DETERMINATION OF TRYPSIN INHIBITOR GENOTYPE IN SOYBEAN SEEDS


Seed grinder

  1. Cut a quarter seed from the cotyledon region opposite the embryo axis, and place each sample into a labeled coin envelope.
  2. Mash seed either by hand or with a seed grinder (see photo) and load into a square well 96-well plate (2ml volume wells).
  3. Add 1.2ml extraction buffer (0.1M Tris-HCl buffer, pH 8.2, 20mM CaCl2). Shake for several hours to overnight at RT. Do not vortex.
  4. Spin at 3,600rpm for 30'. Transfer supernatant to a new deep-well plate for trypsin activity determination.
  5. Mix 40ul seed extract with 600ul buffer (0.1M Tris-HCl buffer, pH 8.2, 20mM CaCl2, 50ul 4.166 x 10-4M trypsin solution) and incubate at room temperature for 6'. For negative control, replace seed extract with 40ul Tris-HCl.
  6. Trypsin activity is determined by mixing 400ul Tris-HCl buffer, 500ul 1.2 x 10-3M D,L-BapNA, and 400ul incubation solution. Incubate 3' at RT.
  7. Stop reaction by adding 200ul 80% acetic acid. Absorbance is determined at 410nm.

Reference
De Carvalho, W.L., M.G. de Almedia Oliveiar, E.G. de Barros, and M.A. Moreira. 1998. "Determination of genotypic classes for trypsin inhibitor in soybean seeds." Biotechnology Techniques. Vol 12. No 12, 859-863.