Drought Stress Treatments

To investigate the genotypic variation in drought tolerance, 12-day barley seedlings were subjected to drought stress in two ways: 1) terminate the supply of nutrient solution for 10 days, termed as water deficit (WD); and 2) elevate the osmotic potential in trays by washing vermiculite with 20% (w/v) PEG8000 (P103734, Aladdin, Shanghai, China) solution (prepared in the background of one-fifth Hoagland solution. Tavakol et al., 2018; Cai et al., 2019) for three times and keep the osmotic potential with 20% (w/v) PEG8000 solution for another 5 days, termed as PEG-simulated drought (PEG). The seedlings grown with Hoagland solution under the same environmental condition were considered as control. The whole experiment was set up in a randomized block design with three treatments being three blocks. In each block, all the trays were swapped randomly every day to minimize the systematic error induced by spatial location. At the end of drought treatments, the shoots of barley seedlings were excised from basal conjunction to determine fresh weight, dry weight and water content. In the experiment of screening, fresh shoots were sampled from barley seedlings grown under either control or drought conditions and immediately weighed to obtain the shoot fresh weight (FW). Thereafter, the weighed shoots were dried in the oven at 75°C to constant weight and then weighed for the shoot dry weight (DW). The water content (WC) in shoot was calculated using the following formula: WC = (FW − DW)/FW × 100. Five replicates were determined for each combination of genotype and drought treatment.

Based on the results from the work of screening for drought tolerance, eighteen barley genotypes, which displayed the consistent performance under both water-deficit and PEG-simulated drought stresses, were selected for further verification of their tolerance to drought stress. Barley seedlings were grown in 0.6 L pots containing vermiculite with nine plants per pot. Each barley genotype was grown in three individual pots. The growth condition and water-deficit drought treatment were the same as described above. The plants well-irrigated with adequate one-fifth Hoagland solution were considered as the control. The plants were grown for 10 d before drought stress started. After onset of water-deficit drought stress for 14 d, the seedlings were sampled for investigation of fresh weight, dry weight, water content, leaf chlorophyll content and fluorescence, leaf stomatal conductance and leaf sap osmolality. In the experiment of verification, shoot fresh and dry weights were determined as described for screening. To determine the relative water content (RWC) in barley leaves, the entire fresh oldest and youngest fully-expanded leaves from barley seedlings grown under control and drought conditions were respectively sampled and weighed immediately for the fresh weight (FW). The weighed leaves were then soaked in deionized water for 2 h at room temperature, then leaves were dried with kimwipes (34155, Kimtech, Kimberly-Clark, USA) softly and their turgid weights (TW) were recorded immediately. Relative water content was calculated using the following formula: RWC = (FW − DW)/(TW − DW) × 100. Five replicates were determined for each combination of genotype and drought treatment.