An improved protocol for flow cytometry analysis of dragon fruit (Hylocereus spp.)—Species with a high polysaccharide content

• Published in: Scientia horticulturae 2017-06, Vol.220, p.130-133
• Main Authors: Li, Daqing, Cisneros, Aroldo, Tel-Zur, Noemi
• Format: Article
• Language: English
• Subjects: Cactaceae; C–DNA content; DNA ploidy; Dragon fruit; Flow cytometry; Nuclei isolation
• ISSN: 0304-4238; 1879-1018
• DOI: 10.1016/j.scienta.2017.03.033

•Flow cytometry analysis in dragon fruit is a challenging task due to the high polysaccharides content of all the tissues.•We developed a more effective nuclear isolation buffer for the isolation of nuclei from new emerging stem tips.•We performed filtration instead of centrifugation, and staining the samples with propidium iodide.•Our flow cytometry protocol is simple, rapid and gives reproducible results. Assessing the nuclear C–DNA content and estimating DNA ploidy in succulent species, such as dragon fruit (Hylocereus spp., Cactaceae), is a challenging task. Dragon fruit tissues are rich in polysaccharides that are released during the nuclei isolation procedure, inducing the formation of clusters of nuclei and hence leading to inaccurate results. Our flow cytometry protocol for dragon fruit species is simple, rapid and reproducible. Our innovations include: modifications to the nuclear isolation buffer to ‘match’ the cytosol composition of the cells; filtration of the samples instead of centrifugation, thereby preventing co-precipitation of nuclei and polysaccharides; and staining of the nuclei with propidium iodide. DNA ploidy of dragon fruit samples were estimated using a reference standard − either external or internal − with a known ploidy level and a known nuclear DNA content. The best results were obtained using new emerging and actively growing stem tips, which have lower concentrations of polysaccharides and a lower frequency of endopolyploid nuclei, thereby simplifying the nuclei isolation procedure.