**2.2 Determination of kunitz trypsin inhibitor genotype**

98 F2 seed from F1 plants for population 1 and 243 F2 seed from F1 plants for population 2 was analysed electrophoretically to determine the presence (SKTI-`+') or absence (SKTI- 'null') of kunitz trypsin inhibitor. A piece of cotyledon from each F2 seed was removed and the remaining embryo germinated to given a F2 mapping population. The separated cotyledon tissue was incubated for 30 min (room temperature) in 1 ml Tris-HCl, pH 8.0, containing 1.56 % v/v β-mercaptoethanol. After centrifugation, 50 l of the supernatant were added to an equivalent amount of 5X sample buffer [10% w/v sodium dodecyl sulphate (SDS), 50% v/v glycerol, 1.96% v/v β-mercaptoethanol, 1 M Tris-HCl, pH 6.8]. The samples were boiling at 97°C for 5 min and then centrifuged. Two microlitre of the supernatant were used for electrophoresis on 12% acrylamide SDS polyacrylamide gel electrophoresis (SDS-PAGE) medium gels in Owl Separation Systems. Inc(Model: P9DS, Portsmouth, NH USA). Electrophoresis was practiced at 120 V for 7 hr. Gels were stained overnight in an aqueous solution of 0.25 g coomassie brilliant blue R250, 10% acetic acid, 45% methanol and destaining solution (5% acetic acid, 14 methanol) for several hours. A Wide-Range SDS-PAGE molecular mass standard (Sigma MarkerTM, Product Code : M4038) containing the 21.5kDa soybean trypsin inhibitor protein, was used to aid recognition of samples lacking the kunitz trypsin inhibitor.
