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.