K amyl

Brown rice from Nip, Olr, F_2, and F₃ progeny plants of Olr and Nip were milled into fine flour by using a flour mill. The fine flour from each of the samples was subsequently used for the determination of the starch, amylose, amylopectin, and protein contents. Starch content, amylose content, and amylopectin contents were determined by using the starch assay kits Megazyme K-TSTA and K-AMYL (Megazyme, Wicklow, Ireland) as described by Wei et al. [24 (link)]. The protein content of the samples was determined following the method described by Kang et al. [25 (link)].

Harvested paddy seeds were air-dried and stored at room temperature. Embryos and pericarps were removed before the experiment, and the endosperms were ground to a powder using a grinding mill. The starch and amylose contents were measured using starch assay kits following the manufacturer’s instructions (K-TSTA and K-AMYL; Megazyme). To determine the amylose content, the powder was soaked for 48 h in 0.4% NaOH (powder:NaOH = 1:3) at room temperature, washed several times with distilled water until no slimy liquid remained, and drained. To determine the amount of soluble sugars, 50 mg of endosperm powder was washed twice in 80% (v/v) ethanol at 80 °C for 40 min and assayed using anthrone reagent^{49 (link)}.

The ratio of amylose to amylopectin and total starch content were determined using an Amylose–amylopectin Assay kit (K-AMYL; Megazyme, Wicklow, Ireland) and a Total Starch Assay kit (K-TSTA; Megazyme, Wicklow, Ireland), respectively, according to the manufacturer’s instructions. Rice endosperm hardness was determined by measuring the pressure at the grain breakage point using a universal testing machine (Z 0.5, Zwick Roell, Ulm, Germany) (Kwak et al., 2017 (link)). To measure damaged starch content, 100 ± 10 mg of rice flour from each mutant and its respective wild type was tested using a Starch Damage Assay kit (K-SDAM; Megazyme, Wicklow, Ireland). For microscopy analysis of the opaque-endosperm phenotype, rice grains were observed on an LED illuminator. Scanning electron microscopy was carried out as described previously (Ryoo et al., 2007 (link)). Starch samples coated with gold were observed under a Stereoscan Leica Model 440 instrument (Leica Cambridge) at an accelerating voltage of 20 kV. The chain-length distribution of starch extracted from mature endosperm was determined according to a previously described high performance anion exchange chromatography method (Ryoo et al., 2007 (link)).