Foxtail Millet Stress Response Protocol

Seeds of foxtail millet cultivar Prasad obtained from National Bureau of Plant Genetic Resources (NBPGR), Hyderabad, India were grown in a plant growth chamber (PGC-6L; Percival Scientific Inc., USA) at 28±1°C day/23±1°C night with 70±5% relative humidity and photoperiod of 14 h. For stress treatments, 21-day-old seedlings were exposed to 250 mM NaCl (salinity), 20% PEG 6000 (dehydration), 100 µM abscisic acid (ABA), 100 µM salicylic acid (SA), 100 µM methyl jasmonate (MJ) or 100 µM ethephon (Et) for 1 h (early) and 24 h (late) based on earlier studies [10] (link), [22] , [66] (link). Unstressed plants were maintained as controls. After the treatments, seedlings were immediately frozen in liquid nitrogen and stored at −80°C until RNA isolation as described elsewhere [66] (link). For obtaining precise and reproducible results, each of these above experiments was repeated twice.