2.2. Ploidy Analysis

For this analysis, the C-values were measured in a sample of mesocarp tissue derived from a pooling of 5 fruits for each genotype and for each time point for five replicates. Nuclei were isolated from approximately 100 mg of frozen mesocarp by gentle chopping with a razor blade in 0.4 mL of CyStain^® PI Absolute P nuclei extraction buffer (Sysmex Partec GmbH, Gorlitz, Germany) supplemented with 1% w/v PVP. Nuclei suspensions were filtered by using 30 μm CellTrics^® (Sysmex Partec GmbH, Gorlitz, Germany). Following the filtration step, 1.6 mL staining buffer was added to each sample and tubes were stored in the dark on ice for 1 h before measurement. The fluorescence intensity of DAPI-stained nuclei was determined using the flow cytometer CyFlow^® Cube Ploidy Analyzer (Sysmex Partec GmbH, Gorlitz, Germany) equipped with an UV-Light Emitting Diode (l = 355–375 nm). Data were plotted on a logarithmic scale and calibration of C values was made with nuclei suspensions prepared from young leaves. On average, 24,000 nuclei were assessed in each run. Ploidy histograms were quantitatively analyzed with the FCS Express 5 Flow software (Sysmex Partec GmbH, Gorlitz, Germany), after manual adjustment to exclude noise. For each ploidy level, significant differences in the number of nuclei were assessed by Student t tests.