Introduction

Abiotic stresses such as salinity, drought, temperature extremes, heavy metals, UV radiation, and nutrient deficiency impair crop growth and productivity and hence threaten global food security (Witcombe et al. 2008). Among these, salinity has affected more soils worldwide. It is reported that about 20% of the world’s crops on irrigated land are affected by salt stress (Ejaz et al. 2012). The reasons include poor drainage, flooding of salt water from coastal land, low quality irrigation water, and accumulation of salts in dry areas (Kijne 2006). Salt stress not only impedes seed germination, but also changes the anatomy and physiology of plants. These circumstances favor the enhanced production of reactive oxygen species (ROS). ROS including superoxide radical (O2 •−), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (OH•) are produced as by-products during cellular metabolism and are fairly regularly removed by antioxidant enzyme activities. Stress conditions lead to disruption of the equilibrium between ROS synthesis and scavenging. Enhanced production of ROS negatively affects cell membrane and cellular functions by causing damage to oxidizing proteins and nucleic acids (Wahid and Ghazanfar 2006). The antioxidant system generally consists of two principle players, the non-enzymatic ones including carotenoids, flavonoids, and α-tocopherol; and the enzymatic ones such as superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and glutathione reductase (Mittler et al. 2004). Therefore, the biochemical ways and means to control and/or scavenge the overproduction of ROS may potentially be exploited to increase a plant’s ability to withstand saline conditions (Gill and Tuteja 2010). A better understanding of these facts may pave the way towards a precise breeding approach for increasing stress tolerance in plants. A number of culture techniques which include field screening and pot experiments have already been employed to study several parameters for stress tolerance (Hayat et al. 2001; Fariduddin et al. 2009; Piñol and Simón 2009). However, the physical and chemical properties of soil and environmental fluctuations make the selection of salt-tolerant varieties a real challenge in such studies. An in vitro approach offers better prospects by circumventing the abovementioned limitations (Queirós et al. 2007; Karan and Subudhi 2012).

Polyhydroxysteroids, a relatively new class of phytohormone, includes brassinosteroids (BRs) such as 24-epibrassinolide (EBR). They cause morphological and physiological responses in plants at micromolar to nanomolar concentrations, and improve plant growth and yield (Rao et al. 2002). Key roles of BRs include modulating cell division, stem elongation, xylem differentiation, leaf development, and reproductive development (Clouse and Sasse 1998); and ethylene biosynthesis, overproduction of DNA, RNA and protein, and changes in the level of endogenous growth regulators such as abscisic acid (ABA; Bajguz 2000). Considerable attention has been given to EBR for its positive effect during stress tolerance in a wide variety of plants such as Chlorella vulgaris (Bajguz 2000), Vigna radiata (Fariduddin et al. 2004), Oryza sativa (Özdemir et al. 2004), Cucumis sativus (Yu et al. 2004), Brassica juncea (Sharma and Bhardwaj 2007; Ali et al. 2008a), Triticum aestivum (Ali et al. 2008b), Glycine max (Zhang et al. 2008), and Vigna unguiculata (El-Mashad and Mohamed 2012). Its exogenous application has enhanced the growth and yield of many plants by modulating protein content, antioxidant enzyme activities, seed germination, seedling growth, proline content, lipid peroxidation, photosynthetic capacity, and water relations (Özdemir et al. 2004; Yu et al. 2004; Sharma and Bhardwaj 2007). Although enhanced plant growth has been observed in many plants in response to BRs in field trials during stress, no study has yet been undertaken of potato in vitro. In view of this background, the aim of the present investigation was to determine a possible ameliorative effect of EBR on the morphological and biochemical aspects of in vitro-grown cultures of potato under salt stress. In addition, the best method of EBR application that could facilitate the alleviation of salt stress was also examined.

Materials and Methods

Procurement of plant material and disinfestation

Healthy tubers of potato cultivars Cardinal and Desiree were obtained from the Seed Centre, University of the Punjab, Lahore. They were placed in sterilized sand in a glasshouse in mid-October. After 2 wk, 10-cm long shoots were cut and used as explants. The excised shoots were thoroughly washed with a household detergent (Unilever Karachi, Pakistan) to get rid of adhering particles. Shoots were then rinsed with distilled water several times, and placed for 5–10 min in a 500-mL Erlenmeyer flask containing a solution of 0.7% sodium hypochlorite (NaOCl; Unilever) and 0.1% Tween-20. Shoots were then washed three times with sterile water in laminar air-flow cabinet to remove traces of NaOCl. Shoot induction and maintenance was carried out on Murashige and Skoog (MS; Murashige and Skoog 1962) basal medium prepared manually from individual reagents (Sigma-Aldrich®, St. Louis, MO). The medium was supplemented with 30 g L−1 sucrose and 0.7% (w/v) agar (Agar Technical No. 3; Oxoid™, Thermo Fisher, Hampshire, UK). The pH of the medium was adjusted to 5.7 prior to autoclaving for 15 min at 121°C (103.42 kPa). Ten-milliliter medium was then poured into 25 × 160-mm pre-autoclaved culture tubes. The chlorinated ends of shoots were trimmed and placed as 8-mm single-node cuttings in each culture tube and incubated at 25 ± 2°C with a 16-h photoperiod (40 μmol m−2 s−1 photon flux density, cool-white fluorescent light, Philips, Karachi, Pakistan) after closing with polypropylene sheets of appropriate size.

Treatment outline and experimental design

In vitro-grown 30-d-old plants were removed from culture vessels, and nodal segments (1-cm long) were cut for pretreatment with 24-epibrassinolide (EBR: Sigma-Aldrich®) solutions. A 4 × 3 × 2 factorial combination was used involving NaCl, EBR, and method of treatment, respectively. In the first method of EBR treatment (PT), nodal segments were kept for 8 h in the filter-sterilized EBR solutions (0, 1, or 2 μM) on an orbital shaker. Control nodal segments were pretreated in the same manner with autoclaved distilled water. All nodal explants were then cultured in MS medium containing 0, 40, 60, or 80 mM NaCl. Ten culture vessels were used for each treatment. In the second method (IM), the same EBR and NaCl concentrations were added directly in the MS medium prior to placing the nodal explants on the media. MS media with abovementioned NaCl treatments were autoclaved and cooled to around 55°C before the addition of filter-sterilized EBR. The specific levels of EBR and pretreatment duration in this study were selected on the basis of pilot experimentation (Khalid and Aftab, unpublished). The culture vessels were kept at 25 ± 2°C with a 16-h photoperiod (40 μmol m−2 s−1 photon flux density, cool-white fluorescent light; Philips) for 30 d. The experiment was repeated twice over a period of 8 mo with the same number of replicates for each experiment. Data were pooled for subsequent analysis.

Morphological and biochemical analysis

After 30 d, results were recorded for various growth and biochemical parameters including shoot length and number, root length and number, the number of nodes and leaves, fresh weight (FW), protein content, and superoxide dismutase (SOD) and peroxidase (POD) activities. Morphological parameters were measured, and 1 g of plant material from each sample was ground in liquid nitrogen using a mortar and pestle to obtain a fine powder. Two milliliters of 0.1 M phosphate buffer containing 0.1 g polyvinypolypyrrolidone and 0.01 mL Triton were added to make a slurry that was then centrifuged at 15,400g. The supernatant was collected and used as a crude enzyme extract for further estimation.

For quantitative estimation of protein, the Biuret method of Racusen and Johnstone (1961) was employed with slight modifications. To test tubes (15 × 150 mm) containing 2 mL of Biuret reagent (Sigma-Aldrich®), 0.2 mL crude enzyme extract (experimental samples) or 0.2 mL distilled water (control) was added. Test tubes were vortexed and kept for 15 min at 25 ± 2°C to complete the reaction. The absorbance was measured at 545 nm. The protein contents were calculated using a standard curve prepared from bovine serum albumin (Merck, Kenilworth, UK).

Determination of superoxide dismutase (SOD; E.C 1.15.1.1) activity was carried out following Maral et al. (1977) with some modifications. Briefly, to 3 mL of reaction mixture (50 mM phosphate buffer pH 7.8, 13 mM methionine, 75 μM Nitroblue tetrazolium, 0.1 mM ethylenediaminetetraacetate, and 2 μM riboflavin; all reagents from Sigma-Aldrich®), 15 μL of crude enzyme extract was added to a 15 × 150-mm test tube (experimental samples), or 15 μL of distilled water was added (control). Both experimental samples and controls were vortexed and then irradiated with 40-W fluorescent cool-white light (40 μmol m−2 s−1 photon flux density) for 10 min. The absorbance was measured at 560 nm, and SOD activity was calculated by using the formula:

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

Determination of peroxidase (POD; EC 1.11.1.6) activity was based on Racusen and Foote (1965). For experimental samples, 10-μL crude enzyme extract was added to 0.1 M Tris-HCL buffer (pH 7.2) containing 1% guaiocol (Sigma-Aldrich®). For control samples, 10-μL distilled water was added to the buffer. Before the addition of 0.3% H2O2, the experimental and control samples were left for 30 min. The absorbance was measured at 470 nm. Calculation for enzyme content is as follows;

$$ \mathrm{Peroxidase}\;\mathrm{content}\left({\mathrm{mgg}}^{-1}\mathrm{of}\;\mathrm{t}\mathrm{issue}\right)=\frac{\mathrm{A}\times \mathrm{d}\mathrm{f}}{\mathrm{EU} \times \mathrm{W}\mathrm{t}\times 1000} $$

where

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

Statistical analysis

Data were analyzed for two quantitative factors (NaCl and EBR) and one qualitative factor (method of EBR treatment) using analysis of variance (ANOVA). The dependant variables included root number and length, shoot number and length, number of nodes and leaves, FW, protein, SOD, and POD. A full factorial multivariate analysis as mentioned above was performed (along with the preparation of three dimensional graphs) using SPSS 20.0 (Sajid and Aftab 2009).

Results

Morphological parameters

Compared to control potato seedlings (without EBR), treatment with various concentrations of NaCl (40, 60, or 80 mM) adversely affected all the studied growth parameters (Table 1). Individual treatments with EBR increased all growth parameters significantly (Table 2; Figs. 1–7). Although both treatment methods also had a significant effect on most growth parameters (except for root length in both cultivars), PT with 1 μM EBR was found to be better for Cardinal and IM containing 2 μM EBR better for Desiree. Mixed results were observed as far as interaction between NaCl, EBR, and method of treatment were concerned. When size was measured, NaCl was found to have a strong effect that drove the morphological parameters (Table 2).

Table 1 Effects of exogenous application of EBR on morphological parameters of potato cultivars Cardinal and Desiree in response to NaCl stress
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Table 2 Multivariate full factorial analysis between fixed factors and dependent variables of S. tuberosum
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Figures 1–10
figure 1

Comparative effect of treatments (pretreated, PT; in medium, IM) viz-á-viz 24-epibrassinolide (0, 1, or 2 μM) and NaCl (0, 40, 60, or 80 mM) on root number/length (1, 2), shoot number/length (3, 4), number of nodes/leaves (5, 6), fresh weight (g; 7), protein (mg g−1; 8), SOD (U mg−1; 9), and POD (mg g−1; 10) in in vitro potato plants (cvs. Cardinal and Desiree).

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Biochemical attributes

In this study, NaCl stress led to alteration in protein contents and levels of antioxidant enzyme activities (Figs. 8–10). An increase in the protein contents was generally observed when either EBR or NaCl were applied alone in comparison with the tested controls. In Cardinal, the quantity of protein was 0.196 and 0.087 mg g−1 in non-treated and pretreated control plants, respectively, which increased up to 0.386 and 0.217 mg g−1 at 1 μM EBR, and 0.294 and 0.138 mg g−1 at 2 μM EBR. A similar trend was observed in Desiree where this value reached 7.133 and 6.737 mgg−1 at 1 and 2 μM EBR. The combination of NaCl and EBR led to decreased total proteins in both treatments (Fig. 8). The results were statistically significant among comparisons of cultivars, media, and methods of treatment (Table 2).

Exogenous application of either EBR or NaCl resulted in an overall enhancement of antioxidant enzyme activities. However, a reduction in the level of SOD was observed when plants were grown with both EBR and NaCl (Fig. 9). The maximum decline was observed in Desiree when plants were supplemented with 2 μM EBR and 40 mM NaCl compared with the respective controls (Fig. 9).

An increase in the activity of POD was recorded with increasing concentration of either NaCl or EBR (PT; Figs. 10a , 10c ). However, their interaction led to an overall decrease in POD contents. The maximum value for protein (68.33 mgg−1) in Desiree was at 80 mM NaCl. The value decreased after exogenous application of 1 or 2 μM EBR, to 45.41 and 46.01 mg g−1 proteins, respectively. These results revealed that while PT stimulated POD activities in both the cultivars, IM yielded mixed results (Figs. 10b , 10d ). It may be observed from the above that an overall trend of the biochemical attributes in general was rather similar though the two tested potato cultivars have shown differential preference for the method of treatment. When compared statistically for the effect size, EBR and methods of treatment influenced SOD and POD more than proteins (Table 2).

Discussion

Both cultivars were significantly influenced by in vitro NaCl, but exhibited differential responses to various NaCl and EBR treatments, with Cardinal being comparatively salt tolerant and Desiree being moderately sensitive. These results are in line with a previous study by Shahbaz et al. (2008) reporting that the inhibitory effects of NaCl stress were ameliorated significantly in response to application of EBR in both wheat cultivars studied (S-24, salt tolerant and MH-97, salt sensitive). However, the salt-tolerant variety showed a better response towards EBR treatment than the sensitive one.

A couple of small-scale methods of exogenous application of EBR have already been reported including foliar application (Fariduddin et al. 2004), pretreatment of seeds (Hayat et al. 2001; Piñol and Simón 2009), and as medium constituent (Arora et al. 2008). Pretreatment of seeds was considered to be the preferred method in O. sativa (Rao et al. 2002; Sharma et al. 2013), Medicago sativa (Zhang et al. 2007), and Zea mays (Arora et al. 2008), whereas the foliar application of BRs was shown to be quite useful in Phaseolus vulgaris (Upreti and Murti 2004), Solanum lycopersicum (Ogweno et al. 2008), T. aestivum (Shahbaz et al. 2008), and G. max (Zhang et al. 2008). Pretreatment of vegetative tissues grown in vitro on the other hand has not been reported so far. An in vitro approach provides an opportunity to manipulate cultures reproducibly under the desired set of experimental conditions.

In a study on Hordeum vulgare, Tabur and Demir (2009) found amelioration of the inhibitory effects of salinity stress on germination and growth of seedlings when seeds were pretreated with 3 μM EBR. In a similar study on O. sativa, Anuradha and Rao (2003) indicated that seed pretreatment with 3 μM EBR not only decreased the influence of salt stress but also improved plant growth and nitrate reductase activity while reducing pigment loss. Although both methods of exogenous EBR application were shown in the present study to be beneficial, pretreatment of nodal explants of Cardinal with 1 μM EBR was most effective. Interestingly, the second method (IM) with 2 μM EBR resulted in the best stress alleviation response in Desiree. These results are in line with several prior studies reporting positive role of BRs for the enhancement of growth either with or without supplemental salt in C. sativus (Yu et al. 2004), Cicer arietinum (Ali et al. 2007), T. aestivum (Ali et al. 2008b), and B. juncea (Fariduddin et al. 2009). It is, therefore, inferred that the use of low EBR concentrations generally alleviates stress in diverse plant species.

As far as the young seedlings were concerned, the increase in shoot length on EBR application may perhaps be a result of enhanced carbohydrate transport from the primary leaf to the upper region, i.e., epicotyl (Nakajima and Toyama 1999). However, there are contrasting reports about the role of BRs in root development. Kartal et al. (2009) described a positive relationship between BRs application and root growth via increased mitotic activity in H. vulgare. On the contrary, Özdemir et al. (2004) reported an inhibitory effect of EBR on root growth in O. sativa. In addition, the response of EBR in root growth was found to be dose dependent. In two independent studies on Arabidopsis (Kim et al. 2007) and Allium cepa (Howell et al. 2007), low EBR concentrations (10−10 and 10−9 M) stimulated root growth, but inhibited root growth at higher doses (10−9, 10−8, and 10−7 M in Arabidopsis, and 10−7 M in A. cepa).

Stress tolerance induced by BRs appears to be a complex phenomenon and probably involves several intrinsic factors. Quantitative analysis of the total proteins in the present study showed an increasing trend in both the potato cultivars when subjected to different NaCl concentrations. This increase was far more in Desiree compared with Cardinal. The reason might be the synthesis of some stress-related proteins (Sharma et al. 2013). Sajid and Aftab (2009) also described that higher amounts of proteins under stress conditions could help plants sustain growth. One of the possible modes of action may simply be to overcome an enhanced production of ROS by such upregulated proteins. It is interesting to note that studies at the gene expression level have also confirmed the association between overexpression of stress-responsive proteins (StDREB1 gene) and stress tolerance in potato. Moreover, StDREB1 provided protection against ROS under stress through regulation of the stress-responsive signaling pathway, i.e., expressing other genes putatively associated with stress resistance, e.g., StCDPK4 and StCDPK5 (Bouaziz et al. 2013). Enhancement of ROS including O2 −, OH, H2O2, and 1O2 − (Munne-Bosch and Penuelas 2003) under various abiotic stresses (salt, heat, drought) is well-known. Among the biochemical defense mechanisms that many plant species have developed, antioxidant enzymes appear to be probably the most effective system at scavenging these enhanced ROS (Farooq et al. 2008). The role of ZmMPK5 (ABA-regulated mitogen-activated protein kinase) on antioxidants was evaluated in response to BRs application in Z. mays (Zhang et al. 2010). The accumulation of H2O2 was shown to upregulate the activities of antioxidant enzymes. Therefore, an upregulation of the antioxidant defense system under an enhanced ROS scenario as evident in the above study (Zhang et al. 2010), as well as others mentioned above including the current investigation, probably does not come as a surprise and in fact seems to hold true in many plant species.

As outlined before, both SOD and POD levels were monitored in the present investigation in order to understand their role in salinity tolerance of potato vis-á-vis EBR treatments. The results from this study only partially corroborate the findings of Shahbaz et al. (2008) and Liu et al. (2009) regarding the antioxidant enzymes SOD and POD in T. aestivum and Chorispora bungeana. Both groups had shown an increased antioxidant level in the above species under abiotic stress that rose even further with the exogenous application of BRs. As far as various salt treatments in the present study were concerned, enhanced SOD and POD levels were observed not only in line with the abovementioned studies but also in agreement with several others (Lima et al. 2002; Ogweno et al. 2008; Liu et al. 2009; Ejaz et al. 2012; Nouman et al. 2014). Arora et al. (2008) interpreted this to be a mechanism for salinity tolerance in Z. mays after BRs application. In the present study, however, the combination of EBR and NaCl decreased the activities of SOD and POD. The reduced activities of SOD and POD might be associated with the removal of the stressful conditions by the EBR treatments in the first place. Not surprisingly, therefore, reduction in POD activity has already been reported in EBR-treated epicotyls of V. radiata (Wu and Zhao 1991) and hypocotyls of C. sativus (Xu and Zhao 1989). Vardhini and Rao (2003) reported a decrease in POD activity after the application of BRs to Sorghum vulgare seeds under osmotic stress. While these findings suggest that activities of antioxidant enzymes might help plants to ameliorate the effects of salt stress, possible co-existence of additional mechanisms operating in potato may not be ruled out without further investigation. It might not be out of context here to mention the possible triggering of other phytohormones in response to EBR pretreatment in potato. Changes in the endogenous ABA levels in response to BRs treatment in C. vulgaris as reported by Bajguz (2000) were probably caused by the same mechanism. The interaction of BRs with gibberellins and auxins has also been reviewed in detail (Mandava et al. 1981; Yopp et al. 1981). Synergistic modes of action to enhance growth thus remain a strong possibility in potato as well.

In conclusion, a useful role of EBR in response to salinity stress in potato has been observed in this study. These results are vital not only for the understanding of the potential role of BRs in the growth and development of potato and other species but also for its use in agriculture at a larger scale. In the present investigation, salt stress markedly decreased growth in both potato cultivars. Although either method of exogenous application of EBR could potentially alleviate the inhibitory effects of stress from in vitro-grown potato plants, pretreatment (PT) of nodal explants with 1 μM EBR was the best choice in Cardinal. Desiree on the other hand responded best in terms of growth parameters with 2 μM EBR in medium (IM). It appears that the use of lower EBR levels in potato has greater potential for increased crop production both in saline and non-saline soils. Detailed insight of the synergistic association of EBR with other plant hormones also needs to be elucidated further. Pretreatment of vegetative parts, i.e., nodes of potato, with EBR provides another method of EBR application having potential for its possible extension in the field. Pretreatment of propagules such as potato eyes is one such possibility. Although further research in this direction will answer these emerging questions, promising results in this study have provided an impetus to move forward with these studies.