Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi http://www.bio.uaic.ro/publicatii/anale_vegetala/anale_veg_index.html s. II a. Biologie vegetală, 2014, 60, 1: 13-24 ISSN: 1223-6578, E-ISSN: 2247-2711 HISTO-ANATOMICAL OBSERVATIONS REGARDING VIOLA L. SPECIES IN THE GÂRBOAVELE RESERVE (COUNTY OF GALAŢI) Anca MEREACRE1*, Angela TONIUC1, Constantin TOMA1 Abstract: The authors research the structure of vegetative organs of five different vernal species of Viola L. which grow in the Gârboavele - Galaţi nature reserve. The histological features, most of them quantitative ones, which differentiate the five species of Viola L., are pointed out: the thickness of the root and rhizomes peridermis, the number of palisadic layers of the leaf lamina, the presence or absence of tector hairs and of oxaliferous cells, their frequency and position. Key words: Viola, histo-anatomy, vegetative organs, Gârboavele-Galaţi reserve. Introduction There are 28 species of Viola L. in the Romanian flora (Grințescu et al., 1955; Sârbu et al., 2013), four of which known to grow in the Gârboavele - Galaţi nature reserve: Viola arvensis Murray, V. elatior Fr., V. hirta L. and V. odorata L. (Mititelu et al., 1968); to the above mentioned ones we bring a 5-th one, recently found by us: V. kitaibeliana Schult. Two of the species are annual ones (V. arvensis and V. kitaibeliana), while three of them are perennial plants (V. elatior, V. hirta and V. odorata), and have a thick rhizome, with or without aerial runners (V. hirta). The anatomic structure of Viola species has been little investigated until now, as can be seen from the two synthesis treaties, the one about the anatomy of dicotyledonous plants (Metcalfe and Chalk, 1972) and the one about the leaves of angiosperms in general (Napp- Zinn, 1973, 1974). When presentations of tissue structures of various vegetative organs are made, the authors mention, among others the Viola genre, too; Viola odorata being most frequently quoted. Burduja and Moţiu (1982) achieve a detailed histo-anatomical study of the aerial parts of Viola hymettia Boiss., a species closely related (often mistaken for ) to V. arvensis, V. tricolor and V. kitaibeliana. The aerial parts of V. arvensis are used (besides V. tricolor) in traditional medicine for their anti-inflammatory, expectorant and diuretic and other properties. The need to know more about the morpho-anatomic features of both species resulted in research work dedicated to these aspects (Meyer, 1916; Toiu et al., 2009, 2010). Reference to structure features of Viola species can be found in works of comparative (Petit, 1887; Howard, 1962; Uphof and Hummel, 1962) or ecological anatomy (Constantin, 1885), but also in some with a special focus on vernal species (Keller, 1934; Ubaidulaev, 1959; Ivanskaja, 1962, 1963; Goryşina, 1965). 1 Faculty of Biology, “Al. I. Cuza” University, Bd. Carol I, no.11, Iaşi – 700506 Romania; [email protected] (corresponding author*) Mereacre, A. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 1: 13-24 Mention must be made that three of the species studied by us (Viola arvensis, V. kitaibeliana and V. odorata) have been analyzed from an anatomical point of view besides other plants growing in Northern Iran (Yousefi et al., 2012). From the above we can conclude that there are few histo-anatomical data referring to Viola L. species, reason why, in our work, we are attempting a comparative analysis of the vegetative organs of the five vernal Viola species mentioned above, which grow in the Gârboavele - Galaţi nature reserve. Materials and methods The material under study was collected in the Gârboavele - Galaţi nature reserve in May 2012, at the anthesis stage. This forest-park is at 90 m altitude, and are predominantly: Quercus pubescens, Q. pedunculiflora, Ulmus foliacea, Tilia tomentosa, to which are added Acer campestre, A. tataricum, Pyrus pyraster, Cotinus coggygria and another species (Mititelu et al., 1968). The plants were fixed and preserved in 70% ethyl alcohol. The vegetative organs (the root, rhizome, aerial runner, aerial stem, and leaf) were sectioned and coloured (in iodine green and ruthenium red) according to the usual histo-anatomical technique in plant research. Cross-sections and the tangential/longitudinal ones have been analyzed under the Olympus CX31 optic microscope and photographed with an Olympus C 5060 camera (Andrei and Predan, 2003). Results and discussions THE ROOT (Plate I, Figs. 1-3) The rhizodermis of V. odorata and V. hirta occasionally presents long absorbent hairs; the rhizodermis, as well as the external layer of the bark of the other three species is exfoliates early. The bark of V. odorata and V. hirta is differentiated in the exodermis, cortical parenchyma and endodermis; the other species keep only the internal layers of the cortical parenchyma (at V. hirta with aerial gaps) and the casparian type of endodermis; some of the parenchymatous cells of V. hirta and V. odorata contain calcium oxalate ursines. The central cylinder is the anatomical area with a secondary structure, due to the activity of the vascular cambium which has produced a thin external phloem ring and a thick central xylem body. The phloem ring consists of sieve-tubes, companiong cells and numerous cells of amiliferous parenchyma (collenchymatous in V. arvensis, arranged in strictly radial rows in V. elatior). Only Viola odorata has an incipient secondary structure, the phloem has few parenchyma cells, and the xylem has very few libriform elements. The central xylem body comprises vessels of varied diameters, irregularly dispersed in the fundamental lignified mass. The axial part of the stem of V. arvensis, V. hirta and V. kitaibeliana is less lignited, wih fewer and smaller vessels which are separated only by cellulosic parenchyma. The species were determined by Ion Sârbu, Ph.D, whom we warmly thank in this paper, too. 14 Mereacre, A. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 1: 13-24 Thus, the early passage from the primary to the secondary structure can be noticed both in perennial and in annual species, but only at the level of the central cylinder, on account of the vascular cambium. THE RHIZOME (Plate I, Fig. 5) Only Viola hirta and V. odorata present rhizomes, both are perennial species, but only one of them has aerial runners with adventitious roots from the nodes (V. odorata). The contour of its cross section is very irregular, with ribs of various size in V. hirta and a symmetrical structure in V. odorata. The structure of both species is typically secondary, resulting from the activity of both lateral meristems, but especially of the vascular cambium. The epidermis and the largest part of the cortical parenchyma have exfoliated. In the deep persistent internal parenchymatic layers of V. odorata some cells contain, like the ones in the pith, calcium oxalate ursines. The phellogen (or cork cambium), differentiated at the level of the internal cortical parenchyma or of the pericycle (V. hirta), has produced one or two areas of suber and the phellem. The vascular cambium has produced the secondary conducting tissues, of a fascicular type in V. odorata and ring type in V. hirta. The secondary phloem ring is relatively thick and contains conducting elements (sieve-tubes, companion cells) in the close vicinity of the cambium and numerous cells of amiliferous parenchyma towards the exterior, disposed in radial rows, some containing calcium oxalate ursines. The secondary xylem ring is much thicker, strongly sclerified and intensely lignified, made up of irregularly dispersed vessels of varied diameters, many libriform fibers and few parenchyma cells. The xylem ring is crossed by some parenchymatic- cellulosic medullar rays In the thickness of the V. hirta ring we can distinguish several (4-5) rings, each of them having more numerous early vessels and libriform fibers with thinner walls, and fewer later vessels and libriform fibers with thicker walls. In the early xylem areas we can see some islands of cellulosic parenchyma. The pith is parenchymatic-cellulosic, and many cells contain calcium oxalate ursines. THE AERIAL RUNNER (Plate I, Fig. 4) The aerial runner, which we find only in V. odorata has a flat-elliptical contour in cross-section, with a slightly concave face (the inferior one), which corresponds to the place where the endogenously formed adventitious roots will come out; here the phloem is reduced and interrupted by the cord which forms the adventitious root. When dealing with thinner and younger material, the contour of the cross-section is circular, the cortical parenchyma is exfoliating, the suber zone has 3-4 layers of suberified cells, the phellodermis area is visibly collenchymatized, and the phloem ring has a different thickness on the circumference of the organ. The structure is typically secondary and asymmetrical for thicker and older material. The aerial runner surface is covered by a relatively thick peridermis, with 4-6 layers of suber (exfoliating) and an equal number of phellodermis layers, which are tangentially elongated and are disposed in radial series, some of them containing calcium oxalate 15 Mereacre, A. et al. 2014/ Analele Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 60, 1: 13-24 ursines. From place to place there are remains of the primary cortical parenchyma, adhering to the suber. The central cylinder is very thick, but irregular thickness along the circumference of the aerial runner, so that it gets the shape of a cordiform contour in cross-section, the xylem getting into direct contact with the suber along one of the sides. We see that the cambium produces an incomplete, but thick, phloem ring (sieve-tubes and companion cells) towards the interior and numerous parenchyma cells, disposed in radial rows and with moderately thickened walls, towards the exterior.