Starch Extraction and Amylose Analysis from Half Seeds

To allow genetic analysis of starch quality in wheat and its relatives, it was necessary to develop techniques suitable for use on endosperm halves of seeds, leaving the embryo half to be grown for the next generation. Seeds were split and the endosperm end was crushed and soaked in 0.5 M NaCl overnight. The solids were ground three times in 0.5 M NaCl, the supernatant starch slurries were pooled and washed through a series of 4 M NaCl, 6 M NaCl/50 %, sucrose, 2 % sodium dodecyl sulphate solution, and acetone before being dried over silica gel. Subsamples of 1 mg of starch were dispersed in ethanol in preweighed microfuge tubes, gelatinised in NaOH solution, diluted to constant concentration, and aliquots were neutralised with citric acid, stained with iodine, diluted with water, and evaluated in an ELISA plate reader at 620 nm. The overall method provided cleaner starch than earlier methods, as shown by higher apparent amylose values, and was highly repeatable. The method was used to demonstrate the variation in amylose content within single heads of an inbred tetraploid wheat. No consistent patterns of variation due to seed location were detected but the overall breadth of variation around the median value of 27 % was ± 5 %.