Whole plants were observed and photographed using an EOS D60 (Canon) or EOS Kiss X7 digital camera (Canon) and a stereoscopic microscope (SZ61; Olympus) equipped with a CCD camera (DP50, Olympus or DSX500, Olympus). Embryos were cleared using Hoyer’s solution (Feng and Ma, 2017 (link)) and observed with a BX51 upright microscope (Olympus) equipped with a CCD camera (DP72, Olympus).
For observation of leaf veins, 10-, 15-, and 20-day-old leaves were fixed in a solution (ethanol:acetate = 3:1) at room temperature. The fixed specimens were washed in an ethanol series (70%, 50%, 30%, and 15%) and rinsed with ClearSee (10% xylitol, 15% sodium deoxycholate, and 25% urea) (Kurihara et al., 2015 (link)).
To observe the cross sections of rosette leaves, leaf samples including the leaf margin were cut to a size of 2 × 2 mm with a razor. Both ends of the leaves were sliced to allow the fixative solution to permeate well into the sample, and then the samples were immersed in a fixative solution (3% glutaraldehyde, and 50 mM Na-Pi, pH 7.0) and degassed thoroughly. The samples were embedded in 5% agar and sliced to 40 μm thickness with a microtome (VT 1200 s, Leica).
For observation of leaf veins, 10-, 15-, and 20-day-old leaves were fixed in a solution (ethanol:acetate = 3:1) at room temperature. The fixed specimens were washed in an ethanol series (70%, 50%, 30%, and 15%) and rinsed with ClearSee (10% xylitol, 15% sodium deoxycholate, and 25% urea) (Kurihara et al., 2015 (link)).
To observe the cross sections of rosette leaves, leaf samples including the leaf margin were cut to a size of 2 × 2 mm with a razor. Both ends of the leaves were sliced to allow the fixative solution to permeate well into the sample, and then the samples were immersed in a fixative solution (3% glutaraldehyde, and 50 mM Na-Pi, pH 7.0) and degassed thoroughly. The samples were embedded in 5% agar and sliced to 40 μm thickness with a microtome (VT 1200 s, Leica).
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