Passiflora Ligularis Juss.) Seeds Análisis Morfológico Y Anatómico De Las Cubiertas De Semillas De Granadilla (Passiflora Ligularis Juss.)

PROPAGATION AND TISSUE CULTURE Morphological and anatomical analyses of the seed coats of sweet granadilla (Passiflora ligularis Juss.) seeds Análisis morfológico y anatómico de las cubiertas de semillas de granadilla (Passiflora ligularis Juss.) Julián Cárdenas-Hernández1,2, Diego Miranda L.1, Stanislav Magnitskiy1, and Carlos Carranza1 ABSTRACT RESUMEN The study of histology and morphology of seeds of genus Pas- El estudio de la histología y morfología de las semillas del siflora has been of high utility for the classification of species. género Passiflora ha sido de gran utilidad para la clasificación In seeds of sweet granadilla, the histological characteristics de especies. Sin embargo, las semillas de granadilla no han and methodologies for their study are unknown. This study sido investigadas. Con este estudio se buscó conocer las was aimed to know the tissue and morphological character- características tisulares y morfológicas de la cubierta de las istics of the seed coats of seeds of sweet granadilla and to be semillas de granadilla y así poder determinar su valor en la able to determine its value in the differentiation of accessions. diferenciación de accesiones. Se analizaron cinco accesiones Five accessions collected in producing zones of the Province colectadas en zonas productoras del departamento del Huila of Huila, Colombia, were analyzed. In morphological analysis, (Colombia). En el análisis morfológico, todas las accesiones all accessions presented falsifoveate ornamentation and entire presentaron ornamentación falsifoveada y borde entero. Las margin. The seeds presented high change in weight and size for semillas presentaron gran variación en peso y tamaño dentro every accession; there stood out the seeds of the accession PmN de cada accesión; se destacaron las semillas de la accesión for presenting major size (7.42 mm long), weight (35.62 mg), PmN por presentar mayor tamaño (7,42 mm de longitud), homogeneity in these variables and a typical orange color. For peso (35,62 mg), homogeneidad en estas variables y un color the histological analysis, a protocol was adapted to realize sec- naranja característico. Para el análisis histológico se adaptó un tions of seed coats in parafin, by means of which one managed to protocolo para realizar cortes de la cubierta de las semillas en obtain sections (7 µ) that in the optical microscope show clearly parafina, mediante el cual se logró obtener cortes (7 µ) que al three well differentiated layers, belonging, possibly, to exoteg- microscopio óptico muestran claramente tres capas bien dife- men (internal layer), mesotesta (medium layer) and exotesta renciadas, pertenecientes posiblemente a la exotesta (externa), (external layer). Every layer presented differences in the form of mesotesta (media) y exotegmen (interna). Cada capa presentó the cells, color and thickness, between the sections of the basal diferencias en la forma de sus células, color y espesor, entre los and medium parts of the seeds, but the differences between the cortes basales y los cortes medios de las semillas, sin diferencias analyzed accessions were not observed. The majority of seeds claras entre las accesiones analizadas. La mayoría de las semillas had a thickness of seed coats that varied among 235 and 475 µ. mostraron un grosor de la cubierta entre 235 µ y 475 µ. Key words: exotegmen, mesotesta, exotesta, macrosclereids, Palabras clave: exotegmen, mesotesta, exotesta, macroesclereids, taniferous cells. células taníferas Introduction Fruits of Passiflora sp. possess multiply seeds adhered to the funiculus on the ovary wall and surrounded by an aril Morphology and anatomy seed research contributes to that covers the seed and constitutes the eatable part of the knowledge of taxonomy, evolution, and ecology of Angio- fruit (Werker, 1997). The seeds of the family Passifloraceae spermae species (Cortez and Carmello-Guerreir, 2008). present seed coats with generally lignified cells that not Passiflora is the most important genus of the family Pas- only affect water absorption but also offer a resistance to sifloraceae and is represented by approximately 500 species. the embryo growth (Cardozo, 1988). This taxon presents a wide natural distribution, with plants grown in the wild in the Americas, Western India, Galapa- Seed coats develop from the integuments that surround gos Islands, Australia, Southeast Asia, Malaysia, Polynesia, the ovule prior to fertilization. Before fertilization, cells of and some islands of the Pacific Ocean (Vanderplank, 1990). the integuments are relatively undifferentiated. However, Received for publication: 15 June, 2011. Accepted for publication: 2 November, 2011. 1 Department of Agronomy, Faculty of Agronomy, Universidad Nacional de Colombia. Bogota (Colombia). 2 Corresponding author. [email protected] Agronomía Colombiana 29(3), 377-385, 2011 development after fertilization can include extensive dif- Materials and methods ferentiation of the cell layers into specialized cell types. In addition, some cell layers in the seed coats may accumulate The collection of five accessions was realized in the pro- large quantities of certain substances, such as mucilage or ducing zone of sweet granadilla (Passiflora ligularis Juss.) pigments that can also contribute to overall seed morphol- in the South of Huila in the municipalities La Argentina, ogy (Moïsa et al., 2005). San Agustín and Palestina, the last one being the largest producer of sweet granadilla at national level. The layers of the seed coats in seeds of Passiflora sp. are developed from bitegmical ovary, in which a radial unequal The fruits were taken to the Laboratory of Crop Physiology elongation of cells could be observed (Werker, 1997). The of Faculty of Agronomy, Universidad Nacional de Colombia aril begins of a meristematic margin continued around the (Bogota), for their physico-chemical characterization. For distal part of the raphe and includes the exostomal region. the analysis of seeds, five accessions found in two commer- The pigments of the aril are located in the chromoplasts cial cultures were collected, one in the district La Mensura (Werker, 1997). (2,012 m a.s.l.) and the other one in the district El Roble (1,807 m a.s.l.) of the municipality Palestina. The chosen MacDougal (1994) realized a taxonomical review of the accessions were named PrJ1, PrJ2, PrJJ, PmFD, and PmN; section Pseudodysosmia of subgenus Decaloba of genus the first three were from the district El Roble and two last Passiflora, and included the ornamentation of seeds among ones were from the district La Mensura. the characters that he used in its descriptions. This allowed considering the species of genus Passiflora to be an excel- Fifty seeds of every accession were taken at random; in each lent object of study to characterize the seed morphology of these, the mass, length, width, thickness, and number and, therefore, illustrate the taxonomical potential that the of fosets were measured, the uniformity of distribution of above mentioned structure presents for this genus (Pérez- fosets was described, the relation lenght-width (L/W) was Cortéz et al., 2005). calculated, and the presence of appendices in the base and the apex of the seeds was measured. In order to do this, On this matter, Pérez-Cortéz et al. (2005) realized an scales of precision and a digital gauge were employed. anatomical description of seed coats of eight species of Passiflora sp., found eight anatomically different patterns The presence of appendices was described in the base and and observed a layer of macrosclereid cells and a layer of the apex of the seeds, enumerating them according to their obliterate cells in all the studied species. Nevertheless, P. presence. For this, some descriptors proposed by Pérez- ligularis was not among the analyzed species. Cortéz et al. (1995, 2002) were applied. The seeds of P. edulis, in the external tissues, present cells Finally, a photographic record of the seeds of every acces- of palisade parenchyma, prismatic and lignified with thick sion was realized with help of a microscope Nikon SMZ800 reticulate walls, which form conical projections inside the with integrated camera DSZMV using the software ele- endosperm. The endotesta has thick cell walls, prismatic ments, in the Laboratory of Entomology of the Faculty of elongated cells in the depressions of the tegmen and cuboids Agronomy, Universidad Nacional de Colombia, Bogota. in the channels (Werker, 1997). In order to undertake histological sectioning, firstly, the The seeds of P. ligularis are black, compressed, relatively protocol for sectioning in microtome in the Laboratory of small, and hard. The seeds and the aril contain about 17.6% Microbiology of Faculty of Agronomy of the Universidad carbohydrates (Trujillo, 1983; Werker, 1997). Pérez-Cortéz Nacional de Colombia (Bogota) was adapted, taking into et al. (2002) describe the seeds of P. ligularis with transverse account the methodology used by Pérez-Cortéz et al. (2005, average section of elliptical form, basal of sharp form or 2009). The principal methodological adaptations occurred sharply truncated, having ornamentation foveated or fal- in the processes of tissue softening with hydrochloric acid sifoveated and entire margin. (18.5%) and fixation of tissues with Mayer Albumen. The objective of the present research was to characterize Using the adapted protocol, sections of 7 µ of seed coats the anatomy of seed coats of seeds of sweet granadilla using were done of the seeds of five chosen accessions. With help five accessions collected in producing zones corresponding of the program Scopephoto 3.0® and optical microscope to the South of Province of Huila, Colombia. Micro BM2000 with built-in camera DCMC510, the photo- 378 Agron. Colomb. 29(3) 2011 graphic record of sections was realized. The entire thickness Some seeds of PrJ2 presented monochrome seed coats of of seed coat was measured as well as the thickness of each of black color (Fig. 2A), also seeds were observed with partially three layers identified with help of the program Imagepro or completely yellow colorations. The distribution of fosets Express 6.3® de Medicybernetic Inc. Both morphological in these seeds was highly uniform (Fig.

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