Abstract: Taking the fresh callus tissue of Amorphophallus konjac K. Koch as materials, the influences of dissociation buffers, fluorescent nucleic acid stains, filtering times, and centrifugation protocols on ploidy detection were compared. The results showed that, MG^b lysis buffer yielded significantly higher counts of A. konjac nucleus compared to MgSO₄ and Tris-MgCl₂ buffers, with a prominent peak and minimal fragment peaks observed. The coefficient of variation (CV) was calculated at 4.47%. Fluorescent staining with DAPI demonstrated comparable efficacy to PI, with the added advantage of reducing background fluorescence noise without RNAase treatment, thereby conserving analysis time. Both the primary filtration and the secondary filtration methods could effectively remove adhered cells and cell debris. The secondary filtration method collected more cell nuclei and had a better effect. Optimal nuclear suspension preparation involved direct light avoidance and in situ staining post-filtration. The proposed flow cytometric protocol for A. konjac ploidy analysis involved: homogenizing 0.25 g of fresh callus tissue in 1 mL MG^b lysis buffer, filtering, staining with 50 μL DAPI in darkness for 30 minutes, followed by immediate analysis.

Key words: Amorphophallus konjac K. Koch, flow cytometry, ploidy detection