Twenty kernels selected from the middle of each ear had their embryo, seed coat and aleurone removed. The resulting endosperms were dried in an oven (42 °C), and then ground into fine powder with a prototype machine (60HZ, 60S). The powder was filtered through an 80-mesh sieve to prepare samples for starch measurement.
For analysis of zeins in immature kernels, endosperms were collected at 18 and 24 DAP, and ground in the lipid nitrogen into fine powder. The extraction and analysis of zein proteins was according to the standard method of our laboratory42 (link). For assaying the total protein content, 60 mg of endosperm powder was used for measurement by the instrument of ELEMENTAR Rapid N exceed. The starch content of endosperm was measured with a Total Starch Assay Kit (K-TSTA; Megazyme) following the standard protocol43 (link). Amylose was quantified according to the Megazyme amylose/amylopectin assay procedure (K-AMYL; Megazyme). Six biological replicates were performed.
For analysis of zeins in immature kernels, endosperms were collected at 18 and 24 DAP, and ground in the lipid nitrogen into fine powder. The extraction and analysis of zein proteins was according to the standard method of our laboratory42 (link). For assaying the total protein content, 60 mg of endosperm powder was used for measurement by the instrument of ELEMENTAR Rapid N exceed. The starch content of endosperm was measured with a Total Starch Assay Kit (K-TSTA; Megazyme) following the standard protocol43 (link). Amylose was quantified according to the Megazyme amylose/amylopectin assay procedure (K-AMYL; Megazyme). Six biological replicates were performed.
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