The germination rate of seeds (percentage of germinated seeds) after-ripened for 21 days (GR21) was estimated for each genotype following standard methods (Alonso Blanco et al. 2003). Briefly, about 75 seeds per genotype were spread on wet filter paper (Whatman, ref. 1001-047) in Petri dishes (Greiner Bio-One, ref. 628102) that were subsequently placed in moisture chambers to maintain high, constant humidity (close to 99%). Moisture chambers consisted of reasonably air-tight, transparent plastic boxes (Ikea), whose bottom was covered by ten layers of paper towel imbibed with tap water. Germination was scored by observing radicle emergence after a week of incubation at 23°C in standard long days (16 h day / 8 h night) and under fluorescent light (Osram L 58W 840 Lumilux, 40 μm/m2/sec).
Three biological replicates of each genotype were first vernalized for eight weeks (4°C, 16 h day / 8 h night, 90% humidity) before being placed in a cold environment (15°C day / 10°C night). The cold treatment was applied from rosette stage to ripening and seed harvest. Seeds were harvested when about 50% of the siliques of a given plant had come to maturity and were subsequently placed in dry environment for after-ripening (30% relative humidity, 16°C, dark).