Axiocam mrc ccd

Embryos were removed from their chorions with watchmaker forceps. For live imaging, the embryos were anesthetized with 0.168 mg/ml tricaine (Sigma, catalog # A-5040) and placed on a 1% agarose plate. For imaging of fixed tissues, the embryos were incubated in a 4% formaldehyde-PBS (1x PBS) solution overnight at 4°C, washed with PBST (1x PBS, 0.1% Tween 20) and then mounted in 1% PBS-low melt agarose (Zymeset). Embryos of different stages were collected for GFP visualization and photography. All imaging was performed on a Zeiss SteREO Discovery.V8 fluorescent microscope equipped with a Zeiss AxioCam MRc CCD camera.

For embryo observations, seeds were embedded in a cryo-embedding medium (SCEM, Leica Biosystems, Wetzlar, Germany) and frozen in liquid nitrogen. The specimen block was cut into 10-μm sections with a CM-3050S cryostat (Leica Biosystems). We used the Kawamoto cryosectioning method (Chiou et al., 2018 (link)) in which the chamber was set at –15°C and the object holder was set at –25°C. The sections were gently mounted on slides. Cross-sections were photographed with a light microscope (Axio Imager.A2, Carl Zeiss, Oberkochen, Germany) equipped with a CCD camera (AxioCam MRc CCD, Carl Zeiss). For coleoptile observations, coleoptiles at 3–7 mm behind the embryo were embedded in 5% agar, and 100 μm sections were made using a vibrating microtome (Leica VT1200S; Leica Biosystems). Cross-sections were photographed with the above microscope. Coleoptile and seed semblances were photographed with a stereomicroscope (Leica M205FA, Leica Microsystems) with a CCD camera (Leica DFC7000T, Leica Microsystems).

The roots used for aerenchyma measurements were the same ones that were previously used for ROL measurements with the O₂ electrode. Root segments at the basal parts (95–105 mm from the root apex) were prepared from the adventitious roots. Root cross-sections were prepared by hand sectioning with a razor blade. Root cross-sections were photographed using a microscope (Axio Imager.A2, Carl Zeiss, Oberkochen, Germany) under white light with a CCD (charge-coupled device) camera (AxioCam MRc CCD, Carl Zeiss). Areas of cortex and aerenchyma in the cross-section were measured using Fiji (version 2.0.0-rc-69/1.52p), and the percentage of the cortex occupied by aerenchyma was calculated from the cross-sectional areas.