For frozen samples, all filled grains from one side of the rachis were pooled while kept frozen. Grains were lyophilized at −15�C for 5 d followed by 24 h at room temperature. All grains from matured, dried samples were lyophilized at room temperature (24 h). Dried grains were crushed, cooled in liquid nitrogen and ground to flour in a SPEX SamplePrep2010 Geno/Grinder� (Metuchen, New Jersey, USA).
Determination of total starch was done using the AA/AMG kit from Megazyme (Bray, Ireland). D-Glucose (1 mg/ml) and Regular Maize Starch from Megazyme (Bray, Ireland) served as controls. Amylose content and amylose: amylopectin ratio was determined using the K-AMYL kit (Megazyme, Ireland). β-glucans were quantified using the K-BGLU kit (Megazyme, Ireland). Five milligram aliquots of flour were used, otherwise manufacturer’s recommendations were followed. Grubbs test for outliers, ANOVA and ANCOVA were made using XLSTAT add-on for Microsoft Excel (Addinsoft, New York, NY, USA). ANOVA was applied to mature samples and ANCOVA to distinct stages during development (HT: 240–393�Cd; recovery: 384–601�Cd; desiccation: 464–681�Cd). For samples taken during development n = 3, for samples taken at maturity n = 5 plants were used for measurements.
Determination of total starch was done using the AA/AMG kit from Megazyme (Bray, Ireland). D-Glucose (1 mg/ml) and Regular Maize Starch from Megazyme (Bray, Ireland) served as controls. Amylose content and amylose: amylopectin ratio was determined using the K-AMYL kit (Megazyme, Ireland). β-glucans were quantified using the K-BGLU kit (Megazyme, Ireland). Five milligram aliquots of flour were used, otherwise manufacturer’s recommendations were followed. Grubbs test for outliers, ANOVA and ANCOVA were made using XLSTAT add-on for Microsoft Excel (Addinsoft, New York, NY, USA). ANOVA was applied to mature samples and ANCOVA to distinct stages during development (HT: 240–393�Cd; recovery: 384–601�Cd; desiccation: 464–681�Cd). For samples taken during development n = 3, for samples taken at maturity n = 5 plants were used for measurements.