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.