Eggplant seeds were surface-sterilized using 70% alcohol solution for one minute before sowing, followed by 5% sodium hypochlorite for three minutes and further seeds were washed with sterile distilled water once accordingly. The eggplant seeds (10 seeds/Petri dish) were sown on three layers of Whatman filter paper (grade 1) in glass Petri dishes (90×25 mm). NaCl (extrapure analytical reagent (AR) grade, were acquired from Sisco Research Laboratories (SRL), Maharashtra, India) and solutions were prepared at 60, 80, 100, 125, 200, 300 and 400 mM concentrations respectively. The pH of the prepared solutions was measured using pHep pH tester, (Hanna Instruments, Woonsocket, RI, USA) and electrical conductivity was measured with a Field Scout electrical conductivity meter, (Spectrum Technologies, Inc. Plainfield, IL, USA). The corresponding data is given in Supplementary Table S1. A control treatment (0 mM NaCl) was also used, and seeds were supplied with solutions prepared with distilled water throughout the experiment. The Petri dishes were organized in a completely randomized design (CRD) with six replications. Each Petri dish received 1 mL of the prepared NaCl solution (0, 60, 80, 100, 125, 200, 300 and 400 mM NaCl) respectively. Petri plates were kept at room temperature (27°C) and normal laboratory light conditions (200–400 μmol m ^-2·s ^-1) photosynthetic photon flux density. After 2 weeks, the percentage of seed germination was calculated to identify the optimum challenging salinity concentration, a 90% reduction to only 10% germination. Seed germination percentage was expressed as a percentage and was calculated by the following formula; Germination percentage (%) = [number of seeds germinated/total seeds]×100. The optimum salinity concentration was 125 mM NaCl ( Figure 1) for the Petri plate experiment. This optimum challenging salinity exhibited less than 7.5% germination recorded at 125 mM NaCl ( Figure 1) and a sub-lethal dose of 100 mM NaCl considered as challenging salinity to the further pot experimental study. Based on the identification of optimum challenging salinity and earlier SIR report in mung bean as supportive data 60, 80, 100 and 125 mM NaCl concentrations were chosen for the SIR cycle. ¹⁶

Eggplant seeds were exposed to an induction cycle as above at 60, 80, 100 and 125 mM NaCl for 0, 30, 60, 90, 120 min. (Extended data, Figure S2). After each time interval, the treated seeds were transferred to Petri dishes, and they were supplied with distilled water until the seeds germinated. Seed germination percentage was expressed as a percentage and was calculated by the following formula: Germination percentage (%) = [number of seeds germinated/total seeds]×100. After analyzing the results, the 120-min interval was determined to be an optimum time considering high germination rate at 120-min interval for the salt induction treatments, when compared with other time intervals from 0, 30, 60, 90 min appropriately.

Surface sterilized eggplant seeds were exposed to the following three treatments: induced (NaCl treatment), non-induced (NaCl treatment) and absolute control (distilled water) by the method in Figure 2. Seeds were treated for a single induction cycle of 120 min with 60, 80, 100 or 125 mM NaCl. In the non-induced treatment, the seeds were exposed to a lethal level salinity (125 mM) for 120 min. All the seeds of induced and non-induced treatments were transferred to Petri dishes and irrigated with distilled water for three days. Treated seeds were maintained at room temperature (27°C) for three days for recovery. The recovery period was determined based upon the previous reports on the temperature [13] and salt induction [14] responses. The absolute control was treated with distilled water only. After three days of recovery, 125 mM NaCl solution (challenging salinity) was applied to all Petri plates (Induced, Non-induced and Absolute control), and the responses were observed. After 11 days of the 125 mM NaCl challenge treatment, the germination percentage, root length, vigour index, seedling biomass and relative water content were determined. The biomass of single seedlings was measured using a balance. After measuring the fresh seedling mass (g), seedlings were subjected to dry biomass analysis. Seedlings were put into a paper packet and placed in a hot air oven (80°C) for 48 h. Seedling dry masses (g) were measured with a balance for calculating the vigour index by the following formula: [Vigour index = germination percentage × seedling dry mass]. ¹⁷ Tissue water content (%) was calculated using the fresh and dry masses of leaves [Tissue water content = (fresh mass − dry mass)/dry mass × 100].

Pot studies was carried out in a shaded nethouse. Seeds from the induced (60, 80, 100 or 125 mM NaCl), non-induced (125 mM NaCl) and absolute control treatments were transferred to protrays and irrigated with distilled water for 15 days and raised as seedlings in the greenhouse. Seedlings were transplanted to pots 15 days after induction (DAI) treatments ended. The pots [ i.e., (25×24 cm) 10 L capacity] contained a soil mixture (15 kg/pot) consisting of red soil (Laterite soil): sand: compost (2:1:1). All the pots were arranged in a completely randomized design with six replications. Pots were irrigated with distilled water (0 mM NaCl) for seven days to avoid transplanting shock. A sub-lethal dose of saline water (100 mM NaCl) was prepared by mixing NaCl with fertilizer (NPK 19-19-19 at a concentration of 1.0 g L ⁻¹). Irrigated with 1 g/L (only fertilizer) or 100 mM NaCl was applied to pots manually throughout the experiment. Irrigations were scheduled at three days interval and each pot received 2 L of water to maintain soil continuously moistened in the pots. The growth conditions inside the shade nethouse were maintained by NMC-Junior, Crop Management Technologies, Netafim Irrigation and as follows: max/min temperature 34/26°C day/night, respectively, relative humidity 80% and average photosynthetic photon flux density (PPFD) 900 μmol m ⁻² s ⁻¹.

The leaf physiological and antioxidant measurements were recorded at 30 days after saline water irrigation. Relative water content (RWC) was calculated using the Weatherly methodology ¹⁸ by the formula: RWC (%) = [(fresh weight-dry weight)/(turgid weight-dry weight)] × 100. Leaf chlorophyll content was measured with a soil plant analysis development (SPAD) meter 502 plus. The Fv/Fm ratio was calculated using the Mini PAM II fluorometer (MINI PAM II- Walz, Effeltrich, Germany). Leaf photosynthetic rate and stomatal conductance were measured with a portable photosynthesis system (LI-6800; LI-COR, Lincoln, NE, USA). Briefly, green leaves of 30 days after saline water irrigated plant and third fully expanded leaf from top to downward were enclosed in the portable photosynthesis system under a light intensity of 800 μmol m ⁻² s ⁻¹ PPFD and 400 μmol mol ⁻¹ CO ₂ at 25°C leaf temperature and relative humidity between 40 and 55%. Leaf osmotic potential of eggplant leaves was measured with a Wescor, VAPRO 5500 vapour pressure osmometer (Wescor, Logan, UT, USA) as described by Ball and Oosterhuis. ¹⁹ The leaf proline content was determined, according to Bates et al. ²⁰ The fifth fully grown leaf sample (0.5 g) was taken and homogenized in 5 mL of sulphosalicylic acid (3%) using pestle and mortar. Around 2 mL ninhydrin reagent and 2 mL of glacial acetic acid were added to it. The whole reaction mixture was kept in 100°C water bath for 30 min. Once the reaction mixture cools down, 6 mL of toluene was added to the reaction mixture and later the mixture was transferred to a separating channel followed by thorough blending. The chromophore comprising toluene was isolated and absorbance was taken at 520 nm in a spectrophotometer against toluene blank. Proline concentration was evaluated by comparing it to a standard curve of proline and calculated as μg g ⁻¹ fresh weight. Hydrogen peroxide (H ₂O ₂) content was estimated using the Loreto and Velikova method, ²¹ 0.5 g of leaf sample was taken and homogenized in 3 mL of 1% ( w/ v) of tri-chloroacetic acid (TCA). The homogenized sample was centrifuged for 10 min at 10,000 rpm at 4°C. Consequently, 0.75 mL from supernatant has been further added to 0.75mL of 10 mM K-phosphate buffer of pH 7.0 and 1.5 mL of 1 M of potassium iodide. The H ₂O ₂ concentration from the supernatant was estimated by comparing its optical density towards a standard calibration curve at 390 nm using a spectrophotometer, and the values expressed as μmol g ⁻¹ fresh weight. The level of malondialdehyde (MDA) content in the sample was estimated by using the method performed by Weisany et al. ²² About 0.2 g of leaf sample were homogenized with 5 mL of 0.1% ( w/ v) trichloroacetic acid (TCA) and centrifuged at 12,000 rpm for 5 min at 4°C. To 1 mL of supernatant 4 mL of 0.5% ( w/ v) thiobarbituric acid in 20% trichloroacetic acid. Further, the samples were kept in water bath boiling at 90°C and then the sample was cooled to stop the reaction. Once again, the reaction mixture was centrifuged at 10,000 rpm for 5 min. The absorbance was evaluated at A532 nm and A600 nm wavelength respectively. The malondialdehyde content was determined by using this formula MDA (nmol g ⁻¹ FM) = [(A532−A600) × V × 1000/ε] × W, and the specific extinction coefficient (ε =155 mM cm ⁻¹), where V is the quantity of grinding medium, W is the fresh weight of the leaf taken. The peroxidase (POD) activity was determined by using procedure described by Hemenda and Klein, ²³ and the reaction mixture contained 25 mmol L ⁻¹ phosphate buffer saline (pH 7.0), 0.05% guaiacol, 10 mmol L ⁻¹ H ₂O ₂ and enzyme. The peroxidase activity was estimated by the increase in absorbance at 470 nm due to guaiacol oxidation. Harvesting of fruits was done on 130 days after saline water irrigation, and fruit yield (g/plant) was recorded. After fruit harvest, leaf area for the whole sampling unit was measured by using a Leaf Area Meter (Model LI-3100 of LI-COR Inc.) and data given in Supplementary Table S2. After harvesting the whole plant was dried in a hot air oven (80°C) for 48 h, and total dry mass (g/plant) was calculated. The treatments were organized in a completely randomized design. The data for each parameter (growth, physiological, biochemical, antioxidants, yield) was statistically analyzed by analysis of variance (ANOVA) with p<0.05 as the measure of significance for each parameter using JMP 2007 (SAS Institute, Cary, NC, USA). Post-hoc comparisons were evaluated using the Tukey Honestly Significant Difference (HSD) test at p<0.05 for the treatment effects.

Growth parameters

The Petri plate experiment results indicated that the seed survival of the eggplant treated with gradually increasing concentrations of NaCl (induced treatment) was 77%, whereas with a level of 125 mM concentration of NaCl (non-induced seedlings) has 57% when observed after 14 days ( Figure 3a). Similarly, the eggplant seedlings treated with a gradual increase of 60 to 125 mM concentration of NaCl had increased root length compared to non-induced and control treatments ( Figure 3b). Induced seedlings had a greater vigour index than the control treatment ( Figure 3c). Thus, the SIR indicated better quality seedlings than the non-induced and control seedlings. Likewise, the tissue water content and fresh and dry biomass of control seedlings were less than induced seedlings ( Figure 3d–f). Therefore, salt induction of eggplant seedlings allowed an improved response to the challenge of 125 mM NaCl.

Physiological parameters

Induced seedlings growing in pots irrigated with 100 mM NaCl solution (sub-lethal level challenging salinity) had significantly greater relative water content over the control and non-induced treatments (15% and 9%, respectively) ( Figure 4a). The leaf osmotic potential of the induced plants (−1.08 MPa) was less negative than the non-induced (−1.45 MPa) and control (−1.60 MPa) treatments under 100 mM NaCl concentration ( Figure 4b). There were no differences in relative water content and leaf osmotic potential among the treatments with 0 mM NaCl irrigation ( Figures 4 a,b). With a few exceptions, leaf photosynthetic rate, stomatal conductance, SPAD chlorophyll content, and the Fv/Fm ratio was significantly higher for induced plants, intermediate for non-induced plants, and lowest for control plants with both 0 and 100 mM NaCl irrigation, and lower for all treatments at 100 mM NaCl than at 0 mM NaCl ( Figure 5).

Antioxidant parameters

The antioxidant parameters indicated that the induced plants synthesized significantly more proline than the non-induced and control plants under 100 mM NaCl irrigation but showed no difference at 0 mM NaCl ( Figure 6a). Lipid peroxidation was less for induced plants at both NaCl irrigation treatments than the control and non-induced plants ( Figure 6b). Hydrogen peroxide content of induced plants was significantly less than the control with both irrigation treatments ( Figure 6c). Leaf peroxidase (POD) activity of induced plants was significantly greater than non-induced and control plants (100 mM NaCl irrigation but all plant types were the same with no NaCl in the irrigation treatment ( Figure 6d).

Growth and yield parameters

The fruit yield and dry mass of the plants were significantly reduced by 100 mM NaCl irrigation irrespective of pre-treatments ( Figure 7). However, fruit yield and total dry biomass of induced plants were significantly greater than of non-induced plants which were greater than control plants in both irrigation treatments. The fruit yield of induced plants was significantly higher (66%) followed by the non-induced plants (24%) compared to control plants under saline irrigation. The total dry mass of induced plant was 94.9% and 88.6% higher than the control plant supplied with 0 and 100 mM NaCl, respectively.

Open Science Framework: Inducing inherent salinity tolerance of eggplants by salt induction response DATA, https://doi.org/10.17605/OSF.IO/NQS95. ⁴⁴

Underlying data SIR.xlsx

Open Science Framework: Inducing inherent salinity tolerance of eggplants by salt induction response DATA, https://doi.org/10.17605/OSF.IO/NQS95. ⁴⁴

Extended Data SIR.pdf (Supplementary figure 1. Seedling germination of eggplants at 7 days after sowing (DAS) under different NaCl treatments. Supplementary figure 2. Standardization of interval time for the salt induction response. Supplementary figure 3. Seed treatment of the induction cycle in different NaCl concentrations at 120 minutes time interval. Supplementary figure 4. Seedling morphology of eggplants at 25 days after induction (DAI) under induced, non-induced and control treatments. Supplementary figure 5. Schematic representation of salinity induction response mechanism. Supplementary table 1. The pH and electrical conductivity (EC) of different NaCl solutions. Supplementary table 2. Leaf area (cm ²) of eggplants grown in pots at 130 days after saline water irrigation under induced, non-induced and control treatments. Supplementary table 3 Soil electrical conductivity (EC) analysis of eggplants grown in pots after harvest (130 days after saline water irrigation) under induced, non-induced and control treatments.)

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).