QUARTER-SEED ASSAY FOR INORGANIC PHOSPHORUS (PI)

In the low phytate soybean lines developed by Wilcox et al. (2000), low phytic acid levels are inversely associated with an elevated level of Pi, which is easier to measure. "Chen's reagent" reacts with Pi to produce a blue color, and the intensity of the blue indicates the concentration of Pi in the extract solution. Therefore, samples which produce a darker blue color are from low-phytate seed, and these are the ones that would be selected for advancement or crossing.

Assay Procedure

  1. Chip quarter seed and place chips into appropriate well of a 96-well plate. Add 0.5ml Pi extraction buffer [12.5% trichloroacetic acid (TCA) and 25mM MgCl2] to each well.
  2. Soak chips 6 hours (or overnight at 4°) to soften tissue. Mash using a glass stirring rod or similar instrument. Clean between samples.
  3. Agitate plate on a vortex to mix extract buffer and seed tissue. Repeat once or twice during incubation (4 hours at RT to overnight at 4°).
  4. Vortex samples one more time and allow tissue to settle for 30'. Prepare Chen's reagent (recipe below). Ten ml Chen's reagent is needed for each 96 samples.
  5. Pipette 90ul of dH2O and then 10ul of extract buffer from each sample well into the corresponding well of a microtiter plate using a multichannel pipettor or a liquid handling robot. Take care when pipetting the extract buffer to make sure that pipette tips did not become plugged with a seed particle.
  6. Add 100ul Chen's reagent to each well and incubate 2 hours at RT. Compare color intensity of progeny samples with high Pi/low phytate control sample(s).

Chen's Reagent

Stock Solutions Quantity For 96 samples (10ml)
6N H2SO4 1 volume 2ml
2.5% ammonium molybdate 1 volume 2ml
10% ascorbic acid * 1 volume 2ml
dH2O 2 volumes 4ml

* Store stock solution at 4°C.

If the solid ammonium molybdate is 81.4% pure, dissolve 3.07g in 100ml dH2O to obtain a 2.5% solution.

When screening a segregating population, include samples of normal parent and low phytate parent seed extracts as controls.

Try to use uniform sized seed chips to minimize variability in results.