Sz2 stu2

For each experiment, cuttings were transferred into a container filled with deoxygenated solid agar (1.5% w/v) and kept at a constant room temperature (25–27 °C) under a photosynthetic photon flux density (PPFD) of about 100 μmol m⁻² s⁻¹ provided by fluorescent tubes (FL20SBR, NEC, Tokyo, Japan). Cuttings were fixed in the container by solid agar, maintaining the water level similar to that when the cuttings were grown in hydroponic or agar media. The target adventitious roots were fixed on the surface of the solid agar and covered with deoxygenated solid gelrite (2% w/v), about 1-cm-thick, to ensure a clear view under a microscope (Figure 1). Experiments were conducted both before and after the water level was raised. Using a micromanipulator, a Clark-type O₂ microelectrode was positioned in the cortex (primary aerenchyma), about 100 μm from the surface of the adventitious roots that developed 10 cm from the root–shoot junction. The positioning of the microelectrode inside the root cortex was aided by a microscope (SZX7, Olympus, Tokyo, Japan) and adjusted using a boom stand (SZ2-STU2, Olympus, Tokyo, Japan).