Appl Biochem Biotechnol DOI 10.1007/s12010-014-0736-2

Effect of Salts (NaCl and Na2CO3) on Callus and Suspension Culture of Stevia rebaudiana for Steviol glycoside Production Pratibha Gupta & Satyawati Sharma & Sanjay Saxena

Received: 17 September 2013 / Accepted: 10 January 2014 # Springer Science+Business Media New York 2014

Abstract Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellowgreen and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l−1 NAA and different concentrations of NaCl (0.05– 0.20 %) and Na2CO3 (0.0125–0.10 %) for 2 weeks, and incubated at 24±1 °C and 22.4 μmol m−2 s−1 light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57 % with 0.10 % NaCl, and 0.025 % Na2CO3, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na2CO3 enhanced the SGs content from 1.36 (control) to 2.61 and 5.14 %, respectively, on the 10th day. Keywords Stevia . Callus . Suspension . Salts . Steviol glycosides . HPLC

Introduction The food and beverages industry has traditionally used sugar as a sweetening agent. However, there is an increasing demand for other sweeteners in response to consumer preference. A P. Gupta (*) : S. Sharma Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India e-mail: [email protected] S. Sharma e-mail: [email protected] S. Saxena The Energy and Resources Institute (TERI), IHC Complex, Lodhi Road, New Delhi 110003, India e-mail: [email protected]

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significant section of consumers are interested in sweeteners which have nutritive values and fulfill the requirements related to non-toxic nature, sugar-like taste profile, low calorific value, heat, and pH stability. At present, there are both synthetic as well as natural sweeteners available in the market addressing these needs. However, in recent years, there has been considerable interest in Stevia-based natural sweeteners that possess many of these desired qualities [53]. Extracts of leaves of Stevia have been used for centuries in South America, Asia, Japan, China, and in some countries of Europe either as a substitute of sucrose in beverages and foods or as a household sweetener. Stevia rebaudiana is a perennial plant belonging to the family Asteraceae and native to Paraguay. Stevia leaves contain nine types of steviol glycosides (SGs) which are about 300 times sweeter than sucrose at their concentration of 4 % (w/v) [29]. These sweet compounds represent about 14 % constituents of dried leaves and are diterpene glycosides which are based on the kaurene skeleton. The nine types of SGs found are; stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, steviolbioside A, and dulcoside A. The primary glycoside is stevioside (ent13-hydroxykaur-16-en-18- oic acid) which is 250–300 times sweeter than sucrose and very stable [17]. Other glycosides found in significant amounts include rebaudioside A, rebaudioside C, and dulcoside A. Rebaudioside A is the most desirable component due to its sweetening potency and superior taste profile [11, 53]. Unlike traditional sugar substitutes such as xylitol or sorbitol, these glycosides are non-toxic, non- mutagenic, and low-calorie compounds [36]. Plant cell suspension culture could be used for large-scale culturing of plant cells from which secondary metabolites could be extracted. However, several tissue culture studies on Stevia rebaudiana regarding induction of callus and suspension culture development using various explants (node, leaves, and internode, etc.) have been conducted [5, 20, 23, 26], but till date, there are no report related to the effect of salts or other elicitors on SGs production from callus and suspension culture. Production of SGs under in vitro conditions is poorly understood, and the results obtained by different authors are highly contradictory. Lee et al. [30] and Hsing et al. [22] reported stevioside in the callus tissue of Stevia while Miyagawa et al. [37], Yamazaki and Flores [52], and Swanson et al. [49] did not obtain SGs in callus and shoot culture. In another report, Bondarev et al. [5] obtained minor quantities of SGs in non-differentiated cell cultures, such as callus and cell suspension and also reported that their content varied during growth cycle of the culture. Biotic (cyanobacteria, chitosan, yeast extract) and abiotic (sodium chloride, methyl jasmonate, fructose) stresses are known to stimulate enhanced yields of secondary metabolite biosynthesis in various plant cells cultures in shorter periods of time [2, 7, 13, 31, 35, 38, 41, 54]. It has been reported that salinity is one of the major environmental stress factors that have adverse effects on plant growth and production. The occurrence of fast climate changes, the adoption of inappropriate irrigation systems, and the use of poor quality water in agriculture are the main cause of salinity [16, 25, 55]. Further, Sodium is one of the major ions which are detrimental to the growth of plants, because of its high concentrations in the environment [9]. In order to obtain high yields suitable for commercial utilization, two salts of sodium (NaCl and Na2CO3) were chosen to create abiotic stress on in vitro culture of Stevia rebaudiana. Plant cells and tissues require an optimum pH for growth and development in cultures. Addition of salts changes the pH of the nutrient medium. The pH affects nutrient uptake as well as enzymatic and hormonal activities in plants [3]. So, the aim of the present study was to evaluate the role of salts (NaCl and Na2CO3) on the growth and development of callus and suspension culture along with production of SGs.

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Materials and Methods Tissue Culture Studies Experimental Material, Callus Induction, and Development/Multiplication Stevia plants were procured from Ritnand Balved Education Foundation, Gurgaon (Haryana), India, for study and maintained in the micromodel complex of IIT Delhi. Leaves excised from in vitro raised shoots obtained after 4 weeks of culturing on Murashige and Skoog (MS) medium with 4.0 mg l−1 of kinetin were used as explants [19]. For callus induction, these in vitro leaves were cultured on MS medium supplemented with 0.75 mg l−1 NAA, 1.0 mg l−1 2,4-D, 3 % (w/v) sucrose and gelled with 0.8 % (w/v) agar [20]. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C and 1.2–1.3 kg/cm2 pressure for 20 min. All the cultures were incubated inside a growth chamber, at 24±1 °C temperature and 22.4 μmol m−2 s−1 light intensity provided by white fluorescent tubes for 16 h. Callus obtained from induction medium was sub-cultured and multiplied on MS+2.0 mg l−1 NAA for further development. Callus and Suspension Culture Development with Salts (NaCl and Na2CO3) One-month-old callus obtained from multiplication medium (MS+2.0 mg l−1 NAA) was used to develop suspension culture. To study the effect of salts on callus and suspension culture for SGs production, four different concentrations of NaCl (0.05, 0.10, 0.15, and 0.20 %) and Na2CO3 (0.025, 0.05, 0.075, and 0.10 %) were added into multiplication medium. Salts (NaCl and Na2CO3) used for experiments are non-toxic, cheap, easily available, and readily soluble in water. In all the treatments, the pH of the medium was adjusted to 5.8. Biochemical Analysis Extraction Procedure Extraction of SGs from callus and suspension culture was achieved following the procedure described by Swanson et al. [49] with minor modifications. The SGs from callus tissue were extracted (3×30 ml) with methanol/water in the ratio 4:1, and pooled extracts were dried in vacuo at 55 °C. Thereafter, residues were dissolved in 10-ml water and defatted twice with equal volume of chloroform. Aqueous layers were partitioned with water saturated n-butanol (3×30 ml) and the combined butanol phases were evaporated in vacuo. The residues were redissolved in acetonitrile/water (4:1), and filtered by membrane filter (nylon 0.45-μm pore size, Millipore Millex-HN). The samples obtained were stored at −20 °C for analysis by high performance liquid chromatography (HPLC). Quantitative Analysis Samples prepared by the extraction procedure described above were used for quantitative analysis for SGs (stevioside and rebaudioside A) using HPLC (Instrument: Agilent technologies 1120 Compact LC made). An isocratic elution of acetonitrile/water (80:20) for 10 min at a flow rate of 1 ml/min was used. Sample (prepared in solvent system) were passed through C18 reverse phase-packed stainless steel BDS column (4×250-mm diameter) and detected by UV/Visible detector at 205 nm.

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Data Analysis All in vitro experiments were repeated thrice with a sample size of 15 (for callusing) and 4 (for suspension culture) each time. Undifferentiated calli and suspension culture obtained from multiplication medium were collected and analyzed for fresh weight, dry weight and growth index (GI) to measure the growth of callus. Fresh Weight and Dry Weight Determination While determining fresh weight, callus was gently pressed on filter papers (Whatman no. 1) to remove excess water. For dry weight, calli were dried overnight in an oven at 60 °C. Growth Index The growth index was determined by using the following formula given by [18]. Growth indexðGIÞ ¼

W F −W 0 W0

Where WF and W0 represents the final and initial callus masses, respectively (either as fresh or dry weight). Quantitative Estimation In case of biochemical analysis, quantitative estimation of stevioside and rebaudioside A in callus and suspension culture was performed by Arbro Pharmaceutical Ltd., Delhi, India, using HPLC. Analytical grade standards with 95 % purity (procured from Natural Remedy Pvt. Ltd., Bangalore) were used for calibration. All data are reported as mean ± standard deviation for two independent samples (n=2) for HPLC. The quantification of SGs (stevioside and rebaudioside A) in callus and suspension culture samples treated with and without salts was done using the following formula: Quantity of Stevioside or Rebaudioside Að%Þ ¼

Test area of sample Amount of standardðmgÞ DilutionðmlÞ Potency of standard     100 Test area of standard DilutionðmlÞ Amount of sampleðmgÞ 100

In an earlier study, Bondarev et al. [5] reported that SGs concentrations in callus and suspension culture were found to be maximum when the samples were collected after 2 weeks. Hence, in various treatments involving callus culture, the samples for SGs analysis were collected on the 15th day while the samples of suspension culture were collected on the 5th, 10th, 15th, and 20th day. In the present study, it was found that control (without salts) suspension culture collected at the 15th day produced maximum amount of total SGs (stevioside + rebaudioside A) in comparison to the samples collected on the 5th, 10th, and 20th day. However, suspension culture treated with salts produced maximum quantities of SGs on the 10th day. Hence, SGs content in suspension cultures treated with salts were estimated on the 10th and 15th day only. Statistical Analysis The influence of various treatments on growth and Steviol glycoside content was analyzed by one-way analysis of variance (ANOVA). The data was analyzed statistically by ANOVA and

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difference between means of the samples was analyzed by Duncan’s multiple range test at a probability level of 0.05. Data values have been presented in tabular form as “means ± standard deviation.”

Results and Discussions Effect of Salts on Callus Culture and Production of SGs Callus Culture Treated with Salts (NaCl and Na2CO3) During the present study, a significant decline in fresh weight, dry weight, and callus GI was observed with the increase in concentration of NaCl in the medium (P≤0.05) (Table 1). At all the concentrations, inclusion of NaCl to the medium proved inhibitory. Among the different treatments with NaCl, maximum fresh weight (101.26±1.10 mg/culture), dry weight (10.27±0.11 mg/culture), and GI (0.50) were observed with lower concentration (0.05 %) of NaCl. In a medium with higher concentrations of NaCl (≥0.15 %) the sensitive callus became soft, necrotic, and stopped growth. However, callus obtained with control and lower concentrations of NaCl (≤0.10) were green and compact and showed less injury. Decline of callus growth due to NaCl stress as noticed in the present study is a usual phenomenon and also observed in many plant tissues which are subjected to stress [43, 48]. The reduction in callus fresh weight might be a consequence of reduced water availability in the culture medium due to increased concentrations of NaCl. Decrease in the relative growth rate and water content of callus proliferating in saline medium compared to callus incubated in salt-free medium was reported in Lycopersicon esculentum Mill. [46] and Thymus vulgaris [56]. Similarly, Rafiq et al. [42] reported the reduction in fresh and dry weights of calli obtained from local mung bean with increased concentration of NaCl in the medium. As with NaCl, retardation in callus growth was also observed with different concentration of Na2CO3 tested. With lowest concentration (0.025 %) of Na2CO3, maximum fresh weight (99.93±1.41 mg/culture), dry weight (10.14±0.14 mg/culture), and growth index (0.49) was seen. All the treatments showed negative response with respect to callus growth. The color of callus changed from green to light green. Significant decrease in fresh weight, dry weight, and Table 1 Effect of salts on growth parameter and steviol glycoside production from callus culture Fresh weight (mg/culture)

Control

113.60a ±1.11 11.36a ±0.11 0.66

0.20±0.01 0.07±0.01

0.05

101.26b ±1.10 10.27b ±0.11 0.50

0.36±0.05 0.14±0.02

0.51±0.07

0.10

93.66c ±1.22

9.55c ±0.12

0.38

0.74±0.04 0.69±0.03

1.43±0.07

0.15 0.20

89.26d ±1.00 84.20e ±1.20

9.19d ±0.10 8.84e ±0.12

0.34 0.29

1.01±0.03 0.24±0.03 0.64±0.05 0.30±0.02

1.25±0.06 0.94±0.08

NaCl

Na2CO3 0.025 99.93b ±1.41

Dry weight (mg/culture)

Growth Stevioside Rebaudioside Total SGs (stevioside + (%) A (%) rebaudioside A) (%) index (GI)

Concentration of salts (%)

0.27±0.02

10.14b ±0.14 0.49

1.34±0.18 0.23±0.17

1.57±0.16

92.46 ±1.00

9.43c ±0.10

0.79±0.08 0.18±0.01

0.97±0.09

0.075 88.13d ±1.30

9.07d ±0.13

0.32

0.78±0.07 0.12±0.01

0.90±0.08

8.71e ±0.12

0.27

0.14±0.01 0.41±0.01

0.56±0.02

0.05 0.10

c

e

83.00 ±1.20

0.37

Values followed by the same alphabet in a column are not significantly different according to Duncan’s Multiple Range Test (DMRT) at p≤ 0.05 level

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calli GI was also observed with all concentrations of Na2CO3 at 5 % level of significance (Table 1). The present result corroborates the findings of Ungar [51] who reported that growth inhibition under saline conditions is usually associated with dehydration at high salinity due to increased water stress and also resultant to loss of cell turgor pressure due to inadequate tissue osmotic adjustment. Steviol Glycoside Production from Salt-Treated Callus Culture Quantitative values of SGs obtained in callus culture treated with NaCl and Na2CO3 are presented in Fig. 1a, b. By increasing the salt concentration up to 0.10 % NaCl, the amount of SGs increased to 1.43 %, which was about 5.3 times higher than the callus culture grown without salt treatment (Fig. 1a). Results obtained in this regard are supported by Jeong and Park [24] who reported that the addition of NaCl less than 0.1 % (w/v) enhanced the ginseng saponin content, which was 1.15 times higher than control value. As with NaCl, Na2CO3 also enhances the SGs content to certain extent. However, the higher concentration of Na2CO3 decreases the total SGs but it was significantly increased over control (Fig. 1b). At 0.025 % of Na2CO3, maximum amount (1.57 %) of SGs was found, which was 5.8 times higher than the callus culture grown without salt (control). As far as the comparison among different salt concentrations is concerned at concentration (≥0.05 %) Na2CO3, the amount of SGs got decreased. In the present study, increased production of SGs (0.27 %) was seen from the callus culture grown without salts (i.e., control) which is contradictory to the results obtained by Miyagawa et al. [37], Yamazaki and Flores [52], and Swanson et al. [49], who did not find the SGs in the callus and suspension culture of Stevia. It has been reported that due to stress, plants exhibit a wide range of responses at the molecular, cellular, and whole plant levels [21]. In the present study, the production of SGs in callus might have been due to some of these responses which need further studies. However, increased production of SGs in callus culture due to stress can be explained by following the observations of [6, 47]. Shaoping et al. [47] reported that increase in stevioside content was positively related to the organization of cells and greening of the cultures, and inversely to the callus growth. They found that compact and green callus cells contained highly vacuolated and fully developed chloroplasts which had dense stroma and plastoglobuli while slow-growing, compact, but yellow calli accumulated lowest Stevioside due to the plastids contain many starch grains with few dispersed lamellae. In the present research work, we also observed a reduced callus growth and yellowish brown callus. Taking these facts into consideration, we can conclude that due to stress, callus growth stopped and secondary metabolite production increased.

0.6

SGs

0.5 0.4 0.3 0.2 0.1 0 Control 0.05 0.1 0.15 Concentrations of NaCl (%)

0.2

b 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

0.7 0.6

GI SGs

0.5 0.4 0.3 0.2 0.1 0

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Quantity of total SGs (%)

GI

Growth index (GI)

0.7

Quantity of total SGs (%)

Growth index (GI)

a

Control 0.025 0.05 0.075 0.1 Concentrations of Na 2CO3 (%)

Fig. 1 Total steviol glycoside (stevioside and rebaudioside A) content in callus culture treated with NaCl (a) and Na2CO3 (b)

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Effect of Salts on Suspension Culture and Production of SGs Suspension Culture Treated with Salts (NaCl and Na2CO3) Cell suspension cultures are mostly suitable for the studies of various physiological, biochemicals, molecular, and cellular response. Analysis of data showed that NaCl had inhibitory effect on all the growth parameters (fresh weight, dry weight, and growth index) studied. With all four concentrations (0.05, 0.10, 0.15, and 0.20 %) of NaCl tested, maximum fresh weight (532.41±1.01 mg/culture), dry weight (46.85±0.14 mg/culture), and growth index (0.89) were obtained with lower concentrations (0.05 %) of NaCl (Table 2). However, a significant difference in fresh weight was observed among all the treatments and control, no significant difference for dry weight was seen between 0.05 % NaCl and control (P≤0.05). At higher salinity, decrease in growth index might be due to hyper osmotic effect caused by NaCl which leads to plasmolysis. In the present study, culture growth declined with an increased concentration of NaCl. The retardation of culture growth might be due to the fact that a certain amount of the total energy available for tissue metabolism is utilized to resist the stress [12]. Further, in support of the above observation, Bohnert and Jensen [4] reported that NaCl decreases cell division and restrict the growth activities. Similarly with NaCl, fresh weight, dry weight, and growth index of suspension culture reduced markedly with increased concentrations of Na2CO3 (Table 2). The negative effects of alkalinity on these parameters were more prominent than that of salinity. Maximum fresh weight (517±1.14 mg/culture) was observed with lowest concentrations (0.025 %) of Na2CO3 followed by 0.05 % Na2CO3 (501±1.14 mg/culture), 0.075 % Na2CO3 (481.4±0.94 mg/ culture) and 0.10 % Na2CO3 (462.4±1.37 mg/culture). A similar trend was also observed for dry weight and growth index of suspension culture treated with different concentrations (0.025–0.10 %) of Na2CO3 after 4 weeks. At lowest concentrations, i.e., 0.025 % Na2CO3 stress, maximum dry weight content (45.80±0.19 mg/culture) and 0.85 growth index were observed. However, a significant decrease in fresh weight, dry weight, and growth index were observed at 5 % level of significance. The results obtained in this regard are corroborates with Roychoudury et al. [45] who reported that salt stress leads to suppression of plant growth and development, membrane leakage, ion imbalance or disequilibrium, enhanced lipid peroxidation, and increased production of reactive oxygen species. In addition, ionic imbalance occurs in the cells due to excessive accumulation of Na+ and Cl− which reduces the uptake of other mineral nutrients, such as K+, Ca2+, and Mn2+ [33] and affects the cell growth. A similar kind of study has been done by Ouyang et al. [39] on the suspension culture of Cistanche deserticola cultured on B5 medium supplemented with growth regulators and various concentrations (0.1–0.5 mmol/l) of sodium acetate, phenylalanine, and phenylacetic acid. They reported that sodium acetate showed a limited effect on cell growth and little changed in biomass at concentration range from 0.02 to 0.5 mmol/l, while the biomass declined sharply when the phenylacetic acid and phenylalanine concentration was ≥0.2 mmol/l. Cultivation Cycle and Steviol Glycoside Production Cell cultures have a higher rate of metabolism than intact differentiated plants because of initiation of cell growth in such type of culture leads to fast proliferation of cell mass and condensed biosynthetic cycle (about 2–4 weeks) [14]. Further, suspension cultures could be used as an important tool for biochemical production, ranging from natural coloring (pigments) to pharmaceutical products [10]. Compared to cell growth kinetics, which is

44.86d ±0.12

43.15f ±0.14

42.33g ±0.21

481.08g ±1.37

462.33h ±1.50

0.075

0.1

0.71

0.73

0.81

0.85

0.82 0.76

0.86

0.89

0.90

10th day

15th day

0.88±0.04 0.33±0.03 0.13±0.03 0.14±0.01

1.08±0.05 0.48±0.01 1.06±0.02 0.15±0.02

0.96±0.05 1.26±0.03 3.27±0.02 1.25±0.06

1.29±0.05 1.33±0.07 3.85±0.01 1.89±0.02

0.51±0.01 0.35±0.02 0.22±0.01 0.12±0.02 0.38±0.01 0.11±0.01 0.29±0.02 0.13±0.01

0.81±0.02 0.27±0.00 1.80±0.03 0.13±0.01

0.53±0.01 0.50±0.01 1.00±0.07 0.18±0.01

0.23±0.01 1.02±0.05 1.13±0.04 0.56±0.04

15th day

Rebaudioside A (%)

1.02±0.07

2.14±0.07

4.23±0.08

5.14±0.06

0.74±0.02 0.67±0.04

2.61±0.05

1.54±0.05

1.36±0.02

10th day

0.47±0.01

0.63±0.03

2.52±0.02

3.22±0.09

0.47±0.04 0.24±0.02

0.55±0.02

0.50±0.02

1.59±0.10

15th day

Total SGs (stevioside + rebaudioside A) (%)

Values followed by the same alphabet in a column are not significantly different according to Duncan’s Multiple Range Test (DMRT) at p≤ 0.05 level

45.81b ±0.20

517.08c ±1.04

501.25e ±1.14

0.025

45.35c ±0.09 43.59e ±0.21

509.08d ±1.12 487.58f ±1.01

0.15 0.2

0.05

45.99b ±0.12

518.50c ±1.14

0.1

Na2CO3

47.10a ±0.08

46.85a ±0.14

537.08a ±1.12

532.41b ±1.01

0.05

NaCl

10th day

Fresh weight (mg/culture) Dry weight (mg/culture) Growth index (GI) Stevioside (%)

Control

Concentration of salts (%)

Table 2 Effect of salts on growth parameter and steviol glycoside production from suspension culture

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usually an exponential curve, most of the secondary metabolites are produced during the plateau phase (stationary phase). It has been reported that many new enzymatic activities are absent during lag or log phases and appear during plateau phase [40]. It has been reported that the cultivation period in cell culture can be reduced by the application of elicitors, although they maintain high concentration of products [44]. An accumulation dynamics of the SGs in Stevia suspension culture (i.e., control, without salt) during its cultivation cycle is shown in Fig. 2. It can be seen that a maximal content of the SGs (about 1.59 % of plant dry weight and 5.6 times higher than control) was found on the 15th day of the cultivation cycle, i.e., at the end of the exponential growth phase and this value significantly decreased on the 20th day of the culture growth. The lack of production during the early stages can be explained by carbon distribution as carbon is mainly required for primary metabolism. On the other hand, when growth stops, carbon is no more required in large quantities for primary metabolism then secondary compounds are more actively synthesized [8, 34]. A positive correlation between the total SGs content in suspension culture and its biomass (based on dry weight) was found in accordance with the data obtained earlier by Bondarev et al. [5] with respect to SGs (stevioside and rebaudioside A). They observed the maximal content of SGs (about 115 μg/g of plant dry mass) on the 14th day of cultivation cycle, i.e., at the end of the exponential growth phase. Steviol Glycoside Production from Salts Treated Suspension Culture The quantity of SGs in suspension culture treated with salts (NaCl and Na2CO3) is shown in Table 2 and Fig. 3a, b. In case of NaCl (cultures collected at the 10th day), a maximum of 2.6 % SGs were produced (2.0 times higher than control) with 0.10 % of NaCl while at other higher concentrations of NaCl, amount of SGs got reduced. Further, it was noticed that SGs production was found lower than control (i.e., 1.59 %) from all the samples (treated with all concentrations of NaCl) collected on the 15th day (Fig. 3a). Our findings, in this regard, are supported by [15], who reported the role of NaCl in stimulation of stress-induced formation of alkaloids in suspension cultures of Coffea arabica. They observed that under stress (7.5 g l−1 NaCl) the relative alkaloid amounts generally shifted from 40 to 70 % caffeine to 80–95 %. 1.8 Dry weight

30

1.6

Total SGs ( Stev and Reb A)

25

1.4 1.2

20

1

15

0.8 0.6

10

Quantity of SGs (%)

Dry weight of suspension culture (mg/culture)

35

0.4 5

0.2 0

0 0

5

10 Days

15

20

Fig. 2 Dynamics of Stevia suspension culture growth and content of SGs (stevioside and rebaudioside A) during its cultivation cycle without salt

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SGs on 10th day

Growth index (GI)

SGs on 15th day 0.9

3

2.5 2

0.85

1.5 0.8

1

0.75

0.5 0

0.7 Control 0.05 0.1 0.15 Concentrations of NaCl (%)

0.2

Total SGs (%)

0.95

GI 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

SGs on 10th day

6

SGs on 15th day

5 4 3 2

Total SGs (%)

b

GI

Growth index (GI)

a

1 0 Control 0.025 0.05 0.075 Concentrations of Na 2CO3 (%)

0.1

Fig. 3 Total steviol glycoside (stevioside and rebaudioside A) content in suspension culture treated with NaCl (a) and Na2CO3 (b)

Similar to NaCl, Na2CO3 also showed the same trend for the production of SGs (i.e., with higher concentrations of salts SGs production decreased). It was observed that with lowest concentration (0.025 %) of Na2CO3, the maximum (5.14 %) SGs production was observed from the samples collected on 10 days (3.8 times higher than control). However, increased concentration of SGs was also obtained from the samples collected on the 15th day (Fig. 3b). Further, it was noticed that with higher concentrations of Na2CO3 (≥0.075 %) the SGs content decreased and reached below to control value (i.e., 1.36 and 1.59 % on the 10th and 15th days, respectively) with all the samples collected either on the 10th or 15th day (Table 2). The increased production of SGs might be due to the occurrence of some physiological changes due to salts stress. However, various research works regarding increased production of active components from cell culture using other chemicals as abiotic stress had been done by many scientists on other medicinal plants. Similar to present work, Lu et al. [32] studied the effect of sodium acetate on suspension culture of Saussurea medusa for the production of flavonoids. They reported that life activities of the cells were enhanced by adding precursors which leads to improving the production of secondary metabolites and found that inclusion of 0.1 mmol/l sodium acetate in the cell suspension cultures could promote the flavonoids biosynthesis on the 6th day. In addition, Ajungla et al. [1] studied the influence of various biotic (Aspergillus niger, Alternaria sps., Fusarium monoliforme, and yeast extract) and abiotic stress (salicylic acid, AlCl3, CaCl2, NaCl, and Na2SO4) on hairy root culture of Datura metel L. for the growth and production of hyoscyamine, scopolamine, and tropane alkaloid. They observed the increased accumulation of both hyoscyamine and scopolamine up to 1.5 and 1.3 times, respectively, more with 129.3 mM NaCl when compared to control. Further, an interesting result regarding rebaudioside A production in suspension culture was also observed. As Tanaka [50] reported that rebaudioside A have the most favorable sensory attributes than other major SGs such as stevioside, steviolbioside, and dulcoside A. Also, rebaudioside A is slightly astringent, less bitter, and had the least persistent aftertaste. In the present work, with various concentrations of salts tested, amount of Rebaudioside A content was found more than the Stevioside content. It was observed that 0.10 % NaCl and 0.025 % Na2CO3 enhanced the rebaudioside A content to 1.6 times (1.80 %) and 3.4 times (3.85 %) over control (Table 2). Increased production of steviol glycosides may be supported by the reports of [5, 27, 28]. They reported that formation of steviol glycosides or at least some steps of this process occurs mainly in chloroplasts. Thus, in Stevia leaf cells, these organelles were found to exhibit a high level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and, in addition, it also contains the enzyme ent-kaurenoic acid 13-hydroxylase which converts entkaurenoic acid to steviol. Further studies related to the role of chemical stress in the biosynthesis of the diterpene steviol glycoside from in vitro culture of Stevia rebaudiana are needed.

Appl Biochem Biotechnol

Conclusions From the present study, it can be concluded that optimum concentrations of both sodium salts viz; NaCl and Na2CO3 played a vital role in enhancing the steviol glycoside content in callus and suspension culture. However, Na2CO3 caused more reduction in the growth and development of callus and suspension culture and also increased production of SGs than NaCl. Further, increased production of rebaudioside A is also an important and novel finding of this research work. This protocol can be utilized for the commercial production of SGs from Stevia rebaudiana at a large scale. Stevia plant represents a new opportunity for researchers and farmers, as it is found to be the excellent source of zero calorie sweeteners with other pharmacological properties. The production of SGs (stevioside and rebaudioside A) from callus and suspension culture applying abiotic stress may solve the problem related to the availability of natural sweetener to certain extent. Applications of different chemicals/elicitors for alteration of plant secondary metabolism offer a novel approach to induce some beneficial changes in the production of photochemical, which can be exploited commercially and further research work in this area are needed. Acknowledgments The authors would like to thank the Science and Society Division (now called SEED) of Department of Science and Technology, India, and the Ministry of Human Resource Development, India for the financial support.

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