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 overn ight. The solids were ground three times in 0.5 M NaCl, the supernatant sta rch slurries were pooled and washed through a series of 4M NaCl, 6M NaCl/50 %, sucrose, 2% sodium dodecyl sulphate solution, and acetone before being d ried over silica gel. Subsamples of 1 mg of starch were dispersed in ethano l in preweighed microfuge tubes, gelatinised in NaOH solution, diluted to c onstant concentration, and aliquots were neutralised with citric acid, stai ned with iodine, diluted with wafer, 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. Th e method was used to demonstrate the variation in amylose content within si ngle heads of an inbred tetraploid wheat. No consistent patterns of variati on due to seed location were detected but the overall breadth of variation around the median value of 27 % was +/-5 %.