Flow Cytometric Ploidy Identification

Fresh leaves were used for ploidy identification using a flow cytometer (FACSCalibar; Beckton Dickinson Co., Franklin lakes, NJ, United States) following the manufacturer’s instructions. About 0.5 cm² of leaf disk was dipped in 400 μL extracting buffer [1% beta-mercaptoethanol, 0.05% Triton X-100, 20 μg mL^–1 RNase A, 15 mM Tris–HCl (pH 8.0), 2 mM Na₂EDTA, 20 mM NaCl, and 80 mM KCl], ground into small particles (Zhang et al., 2011 (link)), and filtrated through a 500-μm mesh sieve. The filtrate was stained with 20 μg mL^–1 propidium iodide (Sigma, Louis, Missouri, United States) and incubated in the dark for 15 min at room temperature. After staining, the nuclei were collected by filtering through a 25-μm nylon mesh. Flow cytometry was performed using the flow cytometer. Diploid “Hanfu” apple (M. domestica, 2n = 2x = 34) was used as a control and internal reference (Ma et al., 2016 (link)). All chemicals were purchased from the TransGen Biotech Co., Ltd. (Beijing, China) unless otherwise indicated.