2.3. SSD technique
In F2 populations, 120–170 plants from each cross combination were
randomly selected to develop SSD lines. The assumption was to obtain
ca. 100 lines for cross combinations with segregation at two Glu-1 loci
and 150 for cross combinations with segregation at three loci (20 plants
were treated as a reserve). In accordance with the SSD technique, each
individual was represented by one seed in the next generation. To
accelerate the attainment of successive generations, in vitro culture of
immature embryos was used. One spike from each plant was detached
15–20 d after flowering and single seed was dissected for in vitro culture
of embryo. Embryos were cultured on B5 medium [24] in boxes (50
embryos per box). After 3–4 d of culture, boxes with developing plantlets
were placed into a vernalization chamber for 8 weeks and then
transferred ex vitro into pots in the greenhouse. This approach allowed
us to produce at least 2 generations per year. In the F6 generation, all
seeds from each plant were harvested and accounted for the SSD line.