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Download Download 2273 citometry_Maquetación 1 28/11/2011 11:32 Página 139 Anales del Jardín Botánico de Madrid Vol. 68(2): 139-154 julio-diciembre 2011 ISSN: 0211-1322 doi: 10.3989/ajbm.2273 Isolation of plant nuclei suitable for flow cytometry from species with extremely mucilaginous compounds: an example in the genus Viola L. (Violaceae) by Eduardo Cires 1*, Candela Cuesta 2, 3*, María Ángeles Fernández Casado 1, Herminio S. Nava 1, Víctor M. Vázquez 4 & José Antonio Fernández Prieto 1 1 Área de Botánica, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Catedrático Rodrigo Uría s/n, E-33071 Oviedo, Spain. Corresponding author: Phone: +34 985 104835; fax: +34 985 104777; e-mail: [email protected] 2 Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Gent, Belgium. [email protected] 3 Área de Fisiología Vegetal, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Catedrático Rodrigo Uría s/n, E-33071 Oviedo, Spain 4 Real Instituto de Estudios Asturianos, Plaza de Porlier 9, E-33003 Oviedo, Spain * Both authors contributed equally to this work Abstract Resumen Cires, E., Cuesta, C., Fernández, M.A., Nava, H.S., Vázquez, Cires, E., Cuesta, C., Fernández, M.A., Nava, H.S., Vázquez, V.M. & Fernández, J.A. 2011. Isolation of plant nuclei suitable V.M. & Fernández, J.A. 2011. Aislamiento de núcleos para cito- for flow cytometry from species with extremely mucilaginous metría de flujo en plantas con alto contenido de compuestos compounds: an example in the genus Viola L. (Violaceae). Ana- mucilaginosos: un ejemplo en el género Viola L. (Violaceae). les Jard. Bot. Madrid 68(2): 139-154. Ana les Jard. Bot. Madrid 68(2): 139-154 (en inglés). Flow cytometry analysis has been widely applied in the determi- El análisis mediante citometría de flujo ha sido aplicado de modo nation of nuclear DNA content and ploidy level in many organ- general para determinar el contenido de ADN nuclear y el nivel isms. Despite being the most appropriate method for DNA con- de ploidía en muchos organismos. A pesar de ser el método más tent measurement, flow cytometry also presents some limita- adecuado para medir la cantidad de ADN, esta técnica también tions. A fairly common, but little-studied problem is the effect presenta algunas limitaciones. Un problema bastante común, on measurements of the presence of secondary metabolites. A aunque poco estudiado, es el efecto de los metabolitos secun- good example is the genus Viola, which is composed of 525-600 darios en los resultados obtenidos. Un ejemplo respecto a la pre- species distributed worldwide. These species have proved to be sencia de estos compuestos se encuentra en el género Viola, problematic for flow cytometric analyses due to the release of compuesto por 525-600 especies distribuidas por todo el mun- extremely mucilaginous compounds into the nuclear suspen- do. Las especies de este género ya han sido previamente descri- sion. In this work, the genome size of 13 species of Viola using tas como problemáticas en los análisis de citometría de flujo de- flow cytometry are presented for the first time. Despite obtain- bido a la presencia de compuestos extremadamente mucilagi- ing histograms with high coefficients of variation, we here pre- nosos en las suspensiones de núcleos. En el presente trabajo se sent an optimized protocol to remove cytoplasmic compounds, analiza por primera vez el tamaño genómico de 13 especies del particularly mucilaginous ones, from plant nuclei that pave the genero Viola mediante el empleo de citometría de flujo. A pesar way for its application to estimate the genome size of other de los altos valores mostrados en los coeficientes de variación de species exhibiting similar problems. Statistical analyses revealed los histogramas, se presenta un protocolo optimizado para eli- significant differences between sections Viola and Melanium, minar compuestos citoplasmáticos, y más concretamente muci- and within each section (P < 0.001). Furthermore, statistically laginosos de las suspensiones nucleares, siendo de aplicación en significant differences were not detected among samples of the la estimación del tamaño genómico de plantas con problemas same species. similares. Los análisis estadísticos mostraron diferencias signifi- cativas entre las secciones Viola y Melanium, así como dentro de cada sección (P < 0,001). Además, no se encontraron diferencias significativas entre aquellas muestras pertenecientes a la misma especie. 2273 citometry_Maquetación 1 28/11/2011 11:32 Página 140 140 E. Cires & al. Keywords: Chromosome number, flow cytometry, genome Palabras clave: Número cromosomático, citometría de flujo, size, mucilaginous compounds, nuclear DNA content, ploidy tamaño genómico, compuestos mucilaginosos, contenido de levels, section Melanium, section Viola. Abbreviations: CV: ADN nuclear, niveles de ploidía, sección Melanium, sección coefficient of variation; DI: DNA index; FCM: flow cytometry; Viola. PI: propidium iodide. Introduction compounds. On the other hand, Loureiro & al. (2007b), in the study of the genera Ulmus L. and Flow cytometry (FCM) is a fast, sensitive technique Celtis L., presented a protocol which ensured high for measuring specific components in large numbers quality analyses with a low coefficient of variation and of cells and organelles. Plant scientists have been at- minimal background debris as well as nuclear fluores- tracted by the numerous advantages of this technique cence stability. However, even Loureiro & al. (2007b) (e.g. ease of sample preparation, speed of analysis, found similar problems in leaves of Ulmus species, and no requirement for dividing cells) and the num- where they got poor results and had to use an alterna- ber of articles related to this technique has been con- tive tissue (samaras) characterized by lower concen- tinuously increasing over the years (Doležel & al., tration of mucilaginous compounds. 2007a). Estimation of DNA content in cell nuclei is The genus Viola L. includes examples of several one of the most important applications of FCM in species with extremely mucilaginous compounds. This plant sciences. In most plants, analyses of relative genus, the largest of the Violaceae family, comprises DNA content of nuclei isolated from young tissues 525-600 species distributed throughout most frost- yield histograms with good resolution. In principle, free regions of the world (Ballard & al., 1999; Yock- every tissue containing vital nuclei should be suitable teng & al., 2003; Karlsson & al., 2008). It is divided for measurement of nuclear DNA content with FCM. into ca. 14 sections and many infrasectional groups However, the presence or absence of endogenous flu- (Becker 1910a, 1910b, 1925) (for a review of infra- orescence inhibitor substances and coatings of debris generic classification see Ballard & al., 1999). Five of influences the quality of the results (Greilhuber & al., those sections are present in Europe: Chamae me - 2007). Plant cells produce a vast array of secondary lanium Ging., Melanium Ging., Xylinosium W. Bec- metabolites which may interfere with staining of par- ker, Delphiniopsis W. Becker and Viola (Valentine & ticular cell constituents and/or exhibit autofluores- al., 1968); all of which have also been described in the cence, thus hampering quantification of signals from Iberian Peninsula (Fernández Casado, 1982; Muñoz fluorescent probes (Doležel & al., 2007b). Although Garmendia & al., 1993). the interference of secondary metabolites with stain- In the present work, the samples selected belong to ing procedures had been recognized for some time in 2 of these sections: Melanium, which includes the so- cytophotometry (e.g. Greilhuber, 1986), it was not un- called pansies and is a morphologically well-defined til Noirot & al. (2000, 2002, 2003, 2005) and Price & group of about 80-100 species; and Viola, which is a al. (2000) published their findings that this effect was Eurasian group of about 25 species with several sub- taken seriously in plant FCM. Moreover, some species sections described (Valentine & al., 1968; Karlsson contained mucilaginous compounds which interfered & al., 2008), of which the following were studied: with analysis, affecting overall histogram quality and Viola, Rostratae Kupffer and Stolonosae Kupffer nuclear fluorescence stability (e.g. Betulaceae, Vio- [=Plagiostigma Godr.]. Taxa of these sections are well laceae, Lythraceae, Malvaceae, Orchidaceae and sub- known for their taxonomic complexity, apparently merged plants) (Suda, 2004; Loureiro, 2007; Greilhu- caused by several factors: (a) scarcity of reliable diag- ber, 2008). Besides hampering the release of nuclei, nostic morphological characters; (b) high phenotypic these compounds confer a high viscosity to the nu- plasticity; (c) frequent interspecific hybridizations; clear suspension, which makes the filtration step diffi- and (d) assumed past reticulate evolution within the cult to accomplish and consequently results in the re- section and subsection (Ballard & al., 1999; Yockteng covery of a low volume of nuclear homogenate. Some & al., 2003; Hodálová & al., 2008; Mered’a & al., authors have found great difficulty in using FCM due 2008; Van den Hof & al., 2008). to these compounds (e.g. Morgan & Westoby, 2005; A high diversity of chromosome numbers has been Talent & Dickinson, 2005; Loureiro & al., 2007a). To proposed within genus Viola (Moore, 1982; Castro- give an example, Morgan and Westoby (2005) found viejo & Valdés-Bermejo, 1991) by showing different for many
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