Journal: bioRxiv

Article Title: Leveraging Crop Wild Relatives and Ploidy Level for Climate-Resilient Annual Ryegrass

doi: 10.1101/2025.10.15.682691

Figure Lengend Snippet: Flow cytometry analysis (left panels) and colchicine treatment procedure (right panels) for induction of tetraploidy in annual ryegrass ( Lolium multiflorum ). Left panels: Histograms show fluorescence intensity of propidium iodide bound to DNA in cell nuclei extracted from leaves of diploid (top) and colchicine-induced tetraploid (bottom) genotypes. Peaks G1 2 x and G2 2 x correspond to diploid nuclei in G1 and G2 phases of the cell cycle, while G1 4 x and G2 4 x represent tetraploid nuclei. The x-axis indicates fluorescence intensity (logarithmic scale), proportional to DNA content; the y-axis shows the number of nuclei analyzed. An internal standard was not used; samples were compared against untreated diploid controls (2n = 2x = 14) for the Marshall cultivar and wild type. Right panels (A–C): A) Approximately 40 germinating seeds were submerged in 0.5% colchicine solution for 3 h. B) Treated seedlings were rinsed with purified water and incubated on filter paper in Petri dishes for 2 days. C) Seedlings showing abnormal growth (leaf curliness, shorter and thicker leaves, darker green color) were transplanted into 7.5 cm pots, maintained under high humidity for several days, and later transferred to the greenhouse for ploidy assessment using flow cytometry.

Article Snippet: The leaf tissue was chopped for 30 seconds with a razor blade, incubated on ice for 30 seconds in the extraction buffer, filtered through Partec 50 μm CellTrics (Partec GmbH, Münster), and stained with 2 ml of propidium iodide and RNase (CyStain PI absolute P, Partec GmbH, Münster).

Techniques: Flow Cytometry, Fluorescence, Purification, Incubation