Pi stain solution

4- or 5-day-old seedlings grown on 0.5X MS media with 1% sucrose and 1% agar were transferred to six well plates containing MS media (Mock) or MS media plus 20 μM of Zeocin (Thermo Fisher Scientific - R25001). Plates were wrapped in aluminum foil and left on a shaker (100 RPM) for 2, 4 and 6 h. After treatment, seedlings were transferred to six well plates filled with PI stain solution (10 mg/ml; Sigma P4170) dissolved in H₂0. After 30 sec, seedlings were washed in ddH₂O, transferred to slides in a droplet of H₂0, sealed with a cover slip and imaged at 10X using a dsRED filter and brightfield of a Zeiss fluorescence microscope. Pollen viability was assessed as described (Li 2011 ). For accuracy and highest yield, the assay was performed with flowers collected between 6 AM and 8 AM. Slides containing pollen grains were imaged using a GFP filter (blue light, wavelength = 495 nm) on a Zeiss fluorescence microscope.