Stevia rebaudiana Bertoni seeds were brought from medicinal plant nursery of Government Cinchona Factory located at Mungpoo, Darjeeling, West Bengal.

Axenically grown cotyledonary leaves were used as explants in the tissue culture experiment. Seeds were first surface sterilized with a 0.1% (w/v) mercuric chloride solution followed by washing in distilled water several times and then soaked in distilled water for 2–3 h. Soaked seeds were kept in a filter paper inside Petri plates moistened with water for germination. Regenerated cotyledonary leaves (6–7 days old) were then thoroughly washed in sterile distilled water followed by surface sterilization in 0.1% (w/v) mercuric chloride for 2–3 min. After that, leaves were rinsed three to four times in sterile double distilled water then inoculated in culture media. Cultures were kept in culture room illuminated with white fluorescent light (2000 lux) under 16/8 h light/dark and 72% RH condition. Subculturing was routinely done after every 4 to 5 weeks of inoculation.

MS medium (Murashige and Skoog, 1962) was used as a basal medium for the in vitro culture experiment. The culture medium was provided with 3% (w/v) sucrose and gelled with 0.8–1% (w/v) agar-agar. The pH of the medium was adjusted to 5.7–5.9 by 0.1(N) NaOH. The gelled medium was poured into culture tubes, plugged with non-absorbent cotton and then autoclaved at 121 °C under 15 lb/sq. inch pressure for 15 min. MS medium supplemented with different plant growth promoters like BA and kinetin singly or in combinations were used for multiple shoot induction [18]. For rooting, regenerated microshoots were transferred into the MS medium containing different concentrations of IBA (0.25 mg/l, 0.5 mg/l, 1 mg/l and 3 mg/l) (Figs. 1–4). Moreover, for priming, the regenerated microshoots were transferred into either different concentration of CCC (0.25 mg/l, 0.5 mg/l, 1 mg/l and 3 mg/l) or in CCC + IBA (at same concentrations) supplemented MS medium (Figs. 5–8).

Well-rooted plantlets were transplanted into a 100 ml beaker filled with autoclaved soilrite-soil mixture (1:1) and kept in the culture environment till the plantlets resumed growth (Figs. 9–12). After that, the plantlets were transferred to the small pot for full growth followed by final transplantation into the garden. Survival percentage of the plantlets was recorded after 1 month of final transplantation (Figs. 13–15).

Extraction of stevioside was carried out following the method described in [19] with slight modifications. Briefly, freshly collected Stevia leaves (200 mg) were dried at room temperature (30 °C) and ground into small particle size (1 mm). Such ground leaves were taken into a 10-mL centrifuge tube and extracted three times with water in a boiling water bath set at 60 °C for 1 h. Each extract was cooled to room temperature and centrifuged (for 15 min at 5000 rpm). The supernatants were filtered through Whatman N^o 1 filter paper and transferred to a 25-mL separating funnel and partitioned against isobutyl alcohol. Then the organic extracts were taken into a pre-weighed beaker and evaporated to dryness under reduced pressure at 50 °C. Evaporated residues (1 mg) were dissolved into a known volume (2 ml) of HPLC grade water and filtered through Millipore microfilter (0.45 μm). The filtrates were then subjected to HPLC analysis.

HPLC was performed by using Water's HPLC system (USA) equipped with a 1525 binary HPLC pump, XTerra RP18 column, 4.6 × 250 mm with 5 μm internal diameter, and 2487 duel λ absorbance detector along with the integrated Empower 2 software. The mobile phase in gradient mode started with 84% acetonitrile (A) and 16% water with 0.05% w/v tri-fluroacetic acid (B). The elution changed to 70% A and 30% B within 25 min. The details of gradient elution are given in Table 1.

The flow rate was 1 ml/min and the detector was set at 210 nm. The injection volumes were 20 μL throughout the experiment. All the solvents and samples were filtered through 0.45 μm filter (Millipore) before use in HPLC. The identification and quantification of stevioside in plant samples were done by retention time, UV spectra and by comparing the peak area of the sample with that of the standard. Calibration curve for standard stevioside was constructed in the concentration range of 0.025 to 2 mg/ml.

All the experiments were carried out in triplicate. Mean, standard deviations and one-way ANOVA with Duncan's Post Hoc tests were performed to determine the significant differences between the data using SPSS version 17 for Windows. Significant differences were considered at P = 0.05.

Microshoots regenerated in MS medium supplemented with different combinations of BA and Kinetin [18] were subjected to rooting in MS containing different concentrations of IBA. For in vitro priming, microshoots (2 cm long) were transferred to the MS medium supplemented with various concentrations of CCC singly as well as in combinations with IBA. Comparing the response induced by IBA and CCC separately, it was found that the number of regenerated roots per microshoot was lower (11.66 ± 1.52 in 3 mg/l CCC, 19.33 ± 1.5 in 3 mg/l IBA) in MS with only CCC (Table 2), but length of the microshoots (4.7 ± 0.30 cm in 3 mg/l CCC, 9.8 ± 0.43 cm in 3 mg/l IBA) and intermodal distance (0.75 ± 0.05 cm in 3 mg/l CCC, 1.63 ± 0.08 cm in 3 mg/l IBA) was reduced significantly following CCC application (Figs. 1,2,16 and 17). Emergence of typical larger leaves was found (max. length 1.35 ± 0.05 cm and max. breadth 1.01 ± 0.07 cm in 3 mg/l CCC, max. length 0.73 ± 0.02 cm and max. breadth 0.51 ± 0.02 cm in 3 mg/l IBA) to develop with CCC priming (Table 2). Moreover, the fresh weight (198 ± 3 mg in 3 mg/l CCC) of the plantlets (without the weight of the roots) and their chlorophyll retaining capacity (65.33 ± 2.51 days in 3 mg/l CCC) were also enhanced significantly after CCC treatment, as compared with MS with IBA (142 ± 1.73 mg and 25.33 ± 1.53 days, respectively in 3 mg/l IBA) (Table 2).

Interestingly, friable calli were found to develop from the base of microshoots when transferred to MS medium containing CCC and IBA, (Fig. 3). These friable calli further differentiated into microshoots (Fig. 4). Data regarding the number of regenerated microshoots is not included here as the paper mainly concentrated on growth and viability of the CCC primed microshoots. Such regenerated microshoots possess characteristically reduced intermodal lengths (0.72 ± 0.03 cm in 3 mg/l IBA + 3 mg/l CCC) as compared with microshoots in MS with either IBA (1.63 ± 0.08 cm in 3 mg/l IBA) or CCC (0.75 ± 0.05 cm in 3 mg/l CCC) (Figs. 5, 6 and 7). It was also recorded that the regenerated microshoots in MS medium supplemented with both IBA and CCC produced leaves with maximum leaf surface (max. length 1.5 ± 0.12 cm and max. breadth 1.2 ± 0.1 cm in 3 mg/l IBA and 3 mg/l CCC) (Fig. 19). Rooting efficiency of these regenerated microshoots was also found to be higher as revealed by more roots per microshoots (25.33 ± 1.52 in 3 mg/l IBA and 3 mg/l CCC) in comparison to microshoots transferred to MS medium with either IBA (19.33 ± 1.5 in 3 mg/l IBA) or CCC (11.66 ± 1.52 in 3 mg/l CCC) (Table 2). Both fresh weight and chlorophyll retaining capacity (279.33 ± 1.52 mg and 71.33 ± 2.51 days, respectively in 1 mg/l IBA and 0.5 mg/l CCC) of the microshoots regenerated in MS medium containing both CCC and IBA were significantly higher in comparison to MS with either IBA (148.34 ± 2.52 mg and 28.33 ± 1.52days, respectively in 1 mg/l IBA) or CCC (169 ± 2.64 mg and 52 ± 3 days, respectively in 0.5 mg/l CCC) (Figs. 20 and 21). The highest fresh weight (329 ± 2.01 mg) and chlorophyll retaining capacity (89.34 ± 1.53 days) were noticed in MS medium containing 3 mg/l IBA and 3 mg/l CCC than either MS with 3 mg/l IBA (142 ± 1.73 mg and 25.33 ± 1.53days, respectively) or 3 mg/l CCC (198 ± 3 mg and 65.33 ± 2.51 days, respectively) (Table 2). Lastly, application of CCC was also found to increase the survival percentage (92.3 ± 3.05% in MS with 3 mg/l IBA and 3 mg/l CCC, 83 ± 2% in MS 3 mg/l CCC compared to 50.66 ± 3.51% in MS with 3 mg/l IBA) of plantlets after transplanting to soilrite-soil mixture (1:1) for hardening and subsequently to the pot (Table 3). This is clear from the results that MS in combination with CCC and IBA played a positive role to manifest all the beneficial changes, which definitely have serious commercial prospects.

The calibration curve of authentic stevioside in the concentration range of 0.025 to 2 mg/ml was found to be almost linear (r² = 0.999) (Fig. 22). The identification and quantification of stevioside content in the plant samples were carried out by comparing the retention time (RT) and peak area of sample with that of the standard. The retention times of standard stevioside (0.025 to 2 mg/ml) and S. rebaudiana leaf extracts were found to be similar respectively (Figs. 23 and 24a–24f). Peak area percentages of standard stevioside (0.025 mg/ml, 0.05 mg/ml, 0.2 mg/ml, 0.4 mg/ml and 0.6 mg/ml) were 44.68%, 56.3%, 74.02%, 86.7%, and 95.1%, respectively almost similar to 45.5%, 55.2%, 74.8%, 87.3% and 94.88% of plant extracts (Tables 4 and 5). HPLC data of standard stevioside and plant extracts proved that CCC priming had promoted the stevioside content as compared with only IBA containing sets (Fig. 25). A significantly higher (2.1 mg/g fresh weight) stevioside content was found in microshoots transferred to MS with only CCC in comparison to MS with only IBA (0.2 mg/g fresh weight) (Table 5). Interestingly, the highest stevioside accumulation (2.9 mg/g fresh weight) was recorded in microshoots grown in MS medium with IBA and CCC combinations compared to MS with either CCC or IBA (Table 5). It was revealed that stevioside production increased almost about 15 times in leaves grown in MS medium with 3 mg/l IBA and 3 mg/l CCC (2.9 mg/g fresh weight) in comparison to MS with only 3 mg/l IBA (0.2 mg/g fresh weight). However, an about 1.4 fold increased stevioside content was noticed in MS medium with 3 mg/l IBA and 3 mg/l CCC (2.9 mg/g fresh weight) as against MS with only 3 mg/l CCC (2.1 mg/g fresh weight) (Fig. 25).