2.3. Hydrogen cyanide estimation

Because previous reports documented that HCN‐P varied based on growth stages and temperatures, HCN‐P was estimated at different growth stages and weather conditions with the goal of finding sorghum pedigrees low in HCN‐P at all times (Bahrani & Deghani, 2004; Barhnhart & Hartwing, 1993; Zahid et al., 2012). At each sampling, the biomass tested was leaves because they are known to accumulate dhurrin more than any other plant part (Gleadow et al., 2016). Younger leaves were selected, particularly the third leaf from the shoot (O'Donnell et al., 2013). One whole leaf was harvested from 10 different plants per plot (Loyd & Gray, 1970) and packaged into a ziplock bag and stored into a cooler from which fresh samples were tested for HCN‐P immediately in the laboratory. Fresh tissue was used because processes such as freeze‐drying samples are known to alter HCN‐P concentrations (Gleadow et al., 2012).

In the establishment year (2019), biomass sampling for HCN‐P occurred in late summer (September 3–6) when the plants were at physiological maturity stage (grains with a black layer at the bottom of the kernel were found in more than half of the plants within a plot). At this time, daily air temperatures ranged from 21 to 38°C, soil temperature ranged from 27 to 30°C, and relative humidity ranged from 28% to 92%. Further, the plots were under drought and heat stress.

In 2020, HCN‐P was first sampled in late spring May (27–29) when sorghum plants were at the five‐ to seven‐leaf stage (vegetative), daily air temperatures ranged from 18 to 30°C, soil temperature ranged from 26 to 27°C, and the relative humidity was 48%–99%. Third and fourth sample dates were collected July (1–3) at 50% flowering (stigma were found in more than half of the plants within a plot) and at physiological maturity (August 18–20) when daily air temperatures ranged from (28–29°C and 30–31°C), soil temperature (27–28°C and 31–32°C), and relative humidity (71%–76% and 65%–71%), respectively.