4 × 106 seedlings per hectare. The sandy loam soil [Typic Fluvaquent, Entisols (US taxonomy)] contains 12.58 g kg− 1
of organic material and 75.19, 45.52 and 99.3 mg kg− 1 of available N, phosphorus and potassium, respectively. Plot dimensions were 4 × 5 m and plots high throughput screening assay were separated by an alley 1 m wide with plastic film inserted into the soil. Each of the treatment had three plots as repetitions in a complete randomized block. The treatment plots received 240 kg ha− 1 at the booting stage. The control plots received no N at the booting stage. All other field conditions and cultivation managements were kept uniform. During the period of wheat anthesis, the anthesis dates were recorded by dotting the glumes and hanging time tags on the wheat plants. Caryopses that bloomed on the same day but developed on different days for the two treatments were chosen for experimentation. Samples were harvested at 15 and 45 DAA. First, 2 mm cubic blocks were
cut by cross-sectioning from wheat caryopses harvested at 15 DAA. The specimens were then fixed with 2.5% glutaraldehyde and 1% paraformaldehyde in a 0.05 mol L− 1 cacodylate buffer solution (pH 7.2) and post-fixation treatment in 1% osmic acid in a 0.15 mol L− 1 sodium cacodylate buffer solution (pH 7.2) for 3 h was applied. The blocks were washed, dehydrated through an ethanol series of 30%–100%, and embedded ERK inhibitor in Spurr’s low-viscosity embedding medium. Sections of 1 μm thick were cut with a glass knife on a Leica Ultracut R (Leica Microsystems, Inc., Wetzlar, Germany), and stained with 0.5% toluidine blue O for 5 min. The sections were visualized and photographed with a Leica Dmls microscope (Leica Microsystems, Inc.). To reflect the nature of caryopsis structure, the findings were compared and confirmed in numerous sections made from
developing grains. Five representative regions of transverse sections of the endosperm were observed for every specimen: subaleurone in dorsal endosperm (SDE), center in dorsal endosperm (CDE), modified aleurone (MA), subaleurone in ventral endosperm (SVE), and center in ventral endosperm (CVE), using three replications O-methylated flavonoid and 20 micrographs representing ten blocks from different regions. Mature grains were harvested at 45 DAA and fractured by applying slight pressure on the middle of the caryopsis with a razor blade. The sample thickness was ~ 3 mm. Caryopses were mounted with the fractured surface facing upwards on a specimen stub and sputter-coated with gold before viewing with a scanning electron microscope (XL30 ESEM, Philips, The Netherlands) at 20 kV to observe the distribution of SGs. The samples at 15 DAA were used to determine the numbers and percentages of SGs. SGs observed in the image were first marked with a specified color using Photoshop CS4 software (Adobe, U.S.A.) and the image was then analyzed to determine the numbers and percentages of SGs using software Image-Pro Plus 6.0 (Media Cybernetics, U.S.A.).