Bol. Soc. Argent. Bot. 53 (3) 2018 V. G. Perrotta and A. M. Arambarri - Cladodes anatomyISSN of 0373-5 Opuntia80 X Bol. Soc. Argent. Bot. 53 (3): 345-357. 2018 DOI: 10.31055/1851.2372.v53.n3.21310 CLADODES ANATOMY OF OPUNTIA (CACTACEAE) FROM THE PROVINCE OF BUENOS AIRES (ARGENTINA) VANESA G. PERROTTA1 and ANA M. ARAMBARRI1,* Summary: The aim of this study was to deepen the knowledge of the cladodes anatomy of nine species of Opuntia that grow in the province of Buenos Aires, Argentina. Fresh and herbarium samples were prepared according to usual methods for light microscope. Histochemical techniques were performed to identify starch, mucilage and oxalate salts. The main traits found were: epidermis smooth and uniseriate, covered by a thin cuticle and epicuticular waxes; large (range 36-57 µm length) stomata in low density (range 21-27/ mm2), located at level respect to the rest of epidermal cells with a deep substomatal chamber; a multiseriate hypodermis with one crystal layer; the cortex with external chlorenchyma and internal hydrenchyma. The eustele presents different size of vascular bundles; in some species the large ones have a secretory duct adjacent to the phloem. Some species showed wide-band tracheids and a few presented fibres in their vascular bundles. Some characters (presence of fibres and ducts) may be a useful tool for plant identification. However, many open questions remain to be investigated such as the correlation of the environment factors with hypodermis, calcium oxalate crystals, and wide-band tracheids. Key words: Buenos Aires province, Opuntia, segment of stems, tissues. Resumen: Anatomía de los cladodios de Opuntia (Cactaceae) de la provincia de Buenos Aires (Argentina). El objetivo de este trabajo fue profundizar el conocimiento de la anatomía de los cladodios de nueve especies de Opuntia que crecen en la provincia de Buenos Aires, Argentina. Se hicieron preparaciones con material fresco y ejemplares de herbario siguiendo métodos habituales para microscopía óptica. Se usaron técnicas histoquímicas para identificar almidón, mucílagos y sales de oxalato. Las principales características halladas fueron: epidermis lisa y uniseriada, cutícula delgada y ceras epicuticulares; estomas grandes (entre 36-57 µm long) en baja densidad (entre 21-27/ mm2), ubicados a nivel con respecto a las restantes células epidérmicas y con una profunda cámara subestomática; hipodermis multiseriada con capa cristalífera; la corteza con clorénquima externo e hidrénquima interno. La eustela presenta haces vasculares de diferente tamaño, en algunas especies los haces mayores presentan un conducto secretor adyacente al floema. Algunas especies presentaron traqueidas con bandas de engrosamiento secundario y muy pocas tuvieron fibras en sus haces vasculares. La presencia de fibras y conducto adyacente al floema pueden ser caracteresútiles en la identificación de especies.Sin embargo, muchos aspectos requieren mayor investigación en relación con los factores ambientales, tales como la hipodermis, los cristales de oxalato de calcio y las traqueidas con bandas de engrosamiento secundario. Palabras clave: Opuntia, provincia de Buenos Aires, segmentos del tallo, tejidos. INTRODUCTION Baes et al., 2010). It has ample distribution in arid regions of Mexico, Brazil, Argentina and Bolivia The family Cactaceae was assigned to order as the principal center of diversity, but also is Caryophyllales, suborder Portulacineae (Ortega- presents in tropical rainforests and temperate areas (Kiesling, 1975, 1988, 2003; Ortega-Baes et al., 2010). In Argentina, the family is represented by 1 Docentes-Investigadores del Laboratorio de Morfología 225 species of which 131 are endemic (Zuloaga et Comparada de Espermatófitas (LAMCE), Facultad de al., 1999). Opuntia Mill. (subfamily Opuntioideae, Ciencias Agrarias y Forestales, UNLP. Avda. 60 y 119, tribe Opuntieae) contains 191 species (Nattero & C.C. 31 (1900) La Plata, Argentina. Telephone 54-0221- Malerba, 2011), and it has a widespread distribution 423-6758, ext. 461 *Author to contact: [email protected]; arambarri@ from northern Patagonia to southern Canada agro.unlp.edu.ar (Kiesling, 1975). In Argentina, are growing 30 345 Bol. Soc. Argent. Bot. 53 (3) 2018 species (Kiesling, 1975, 1999, 2005), and in the reconstituted by immersion in water and placed province of Buenos Aires, Cabrera & Fabris (1965) in an oven at 30ºC for 24-72 h, then fixed in described seven species of Opuntia. Zuloaga et FAA. To perform anatomy study a small piece al. (2008) cited seven species. Later, Opuntia from the middle part of the cladode was used. To bonaerensis was recognized, and Long (2012) study characteristics in surface view transparent described a new species, Opuntia ventanensis. material was prepared by the following technique During the last years, many anatomical studies of LAMCE Laboratory: at room temperature, for the family, subfamilies and genera have been samples of cladodes were submerged in a solution performed (e.g. Conde, 1975; Mauseth & Plemons- of sodium hydroxide (5%) add sodium hypochlorite Rodríguez, 1998; Loza-Cornejo & Terrazas, 2003; (5%), ratio 1: 1 for 2-5 days. At completion of the Arruda et al., 2005; Mauseth, 2005, 2006; Melo-de- bleaching process, five washed in distilled water Pinna et al., 2006; Pereira de Arruda & Melo-de- were carried out to remove the sodium hydroxide Pinna, 2015) introducing novelties, but only a few and hypochlorite. Samples were then transferred contains some structural data of Argentinian Opuntia into a solution of chloral hydrate (5%) for 24-48 h. species (Vergez Manghi et al., 2015). Therefore, our To analyze the structure, small pieces of cladodes objective was to deepen the anatomy knowledge were sectioned transversally, bleached in sodium of segment of stems (cladodes) of the species of hypochlorite (50%), washed three times with Opuntia from the province of Buenos Aires. distilled water, and stained with alcoholic solution of safranin (80%). Slides were mounted on glycerin jelly (Johansen, 1940). MATERIALS AND METHODS In surface view external characteristics of epidermal tissue, and internal traits of hypodermis Plant materials studied and subepidermal druses were examined. The Accessions from the following herbaria have quantitative analysis was performed on four areas been studied (listed in the Appendix): BA, BAA, on both cladode surfaces. The epidermal cells size, LP, SI (acronyms according to Thiers, http:// anticlinal cell walls width; stomata guard cells sweetgum.nybg.org/ih). In addition, fresh samples length and width, and the diameter of subepidermal were surveyed (included in Appendix). To identified druses between the tips of the crystal were taken. the Opuntia collected, and for nomenclature were The number of epidermal cells, stomata, and consulted the following documents and data base: subepidermal druses per field was determined; Flora de la provincia de Buenos Aires (Cabrera those cells or druses which were in the border of the & Fabris, 1965), Flora ilustrada de Entre Ríos field were not considered in case that more than half (Kiesling, 2005), Instituto de Botánica Darwinion, of structure was out. www.darwin.edu.ar, Long (2012), Font (2014), In transverse sections of the cladode, the Realini et al. (2014), and Las Peñas et al. (2017). tissues and the internal structure were analyzed. Fresh samples of cladodes were preserved in Thereby, quantitative features, such as thickness formalin-acetic-alcoholic solution (FAA 70%) of external periclinal epidermal cell walls plus (Johansen, 1940) in the Laboratorio de Morfología cuticle, hypodermis width and diameter of druses Comparada de Espermatófitas (LAMCE). A located in the cortex and pith were measured. All sample of the collected material was cultivated measurements were expressed in micrometer (µm). in greenhouse, Botanical Garden and Arboretum The average dimensions (e. g. cells, stomata, and “C. Spegazzini”, Facultad de Ciencias Agrarias druses) were established on the basis of more than y Forestales, Universidad Nacional de La Plata. 50 measurements per sample. In Fig. 1 is showed the distribution of the studied Histochemical localization of cell components material in the Buenos Aires province. was performed for starch with iodine-potassium iodide (IKI) (Ruzin, 1999); mucilage was Anatomical and histochemical analyses determined with a color reaction with blue brilliant To avoid alterations on fresh samples, they were of cresyl (1%) (Zarlavsky, 2014), and to distinguish immediately fixed in FAA solution (Johansen, oxalate salts from carbonate we used acetic acid 1940). Dried small pieces of cladodes were (5%) test (Yasue, 1969). 346 Bol. Soc. Argent. Bot. 53 (3) 2018 V. G. Perrotta and A. M. Arambarri - Cladodes anatomy of Opuntia Fig. 1. Geographical points where the studied species of Opuntia were collected in the province of Buenos Aires (Argentina). Microscopic studies were performed by means with digital camera and Micrometrics SE Premium of a CETI T light microscope equipped with a software also was used. camera lucida used to count cells, stomata and Terminology is according to Metcalfe & druses in surface view. The images were captured Chalk (1979), and for stomata type we used with a Moticam 1000 attached to the eyepiece of the classification of Ash et al. (1999). Scientific a microscope Gemalux XSZ-H, and Motic Image names are according to the Instituto de Botánica Plus 2.0 software. A Nikon E200 LED, equipped Darwinion, www.darwin.edu.ar. 347 Bol. Soc. Argent. Bot. 53 (3)