4.2. Growth Conditions and Evaluation of Nitrogen Fixation by Acetylene Reduction Assay

The red clover seeds were scarified and germinated on wet perlite. Sprouted seeds were planted in individual pots filled with perlite and inoculated with rhizobia by adding 1 mL of Rhizobium leguminosarum bv. trifolii inoculum, which was provided by the Crop Research Institute (Prague, Czech Republic). Different rhizobia strains were applied for diploid and tetraploid varieties as recommended by the collection’s curator. Plants were grown hydroponically in a greenhouse within individual pots filled with perlite. They were watered with a nutrient solution containing 870 mg/L K₂HPO₄, 135 mg/L FeCl₃∙6H₂O, 735 mg/L CaCl₂∙2H₂O, 246 mg/L MgSO₄∙7H₂O, 0.123 mg/L Na₂MoO₄∙H₂O, 0.486 mg/L H₃BO₃, 0.055 mg/L CuSO₄∙5H₂O, 0.25 mg/L MnCl₂∙4H₂O, and 0.06 mg/L ZnSO₄∙7H₂O. No nitrogen was supplied exogenously, and the pH was 6.5–6.8. The solution was replenished as necessary and exchanged once a week. ARA was used for evaluating the efficiency of nitrogen fixation in individual plants through analyzing nitrogenase activity [56]. ARA was carried out approximately 100 days after sowing. The results were expressed as concentration of ethylene C_E (μmol/mL) in a jar after 0.5 h of incubation.