EMS Treatment

EMS treatment was performed as described (Xin et al., 2008). Briefly, dry BTx623 seeds in batches of 100 g (∼3300 seeds) were soaked with agitation for 16 h at 50 rpm on a rotary shaker in 200 mL of tap water containing 0.1 to 0.3% EMS (v/v). The treated seeds were thoroughly washed in ∼400 mL of tap water for 5 h at ambient temperature; wash water was changed every 30 min. Mutagenized seeds were air-dried and prepared for planting. The air-dried seeds were planted at a density of 120,000 seeds per hectare. Before anthesis, each panicle was covered with a 400-weight rainproof paper pollination bag (Lawson Bags) to prevent cross-pollination. Each bag was injected with 5 mL Chlorpyrifos (Dow AgroSciences) at 0.5 mL/L to control maize (Zea mays) earworms that might hatch within the bag and destroy the seeds. Sorghum (Sorghum bicolor) panicles were harvested manually and threshed individually, and M2 seeds were planted as one row per head. To ensure high mutagenesis efficiency, only panicles that set 10% or fewer seeds were allowed to propagate. Three panicles from each M2 head row were bagged before anthesis. Only one fertile panicle was used to produce the M3 seeds. For DNA extraction, duplicate leaf samples were collected from the same fertile plant, and both the leaf samples and the panicle were barcoded. Seeds from the barcoded M2 plants were harvested as M3 families of seeds. For phenotypic evaluation, each M3 family of seeds was planted as one row in the field. Many of the mutant lines exhibited diminished seed production during the M3 generation. Thus, 10 panicles were bagged for each M3 head row and pooled as M4 seeds, which will be distributed to end users upon request.