2.3.2 Sampling in Exp. 2
Five superior and inferior grains from each treatment were sampled at DAS 78, 88, 98, 108 d after vitro culture in Exp. 2. All grains were dried at 70℃ to constant weight and weighed. The kernel weight and grain filling parameters were obtained by methods of Exp. 1.
2.3.3Measurements of soluble sugar and starch content in Exp. 3
The five grains sampled at DAP (Days after pollination) 10, 30, 40 after vitro culture for the measurement of soluble sugar and starch content with the anthrone-sulfuric acid method as follows. The grain pulverized to pass a 1 mm screen, 1 mg samples were extracted with of 80% ethanol. Extraction was done three times, followed by centrifugation at 4500 r/min for 10 min, supernatant was incubated at 100 °C for 20 min. The 0.2 mL supernatant was mixed with 1.25 mL of Anthron reagent (100 mg Anthron in 50 mL sulfuric acid). The mixture was heated in a boiling water for 10 min and cooled in water. OD was measured at 630 nm by using 1510 Microplate Reader (ThermoFisher, Shanghai, China).
The residue of the soluble sugar was mixed with 7 mL 3 mol L-1 of HCL, then heated in a boiling water for 10 min and cooled in water, followed by centrifugation at 4500 r /min for 10 min. The supernatant was mixed with 7 mL 3 of mol L-1NaOH in 50 mL volumetric flask, and dilute with water to 50 mL. The starch content were quantified by using 1510 Microplate Reader (ThermoFisher, Shanghai, China) at a wavelength of 625 nm after reaction with 0.5 mL solution from volumetric flask and 2.0 mL Anthron reagent at 45ºC for 10-15 min.