Pelargonium roseum Willd. is an herbaceous species, belonging to the Geraniaceae family, cultivated in Romania as an ornamental species. The present study aims to establish the identity of this species, by the microscopic analysis of the vegetative and reproductive organs. The main method used for the study of the species was optical microscopy. Macroscopic and microscopic characters of the vegetative (root, stem, leaf) and reproductive (flower) organs were identified. Data obtained in this study are important criteria for the identification of the species

CRIŞAN, Gianina

GÂLEA, Ionela Carmen

IELCIU, Irina

TĂMAŞ, Mircea

English

Publicatii USAMV Cluj-Napoca (University of Agricultural Sciences and Veterinary Medicine / Universitatea de Ştiinţe Agricole şi Medicină Veterinară din Cluj-Napoca)

115  Hop and Medicinal Plants, Year XXV, No. 1-2, 2017 ISSN 2360-0179 print, ISSN 2360-0187 electronic  HISTO-ANATOMICAL STUDIES ON THE VEGETATIVE AND REPRODUCTIVE ORGANS OF PELARGONIUM ROSEUM WILLD. (GERANIACEAE)  GÂLEA Ionela-Carmen 1, Irina IELCIU2,*,  Gianina CRIŞAN2, Mircea TĂMAŞ 1 University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 38 Gheorghe Marinescu street, Târgu-Mureș, Romania; 2 “Iuliu Haţieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Botany, 3rd floor, 23 Gheorghe Marinescu street, Cluj-Napoca, Romania;  *Corresponding author, e-mail: ielciu_irina@yahoo.com  Abstract. Pelargonium roseum Willd. is an herbaceous species, belonging to the Geraniaceae family, cultivated in Romania as an ornamental species. The present study aims to establish the identity of this species, by the microscopic analysis of the vegetative and reproductive organs. The main method used for the study of the species was optical microscopy. Macroscopic and microscopic characters of the vegetative (root, stem, leaf) and reproductive (flower) organs were identified. Data obtained in this study are important criteria for the identification of the species.   Keywords. Geraniaceae, macroscopic and microscopic characters, Pelargonium roseum Willd., vegetative and reproductive organs.   Introduction  Pelargonium roseum Willd., rose geranium, has South-African origins and was introduced in Europe in the 18th century (Gâlea, 2009). It is an ornamental species, which can reach heights of 120cm. It has woody, straight and branched stem. The leaves are pinnatisect, covered in short, rough, glandulous and villous trichomes, which confer to the plant a strong, pleasant smell, similar to the one of the rose flowers, of which the traditional name is derived (Săvulescu, 1953). The species, among other species of the Pelargonium genus, is known and used in traditional medicine for the presence of the volatile oil (Kopp et al., 1960, Bojor et al., 2003; Diener et al., 1989). 116  Most of the existing data in the scientific literature provide information on the trichomes of Pelargonium species. Considering that the volatile oil is stored in the trichomes of the leaf, Walt and Demarne (1988) studied the trichomes of the species Pelargonium graveolens L'Her. and Pelargonium radens H.E.Moore, using electronic microscopy (Balchin, 2002). Oosthuizen studied the trichomes of the species P. radens, describing 3 types of non-glandular trichomes and 2 types of glandular ones. He extended his work on 133 Pelargonium species and subspecies, revealing 11 different types of trichomes (Oosthuizen, 1983; Oosthuizen et al., 1984). Previous studies aim the morpho-anatomic structure of petiole and leaf of some Pelargonium species (Lancu et al., 2013; Romitelli et al., 2013).  The present study aims to perform a detailed observation of the following histo-anatomical details, both for the vegetative and for the reproductive organs:  The root: thickness of the suber, zoning of the phloem, thickness and composition of the xylem, degree of lignification and presence of the calcium oxalate crystals;  The stem: structure of the epidermis (shape and size of the cells, degree of thickening of the external wall, tector and secretory trichomes), of the suber, of the phellogen, of the cortex, of the sclerified pericycle, of the phloem, of the cambium and of the xylem fibres;  The leaves: both the upper and the lower epidermis (shape and size of cells, the structure of the lateral walls, the presence and frequency of the stomata, the type of stomata, the structure of the palisade and of the spongy mesophyll);  The flower: on superficial sections, both the epidermis of the perianth.  Materials and methods   The vegetal material was harvested from the Medicinal Plant Botanical Garden of the University of Medicine and Pharmacy in Târgu-Mureş and was identified at the Pharmaceutical Botany Department of the Faculty of Pharmacy Cluj-Napoca. A voucher specimen is deposed Pharmaceutical Botany Department of the Faculty of Pharmacy Cluj-Napoca (voucher no 84.3.5.1-5).  Optical microscopy. Cross sections of the vegetative (root, stem, leaf) and reproductive (flower) organs were performed on vegetal material preserved 117  in 700 ethanol, using the classical sectioning technique (Dobjanschi et al., 2005; Rus et al., 2015; Păltinean et al., 2015). Vegetal material was sectioned with the botanical razor, after the prior inclusion in the elder pith. The sections were stained for highlighting histo-anatomical details, with malachite green Merck® and subsequently were included in a gelatin-glycerin inclusion medium (Gâlea, 2009, Rus et al., 2015; Păltinean et al., 2015). Analysis of the specimens obtained was performed at the Novex trinocular microscope, using the 4X, 10X and 25X objectives and the observed images were captured using a Panasonic DMC – LZZ digital camera (Gâlea, 2009).  Results and discussion  The root. The sectioned organ presents a secondary structure, as a result of the activity of phellogen (fig.1.1) and cambium (fig.1.2). On the outer side, the suber is found (fig.1.3), formed of 4-5 layers of overlapped cells, tangentially elongated, with suberified walls. In the cortex, calcium oxalate crystals are found as druses (fig.1.4). The central cylindrer is not clearly delimited by a pericycle. The secondary phloem is observed (fig.1.5), being disposed towards the exterior part, in a circular shape. The secondary xylem (fig.1.6) occupies most of the center of the root, being interrupted by two parenchymatous strings, produced by the cambium, the secondary medullary rays (fig.1.7). The secondary xylem is formed of vessels disposed in radial strings and fibres. Towards the center of the section, the primary xylem can be found (fig.1.8). The radicle also presents a secondary structure. At the outer side, it is surrounded by the suber cells, tangentially overlapped (fig.1.9). The cortex (fig.1.10) contains calcium oxalate crystals (fig.1.11). The center of the section is occupied by the secondary xylem (fig.1.12), which has the secondary phloem (fig.1.13) above. 118   Fig. 1. Secondary structure of the root of Pelargonium roseum Willd. (cross section) – 4X overview (a), 10X structural details (b,c) – and secondary structure of the radicle of Pelargonium roseum Willd. (cross section) – 4X overview (d)   The stem. The stem was sectioned at the basal, median and upper level, in order to establish the differences between the histo-anatomical structures of this organ at these three different levels, given the fact that the macroscopic structure of this organ is irregular. Differences appear in the pericycle, which is sclerified and appears as strings in the basal stem and as a continuous layer in the medial stem, while in the upper part, it appears incompletely formed and not sclerified.  The basal part of the stem has epidermis with thickened walls (fig.2.1) and contains many tector and secretory trichomes (fig.2.2, c). Below the epidermis, the suber (fig.2.3) appears under development by the repeated divisions of the phellogen (fig.2.4). Under the phellogen, the cortex (fig.2.5) is found and the central cylinder is delimited by the strings of pericycle (fig.2.6), which protects the primary (fig.2.7) and secondary phloem (fig.2.8). Between the phloem and the secondary xylem (fig.2.9), situated towards the center of the section, the cambium (fig.2.10) is found. The xylem is composed mostly of xylem fibres, the vessels being in a small number. In the center of the section, the medular parenchyma is lignified (fig.2.11).   The median part of the stem has the vascular bundles arranged on a circular contour. The epidermis (fig.3.1) also has thickened walls and a larger number of tector a secretory trichomes (fig.3.2). The secretory trichomes appear frequently and are larger. Below the epidermis, a collenchymatic layer (fig.3.3) appears, followed by a parenchymatic cortex (fig.3.4). Druses of calcium oxalate (fig.3.c) are found. The central cylinder is delimited by the continuous pericycle (fig.3.5), which protects the primary vascular bundles (fig.3.e), of different sizes, separated by 119  parenchyma strings (fig.3.6). Between the xylem (fig.3.7) and phloem (fig.3.8), the cambium (fig.3.9) appears to edify the secondary structure.   The upper part of the stem has the structure incompletely developed. The epidermis (fig.4.1) is also covered in tector and secretory trichomes (fig.4.2, c) of different sizes. Secretory trichomes have 1-3 basic cells and a big secretory cell in the top (fig.4.c). Below, the cortex (fig.4.3) has the first layer with slightly collenchymatic cells (fig.4.4). The pericycle (fig.4.5) is not yet sclerified. The primary vascular bundles (fig.4.b) are formed of xylem (fig.4.6) and phloem (fig.4.7).    Fig. 2. Anatomical structure of the basal part of the stem of Pelargonium roseum Willd. (cross section) – 4X, 10X structural details (a,b) and secretory and tector trichomes of the basal part of the stem of Pelargonium roseum Willd. (superficial section) – 25X (c)   Fig. 3. Anatomical structure of the median part of the stem of Pelargonium roseum Willd. (cross section) – 4X overview (a), 10X,25X structural details (b,c, d,e)  120   Fig. 4. Anatomical structure of the upper part of the stem of Pelargonium roseum Willd. (cross section) – 4X, 10X structural details (a, b) and secretory and tector trichomes of the upper part of the stem of Pelargonium roseum Willd. (superficial section) – 25X (c)  The leaf. The petiole has circular contour (fig.5.a) in cross section. The anatomical structure is similar to the median part of the stem. The first layer is represented by the epidermis (fig.5.1), followed by the parenchymatic cortex (fig.5.2), where the calcium oxalate druses (fig.5.3) are found. The central cylinder is delimited at the exterior side by the pericycle (fig.5.4, b), formed of a few layers of collenchymatic cells. Below the pericycle, the primary collateral vascular bundles (fig.5.5, c) are found on a circular contour, separated by strings of parenchyma (fig.5.6).   Fig. 5. Anatomical structure of the petiole of Pelargonium roseum Willd. (cross section) – 4X overview (a), 10X, 25X structural details (b, c, d)   121  The lamina has a well-developed prominence on the inferior part (fig.6.1), correspondent to the central vein. The bifacial anatomical structure of the lamina is protected on both sides by the two epidermis: the upper epidermis (fig.6.2) and the lower epidermis (fig.6.3). Between the two, the mesophyll (fig.6.4) is found, having a parenchymatic structure, with many calcium oxalate druses (fig.6.5). A single-layer palisade mesophyll (fig.6.6), under the upper epidermis and a multi-layer spongy mesophyll (fig.6.7), above the lower epidermis are found. Both the upper and the lower epidermis contain tector (fig.6.8) and secretory (fig.6.9) trichomes. Secretory trichomes have pluricellular basis and a globulous secretory cell in the top. In the proeminence, a collateral vascular bundle (fig.6.10) is found, surrounded by a few layer of parenchymatic cells (fig.6.11). The upper and lower epidermis of the leaf present thin and wavy cell walls (fig.7.1), with cellulosic structure. The anexes of the epidermis can be found both on the upper epidermis and on the lower one. The stomata (fig.7.2) are anomocytic and the trichomes are tector and secretory (fig.7.3) ones.  The trichomes of the leaf are similar to the trichomes found at the surface of all the vegetative and reproductive organs. Tector trichomes are formed of 2 cells, with smooth walls, having no thikenings and the terminal cell is sharpened. Secretory trichomes have 2 shapes, depending on the size of the basis, which can be formed of 2 or 3 cells. The bi-cellular trichomes become tri-cellular ones, as the leaf matures. Both types of glandular trichomes have at the top a secretory cell, covered with a cuticle. Below the cuticle, the secretion of the cell, an essential oil is deposed.   Fig. 6. Anatomical structure of the leaf lamina of Pelargonium roseum Willd. (cross section) – 4X overview (a), 10X, 25X structural details (b, c, d) 122    Fig. 7. Anatomical structure of the upper leaf epidermis of Pelargonium roseum Willd. (superficial section) – 4X view  The flower. Petals and stamens were examined. On the surface of the petals, tri-cellular, simple glandular trichomes were revealed (fig.8.a, b). On the surface of the stamens, pollen grains were observed, having a concave, triangular-like shape, with rounded tips and 3 apertures (fig.8.c). The external layer of the pollen grain, the exine, has thorns.     Fig. 8. Anatomical structure of the flower of Pelargonium roseum Willd. (superficial section) – 4X view  123  Conclussions  Microscopic analysis allowed the identification of the anatomical structures of the vegetative (root, stem, leaf) and reproductive (flower) organs. Results obtained in this study are important for the macroscopic and microscopic identification of the species. This study is bringing originality and represents a supplementary argument for the future investigation of the main active substances of this plant and their cellular localization.  References  1. Balchin, L.M. (2002). Geranium and Pelargonium, Ed. Taylor and Francis, London. 6-29, 118-216. 2. Bojor, O., Popescu, O. (2003). Fitoterapia tradiţională şi modernă, 3rd edition, Ed. Fiat Lux. 321-325. 3. Diener, H. (1989). Arzneipflanzen und Drogen, VEB Fachbuchverlag, Leipzig. 336.   4. Dobjanschi, L., Tămaş, M. (2005). Cercetări histo-anatomice asupra speciilor indigene de Solidago. Clujul Medical. 1: 188-191. 5. Kopp, E., Kotilla E. (1960). Experienţe de cultură şi obţinere a uleiului esenţial din Pelargonium roseumWilld. Farmacia. 6: 423-428. 6. Lancu, C.E., Cioanca, O., Mircea, C., Hancianu, M. (2013). Contributions regarding the leaf histo-anatomy of some Pelargonium species. Rev Med Chir Soc Med Nat. 117(3): 812-818. 7. Oosthuizen, L.D. (1983). The taxonomic value of trichomes in Pelargonium L’Herit. (Geraniaceae). S Afr J Bot. 49: 221-242.  8. Oosthuizen, L.D., Coetzee, J. (1984). Trichome initiation during leaf growth in Pelargonium scabrum. S Afr J Bot. 3: 50-54.  URL: http://www.pelargonium.si/literature/Trichome-initiation-during-leaf-growth-in-Pelar_1984_South-African-Journal-o.pdf 9. Păltinean, R., Crişan, G., Balica, G., Ielciu, I., Ştefănesu, C., Tămaş M. (2015). Histo-anatomical researches on the vegetative organs of five Romanian Fumaria species. Farmacia. 63(2):262–266.  URL: http://www.revistafarmacia.ro/201502/art-16-Paltinean_262-266.pdf 10. Romitelli, I., Martins, M.B.G. (2013). Comparison of leaf morphology and anatomy among Malva sylvestris ("gerânio-aromático"), Pelargonium graveolens ("falsa-malva") and Pelargonium 124  odoratissimum ("gerânio-de-cheiro"). Revista Brasileira de Plantas Medicinais. 15(1): 91-97.  URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-05722013000100013 11. Rus, L.M., Ielciu, I., Păltinean, R., Vlase, L., Ştefănescu, C., Crişan G. (2015) Morphological and Histo-Anatomical Study of Bryonia alba L. (Cucurbitaceae). Not Bot Horti Agrobot. 43(1):47–52.  URL: http://www.notulaebotanicae.ro/index.php/nbha/article/view/9713/7811 12. Săvulescu, T. (1953). Flora Republicii Populare Române, Editura Academiei R.P.R., Bucureşti. 6: 160. 

HISTO-ANATOMICAL STUDIES ON THE VEGETATIVE AND REPRODUCTIVE ORGANS OF PELARGONIUM ROSEUM WILLD. (GERANIACEAE)

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HISTO-ANATOMICAL STUDIES ON THE VEGETATIVE AND REPRODUCTIVE ORGANS OF PELARGONIUM ROSEUM WILLD. (GERANIACEAE)

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