Salt Stress by Nacl Alters the Physiology and Biochemistry of Tissue Culture-Grown Stevia Rebaudiana Bertoni

Home , Steviol glycoside

Turkish Journal of Agriculture and Forestry Turk J Agric For (2019) 43: 11-20 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1711-71

Salt stress by NaCl alters the physiology and biochemistry of tissue culture-grown Stevia rebaudiana Bertoni

1,2, 2 Rabia JAVED *, Ekrem GÜREL  1 Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan 2 Department of Biology, Faculty of Arts and Sciences, Abant İzzet Baysal University, Bolu, Turkey

Received: 16.11.2017 Accepted/Published Online: 30.06.2018 Final Version: 06.02.2019

Abstract: This study reports the response to salinity stress (100 mM, 200 mM, and 300 mM NaCl concentration) exposure of the commercially valuable medicinal plant Stevia rebaudiana during micropropagation for 4 weeks. The significant enhancement of physiological parameters, steviol glycosides (SGs), i.e. rebaudioside A and stevioside, as examined by high-performance liquid chromatography, and nonenzymatic antioxidant activities, i.e. total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power, and DPPH-free radical scavenging activity, was observed during the shoot formation process under up to 100 mM NaCl stress. Callus formation produced similar results regarding physiology and antioxidant assays, except that it produced merely a negligible amount of SGs. Contrarily, root formation showed marked susceptibility to 100 mM, 200 mM, and 300 mM NaCl concentrations and reduced growth parameters, sweetening compounds, and secondary metabolites. Hence, NaCl plays the role of abiotic stress elicitor, causing accumulation of reactive oxygen species and thus altering metabolic processes and physiology of Stevia under in vitro culture conditions.

Key words: Antioxidant activities, in vitro culture, NaCl stress, physiology, Stevia rebaudiana, steviol glycosides

1. Introduction et al., 2014). Tissue culture has long been used to increase Stevia rebaudiana, commonly called “honey leaf”, is a the SG content in the leaves of S. rebaudiana by employing member of the family Asteraceae (sunflower) and is different types of stresses (Bondarev et al., 2003; Sivaram indigenous to South America, specifically Brazil and and Mukundan, 2003; Thiyagarajan and Venkatachalam, Paraguay (Soejarto, 2002). It is famous for the production of 2012; Gupta et al., 2014; Pandey and Chikara, 2015; natural sweeteners, i.e. steviol glycosides (SGs), including Gupta et al., 2016). The growth of shoots, SGs, phenolics, rebaudioside A (Reb A), rebaudioside C, stevioside (ST), flavonoids, and antioxidant activities rise on exposure to dulcoside, and steviol bioside, which possess 300 times abiotic stress agents such as ZnO and CuO nanoparticles, more sweetness than sucrose (Wölwer-Rieck, 2012). These different plant growth regulators (PGRs), and hydrogen bioactive molecules make Stevia protective against cancer, peroxide in optimized environmental conditions (Rafiq diabetes, high blood pressure, obesity, and inflammation et al., 2007; Javed et al., 2017a, 2017b, 2017c). Moreover, (Liu et al., 2003; Dey et al., 2013). Large amounts of the effect of ZnO and CuO nanoparticles on callus phenolics, flavonoids, and other metabolites have also cultures of S. rebaudiana has been reported (Javed et al., been detected in dry leaves of S. rebaudiana (Tadhani 2017d). Javed et al. (2017e) studied the comparison of et al., 2007). Due to the presence of nonmutagenic and stimulation of physiological and biochemical parameters nontoxic secondary metabolites, S. rebaudiana has gained in shoot cultures of S. rebaudiana under the stress of paramount importance as an alternative noncaloric capped ZnO and CuO nanoparticles (ZnO-PEG, CuO- sweetening crop in many countries (Bopp and Price, 2001; PEG, ZnO-PVP, CuO-PVP) and uncapped ZnO and CuO. Sekihashi et al., 2002). The stress elicitors function in accumulation of reactive Since the germination percentage of Stevia seeds is very oxygen species (ROS) and change of metabolism until the low and its vegetative propagation by stem cuttings is not maximum threshold is achieved; thereafter, phytotoxicity productive, researchers have adapted micropropagation for causes damage to plant cells (Badran et al., 2015; Soufi et producing large amounts of true-to-type progenies (Khalil al., 2016; Javed et al., 2017a, 2017b, 2017c, 2017d). * Correspondence: [email protected] 11

This work is licensed under a Creative Commons Attribution 4.0 International License. JAVED and GÜREL / Turk J Agric For

Various studies of salinity stress on S. rebaudiana have A total of 4 treatments were prepared containing 0 mM, been reported previously. For instance, Gupta et al. (2014) 100 mM, 200 mM, and 300 mM NaCl. The pH of the reported the effect of NaCl and Na2CO3 on the callus and media was adjusted to 5.7–5.8 and plant agar [0.8 % (w/v)] suspension culture of S. rebaudiana for SG production. was added for solidification of media. The culture media

Additionally, the influence of NaCl, Na2CO3, proline, and were then autoclaved for 15 min under a pressure of 1.06 –2 PEG on SG production of S. rebaudiana was studied by kg cm at 121 °C temperature. Gupta et al. (2016). Furthermore, the expression level of 2.2. Growth conditions of callus, shoot, and root vital genes in the S. rebaudiana biosynthetic pathway was formation studied along with growth parameters and SG contents, Seeds of S. rebaudiana were purchased from Polisan and NaCl was found as an enhancer of gene transcription Tarım, İstanbul, Turkey. According to the method of of the SG biosynthetic pathway (Pandey and Chikara, Javed et al. (2017b, 2017d), the seeds were cultured on 2015). The gene expression, morphological parameters, basal MS medium after being disinfected with 0.1% and biochemistry of compounds were also evaluated in S. (w/v) mercuric(II) chloride (HgCl2) for 15 min. The leaf rebaudiana by Fallah et al. (2017), producing remarkable explants and axillary shoot nodes were excised from effects. 4-week-old seedlings and incubated in media having Zeng et al. (2013) evaluated oxidative enzyme activities PGRs: a combination of 0.5 mg/L kinetin (KN) and 0.5 (superoxide dismutase, peroxidase, and catalase), Reb A/ mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) along with ST ratio, and K+/Na+ ratio in field-grown Stevia plantlets in different concentrations of NaCl, i.e. 100, 200, and 300 mM. response to salt stress. The effects of salinity stress onStevia This was done for callus formation. However, no PGR was plants grown in vitro were previously reported by Pandey added in the case of shoot formation. The experiment was and Chikara (2014) and Rathore et al. (2014). Their studies conducted in triplicate in a growth room chamber having reported changes in growth, chlorophyll content, proline a 16-h light/8-h dark photoperiod, provided by cool- accumulation, and protein and sugar contents. Moreover, white fluorescent lighting of 35 µmol m–1 s–1 irradiance the influence of salt and drought stress onS. rebaudiana and 24 ± 1 °C temperature at 55%–60% relative humidity. growth dynamics was revealed by Mubarak et al. (2012). A Each treatment had 15 explants that were cultivated for field study of NaCl stress in S. rebaudiana revealing effects the required time period. The callus was harvested after on growth, photosynthetic pigments, diterpene glycosides, 45 days while shoots were harvested after 30 days of and ion content in roots and shoots was performed by cultivation. Finally, the physiological parameters involving Shahverdi et al. (2017). Furthermore, Cantabella et al. amount of callus, FW of callus, and DW of callus were (2017) studied salt effects on mineral nutrition along observed regarding callus formation. Similarly, the mean with antioxidative metabolism and SG contents in S. length of shoots, mean number of nodes and leaves, and rebaudiana under in vitro conditions. However, none of FW and DW of shoots produced by shoot formation these previous studies elucidated the effect of salt stress on were measured. Furthermore, the developed shoots were nonenzymatic antioxidant activities. Hence, the aim of the inoculated in the root formation medium (without any current study is to examine the differential effects of NaCl PGRs) having 15 shoots per treatment and different NaCl stress on different physiological parameters (including concentrations for 4 weeks in the growth chamber under amount of callus; fresh weight (FW) and dry weight (DW) similar conditions as described above. of callus; length of plantlets, shoots, and roots; number 2.3. Extraction and analysis of steviol glycosides of nodes, leaves, and roots; and FW and DW of plantlets, Steviol glycosides were extracted from the calli and leaves shoots, leaves, and stems), SG contents (Reb A and ST), of in vitro regenerated shoots and plantlets grown under and nonenzymatic antioxidant activities (including total NaCl stress (Javed et al., 2017b). In this process, the phenolic content (TPC), total flavonod content (TFC), calli, shoots, and plantlets propagated from all different total antioxidant capacity (TAC), total reducing power treatments were carefully washed with sterile distilled (TRP), and DPPH-free radical scavenging activity) of S. water. Then the plant material was soaked on Whatman rebaudiana during callus formation as well as shoot and filter paper No. 1 and dried in an oven at 60 °C for 48 h. root formation. Analysis of steviol glycosides was performed by carrying out high-performance liquid chromatography 2. Materials and methods (HPLC) of the samples. According to Javed et al. (2017b), 2.1. Preparation of medium containing NaCl for callus, the procedure involved addition of 20 mg of sample shoot, and root formation from each treatment to 1 mL of 70% (v/v) methanol in a Murashige and Skoog (MS) medium (1962) and 3% (w/v) microcentrifuge tube. After incubation in an ultrasonic sucrose were used for the preparation of culture medium. bath at 55 °C for 15 min, samples were centrifuged at 25 °C

12 JAVED and GÜREL / Turk J Agric For and 12,000 rpm for 10 min. The pellet was discarded and was used as the standard and the results were expressed as supernatant was filtered using 0.22-µm PTFE Millipore µg quercetin equivalent per mg (µg QE/mg). syringe filters. Finally, HPLC analysis was performed by 2.4.3. Determination of total antioxidant capacity running all of the samples in triplicate. Total antioxidant capacity was evaluated by the procedure According to Javed et al. (2017b), chromatography was of Jafri et al. (2014) after slight modifications. An aliquot performed using an autosampler (WPS-3000-SL Dionex of 100 µL from the stock solution of each sample (4 mg/ Semi Prep Autosampler) injecting 10 µL of each sample, mL in DMSO) was mixed with 900 µL of reagent solutions a binary pump (LPG 3400SD Dionex) solvent delivery –1 containing 0.6 M sulfuric acid, 4 mM ammonium system working at a flow rate of 0.8 mL min , and a dual molybdate, and 28 mM sodium phosphate. The reaction wavelength absorbance detector operating at 210 and 350 mixture was incubated at 95 °C for 90 min and then cooled nm (MWD-3100 Dionex UV-Vis Detector). The column, at room temperature. All samples were run in triplicate and an Inertsil ODS-3 (GL Sciences Inc., Japan) of 150 × 4.6 their absorbance was measured at 695 nm using a Biotek mm in length with 5 µm particle size, was kept warm at ELX800 microplate reader. Ascorbic acid was used as the 40 °C in a column oven system (TCC-3000SD Dionex). At standard and the results were expressed as µg ascorbic acid the end, isocratic flow was performed using an acetonitrile equivalent per mg (µg AA/mg). and 1% (w/v) phosphoric acid buffer mixture (68:32) for 20 min. 2.4.4. Determination of total reducing power Total reducing power of samples was calculated according 2.4. Preparation of extract and antioxidant assays to the procedure of Jafri et al. (2014) after slight According to Javed et al. (2017b, 2017d), the calli and leaf extracts of S. rebaudiana were prepared by drying the modifications. First 100 µL of each sample (4 mg/mL in calli and leaves, and then taking 0.1 g of their fine powder DMSO) was mixed with 200 µL of phosphate buffer (0.2 obtained under different NaCl concentrations. Methanol M, pH 6.6) and 250 µL of 1% w/v potassium ferricyanide. in an amount of 500 µL was used for the dissolution of The resulting mixture was incubated at 50 °C for 20 min. powder. It was vortexed for 5 min and then sonicated for Thereafter, the reaction was acidified with 200 µL of 10% 30 min followed by 15 min of centrifugation at 10,000 w/v trichloroacetic acid and centrifugation was performed rpm. The pellet was discarded and supernatant was stored at 3000 rpm for 10 min. The pellet was discarded and the to evaluate different antioxidant activities. obtained supernatant (150 µL) was mixed with 50 µL of 0.1% w/v ferric chloride solution. All samples were run in 2.4.1. Determination of total phenolic content triplicate and their absorbance was measured at 700 nm The method of Jafri et al. (2014) was performed with using a Biotek ELX800 microplate reader. Ascorbic acid slight modifications to estimate the total phenolic content was used as the standard and the results were expressed as in callus and leaf extracts of Stevia. The process involved transfer of an aliquot of 20 µL (4 mg/mL) of dimethyl µg ascorbic acid equivalent per mg (µg AA/mg). sulfoxide (DMSO) stock solution of each sample to the 2.4.5. Determination of DPPH-free radical scavenging respective well of a 96-well plate and then the addition activity of 90 µL of Folin–Ciocalteu reagent to it. The plate was Since the overproduction and accumulation of free radicals incubated for 5 min and then 90 µL of sodium carbonate is damaging to plant cells, the ability of antioxidants was added to the reaction mixture. All samples were run produced to prevent oxidative damage was elucidated by in triplicate and their absorbance was obtained at 630 nm 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. This assay using a Biotek ELX800 microplate reader. Gallic acid was was performed according to the protocol of Haq et al. used as the standard and the results were expressed as µg (2012) after slight modifications. First 10 µL (4 mg/mL) gallic acid equivalent per mg (µg GAE/mg). of Stevia callus and leaf extracts was mixed with 190 µL of 2.4.2. Determination of total flavonoid content DPPH (0.004% w/v in methanol). The resulting reaction The aluminum chloride colorimetric method of Jafri et mixture was incubated in darkness for a period of 1 h. All al. (2014) was performed after slight modifications to samples were run in triplicate and their absorbance was determine the total flavonoid content of different callus measured at 515 nm wavelength using a Biotek ELX800 and leaf extracts of Stevia. First 10 µL of 10% (w/v) microplate reader. Ascorbic acid was used as the positive aluminum chloride, 10 µL of 1.0 M potassium acetate, control while DMSO was the negative control. and 160 µL of distilled water were added to an aliquot of % inhibition of test sample = % scavenging activity = (1 –

20 µL (4 mg/mL) of DMSO stock solution of each sample Abs / Abc) × 100 contained in the respective well of 96-well plates. It was Here, Abs indicates the absorbance of DPPH solution kept at room temperature for 30 min. The samples were with the sample, and Abc is the absorbance of only DPPH run in triplicate and their absorbance was measured at 415 solution. The IC50 was calculated by using Table curve nm using a Biotek ELX800 microplate reader. Quercetin software 2D Ver. 4.

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2.5. Statistical analysis control treatment, i.e. 3.59 and 0.84, respectively. It was The design of experiments was randomized and the followed by the steviol glycosides obtained under 100 mM, statistical analysis of data was performed using SPSS 200 mM, and 300 mM on a respective basis. 17.0 (SPSS Inc., Chicago, IL, USA). Statistical differences 3.3. Determination of antioxidant activities were determined using ANOVA, and the significance The antioxidant activities in calli were measured and it of differences between means ± standard error (SE) was found that antioxidant activities were possessed by values was obtained using Duncan’s multiple range tests the calli grown under control, 100 mM, and 200 mM NaCl performed at P < 0.05. stress as shown in Table 4. Calli were not formed under 300 mM NaCl and the leaf explants were found to be dead. A 3. Results significantly higher amount of all antioxidant activities, i.e. 3.1. Determination of physiological parameters TPC, TFC, TAC, TRP, and DPPH-free radical scavenging Table 1 gives the detailed comparison of amount of callus activity, was revealed by the calli obtained under 100 mM (cm), FW of callus (g), and DW of callus (g) in MS medium NaCl, followed by control treatment and then 200 mM provided with 0 mM, 100 mM, and 200 mM NaCl. The NaCl concentration. data for 300 mM NaCl concentration are absent because All antioxidant activities, i.e. TPC, TFC, TAC, TRP, the calli died at this concentration. The amount, FW, and DPPH-free radical scavenging activity, were higher and DW of calli were revealed to be significantly higher under the stress of 100 mM NaCl as shown in Table 5. The under the control and 100 mM of NaCl stress, followed TPC, TFC, TAC, TRP, and DPPH-free radical scavenging by 200 mM NaCl stress. Figure 1 clearly shows all of these activities at 100 mM of NaCl stress were 7.48 µg/mg of physiological parameters after 4 weeks of callus formation. DW, 5.04 µg/mg of DW, 14.63 µg/mg of DW, 10.39 µg/mg Regarding shoot formation, Table 2 shows that the of DW, and 49.48%, respectively. Antioxidant activities mean length of shoots (cm), number of nodes and leaves, were also possessed by the control treatment. However, no FW of shoots (g), FW of leaves (g), FW of stems (g), DW antioxidant activity was seen under 200 mM and 300 mM of shoots (g), DW of leaves (g), and DW of stems (g) were NaCl concentrations as the shoot nodes could not cope significantly higher under 100 mM of NaCl stress, i.e. 4.1 with this stress and hence died. cm, 4.6, 13, 0.43 g, 0.30 g, 0.09 g, 0.03 g, 0.02 g, and 0.02 It is revealed from Table 6 that all of the antioxidant g, respectively. These results were followed by control activities, i.e. TPC, TFC, TAC, TRP, and DPPH-free treatment results, i.e. 2.83 cm, 2.66, 10, 0.16 g, 0.22 g, radical scavenging activities, were obtained by NaCl stress 0.03 g, 0.02 g, 0.01 g, and 0.01 g on a respective basis. The of 100 mM, 200 mM, and 300 mM as well as by the control treatment. Despite the antioxidant activities revealed by all shoots died under 200 mM and 300 mM salt stress. The three conditions of stress, significantly higher antioxidant data of Table 1 are clearly understandable from Figure 2. activities were shown by the regenerants grown under no The physiological parameters for root formation were stress, i.e. the control treatment. The data of the control also analyzed, and it is revealed from Table 3 that the treatment, 5.83 µg/mg of DW, 2.65 µg/mg of DW, 11.5 µg/ length of plantlets and roots (17 cm and 1.83 cm); number mg of DW, 10.5 µg/mg of DW, and 64.04%, were followed of roots, nodes, and leaves (7.33, 12.33, and 26.6); FW of by 100 mM, 200 mM, and 300 mM NaCl concentration plantlets, leaves, and stems (1.02 g, 0.38 g, and 0.22 g); and treatments, respectively. DW of plantlets, leaves, and stems (0.15 g, 0.06 g, and 0.04 g) were significantly higher in the control treatment. The 4. Discussion data of the control treatment were followed by 100 mM, The physiological parameters of callus formation and 200 mM, and 300 mM NaCl concentrations, respectively. shoot formation were significantly enhanced when MS Figure 3 shows dense roots in the control treatment and medium was supplemented with up to 100 mM NaCl then a continuous decline in root formation from 100 mM concentration, whereas a decline in all parameters was to 200 mM and 300 mM of NaCl stress. observed with increasing NaCl concentration during the 3.2. Determination of steviol glycosides formation of regenerants. The best growth was obtained The steviol glycosides (Reb A and ST) were not obtained in the case of the control during root formation. The most from the grown calli. However, the shoots and regenerants probable reason for growth reduction in the case of root possessed significantly greater contents of SGs, as shown formation is the change in metabolic activities resulting in in Figure 4 and 5. In the case of shoot formation, the reduction in cell division, elongation, and differentiation highest amount of Reb A and ST was seen in the shoots of roots. Mubarak et al. (2012) obtained significantly less raised under 100 mM of NaCl stress, i.e. 2.04 and 1.73, growth after exposure to rising salt stress. The control respectively. The control treatment produced 1.27 and 1 treatment produced the best results with respect to shoot amounts of Reb A and ST, respectively. The regenerants number, shoot length (cm), node number, leaf number, produced higher amounts of Reb A and ST under the root number, root length (cm), and % survival rate.

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Table 1. Comparison of physiological parameters in 6-week-old calli produced from leaf explants on MS medium supplemented with different concentrations of NaCl.

Conc. of NaCl (mM) Amount of callus FW of callus (g) DW of callus (g) Control 1.5 ± 0.20a 0.09 ± 0.05a 0.02 ± 0a 100 1.5 ± 0a 0.09 ± 0.02a 0.02 ± 0a 200 1.0 ± 0b 0.03 ± 0b 0.01 ± 0b

±: Standard error; means with the same letters within columns are not significantly different according to Duncan’s multiple range test at confidence level of 95%.

Figure 1. Calli grown on MS media containing PGRs, i.e. combination of 0.5 mg/L of KN and 0.5 mg/L of 2,4-D, under 0, 100, 200, and 300 mM NaCl stress after 6 weeks.

Figure 2. Shoots grown from shoot nodes raised from seedlings without using PGRs under 0, 100, 200, and 300 mM NaCl stress after 4 weeks.

15 JAVED and GÜREL / Turk J Agric For a b DW of of DW stems (g) 0.04 ± 0.01 0.09 ± 0.01 0.03 ± 0 0.01 ± 0 DW of stems of DW (g) 0.01 ± 0 0.02 ± 0.01 b a FW of FW of stems (g) 0.22 ± 0.12 0.62 ± 0.05 0.14 ± 0.04 0.07 ± 0.02 a b b b 50.38 64.04 30.58 DPPH (%) DPPH FW of stems FW of (g) 0.03 ± 0.01 0.09 ± 0.01 DW of of DW leaves (g) 0.06 ± 0.02 0.05 ± 0.01 0.05 ± 0 0.05 ± 0 b a a d b c a c b DW of leaves of DW (g) 0.01 ± 0 0.02 ± 0 FW of FW of leaves (g) 0.38 ± 0.11 0.29 ± 0.07 0.23 ± 0.01 0.33 ± 0.04 b a a b b 9.9 ± 0.01 10.5 ± 0.01 9.68 ± 0.01 TRP (µg/mg of DW) of TRP (µg/mg c FW of leaves FW of (g) 0.22 ± 0.01 0.30 ± 0.03 0.15 ± 0.02 0.09 ± 0.02 0.09 ± 0.01 0.08 ± 0 DW of of DW plantlets (g) a a b c d b c b a 10.46 ± 0 11.5 ± 0.01 8.52 ± 0 TAC (µg/mg of DW) of (µg/mg TAC 1.02 ± 0.18 0.52 ± 0.10 0.46 ± 0.12 0.42 ± 0.06 FW of FW of plantlets (g) DW of shoots of DW (g) 0.02 ± 0 0.03 ± 0 a c d b a b a b c 14.6 ± 1.33 11.3 ± 1.76 20 ± 1.15 26.6 ± 0.76 No. of leaves of No. 0.16 ± 0.03 0.43 ± 0.03 FW of shoots FW of (g) b d c 4.4 ± 0.02 5.83 ± 0.02 4.1 ± 0.01 TPC (µg/mg of DW) of TPC (µg/mg b a 6 ± 0.57 4.66 ± 0.33 8.66 ± 0.88 12.33 ± 0.33a No. of nodes of No. No. of leaves of No. 10 ± 1.18 13 ± 0 b b a c b a c c 7.33 ± 1.33a 0.66 ± 0 0 No. of roots of No. 2.65 ± 0.01 1.72 ± 0.02 TFC (µg/mg of DW) of TFC (µg/mg 1.88 ± 0.01 a b 2.66 ± 0.4 4.6 ± 0.33 No. of nodes of No. c c b 1.83 ± 0.16 0.33 ± 0 0 Length of roots (cm) a b c d Comparison of antioxidant activities in 6-week-old calli produced from leaf explants on MS medium supplemented supplemented MS medium on explants leaf from calli produced activities in 6-week-old antioxidant of Comparison a 2.83 ± 0.24 Length shoots of (cm) 4.1 ± 0.16 100 200 Conc. of NaCl (mM) Conc. NaCl of Control Table 4. Table NaCl. of concentrations different with ±: Standard error; means with the same letters within columns are not significantly different according to Duncan’s multiple multiple to Duncan’s according different significantly not are columns within letters the same with error; means Standard ±: of 95%. level confidence at test range Length of plantlets (cm) 17 ± 1.15 10.16 ± 0.44 6.83 ± 0.44 5.16 ± 0.72 Control Conc. of NaCl Conc. NaCl of (mM) 100 ±: Standard error; means with the same letters within columns are not significantly different according to Duncan’s multiple range test at confidence level of level confidence at test range multiple to Duncan’s according different significantly not are columns within letters the same with error; means ±: Standard 95%. Comparison of physiological parameters in 4-week-old regenerants produced from shoots on MS medium supplemented with different concentrations concentrations different with supplemented MS medium on shoots from produced regenerants in 4-week-old parameters physiological of 3. Comparison Table NaCl. of Conc. of NaCl Conc. NaCl of (mM) Control 100 200 300 Comparison of physiological parameters in 4-week-old shoots produced from nodal stem explants on MS medium supplemented with different different with supplemented MS medium on nodal explants stem from produced shoots in 4-week-old parameters physiological of 2. Comparison Table NaCl. of concentrations ±: Standard error; means with the same letters within columns are not significantly different according to Duncan’s multiple range test at confidence level of 95%. level confidence at test range multiple to Duncan’s according different significantly not are columns within letters the same with error; means ±: Standard

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Figure 3. Roots grown from shoots raised on PGR-free media under 0, 100, 200, and 300 mM NaCl stress after 4 weeks.

2.5 Amount of Reb A 2.04 2 Amount of ST 1.73 )

( % 1.5 1.27 f SGs

o 1

un t 1 o m A 0.5

0 Control 100mM Conc. of NaCl Figure 4. Effect of NaCl concentrations (0 and 100 mM) on shoots after 4 weeks regarding Reb A content, represented with red bars, and ST content, blue bars. Error bars are shown as standard deviation for each bar.

4 3.59 Amount of Reb A 3.5 Amount of ST 3 2.56 ) 2.5 ( % 1.94 1.91

f SGs 2 o

un t 1.5 o m A 1 0.84 0.51 0.42 0.4 0.5

0 Control 100mM 200mM 300mM Conc. of NaCl Figure 5. Effect of NaCl concentrations (0, 100, 200, and 300 mM) on regenerants after 4 weeks regarding Reb A content, represented with blue bars, and ST content, red bars. Error bars are shown as standard deviation for each bar.

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Table 5. Comparison of antioxidant activities in 4-week-old shoots produced from nodal shoot explants on MS medium supplemented with different concentrations of NaCl.

Conc. TFC (µg/mg of DW) TPC (µg/mg of DW) TAC (µg/mg of DW) TRP (µg/mg of DW) DPPH (%) Control 4.44 ± 0.01b 6.97 ± 0.01b 13.77 ± 0b 10.22 ± 0.02b 48.03 100 mM 5.04 ± 0.01a 7.48 ± 0a 14.63 ± 0a 10.39 ± 0.01a 49.48

±: standard error; means with the same letters within columns are not significantly different according to Duncan’s multiple range test at confidence level of 95%.

Table 6. Comparison of antioxidant activities in 4-week-old regenerants produced from shoots on MS medium supplemented with different concentrations of NaCl.

Conc. of NaCl (mM) TFC (µg/mg of DW) TPC (µg/mg of DW) TAC (µg/mg of DW) TRP (µg/mg of DW) DPPH (%) Control 2.65 ± 0.01b 5.83 ± 0b 11.5 ± 0.01a 10.5 ± 0.01b 64.04 100 3.89 ± 0.02a 5.91 ± 0a 10.86 ± 0.01b 10.6 ± 0.01a 63.91 200 1.94 ± 0.01c 5.31 ± 0c 7.22 ± 0d 10.14 ± 0.01d 67.45 300 1.7 ± 0.01d 5.13 ± 0.01d 9.52 ± 0.01c 10.49 ± 0.01c 69.82

±: standard error; means with the same letters within columns are not significantly different according to Duncan’s multiple range test at confidence level of 95%.

Almost all of the parameters declined with increment in NaCl concentrations during root formation. Our results salinity from the control to 5000 ppm, 7500 ppm, and for root formation are consistent with the findings of 10,000 ppm concentrations of NaCl. The highest NaCl Zeng et al. (2013) that showed continuous decline in SG concentration produced a lethal effect leading to death contents with the increase in salinity stress imposed by of in vitro-grown plantlets. These results agree with our NaCl to Stevia plants. The significant reduction of SGs at data on root formation but disagree with our callus and 90 mM and 120 mM salt concentration was illustrated by shoot formation results. Zeng et al. (2013) investigated this study. In addition to this, a significant increase of SG the reduction in physiological parameters upon increase contents from 25 mM to 50 mM, 75 mM, and 100 mM of NaCl stress up to 90 mM, which is consistent with was observed in S. rebaudiana by Pandey and Chikara our root formation results, but not our callus and shoot (2015). Gupta et al. (2016) showed an increase of SG formation findings. The study conducted by Pandey and content upon increasing NaCl salt concentration in in Chikara (2014) illustrated an increase of all growth and vitro-grown S. rebaudiana plants. S. rebaudiana was found development parameters with up to 100 mM of NaCl stress to be a moderately salt-tolerant species by Hussin et al. for Stevia shoots and roots. These results are in agreement (2017), which is consistent with our findings of shoot with our findings regarding callus and shoot formation, formation whereby after reaching a certain threshold the while they disagree in the context of root formation. SG increment stopped. Moreover, a significant decrease In addition, Pandey and Chikara (2015) examined the of SG contents was observed from 100 mM to 200 mM significant effect of 25 mM, 50 mM, 75 mM, and 100 mM and 300 mM NaCl concentration during root formation. NaCl concentrations on different growth parameters ofS. Fallah et al. (2017) showed a significant decrease of SGs rebaudiana and found their decrease with increasing NaCl with increasing NaCl stress in S. rebaudiana. The highest concentrations up to 100 mM. Gupta et al. (2016) reported amounts of SGs were formed in the case of the control an increase in growth by adding lower concentrations of treatment in this study. Moreover, in our study, SGs NaCl, but higher concentrations of NaCl did not support were not detected in calli obtained by callus formation. the growth enhancement of S. rebaudiana. A significant The reason for this might be that energy was utilized in decline of physiological parameters was observed with the maintaining metabolism of enzymatic and nonenzymatic rise of NaCl stress (from 0 to 20 mM, 40 mM, 60 mM, and antioxidants rather than the synthesis of complex SG 80 mM) in S. rebaudiana by Fallah et al. (2017). molecules and secondary metabolites. The SG (Reb A and ST) content was found to increase Metabolic pathways are altered as a result of salinity/ in the case of shoot formation at up to 100 mM of NaCl NaCl stress in the case of callus, shoot, and root formation stress; however, the SG content decreased with increasing of S. rebaudiana. The disturbance of metabolic processes

18 JAVED and GÜREL / Turk J Agric For leads to increased phenols, flavonoids, total antioxidant shoots at up to 100 mM NaCl concentration augmented to activity, total reducing power, and DPPH-free radical MS growth medium. However, the obtained roots indicated scavenging activity. All phytochemicals increase in the salt susceptibility with the rise of NaCl concentration calli and shoot leaves formed in MS medium containing from the control to 100 mM and above. Hence, this study up to 100 mM NaCl concentration. However, a decline in suggests that S. rebaudiana plants possess the capability to phytochemical activity is observed in regenerant leaves grow in saline soils because of the significant tolerance of during root formation taking place in MS medium having this plant to the deleterious effects of salt stress, but only 100 mM, 200 mM, and 300 mM NaCl concentration. Stevia up to the threshold level of 100 mM NaCl. Therefore, we has previously shown such types of responses regarding may consider this plant to be moderately halotolerant. the accumulation of phenols in shoots containing up to 75 mM NaCl concentration (Rathore et al., 2014). Moreover, an increment of enzymatic antioxidant activities in soil- Acknowledgments grown shoots was estimated with increasing concentrations Rabia Javed is grateful to the Scientific and Technological of NaCl salt (Zeng et al., 2013). The most probable Research Council of Turkey (TÜBİTAK) (Program No. mechanism involves formation of ROS causing oxidative 2216) for providing financial support to conduct this stress by supplementing NaCl to the medium. This causes research. The authors are also thankful to the Department plant cells to form more secondary metabolites. of Biotechnology, Quaid-i-Azam University, Pakistan, and In conclusion, the in vitro growth of S. rebaudiana calli the Department of Biology, Abant İzzet Baysal University, and shoots revealed significant salt tolerance by calli and Turkey, for providing all the required facilities.

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