Murashige and skoog medium basal salt mixture

Surface-sterilized seeds of Arabidopsis Col-0, APUM5-RNAi, and 35S-APUM5 plants were grown on plates containing 1/2 Murashige and Skoog Medium Basal Salt Mixture (Duchefa, Haarlem, The Netherlands), 1.5% sucrose, and 0.8% phytoagar with or without various concentrations of NaCl, mannitol, and ABA at 22°C under a long day condition to determine the effect of osmotic stress and ABA on germination. The percentage of germinated seeds was scored daily in three independent experiments (60–70 seeds per experiment).
A root growth assay was carried out by transferring 3-day-old seedlings to the minimal medium supplemented with different concentrations of NaCl, mannitol, and ABA to determine the effect of osmotic stress and ABA on root growth. After 7 or 10 days, root growth was measured in three independent experiments (30–40 seedlings per experiment).

For all experiments, rice (Oryza sativa) seedlings were grown vertically in sterile square petri dishes (Corning, 431301; 20 cm × 20 cm) under controlled conditions (day/night temperature of 28/25°C, a 12 h photoperiod, and a light intensity of 500 μEm^-2s^-1). First, the seeds are surface-sterilized: seeds are rinsed in 70% ethanol for ∼1 min. Then, ethanol is replaced by a solution composed of 40% bleach in distilled water containing 0.4% Tween 80 (Sigma-Aldrich P4780-500 mL). The seeds are soaked in this solution for 30 min with gentle agitation, and then rinsed at least four times with sterile distilled water. The sterile seeds are then sown on square petri dishes containing 250 mL of half strength Murashige and Skoog (MS/2) solid medium with the radicle oriented downward. Ten seeds are plated on each petri dish. The MS/2 solid medium is composed of 2.15 g/L of Murashige and Skoog medium basal salt mixture (Duchefa Biochemie, M0221), 75 mg/L Murashige and Skoog vitamin mixture (Duchefa Biochemie, M0409) and 8 g/L of agarose type II (Sigma-Aldrich, A6877). For salt-stress experiments, 7 g/L of NaCl (120 mM) is added to MS/2 medium before autoclaving. Radicles of the plantlets were harvested after 6 days of growth.