ACTA AGROBOTANICA Vol. 66 (4), 2013: 35–44 Original Research Paper DOI: 10.5586/aa.2013.049 STRUCTURAL FEATURES OF FLOWER TRICHOMES IN DRUG EYEBRIGHT (Euphrasia stricta D. Wolff EX J. F. Lehm.) Weronika Haratym, Elżbieta Weryszko-Chmielewska Department of Botany, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland e-mail:[email protected] Received: 10.06.2013 Abstract seed set, its population abundance has been declining Euphrasia stricta D. Wolff ex J. F. Lehm. (Orobancha- from year to year. In Poland, taxonomic and phytoge- ceae) is a representative of plants that are widely used in folk ographical investigations of the drug eyebright were medicine, phytomedicine, and homeopathy. The medicinal raw initiated again in 2001 primarily in the south of the co- material derived from the drug eyebright is applied primarily in untry, since data obtained several decades ago proved treatment of ophthalmic diseases. outdated, given the current state of the art [7,8,9]. The investigations of trichomes in drug eyebright Species from the genus Euphrasia are perennial (Euphrasia stricta D. Wolff ex J. F. Lehm) were conducted or annual plants with a semi-parasitic life style. Although in 2010–2011. Using light microscopy and scanning electron they are capable of independent life, they achieve a hi- microscopy, their location and morphological and anatomical gher growth rate while producing haustoria penetrating features were identified. Three types of non-glandular tricho- the root systems of other plants, in particular represen- mes were found: short unicellular, long 1–2 celled, and long 2-celled with wall ornamentation. Additionally, 7 types of glan- tatives of the Fabaceae family [10,11]. Moreover, drug dular trichomes were found; these included: unicellular clavate, eyebright individuals have been reported to parasitize 2–3-celled clavate, capitate with a unicellular head and a 3-cel- one another with dominance of some specimens over led stalk, capitate with a unicellular head and a 2-celled stalk, others [10]. Drug eyebright plants usually reach a height capitate with a 2-celled head, conical papillae, and ribbon-like of 30 cm. They have an erect, highly branched stem and trichomes with wall thickening. ovate or elliptic toothed leaves with an opposite arran- gement on the stem. Flowers are borne on the apical Key words: Euphrasia stricta, flower, glandular trichomes, parts of shoots between July and September. They are non-glandular trichomes, location, micromorpho- small (ca. 10 mm), white or light purple with dark pur- logy, LM, SEM ple stripes and a yellow spot on the lower lip. The fruit is a capsule [12,13]. Investigations of the flowering and pollination biology in E. willkommii have demonstrated INTRODUCTION that the plant species is relatively seldom visited by pol- Until recently, the drug eyebright Euphrasia L. linators. As assumed by Rawski (1948), this is related has been classified as a representative of the family to the moderate nectar secretion and poor pollen reward, Scrophulariaceae [1,2]. In the new taxonomic system which is hardly attractive to insects. Additionally, the presented on the Angiosperm Phylogeny Website, the small-sized tube-shaped flowers emerging too close to genus belongs to the family Orobanchaceae, i.e. one the ground prevent the process of pollination by larger of the clades in the order Lamiales. The genus com- insects. Species that were reported to be carrying pollen prises 350 species. In the Polish flora, only 10 Euph- were ants (Hymenoptera) and thrips (Thysanoptera). rasia species occur in natural localities [2,3,4,5]. Due A common phenomenon observed in the plant species to the specific biology, emergence of seasonal races, is self-pollination. At the final stage of flower’s life, the and easy hybridisation, the genus Euphrasia poses dif- corolla elongates, which makes adnate anthers change ficulty to plant systematics [6]. In recent years, many their position. At the same time, the style does not chan- species from the genus Euphrasia have been reported ge its length. In consequence, anthers approach the stig- to be threatened with extinction. Despite flowering and ma, which facilitates pollination [14,15]. © The Author(s) 2013 Published by Polish Botanical Society 36 Weronika Haratym, Elżbieta Weryszko-Chmielewska Plants of the Euphrasia L. genus are widely RESULTS known in folk medicine, phytomedicine, and home- opathy as anti-inflammatory, hypotensive, and rege- General habit of the flower nerative agents. Aqueous tinctures or alcohol extracts The flower habit was observed under a stereo- from the above-ground parts of the eyebright are used scopic light microscope. The hermaphrodite zygomor- for treatment of various eye problems such as cataract, phic flowers are borne on apical parts of shoots and glaucoma, conjunctivitis, red, inflamed, irritated, and arranged at an acute angle thereto. At the base, they are sore eyes, corneal ulceration, sharp pain in the eyes, surrounded by bristle-toothed bracts (Fig. 1a). or spastic photophobia. Furthermore, they are recom- The dark green cylindrical calyx is gamosepalous mended for people who suffer from stomach disorders, with four sharp teeth. Its surface bears dark purple stri- jaundice, headache, and diseases of the upper respirato- pes (Fig. 1b). The flower corolla is bilabiate (Fig. 1a,c). ry tract [16,17,18,19]. The lower lip is formed of 3 fused petals. The surface Although there is a lot of information about the of marginal lobes bears 2 dark purple stripes and 1 light medicinal properties of the eyebright, no specific data purple medial stripe. The central lobe has 3 light purple about the epidermal structures on the flower can be fo- stripes as well. A yellow spot is visible in the central und. The aim of the present study was to examine the lo- part of the middle corolla petal. The entry to the flower cation, and micromorphological and anatomical features throat is also marked with yellow colouration (Fig. 1c). of non-glandular and glandular trichomes on the surface The upper lip consists of 2 fused corolla petals. of the calyx, corolla and anthers of Euphrasia stricta. It is white initially and becomes light violet during de- velopment. Each lobe of the upper lip bears dark pur- ple stripes – 3 on each lobe and 1 light purple stripe MATERIALS AND METHODS along the petal fusion line. Yellow colouration is visi- The research material comprised buds and de- ble at the entry to the corolla tube (Fig.1c,d). veloped flowers of Euphrasia stricta D. Wolff ex J.F. The four stamens with filaments of unequal Lehm. Plant fragments were sampled from their natu- length are didynamous and fused with the upper lip. ral habitats on xerothermic grasslands in the Czechów The dark purple stamen heads are fused. Anthers are district, Lublin, between late July and mid-September equipped with sharp-tipped appendages in the basal 2010 and 2011. part. The filaments are white with yellow colouration Preliminary analyses of the ecological features only at the entry to the corolla tube. Their surface bears of flowers and location of various types of trichomes long mechanical trichomes (Fig. 1d). The pistil has a were performed using a stereoscopic microscope co- small green ovary, a long style, and a stigma, which upled with a NIKON COOLPIX 4500 camera. emerges from the flower throat above the stamens and Hand-made cross sections through sepals and co- is covered by dense hairs (Fig. 1e). rollas were fixed in 50 % glycerol and observed under a Corolla trichomes Nikon Eclipse 400 light microscope. The types and topo- graphy of trichomes were identified. Additionally, their The petals bear only one type of non-glandu- morphological and anatomical traits were assessed and lar trichomes and a few types of glandular trichomes. the length and width of the structures were measured. 2-celled mechanical trichomes exhibit the highest den- To obtain permanent preparations, flower frag- sity on the abaxial surface of the upper lip (Table 1). On ments were fixed in a 4% glutaraldehyde solution in average, they are 346,43 μm long and 17,97 μm wide 0,1M phosphate buffer, pH 7, at a temperature of 4ºC (Table 2). Two types of clavate trichomes are located for 12 hours. Next, the material was washed in the near the yellow spot on the lower lip. These hairs are same buffer 4 times at 20-minute intervals, fixed in a fairly large and visible under a stereoscopic microscope 1% solution of osmium tetroxide for 1 hour, and rinsed (Fig. 1f). One of the types is represented by multicellu- again in phosphate buffer. The preparations were de- lar trichomes with a thin cell wall and visible chromo- hydrated in 30, 50, 70, 90, and 95% alcohol series and plasts inside the cells. They reach a length up to 262,5 immersed twice in absolute alcohol. After dehydration, μm and a width of 40 μm (Figs 1g,2g). Close to these the material was embedded in LR White resin and se- hairs, there are unicellular clavate trichomes, which are mi-thin sections were cut using a Reichert Ultracut-S longer (up to 343,4 μm) than the multicellular ones. ultramicrotome. The preparations were stained with a Their cell wall is covered by a cuticle, whose sculpture 1% aqueous methylene blue – Azur B solution and ob- consists of short striae (Figs 1h; 2g–i). Remains of se- served under a light microscope. cretion were observed on some trichomes (Fig. 2i). Some material was critical-point dried after The adaxial surface of the lower and upper dehydration. Next, the preparations were coated with lips is covered by densely distributed conical papillae gold and analysed under a TESCAN VEGA II LMU (Figs 1i,j; 2d–f). Their maximum length reaches 15 μm.