Single Seed Selection for Low Phytate Lines

Most seed phosphorus (P) is bound in phytate which is unavailable to monogastric animals depriving them of P and causing eutrophication from P in animal waste. It is valuable to reduce the phytate levels of seeds used for food and feed. Low phytate mutant seeds such as soybean [Glycine max (L.) Merr.] MIPS (D-myo-inositol 3-phosphate synthase) mutants have correspondingly increased inorganic phosphate (Pi). Measurement of seed Pi levels is an established technique for screening for low phytate mutants but to date it has not been performed non-destructively from single seed samples. A protocol was developed greatly reducing the sample size thereby reducing the cost and time and saving a generation in the selection of low phytate mutant seeds based on the high Pi phenotype. Pi was extracted using 12.5 % trichloroacetic acid. Pi reacts with ammonium molybdate in acidic solution to form phosphomolybdic acid, which upon reduction with ascorbic acid produces an intensive blue color. This new technique was tested using 1-2 mg samples, 96-well plates with 8 MIPS mutants (GM-lpa-TW1), 8 wild-type and 8 seeds segregating for the MIPS mutation. Selection of low phytate mutant seeds based on the high Pi level measured via a colorimetric assay based on a reaction of extracted Pi with ammonium molybdate forming a blue color. This was confirmed by DNA analysis of seed chips. This protocol is applicable to many other crop seeds including corn, wheat, rice and sorghum.