Plant material, growth conditions and NaCl treatment
Pongamia seeds (accession TOIL 12) were obtained from Tree India Limited (TOIL), Zaheerabad, Hyderabad, Telangana. Freshly collected seeds were sterilized with 1% (v/v) hypochlorite solution for 5 min. Further, the seeds were kept for germination on moist cotton bed at 25°C in dark for 10 days after thoroughly washed with autoclaved distilled water. Germinated seeds were grown hydroponically in Hoagland No. 2 basal salt mixture (Himedia) solution (pH 5.75 ± 0.02) for 30 days. The seeds were kept for germination on moist cotton bed for 10 days at 25°C in dark after thoroughly washed with autoclaved distilled water. After radicle emergence, the germinated seeds were placed just above the nutrient medium (Hoagland No. 2 basal salt mixture (Himedia) solution (pH 5.75 ± 0.02)) level with help of parafilm in 50 ml falcon tubes (Genaxy) for 10 days. Further, the plants were grown initially in long cylindrical glass tubes (5 cm diameter X 30 cm length) for 10 days and then transferred to long cylindrical glass tubes (5 cm diameter X 60 cm length) before giving the salt stress treatment. The glass apparatus were designed in such a way to grow the plants without root limitation. We maintained following culture conditions 24°C temperature, 16 h light and 8 h dark, and relative humidity maintained approximately at 60%. In order to avoid hypoxic conditions nutrient solution was renewed on daily basis. Salinity stress treatment was given according to Marriboina & Attipalli, (2020b). For salinity stress, two different salt concentrations (300 mM NaCl and 500 mM NaCl (sea water equivalents)) were used. For stress treatment, 30 days old plants (n = 20 to 30) were chosen and exposed to 300 and 500 mM NaCl stress with increment of 100 mM NaCl per day (Marriboina & Attipalli, 2020b). Control plants were maintained in a fresh Hoagland’s solution. Plants were harvested at an interval of 1, 4 and 8 days. Fresh weights of leaves and roots were measured immediately after harvest. Dry weights of control and treated plant tissues were determined after drying at 70°C for 3 days. Control and treated samples were harvested at each time intervals, flash frozen in liquid nitrogen and stored at -80°C till analysis.