MATERIALS AND METHODS

Seeds and seed processing

Seeds from a double haploid C. sativa cultivar (DH55) were obtained from plants grown under greenhouse conditions (harvesting at 85-100 d after planting) and stored at 4°C. Seed from two biological replicates grown in November-February 2014 and April-July 2015 was used.
Mucilage interferes with aqueous extraction of proteins and was removed (or reduced) before seed disruption. First, whole seeds were placed in deionized water at pH 4.5 (1 g seed:10 mL water) containing Viscozyme® (Sigma-Aldrich Canada Co.) at 0.1 mL enzyme/g seed and incubated at 40°C for 3 h with mixing using an overhead stirrer (850 rpm). The seed slurry was then passed through a #25 sieve (710 µm mesh size) to recover seeds. The seeds were washed 5 times with deionized water (1:10 w/v) to remove residual enzyme and mucilage, and then dried overnight in a forced air oven at 35°C. The seeds were then de-oiled according to AOCS method AM 2-93 (Firestone, 1997) using hexane and the resultant meal was air-dried in a fume hood for 12-16 h. Mucilage-reduced, oil-free meal was stored in airtight containers at ambient temperature.