal- the phelloderm, whereas the smallest ones in the xylem area. s k i , 2000; L ewkowicz-Mosiej, 2003). Extracts The secretory cavities located in the stems and leaves developed from roots and rhizomes are given in upper airway dis- by schizogenesis, whereas the rhizome reservoirs were prob eases and digestion disorders (O ż arowski and Jaro- ably formed schizolisygenously. The cells lining the reservoirs niewski, 1987; L e w kowicz-Mosiej, 2003). formed a one four layered epithelium. Observed in TEM, the In China and Japan it is used in treatment of cholera secretory cells of the mature cavities located in the rhizomes and malaria, and externally it is applied as an anti- were characterised by the presence of a large central vacuole, dote against snakes and venomous insects (Perry and whereas the protoplast was largely degraded. Fibrous elements of osmophilic secretion and numerous different coloured vesi Metzger, 1980). cles could be distinguished in it. The cell walls formed, from It is known from reports of other authors that the side of the reservoir lumen, ingrowths into the interior of the secretory ducts, running through the petiole to the leaf epithelial cells. Between the cell wall and the plasmalemma of blade, occur in the stems of species from the genus Inu- the glandular cells, a brighter periplasmatic zone with secretory la (Metcalfe and Chalk, 1957). In elecampane roots, vesicles was observed. 2 μm long secretory cavities were observed (D eryng, 1961). Bukowiecki and Furmanowa (1972) re- Key words: secretory cavities, stem, leaf, rhizome, Inula hele- port that secretory cavities were found in rays, phloem nium, Asteraceae and xylem of the underground organs. However, we lack detailed information on the structure and topography of endogenous secretory INTRODUCTION tissues of Inula helenium. The aim of this study was Elecampane is an aromatic perennial originat- to carry out an anatomical examination of the vegeta- ing from Central Asia, long run wild and cultivated in tive organs and the ultrastructure of cells, what may Europe, Asia Minor, Japan and North America. In Po- fill in the gap in this area. 2 Aneta Sulborska MATERIAL AND METHODS Nikon microscope. To examine the cell ultrastructure, the material was cut into 60 nm thick sections by using The subject of the investigation was Inula heleni- the Reichert Ultracut S ultramicrotome. The ultrathin L. (Asteraceae) plants from the tribe (We y - um Inuleae sections were treated with an 8% solution of uranyl ac- m a r, 1970; H e ywood et al. 1977), coming from the etate in 0.5% acetic acid and lead citrate. Observations Botanical Garden of the Maria Curie-Skłodowska Uni- and documentation were made by using the BS-500 versity in Lublin. Anatomical observations were made Tesla electron microscope. in the years 1999–2003 from the start of vegetation of the investigated plants until its end. In order to determine the type of secretory tis- RESULTS sues and their location in the plants, stems, leaves and In the cross section, the above-ground stems of rhizomes were examined. Stem fragments (2.5 cm long) elecampane had a rounded and slightly flattened con- were sampled from three places: at the base of the shoot, tour. Beneath the epidermis, several layers of collenchy- from the middle portion and from the portion under the ma and cortex parenchyma were located (Fig. 1). The inflorescence, taking into account different plant growth collateral bundles were surrounded by sclerenchyma fi- stages. The tissues of the leaf petioles and midribs, as bres, making up expanded caps from the peripheral side well as the rhizome fragments were analysed. Initial of the stem (Figs. 1, 2 A). The interior of the stem was observations were made on fresh material and fixed in filled with a wide cylinder of pith parenchyma. The se- 70% ethanol. cretory cavities were located in the region of the phloem The number of secretory cavities was determined and xylem bundles at the level of the xylem, phloem or and their measurements were made (n=30). In the cross sclerenchyma caps (Figs. 1, 2 A). At the vegetative stage sections, the size of the cavities was measured along their of shoot growth, the conducting bundles, accompanied longer axis (together with the epithelium), as well as the by the cavities, were separated by 2–3 vascular bun- length of the reservoir lumen (to simplify descriptions, dles by which no secretory reservoirs were observed. the term “diameter length” is used in this paper). In the On the other hand, in the stems of the flowering plants cross sections, the number of the epithelial cells, sur- each bundle was usually accompanied by the cavities, rounding the cavity lumen, was also determined. In the though there were also bundles by which no reservoirs case of the several-layered secretory tissue, the number occurred. In the region of a single vascular bundle, two of cells of the first layer of cells was given, counting cavities were most often located, one cavity on each from the lumen. side of the bundle (Figs. 1, 2 A), and sometimes only From fresh material and fixed in 70% ethanol, one secretory reservoir occurred. It also happened that cross sections of the stems, leaf petioles, midribs and four cavities accompanied a single phloem and xylem rhizomes, as well as longitudinal sections of the under- bundle, two on each side of the bundle. Apart from the ground stems, were made manually. Sections embedded main ring of the phloem and xylem bundles, additional in glycerine were prepared, a part of which was stained smaller bundles were observed, located in the outer part with Sudan III and safranin. Observations and photos of the stem, by which no cavities occurred. In the young were made in a Eclipse 400, Nikon microscope. shoots of elecampane, the reservoirs were poorly dis- Semithin sections (0.5 μm thick) and ultrathin tinguished against the background of other cells, due sections (60 nm thick) were also prepared. The stem to their small sizes (Fig. 3). and rhizome fragments were fixed in 2% paraformal- The shape of the secretory cavities in the stem dehyde with 2.5% glutaraldehyde in 0.1 M cacodylate cross section was close to rounded or oval (Figs. 2 B–7). buffer with a pH of 7.4 at a temperature of 20oC for 2 The cavity lumen was surrounded by a single epithelium hours, and then for 12 hours at a temperature of 4ºC. whose cells, along with the growth of the organ, split After the washing of the specimens for 24 hours in 0.1 parallel to the cavity lumen forming 2–3 glandular lay- M cacodylate buffer at a temperature of 4ºC and postfix- ers. The cavities developed by schizogenesis, and their ing them in 1% OsO4, they were transferred to distilled lumen enlarged by the gradual separation of the cells water and stained in a 0.5% aqueous solution of uranyl (Figs. 3, 5, 6). Thick cytoplasm was visible in the epi- acetate. At the next stage, the material was dehydrated thelial cells from the side of the reservoir lumen (Figs. in alcohol series and by using increasing concentrations 3, 5). In the sections cut manually, the sclerenchyma tis- of propylene oxide in alcohol, going over to pure pro- sue was observed surrounding the cavities or cells of pylene oxide. The specimens were embedded in Spurr the glandular layer with partly lignified walls (Fig. 4), Low Viscosity resin for 12 hours at a temperature of what was confirmed by staining with safranin (Fig. 2 B). 70ºC. The semithin sections (0.5 μm) were stained with But in the 0.5 μm thick sections, wall lignification was 1% methylene blue with 1% azur II in a 1% aqueous poorly visible, or it was not observed at all (Figs.