Introduction

Soil salinity is a major factor that limits crop yield. It is becoming a global problem and affects about 20% of the irrigated agricultural land (Zhu 2001). During salinity stress, not only do the plants experience the shortage of water but they also undergo ion disequilibrium, which may disrupt homeostasis and lead to oxidative damage (Karimi et al.2005; Gill and Tuteja 2010). Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide (O2−), singlet oxygen (1O2), and hydroxyl radical (·OH), are normally produced as by-products during plant cell metabolism and support the biosynthesis of complex organic molecules (Foyer and Shigeoka 2011). Under stress conditions, the production of ROS increases, which disrupts a balance that normally exists between the production of ROS and their detoxification. This off-balance (more ROS at a particular given time) not only inactivates various enzymes but also causes damage to vital cellular macromolecules, such as lipids, proteins, and DNA, which poses a serious threat to the existence of plants. Under these circumstances, cells may produce enhanced copies of antioxidative enzymes such as superoxide dismutase, catalase, peroxidase, and nitrate reductase, to scavenge surplus ROS in an improved manner and streamline cellular metabolism and plant growth under stress.

Phytohormones play a crucial role in the growth and development of plants by regulating many processes. Phytohormones may enhance stress tolerance and minimize the yield loss of plants caused by abiotic stress (Ilias et al.2007). Various studies have determined the roles of indole-3-acetic acid (IAA) and gibberellic acid (GA3) in plants under stress conditions (Amzallag et al.1990). Alone or in combination, they improve plant growth by either improving germination or reducing oxidative damage by controlling activities of antioxidative enzymes (Kaur et al.2000; Senthila et al.2005; Shah and Ahmad 2007).

Potato is the fourth most important crop by volume of production after maize, wheat, and rice (FAO 2008). Potato is considered to be moderately salt tolerant but is sensitive during tuber bud initiation, which leads to a decrease in the average tuber size (Teixeira and Pereira 2007). The acquisition of salt tolerance in potato plants may help to improve its growth and ultimately crop yield. It is important to blend various approaches to develop a successful experimental plan that results in improved yield using biotechnology methods such as genetic engineering and chemical modifications. The use of plant tissue culture techniques has also been used to screen stress-tolerant varieties of various crops (Tal 1983). Plant tissue culture can provide an opportunity to manipulate in vitro cultures reproducibly under a desired set of experimental conditions. Exogenous application of various biomolecules including PEG, glycine-betaine, proline, sorbitol, mannitol, ascorbic acid, brassinosteroids, IAA, and GA3 have been reported to induce stress tolerance in plants (Datta et al.1998; Al-Hakimi and Hamada 2001; Ashraf and Fooland 2005; Qasim et al.2006; Anuradha and Rao 2007; Chauhan et al.2009; Khalid and Aftab 2016; Kaur and Gupta 2018). Exogenous application of IAA and GA3 that affects various growth parameters and antioxidant enzyme activities has not been thoroughly reported in potato plants grown in vitro under salt stress. The purpose of the present study was to evaluate the impact of selected levels of NaCl on growth and development of potato plants and to determine the effect of the exogenously applied growth regulators IAA and GA3 on various growth and biochemical parameters and their response to stress. The study also aimed to determine the relationship (if any), between the activities of superoxide dismutase and peroxidase, and the growth of calluses/plants under stress.

Materials and Methods

Plant material

Solanum tuberosum L. tubers (cvs. Cardinal and Desiree) were obtained from the Seed Centre, University of the Punjab, Lahore. They were planted in 8 × 12 cm pots and grown in a greenhouse at 27°C, during October 2014. After 2 wk, 10-cm-long shoots were cut and used as the explant source for additional experiments. The shoots were initially disinfected by washing with detergent (Unilever Karachi, Pakistan) to remove any adhered dust particles. Next, the shoots were rinsed three times with distilled water and kept in a solution of 0.7% (v/v) sodium hypochlorite (Unilever, Karachi, Pakistan), and 0.1% (v/v) Tween 20 (Sigma-Aldrich, St Louis, MO) for 5–10 min in an Erlenmeyer flask (500 mL, Pyrex, Corning Inc., Corning, NY). The shoots were washed three times with sterile (autoclaved) distilled water in a laminar air-flow cabinet to get rid of traces of the sodium hypochlorite. Murashige and Skoog (MS) medium (Murashige and Skoog 1962) was used for shoot induction and additional culture maintenance. The medium was prepared by weighing appropriate quantities of the individual required chemicals (Sigma/Merck grade) for the preparation of stock solutions and later mixing the solutions according to the requirements. Sucrose (30 gL−1) and agar (0.7% (w/v); Oxoid, Hampshire, UK) were added after adjusting the pH of the medium to 5.7. The medium was autoclaved for 15 min at 121°C (103.42 kPa), and 10 mL of the medium was aliquoted in each of the culture tubes (25 × 160 mm; Pyrex). After trimming the chlorinated shoot ends, 8-mm-long single-node cuttings were placed in each culture tube and incubated for a 16-h photoperiod (40 μmol m−2 s−1 photon flux density cool-white fluorescent light, Philips, Pakistan), at 25 ± 2°C for shoot induction. For callus induction, the internodes were excised from the disinfected shoots and inoculated on MS medium supplemented with 1 mM 2,4-dichlorophenoxyacetic acid (2,4-D; Sigma-Aldrich) and incubated at 25 ± 2°C in the dark for 60 d.

Treatment outline and experimental design

A 4 × 3 factorial combination of NaCl (0, 40, 60, and 80 mM) and each growth regulator, which included IAA (0, 7, and 14 μM; Sigma-Aldrich) and GA3 (0, 14, and 21 μM; Sigma-Aldrich) was used. The specific levels of IAA/GA3 were selected based on the results of a pilot experiment (Khalid and Aftab, unpublished). MS medium with NaCl treatments (0, 40, 60, and 80 mM) were autoclaved and cooled-down to around 55°C, before the addition of respective levels of filter-sterilized IAA/GA3 solutions (dissolved in ethyl alcohol and diluted with distilled water). Ten culture tubes were used for each treatment. In vitro-grown 30-d-old potato plants cvs. Cardinal and Desiree were excised from the culture tubes, and 1 cm long single nodes were cut for inoculation. The culture tubes were kept at 25 ± 2°C for a 16-h photoperiod (40 μmol m−2 s−1 photon flux density cool-white fluorescent light) for 30 d, and morphological and biochemical parameters were evaluated. For studies on calluses, the callus induction medium was supplemented with respective IAA/GA3 concentrations and later inoculated with pre-weighed calluses. They were kept in the dark at 25 ± 2°C. The data for callus morphology and fresh weights were recorded after 30 d of inoculation. The experiment was repeated three times, over a period of 8 mo with the same number of replicates for each experiment. Data were pooled together from these experiments for subsequent analyses.

Morphological and biochemical analyses

Data were obtained for both growth and biochemical parameters for plantlets, which included root and shoot number and length, number of nodes/leaves, fresh weight (FW), total soluble proteins, and activities of superoxide dismutase (SOD) and peroxidase (POD), after treatment for 30 d. For morphological analysis, plants were uprooted from the culture vessels and root and shoot lengths were determined. Fresh weight was recorded by weighing the whole plants on an electric balance (Scientech 5220). Other morphological parameters, which included number of leaves/nodes were determined at the same time. For calluses, the morphology, increase/decrease in fresh weight, and callus proliferation responses were recorded.

For biochemical analysis, 1 g of plant material was ground in liquid nitrogen using a mortar and pestle to obtain a fine powder. Two milliliters of phosphate buffer (0.1 M) containing 0.1 g polyvinypolypyrrolidone (PVP; Sigma-Aldrich) and Triton (0.01 mL; Sigma-Aldrich) were added to make a slurry, which was then centrifuged at 4°C for 30 min at 15,400×g. The supernatant collected was used for additional estimation as a crude enzyme extract.

For the estimation of total soluble protein, the Biuret method (Racusen and Johnstone 1961) was followed with minor modifications. Two test tubes (15 × 150 mm) were labeled as “control” and “experimental.” In both tubes, 2 mL Biuret reagent was added. In the experimental tube, 0.2 mL crude enzyme extract was added, and in the control tube, 2 mL distilled water was added. Both tubes were vortexed and kept for 15 min at 25 ± 2°C to complete the reaction. The optical density (OD) was measured at 545 nm. The total soluble protein was calculated following a standard curve that was prepared by bovine serum albumin protein (Robinson 1979).

Quantitative estimation of the superoxide dismutase (SOD; E.C 1.15.1.1) activity was carried out using the method of Maral et al. (1977) with minor modifications. Briefly, to the 3-mL reaction mixture, which consisted of 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μM nitroblue tetrazolium, 0.1 mM ethylenediaminetetraacetate, and 2 μM riboflavin, 15 μL crude enzyme extract was added to the test tube labeled experimental, whereas distilled water (15 μL) replaced the crude enzyme in the control. Both samples were vortexed briefly following irradiation for 10 min with 40-W fluorescent cool-white light (40 μmol m−2 s−1 photon flux density). The absorbance was measured at 560 nm, and the SOD activity was calculated by the following formula:

$$ \%\mathrm{inhibition}=\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}\ \mathrm{sample}-\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{experimental}\ \mathrm{sample}}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{experimental}\ \mathrm{sample}}\times 100 $$

POD (EC 1.11.1.6) activity was measured using the Racusen and Foote (1965) method with some modifications. Two test tubes that were labeled control and experimental were used. To both tubes, 2.5 mL Tris-HCL buffer (0.1 M, pH 7.2) and 0.2 mL guaiacol (1%, v/v, purity 98%; Sigma-Aldrich, St Louis, MO) were added. In the experimental tube, 10 μL crude enzyme extract was added, while 10 μL distilled water was added in the control. Both samples were kept for 30 min at room temperature before the addition of H2O2 (0.3%, v/v; 0.2 mL). The absorbance was measured at 470 nm. The enzyme content was calculated as follows:

Peroxidase content (mg g−1 of tissue)\( =\frac{A\times \mathrm{df}}{\mathrm{EU}\times \mathrm{Wt}\times 1000.} \)

where A is absorbance, df is the dilution factor, EU is the extract used, and Wt is fresh weight of the sample tissue.

Statistical analysis

Data were analyzed statistically using analysis of variance (ANOVA). The dependent variables included root and shoot number and length, number of nodes/leaves, fresh weight of plants/calluses, callus morphology, proliferation response, protein, SOD, and POD. A full-factorial multivariate analysis was performed using SPSS 20.0.

Results

Effect of IAA and GA3 on growth and biochemical parameters of potato under salt stress

Nodal explants All of the tested growth parameters were negatively affected when nodal explants were grown on saline medium. However, the deleterious effects of salt stress were successfully alleviated with various concentrations of IAA and GA3 (Figs. 1 and 2). In the cultivar Cardinal, a significant difference in shoot length was observed (Fig. 1). The reduction in growth at 40, 60, and 80 mM NaCl was successfully alleviated after treatment of IAA in both the cultivars (Fig. 1). In cv. Desiree, the root growth was completely inhibited at 80 mM NaCl, which was improved after IAA treatment (Fig. 1). Total protein content, SOD, and POD activities generally decreased with an increase in the NaCl concentration, which thereby increased with exogenous application of IAA (Fig. 1).

Figure 1.
figure 1figure 1

Comparative effects of indole-3-acetic acid (IAA: 0, 7, or 14 μM) and NaCl (0, 40, 60, or 80 mM) on morphological and biochemical parameters of Solanum tuberosum L.: (a) root number, (b) root length, (c) shoot number, (d) shoot length, (e) number of nodes, (f) number of leaves, (g) fresh weight, (h) protein, (i) Superoxide dismutase (SOD), and (j) Peroxidase (POD) in in vitro potato plants (cvs. Desiree and Cardinal).

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Figure 2.
figure 2figure 2

Comparative effects of gibberellic acid (GA3; 0, 14, or 21 μM) and NaCl (0, 40, 60, or 80 mM) on morphological and biochemical parameters of Solanum tuberosum L.; (a) root number, (b) root length, (c) shoot number, (d) shoot length, (e) number of nodes, (f) number of leaves, (g) fresh weight, (h) protein, (i) Superoxide dismutase (SOD), and (j) Peroxidase (POD) in in vitro potato plants (cvs. Desiree and Cardinal).

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The data presented in Fig. 2 indicate that growth was reduced with NaCl alone, and the exogenous application of GA3 alleviated these harmful effects in both of the tested cultivars. The number/length of roots was reduced with increasing salt concentrations and completely inhibited at 80 mM, which could not be improved by GA3 application (Fig. 2). The long internodes were observed when nodal explants were supplemented with various GA3 concentrations. Total protein content, SOD, and POD activities were reduced when the NaCl concentration was increased. However, an increase in the above-mentioned parameters was recorded with exogenous application of GA3 (Fig. 2).

Callus cultures

IAA affected the growth of calluses positively by increasing the fresh weights from 15.51 to 19.6% (7 μM) and 18.75% (14 μM; Table 1). The calluses were off white and friable. Under salt stress, the fresh weights decreased and the calluses were necrotic at 80 mM (Fig. 3). A similar response was observed in cv. Desiree (Table 2), in which the exogenous application of various concentrations of IAA improved the fresh weights of calluses, which could be observed from their morphological parameters because the dark brown/necrotic callus changed into a light brown color with exposure to IAA (7 or 14 μM; Table 2).

Table 1. Effect of various concentrations of NaCl and IAA on the callus proliferation response in Solanum tuberosum L. cv. Cardinal
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Figure 3.
figure 3

Off-white callus cultures of Solanum tuberosum L. cv. Cardinal: control (A); 7μM Indole-3-acetic acid (IAA) (B); and 14 μM IAA (C) with 0 mM NaCl (i), 40mM NaCl (ii), brownish yellow callus with 60 mM NaCl (iii), and yellowish brown calluses with 80 mM NaCl (iv).

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Table 2. Effect of various concentrations of NaCl and IAA on the callus proliferation response in Solanum tuberosum L. cv. Desiree
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Exogenous application of GA3 (14, 21 μM) also improved callus morphology and the fresh weights of both cultivars. A reduction of 8.16% in fresh weight of the calluses at 80 mM NaCl (cv. Cardinal) was increased to 3.92% (14 μM) and 2.08% (21 μM), respectively (Table 3). Likewise, an 11.53% decrease (60 mM NaCl) in cv. Desiree was reduced to 3.7% (14 μM) and 2.12% (21 μM; Table 4). In addition, the callus morphology was also positively affected by the exogenous application of GA3 (Fig. 4).

Table 3. Effect of various concentrations of NaCl and GA3 on the callus proliferation response in Solanum tuberosum L. cv. Cardinal
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Table 4. Effect of various concentrations of NaCl and GA3 on callus proliferation response in Solanum tuberosum L. cv. Desiree
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Figure 4.
figure 4

Off-white, yellow callus cultures of Solanum tuberosum L. cv. Desiree; control (A); 14 μM Gibberellic acid (GA3) (B); and 21 μM GA3 (C) with 0 mM NaCl (i), yellow calluses with 40 mM NaCl (ii), yellowish-green calluses with 60 mM NaCl (iii), and brown calluses with 80 mM NaCl (iv).

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Discussion

With exposure to salinity stress, a growth reduction in salt-sensitive plants is usually the first noticeable response (Parida and Das 2005). A decline in morphological parameters (root and shoot length, number of nodes/leaves, and fresh weight) was observed with a gradual increase in salt in the MS medium. The most drastic effect of NaCl stress was at the highest tested concentration (80 mM), in which the plantlets exhibited reduced shoot and root length but with an increased shoot number. These results are in agreement with the results of Ahmad et al. (2012) who reported a similar response of in vitro-grown potato under NaCl stress. Reduction of growth due to inadequate water uptake because of low osmotic potential is a common indicator of salt stress (Munns 2002; Borsani et al.2003). Initially, salt stress decreases the water absorption capacity of the root system, which is followed by an increased water loss from the leaves. As a result of osmotic stress, physiological changes occur in plants, in which nutrient imbalance, membrane interruption, and reduced photosynthetic capacity have been observed (HanumanthaRao et al.2016). Callus cultures also exhibited a reduction in the fresh weight of both cultivars with an increase in salt stress. These results are in agreement with a previous study conducted by Ochatt et al. (1999), in which a decrease in callus growth was observed at higher NaCl concentrations in Solanum tuberosum L. In another study, Liu and Van-Staden (1999) also reported a similar reduction in the fresh weight of calluses after 28 d, and callus growth was inhibited with a concentration of 100 mM salt. One reason for this salt-induced growth reduction could be the adverse effect of salt on roots, as it reduces uptake of water and nutrients. Hormonal imbalance is another probable reason, which leads to reduced growth in response to salinity. The repressive effect of salt stress on plant growth could also be attributed to the decline in endogenous levels of plant hormones (Hamayun et al.2010).

IAA and GA3 are phytohormones that promote cell expansion and elongation, vascular tissue development, maintain apical dominance, regulate phototropic and gravitropic behavior, and therefore ultimately enhance plant growth (Hamayun et al.2010). In the present study, exogenous application of IAA and GA3 enhanced callus/nodal explant growth and successfully counteracted the adverse effects of salt stress. The tested concentrations of both of the growth regulators significantly enhanced plant growth by increasing shoot and root length, the number of roots/node, and fresh weight in non-stressed and NaCl-stressed plants compared with the control. This growth enhancement could have helped the plants cope with stress by delaying the onset of the salinity tolerance threshold (Dalton et al.2000). In the present study, a relatively lower concentration of IAA, such as 7 μM, or a slightly higher level of GA3 (21 μM), were found to enhance growth in both normal and stress conditions. However, IAA influenced root growth positively, which is in agreement with several earlier studies, in which the role of IAA in the formation of lateral and adventitious roots was reported (Arteca 1996; Rahman et al.2002). These results are also in agreement with the results of Chakrabarti and Mukherji (2003), Akbari et al. (2007), and Egamberdieva (2009) who described the positive role of IAA on seed germination and seedling growth under NaCl stress. Plant hormones act as a signal for an expression of numerous genes that contribute to salt tolerance (Shakirova et al.2003). An immediate effect of salt stress is the reduction in the rate of leaf surface expansion, with additional growth reduction at higher salt concentrations (Wang and Nil 2000). Such a reduction in growth may be overcome by the exogenous application of certain plant growth regulators. Veselov et al. (2008) reported that exogenous IAA application not only stimulated growth but also alleviated the adverse effects of salt stress in maize. They attributed this response to an increase in the expression of ZmEXPA1 expansin genes that promoted leaf cell extension.

Proteins are among the potential biochemical indicators of salinity tolerance. In this study, a progressive decrease in soluble protein content after exposure to NaCl was observed. These results are similar to the results of Wen et al. (2010), in which decreased levels of soluble protein content in potato were recorded under salt stress. The negative effect of salinization on protein content could be attributed to the osmotic effect (Yurekli et al.2004) or due to a decreased availability of amino acids and denaturation of the enzymes involved in the amino acid and protein synthesis under saline conditions (Parida and Das 2005; Khalid 2017). Another possible reason could be the loss of potassium ions (K+) under salt stress, as these are necessary for protein synthesis (Ayala-Astorga and Alcaraz-Meléndez 2010). It has been reported by Wang et al. (2003) that plants accumulate certain proteins that are likely protective in nature to survive under stressful conditions. Compared with the control used in this study (0 mM NaCl and 0 μM IAA in MS medium), the exogenous application of IAA increased the protein content in both the non-stressed and NaCl-stressed potato plants. This increased protein content could help plants maintain growth under stress conditions. These results are in agreement with the results of Agastian et al. (2000) and Fidalgo et al. (2004) who reported that stress-induced proteins play a major role in stress tolerance. High protein levels accumulate in plants under stress conditions and provide a storage form of nitrogen (Singh et al.1987) and possibly play a role in osmotic adjustments (Ashraf and Harris 2004; Parida et al.2004). In this study, increased protein content in response to IAA treatment could be due to a stimulating effect of auxin on the activation of K+ uptake channels (Claussen et al.1997), which might have enabled the plants to withstand the harmful effects of salt stress through osmotic adjustments or by balancing ionic homeostasis. This upregulation of proteins could be due to inclusion of salt stress proteins, as reported by Kim et al. (2004). Exogenous application of GA3 also induced regulation of salT gene, which enhanced synthesis of proteins in response to salt stress (Wen et al.2010).

Salt stress enhances the production of reactive oxygen species (ROS) and causes oxidative stress (Parida and Das 2005; Tanveer 2019). These ROS react with vital bio-molecules and cause pigment co-oxidation, lipid peroxidation, membrane disruption, protein denaturation, and DNA mutation (Molassiotis et al.2006). In this study, the exogenous application of IAA alleviated the oxidative damage by increasing the superoxide dismutase (SOD) and peroxidase (POD) activities, in both the NaCl-stressed/non-stressed plants of potato compared with the control. Similar increases in the activity of SOD were reported by Rahnama and Ebrahimzadeh (2005) and Esfandiari et al. (2007) in salt-tolerant potato and wheat cultivars, respectively. In another study, Senthila et al. (2005) reported a similar increase in POD activity under salt stress in response to IAA. An increase in POD activity was also suggested to play a pivotal role in scavenging H2O2 in a salt-tolerant potato cultivar (Aghaei et al.2009). The current studies are in agreement with results reported in earlier research (Meloni et al.2001; Rahnama and Ebrahimzadeh 2005; Li 2009) who suggested that increased activities of antioxidant enzymes confer greater resistance in plants against stress-induced damage. SOD probably functions as the first line of defense against ROS, but its end product is the toxic H2O2. POD could provide a selective advantage for defense and play a role in scavenging H2O2. Higher POD activity decreases H2O2 levels in the cells and increases the stability of membranes and CO2 fixation, as several enzymes of the Calvin cycle within the chloroplast are sensitive to H2O2 (Esfandiari et al.2007). Similar results were observed in maize by Tuna et al. (2008). Manchandia et al. (1999) reported two- to fourfold increases in POD activity in cotton calluses with exposure to salt stress. However, in Vigna radiata, SOD and POD activities were reduced with GA3 treatment under salt stress (Chakrabarti and Mukherji 2003), which indicated the complexity of biochemical events underlying salt tolerance and the various approaches diverse group of plants may have under stress conditions.

The results of this study demonstrate that IAA and GA3 can alleviate the salt-induced damage under in vitro conditions in potato. These growth regulators likely act to maintain the endogenous hormonal levels and/or increase the activities of the antioxidative enzymes, which promote growth even under stress. However, to harness potential benefits of this study and to determine the application for agronomic practices, these results need to be extended to future studies conducted in the greenhouse and in field conditions.