Mzfliii stereomicroscope

Histochemical GUS assays were performed as described by Takata and Taniguchi [15] . Plant tissues such as leaves, stems and roots were harvested from transformants growing for 32 days in soil mix and fixed in cold 90% acetone for 30 min. Tissues were washed with 50 mM sodium phosphate (pH 7.0) and then incubated in GUS staining solution [50 mM sodium phosphate (pH 7.0), 0.5 mg ml -1 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% Triton X-100] at 37 °C for 16 h. Samples postfixed in 2.5% (v/v) glutaraldehyde in 50 mM sodium phosphate (pH 7.0) were bleached in ethanol:acetic acid (6:1, v/v) for leaf blades and 70% ethanol for leaf veins, petioles, stems and roots. Leaf blades were cleared with chloral hydrate/ glycerol solution. Other tissues were infiltrated through a series of 25, 33, 50, 66, 75, and 100% 2.3 M sucrose in 100 mM sodium phosphate (pH 7.0). Leaves and stems were embedded in SCEM-L1 mounting medium (Leica, Solms, Germany) and frozen in liquid nitrogen-cooled hexane. Cryosections of 50 lm were cut using a Leica CM3050 S cryomicrotome (Leica). Roots were sectioned at a thickness of 80 lm using a vibratome (Dosaka EM, Kyoto, Japan). Samples were imaged by a Leica MZ FLIII stereomicroscope and a Leica DMR microscope (Leica).