Isolation of plant nuclei suitable for flow cytometry from species with extremely mucilaginous compounds: an example in the genus <i>Viola</i> L. (Violaceae)

Abstract

Cires, E., Cuesta, C., Fernández, M.A., Nava, H.S. Flow cytometry analysis has been widely applied in the determination of nuclear DNA content and ploidy level in many organisms. Despite being the most appropriate method for DNA content measurement, flow cytometry also presents some limitations. A fairly common, but little-studied problem is the effect on measurements of the presence of secondary metabolites. A good example is the genus Viola, which is composed of 525-600 species distributed worldwide. These species have proved to be problematic for flow cytometric analyses due to the release of extremely mucilaginous compounds into the nuclear suspension. In this work, the genome size of 13 species of Viola using flow cytometry are presented for the first time. Despite obtaining histograms with high coefficients of variation, we here present an optimized protocol to remove cytoplasmic compounds, particularly mucilaginous ones, from plant nuclei that pave the way for its application to estimate the genome size of other species exhibiting similar problems. Statistical analyses revealed significant differences between sections Viola and Melanium, and within each section (P < 0.001). Furthermore, statistically significant differences were not detected among samples of the same species. ResumenCires, E., Cuesta, C., Fernández, M.A., Nava, H.S., Vázquez, V.M. & Fernández, J.A. 2011. Aislamiento de núcleos para citometría de flujo en plantas con alto contenido de compuestos mucilaginosos: un ejemplo en el género Viola L. (Violaceae). Ana les Jard. Bot. Madrid 68(2): 139-154 (en inglés).El análisis mediante citometría de flujo ha sido aplicado de modo general para determinar el contenido de ADN nuclear y el nivel de ploidía en muchos organismos. A pesar de ser el método más adecuado para medir la cantidad de ADN, esta técnica también presenta algunas limitaciones. Un problema bastante común, aunque poco estudiado, es el efecto de los metabolitos secundarios en los resultados obtenidos. Un ejemplo respecto a la presencia de estos compuestos se encuentra en el género Viola, compuesto por 525-600 especies distribuidas por todo el mundo. Las especies de este género ya han sido previamente descritas como problemáticas en los análisis de citometría de flujo debido a la presencia de compuestos extremadamente mucilaginosos en las suspensiones de núcleos. En el presente trabajo se analiza por primera vez el tamaño genómico de 13 especies del genero Viola mediante el empleo de citometría de flujo. A pesar de los altos valores mostrados en los coeficientes de variación de los histogramas, se presenta un protocolo optimizado para eliminar compuestos citoplasmáticos, y más concretamente mucilaginosos de las suspensiones nucleares, siendo de aplicación en la estimación del tamaño genómico de plantas con problemas similares. Los análisis estadísticos mostraron diferencias significativas entre las secciones Viola y Melanium, así como dentro de cada sección (P < 0,001). Además, no se encontraron diferencias significativas entre...

Flow cytometry – a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species

“…GS estimation of five species belonging to the family Hyacinthaceae, characterized by high mucilage content, was established after modifications to the frequently used Galbraith's isolation buffer (Galbraith et al, 1983), by increasing pH and detergent concentration, and replacing sodium citrate with citric acid (Nath et al, 2014). Optimized protocols for sample preparation of 13 Viola and Hylocereus species, whose tissues are also rich in polysaccharides, were proposed by Cires et al (2011) and Li et al (2017), respectively. An alternative to modifying the sample preparation procedure is to find tissues/plant parts free of undesirable compounds, for example a seed Jedrzejczyk and Sliwinska, 2010).…”

Genome size variation and polyploidy incidence in the alpine flora from Spain

“…corniculatus, considering the reported values for diploids and tetraploids of this species (Bennett & Smith, 1976), our estimations fall in the middle of the variation reported so far, suggesting the presence of DNA triploid individuals with 18 chromosomes; still as no infra-specific categorization was given in those works, this assumption should be confirmed in the future using classical karyology. For V. riviniana, despite of the difficulty to obtain histograms with low CV values, our estimation is approximately half that observed before for tetraploid individuals (Cires & al., 2011), which suggests that the sampled individuals are diploid with 20 chromosomes. Despite diploids have never been reported for this species, there are previous evidence of some variability in the number of chromosomes (2n = 35, 40, 45, 46, 47 chromosomes;Cires & al., 2011).…”

“…For V. riviniana, despite of the difficulty to obtain histograms with low CV values, our estimation is approximately half that observed before for tetraploid individuals (Cires & al., 2011), which suggests that the sampled individuals are diploid with 20 chromosomes. Despite diploids have never been reported for this species, there are previous evidence of some variability in the number of chromosomes (2n = 35, 40, 45, 46, 47 chromosomes;Cires & al., 2011).…”

Chromosome Characterization of Festival Strawberry (Fragaria x ananassa D. var. Festival) Result of Polyploidization

“…Flow cytometry is applied method for approximate quantity DNA in nucleus cell and determines ploidy level in the organism (Cires et al, 2011). Basic principles of flow cytometry are the analysis of fluorescent microscopy and optical properties from the movement of particles in suspension.Then the suspension will pass flow chamber and radiated light source and produce fluorescent signal that is detected by series of chronic mirrors then photomultiplier will convert electrical signals into digital value and displayed in histogram form (Ochatt, 2008) (Doležel & Bartoš, 2005).…”