Thunder imager 3d tissue epifluorescence microscope

For pollen viability analysis, we only used colchicine-treated plants. After genotyping, we performed flow cytometry in petioles to identify truly neotetraploid and diploid flowers. Then, we use commercial Alexander staining (Morphisto 13441) to stain either released pollens or anthers that were imaged using Leica Thunder Imager 3D Tissue epifluorescence microscope. Details about the pollen viability data are included in Dataset S4.

To immunolocalize HEI10 in our chromosome spreads, we first selected slides containing enough cells in late prophase I (mostly diakinesis but also some diplotene). Then, we followed the protocol described in ref. 63 (link). Briefly, we performed a 45-s microwave treatment at 850 W in our slides containing spreads (from recently fixed material) immersed in citrate buffer pH 6. Immediately after microwave, we washed the slides in PBS-T (0.01% Tween-20 diluted in PBS buffer) for 5 min and then incubated in presence of an antibody against A. thaliana HEI10 (Biomatik) raised in rabbit serum in dilution 1:200 overnight. Then, after 3 washes in PBS-T we incubated using Alexa488-anti-rabbit secondary antibody (Thermo Fisher Scientific A-11034) and mounted the slides. Cells were visualized and imaged using Leica Thunder Imager 3D Tissue epifluorescence microscope. HEI10 foci counting data are detailed in Dataset S1.