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

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