3.5. Ploidy Level

Analysis of ploidy level was performed in 2020 using flow cytometry analysis (FCM). Young leaf samples were randomly collected from two plants of each genotype. Leaf tissue (0.25−0.5 cm²) was chopped in a Petri dish in 0.75 mL nuclei isolation Partec buffer (Sysmex Partec GmbH, Münster, Germany) to which 50 μg mL⁻¹ 4′,6-diamidino-2-phenylindole (DAPI) and 1% polyvinylpyrrolidone (PVP) were added. After adding 0.75 mL of the isolation buffer, the samples were filtered through a 30 μm filter and then incubated for 45–60 min. in darkness at room temperature. The fluorescence of the nuclei was measured using ploidy analyser CyFlow Ploidy (Sysmex Partec GmbH, Münster, Germany) with UV-LED 365 nm. The data were analyzed by means of software CyView (Sysmex Partec GmbH, Münster, Germany). Samples with at least 2000 nuclei were measured for two leaves of each plant. As external standard of known ploidy levels, diploid cultivar ‘Santa Rosa’ [5] and hexaploid P. domestica ‘Eruni’ were used [7].