Anales del Jardín Botánico de Madrid, Vol 68, No 2 (2011)

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


https://doi.org/10.3989/ajbm.2273

Eduardo Cires
Área de Botánica, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Spain

Candela Cuesta
Department of Plant Systems Biology - Área de Fisiología Vegetal, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Belgium

María Ángeles Fernández Casado
Área de Botánica, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Spain

Herminio S. Nava
Área de Botánica, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Spain

Víctor M. Vázquez
Real Instituto de Estudios Asturianos, Spain

José Antonio Fernández Prieto
Área de Botánica, Universidad de Oviedo, Departamento de Biología de Organismos y Sistemas, Spain

Abstract


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.

Keywords


Chromosome number; flow cytometry; genome size; mucilaginous compounds; nuclear DNA content; ploidy levels; section Melanium; section Viola

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References


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