Chemicals and reagents
Squalene, cholesterol, β-sitosterol, salicylic acid, methyl jasmonate, jasmonic acid, abscisic acid (ABA) and standard diosgenin (>99% purity) were procured from Sigma-Aldrich, USA. HPLC grade chemicals like ethanol, hydrochloric acid, chloroform and analytical grade methanol were procured from HiMedia Laboratories Pvt. Ltd. (India). Plant growth regulators such as N6-benzyladenine (BA) and Indole-3-butyric acid (IBA) were purchased from Sigma-Aldrich, India.
Plant material and culture conditions
Mother plants of D. deltoidea were collected in the month of July, 2017 from Baramulla, Kashmir located at an elevation of 1690 m. The authentication of plant species was done by the curator, submitted the specimen in KASH Herbarium, Centre for Biodiversity and Taxonomy, University of Kashmir (Voucher Specimen No. 2614-KASH).
Explant selection and sterilization
Healthy shoots obtained from mother plants were kept in greenhouse. The explants were rinsed in running tap water (10 min), followed by washing in tween-20 (10 min) and finally washed with distilled water thrice. Surface sterilization of explants was performed inside the laminar air flow chamber with 0.1 % (w/v) of mercuric chloride (HgCl2) for 3 min and then thoroughly rinsed with sterilized distilled water in order to remove the HgCl2 traces. Sterilized nodal segments were carved into proper size (1.5-2.0 cm) before inoculation.
Culture conditions and shoot initiation
Nodal segments were inoculated onto the Murashige and Skoog (1962) medium supplemented with 3 % sucrose, 0.8 % agar (w/v), BA (2.0 mg/l) and IBA (1.5 mg/l) for direct shoot organogenesis. The pH of the medium was adjusted to 5.8 with 1 N NaOH or 1 N HCl. The medium was autoclaved at 121 ºC for 15 minutes. The cultures were maintained at (25±2 °C) with photoperiod (16 h light/8 h dark) and photosynthetic photon flux (PPF) of 40-50 µmol m-2s-1 provided by cool white fluorescent tubes. After 21 days, plantlets were transferred into liquid MS medium with similar composition of PGRs for 5 weeks and then used for further process. Optimal harvest time was evaluated in terms of biomass accumulation at 7 weeks of culture in liquid media when plant biomass reached a maximum level of 1.95 g DW (6.6 g FW) and further increase or decrease in harvest time has led to reduction of biomass accumulation.
Screening of the best elicitors
To establish an efficient elicitation strategy, the four stress elicitors salicyclic acid (SA), methyl jasmonate (MeJA), jasmonic acid (JA) and abscisic acid (ABA) were used to screen high efficient elicitors by making 100 µM concentration each by dissolving them in aqueous ethanol (50%) and filter sterilize them through a syringe filter (0.22 µM). The multiple shoots (5 g FW) was allowed to grow at different elicitors concentrations of SA (100 µM), MeJa (100 µM), ABA (100 µM) and JA (100 µM) at exposure time duration (4 h) in the MS liquid media with BA (2.0 mg/l) and IBA (1.5mg/l). The multiple shoot (5 g FW) of 5th week of culture were transferred to the liquid media supplemented with BA (2.0 mg/l) and IBA (1.5mg/l) aseptically. After elicitation, the shoots were harvested on the 7th week for production of biomass and diosgenin. All sets were done in triplicates and for each trial control cultures were sustained and 50% ethanol (v/v) was used in control cultures.
Effect of elicitors on biomass and diosgenin production
SA and MeJa were used as elicitors. Stock solutions of SA and MeJa were prepared individually by dissolving them in aqueous ethanol (50% ethanol: 50% water v/v) and filter sterilize through a syringe filter (0.22 µM). The multiple shoots (5 g FW) were allowed to grow at different concentrations of SA (100 µM and 200 µM) and MeJa (100 µM and 200 µM) at different time duration (4, 8, 16 h) on 5th week of culture with MS liquid media with BA (2.0 mg/l) and IBA (1.5mg/l). After treatment of SA and MeJa in different time period the multiple shoots were immediately transferred to the liquid MS medium supplemented with BA (2.0 mg/l) and IBA (1.5mg/l) aseptically. After elicitation, the shoots were harvested on the 10th day for production of biomass and diosgenin. All sets were done in triplicates and for each trial control cultures were sustained and 50% ethanol (v/v) was used in control cultures.
Effect of precursors on biomass and diosgenin production
Squalene, β-sitosterol and cholesterol, the precursors in diosgenin pathway were used in different concentrations (100 and 200 µM) respectively. Stock solution of squalene, β-sitosterol and cholesterol were prepared in 99% ethanol and filter sterilized with 0.22 µM of syringe filter. Filter-sterilized squalene, β-sitosterol and cholesterol were added to the liquid MS medium fortified with BA (2.0 mg/l) and IBA (1.5 mg/l) aseptically on the 5th week of culture. Cultures were harvested after 5th and 10th day after the addition of precursor and were done in triplicates.
Statistical optimization of combined addition of elicitor and precursor
After the identification of the most potent elicitors and precursor, statistical optimization tools (Response Surface Analysis) was used to establish the optimum concentrations of the selected elicitors and precursors. These were then used as a combined strategy for enhancing the diosgenin and biomass yield. A 13 full factorial Central Composite Design (CCD) for independent variables was employed for optimization (Table 3). Filter-sterilized salicyclic acid and β-sitosterol were added to the liquid MS medium fortified with BA (2.0 mg/l) and IBA (1.5 mg/l) aseptically on the 5th week of culture. Cultures were harvested after 10th day. A second order polynomial model was developed based on studies of the responses of different effectors.
Fresh weight of control shoots and treated shoots were recorded after harvesting. In vitro harvested shoots were freeze-dried and lyophilized and dry weight measurement was recorded.
The shoots were pulverized into a fine powder after drying and 1 g DW of fine biomass powder of each set was macerated with aqueous ethanol (50% v/v) for 24 h at room temperature. The extract was filtered through Whatman filter paper No 1 and was dried with the help of rotary evaporator at 40 ºC. 20 ml of HCL (10%) was mixed to the dried residue and hydrolysed at 98 ºC for 1 h. After cooling, chloroform (15 ml) was added two times for washing and the collective mixture was extracted and isolated, lower layer i.e. chloroform layer was collected and other 20 ml chloroform was used to extract upper layer. Chloroform layers were combined and concentrated to dryness. An appropriate amount of methanol was added to the residue and final concentration was filtered through 0.2 µM syringe filter and preserved in refrigerator (4 °C) for further analysis. 
An Agilent 7890A gas chromatography coupled to a 5875C mass spectrometer detector (XL MSD) with triple axis and mass hunter work station software (USA) was used for the analysis of diosgenin. Chromatography was performed on DB-5: 30 m x 0.25 mm i.d. x 0.25 µM film thickness column. Helium works as a carrier gas at a flow rate of 0.5 mL/min. The Gas chromatography oven temperature was raised from 200 °C for 2 min to 280 °C for 20 min at a heating rate of 10 °C/min. The injection volume was 5 µl using split ratio (1:1). The Mass spectra were recorded in electron impact mode with ionization energy of 70 eV and scan rate of 0.5 s/scan with scan range of 50-600 amu. Inlet and transfer line temperature were set 250 0C. Component identification was achieved by Wiley, NIST libraries. Compounds were also recognized by peak enrichment on co-injection with available authentic standards. Peak area percentages were achieved electronically from the TIC response without the use of correction factors. GC-MS chromatogram of diosgenin is shown in Figure 2 (a, b).
The values were represented in triplicates as mean ± standard deviation. Statistical analyses were done by implementing analysis of variance (ANOVA) with Tukey’s test using SPSS (p < 0.05).