Growth Condition and Phenotypic Evaluation
Healthy and good quality seeds of all genotypes were used for phenotypic
studies and other investigations. These seeds were first disinfected by
using 70% (v/v) ethanol, and then surface-sterilized for 4h in a
tightly sealed chamber using chlorine gas (100 ml NaClO + 15 ml HCl). To
determine the optimum PEG 6000 concentration for drought-stress
treatment, we tested different PEG concentrations viz., 10%, 15% &
20% (w/v). About 50-70 seeds of each genotype were placed in petri
dishes (diameter is 15 cm) containing two filter paper at the bottom,
and were filled with 20 mL of PEG 6000 solution.
Seed germination was evaluated under 15% PEG (w/v) and control
(distilled water) conditions, respectively. Completely randomized design
(CRD) were used for experiment trial with three biological replicates.
Petri-plates containing sterilized seeds were incubated in a growth
chamber for five days at temperature of 25°C and relative humidity (RH)
of 60% under a 16 h/8 h (light/dark) cycle. Seeds with young radicle
length approximately ~1–2mm were considered as
germinated seed. Seeds germination was observed every day for the
estimation of the total germination percentage (GP). After four days,
root length (RL) of each genotype was calculated under drought and
control conditions. Primary roots from each genotype under both
conditions were collected, and samples were named as DTL-C, DTL-T,
DSL-C, DSL-T, DSP-C, DSP-T, DTP-C and DTP-T. From each genotype two
biological replicates were used, and root tissue were quickly frozen in
liquid nitrogen and stored at −80°C for RNA extraction. Following
formula was used to calculate Germination rate (GR) :
GR= (germination rate under drought stress)/ (germination rate in
control) ×100%