Truechrome 2

To assess the anatomy of wheat seeds, the seeds were fixed in FAA (38% formaldehyde:glacial acetic acid:70% alcohol = 1:1:18 volume ratio) solution, sliced and embedded in paraffin. The slices were then processed as follows: Fist, the sections were sequentially placed in xylene I for 20 min, xylene II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 75% alcohol for 5 min, and tap water for 5 min to deparaffinize the paraffin sections until dehydration. Second, the slices obtained using an ultramicrotome (Ultracut R, Leica, Germany) were sequentially stained with periodic acid, Schiff’s reagent, and naphthol yellow S (Wuhan Servicebio Technology Co., Ltd.). Third, the slices were dehydrated and mounted with anhydrous ethanol, xylene and neutral gum. Fourth, the slices were observed under a light microscope (DMLS, Leica, Solms, Germany) and photographed with a camera (TrueChrome II, Tucsen, China). Photoshop and Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) were used to count the numbers of amyloplasts based on the micrographs (three replicate seeds and 10 micrographs of each seed).

Caryopses at different development stage were collected and cut transversely into 2 mm thin slices from the middle with a razor blade and immediately immersed in 2.5% glutaraldehyde fixative at 4 °C for 4 h. Then, the samples were rinsed thrice with 0.1 M phosphate buffer (pH 7.2) and dehydrated in a graded ethanol series followed by propylene oxide replacement. Afterward, the samples were infiltrated and embedded in Spurr’s resin and polymerized at 70 °C for 12 h. Eventually, the samples were cut into 1 μm thickness slices using an ultramicrotome (Ultracut R, Leica, Germany), pasted on glass slides, stained with 1% toluidine blue for 5 min, rinsed, dried, observed under a light microscope (DMLS, Leica, Solms, Germany) and photographed by a camera (TrueChromeII, Tucsen, China). Each rice cultivar selected three caryopses as repeats, each of which was from different rice panicles.