GUS staining and histological analysis
Histochemical GUS staining was performed with a GUS Staining Kit (G3061,
Solarbio Co., Beijing, China) following the manufacturer’s instructions.
Samples were fixed in 90% acetone at –20°C, rinsed four times with 0.1
M sodium phosphate buffer (pH 7.4), and then incubated in X-Gluc
solution (0.1 M sodium phosphate (pH 7.4), 3 mM potassium ferricyanide,
0.5 mM potassium ferrocyanide, and 0.5 g L–15-bromo-4-chloro-3-indolyl-β -d-glucuronide cyclohexilammonium
salt) at 37°C. After staining, chlorophyll was removed from the samples
by incubating them in methanol; they were then mounted in a clearing
solution (a mixture of chloral hydrate, water, and glycerol in a ratio
of 8:2:1). Observations were made using a stereomicroscope (MZ16F, Leica
Microsystems, Germany) or a microscope equipped with Nomarski optics
(BX51, Olympus Co., Tokyo, Japan). To characterize vascular patterns,
cotyledons were fixed in a mixture of ethanol and acetic acid in a ratio
of 9:1, dehydrated through a graded series of ethanol, and then mounted
with a clearing solution (Konishi & Sugiyama, 2003).