Elicitation effect on the production of asiaticoside and asiatic acid in shoot, callus, and cell suspension culture of Centella asiatica

Establishment of shoot culture

In vitro grown culture of C. asiatica, accession number 347492, was collected from National Bureau of Plant Genetic Resources, New Delhi, India. The shoot cultures were grown in Murashige and Skoog (MS) media supplemented with 1 mg/l 6-benzylaminopurine (BAP) (Roy et al., 2016). Nodes were inoculated vertically into culture tubes and incubated at 26°C ± 2°C under 16-hour photoperiod and light intensity of 3,000 lux with 55%–60% relative humidity. Readings and visual data were recorded.

Establishment of callus culture

Leaf explants of C. asiatica were excised from basal shoot culture and edges of the leaves and inoculated onto solid MS medium supplemented with 1 mg/l BAP and 1.5 mg/l naphthalene acetic acid (NAA). The culture was incubated at 26°C ± 2°C under 16-hour photoperiod and light intensity of 3,000 lux with 55%–60% relative humidity. Callus growth and visual data were recorded.

Establishment of cell suspension culture and growth curve analysis

To establish cell suspension culture, approximately 2 g of fresh callus was weighed and finely chopped into small pieces and transferred to 250 ml Erlenmeyer flask containing 50 ml of liquid MS medium supplemented with 3% (w/v) of sucrose, 1 mg/l BAP, and 1.5 mg/l NAA. It was kept in a rotary shaker at 100 rpm in dark conditions at 25°C for 45 days. For growth curve analysis, 5-ml sample was retrieved using sterilized pipette at 3-day interval until the 45^th day. To measure wet cell weight, cells were filtered through nylon membrane, washed with distilled water, and weighed immediately. Dry cell weight was determined by drying the collected cells in nylon membrane at 50°C until a constant weight was attained.

Elicitation studies

To study the effect of MeJA on shoot multiplication, MS media was supplemented with 1 mg/l BAP along with different concentrations of MeJA, i.e., 50, 100, 150, 200, and 250 µM. Observations were recorded after 6 weeks of culture period. In callus culture, MS media was supplemented with 1 mg/l BAP and 1.5 mg/l NAA along with 100 µM MeJA. The cell suspension culture was supplemented with the same amount of BAP, NAA, and MeJA.

Quantitative estimation of asiaticoside and asiatic acid

Approximately, 0.5 g of air dried shoots grown in MS media with 1 mg/l BAP (control) and air dried callus grown in MS media with 1 mg/l BAP and 1.5 NAA (control) was taken. Samples were crushed to fine powder and dissolved in 5 ml of methanol:water (9:1) solution. Then, samples were sonicated for 10 minutes and after cooling, samples were filtered through polytetrafluoroethylene (PTFE) syringe filter (0.22 µM) and transferred to glass vials. Similar procedure was used for MeJA-elicited shoot and callus extracts.

For preparing cell suspension culture samples, control cultures were transferred into sterile tubes and centrifuged at 1,500 g for 15 minutes. Approximately, 0.5 g of cells was added in 5 ml of methanol:water (9:1 ratio). After sonication, samples were centrifuged at 1,500 g for 10 minutes, and supernatant was filtered through PTFE syringe filter (0.22 µM). Same procedures were followed for MeJA-treated suspension culture. Samples were stored at −20°C for further analysis. About 1 mg each of asiaticoside standard (Sigma Aldrich) and ATA standard (Sigma Aldrich) was dissolved in 1 ml of methanol:water (9:1) solution. For calibration, standards of asiaticoside and ATA were prepared at different concentrations, i.e., 1, 0.5, and 0.1 mg/ml. For HPLC analysis, C18 column was used, mobile phase was a mixture of methanol and water (70:30) for asiaticoside and (80:20) for ATA. Twenty microliters solution was injected into the column, with a flow rate of 0.5 ml/minute at 26°C. The absorbance was read at 214 nm wavelength.

Statistical analysis

In vitro experiments were performed in triplicates and HPLC analysis of samples was performed in duplicates. The data analysis was performed using one-way analysis of variance using Microsoft Excel 2007. Results were shown in mean ± standard error (M ± SE) with p ≤ 0.05 and F_stat > F_{critical value} considered as statistically significant.

Shoot, callus, and cell suspension culture

Shoot cultures grown in different concentration of MeJA (50, 100, 150, 200, and 250 µM) resulted in decreased shoot proliferation compared to control (untreated) one (MS + 1 mg/l BAP) after 6 weeks of incubation (Table 1). The highest shoot multiplication was observed in control one with a shoot number of 31 ± 1 (Fig. 1a). Among the elicitor-treated cultures, shoot cultures supplemented with 1 mg/l BAP + 100 µM MeJA had the highest shoot number of 24 ± 1 (Fig. 1b).

Callus induction from leaf explants of C. asiatica in MS media supplemented with 1 mg/l BAP and 1.5 mg/l NAA was observed (Fig. 1c). After 6 weeks of incubation, callus was green colored and compact. However, after 10 weeks of incubation, it turned brown colored. Callus culture treated with 100 μM MeJA resulted in poor callus induction and turned yellow.

For establishment of cell suspension culture, MS liquid media supplemented with 1 mg/l BAP and 1.5 mg/l NAA was used. The results after 6 weeks of incubation is shown in Figure 1d. Growth curve was determined using dry weight (Fig. 2a) and wet weight analysis (Fig. 2b) for both control and MeJA-elicited cell suspension culture for 45 days.

Table 1. Effect of MeJA concentrations on growth of Centella asiatica (accession number 347492) supplemented with 1 mg/l 6-benzylaminopurine (BAP), and different concentrations of MeJA (50, 100, 150, 200, and 250 µM). Shoot number and shoot length were recorded after 6 weeks of culture period. Analysis was done in triplicate. [Click here to view]

Figure 1. Shoot culture of Centella asiatica grown in MS media supplemented with (a) 1 mg/l BAP (control) (b) 1 mg/l BAP with 100 μM MeJA (elicited) after 6 weeks of culture period (c) Callus culture treated with 1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA after 6 weeks of culture period and (d) Cell suspension culture treated with 1 mg/l BAP + 1.5 mg/l NAA+100 µM MeJA after 6 weeks of culture period. [Click here to view]

In the present study, MeJA has an overall negative effect on shoot proliferation of C. asiatica this might be because MeJA disrupts the cortical microtubules of the plants which lead to growth inhibition, induction of leaf senescence, and promotion of ethylene production (Saniewski et al., 1987). Previous studies reported that increase concentration of MeJA inhibits growth of the whole plant and cell culture (Kim et al., 2004; Mangas et al., 2006). Bonfill et al. (2006) reported that Centella plantlet treated with MeJA showed reduction in growth with over 50% in fresh weight (aerial parts) and also showed symptoms of leaves necrosis at the end of culture period. Although treatment with MeJA resulted to growth inhibition, it was able to increase the secondary metabolites in in vitro cultures (Kim et al., 2004) as same happened in our study.

Figure 2. Growth curve of (a) dry weight and (b) wet weight for both control and MeJA-elicited cell suspension cultures treated with 1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA up to 45 days. Suspension cultures (treated and untreated) were separated from cells using centrifugation, and wet weight and dry weight were determined and plotted against number of days. [Click here to view]

In case of callus culture, change in color of callus may be due to the secretion of phenolic compounds in explant or explant tissues (Dan et al., 2000). Previous studies reported that MS medium supplemented with 1 mg/l BAP and 1 mg/l NAA resulted in strong callus induction from petiole explants of Centella (Hoang and Nguyen, 2010). MeJA completely ceased the callus growth of Taxus media var. Hatfieldii (Furmanowa et al., 1997) and callus cultures of R. cordifolia in a dose-dependent manner (Bulgakov et al., 2002).

For cell suspension culture, a usual type of lag and stationary phase were not observed in the growth curve of both control and MeJA elicited cells. Exponential phase of the cells in the control culture lasted for 39 days and reached death phase by day 42, whereas exponential phase for MeJA elicited cells lasted for 36 days and reached death phase by day 40. Growth curve for MeJA-elicited and control cells were almost similar to each other but exponential phase of elicited cells was shorter than control cells. The growth curve of control and MeJA-elicited cell suspension culture lacks a typical lag phase, and the stationary phase may be due to their shorter duration (Hoang and Nguyen, 2010). The control cells reached death phase by 42 days may be due to the nutrient depletion in the culture media (Bonfill et al., 2011). Previous studies reported that viability of cells reduced by more than half of initial population of C. asiatica suspension cells at the end of the culture period when elicited with 100 μM MeJA (Bonfill et al., 2011).

Quantitative estimation of asiaticoside and asiatic acid by HPLC

The approximate retention time for standard asiaticoside (ASD) was 8.9 minutes (Fig. 3a). It was observed that after treatment with 100 µM MeJA, there was a significant increase in asiaticoside content by 39-fold in shoot culture and about 69-fold in callus extracts (Table 2).

However, the asiaticoside content in cell suspension culture was reduced with MeJA addition. The asiaticoside content in cell suspension culture was reduced after MeJA elicitation. Chromatogram of asiaticoside content before and after elicitation in shoot culture, callus culture and cell suspension culture are shown in Figure 3b–g. In C. asiatica, βAS (β-amyrin synthase), an important enzyme in the biosynthesis of triterpene saponins gets activated when elicited with MeJA, thereby boosting up the production of asiaticoside in shoot and callus culture (Kim et al., 2005). Previous studies indicated that the highest expression of cycloartenol synthase) mRNA transcript in leaves of C. asiatica regulates phytosterol synthesis and gets decreased upon addition of MeJA to the medium (Kim et al., 2005). This explains that MeJA inhibits the formation of phytosterol and promotes the asiaticoside pathway in shoot and callus cultures. Previous studies had shown that 0.1 mM MeJA is sufficient to upregulate the levels of squalene synthase mRNA and βAS mRNA, and thus enhance the triterpene saponin content in C. asiatica (Mangas et al., 2006). Asiaticoside was reduced in cell suspension culture after MeJA treatment, maybe because the effect of MeJA is dependent on factors like elicitor’s specificity, concentration, duration of treatment, and growth phase of the culture. Previous studies reported that asiaticoside synthesis in Centella is highly tissue-specific or organ specific, occurring mainly in the leaves cultured with 100 µM MeJA elicitor (Kim et al., 2004). In certain cases, the metabolic flux of specific metabolic pathways gets manipulated in response to elicitors and signal molecules of plant defense responses (James et al., 2008). Previous studies reported that callus and cell suspension of Centella from Indian origin synthesized asiaticoside (Nath and Buragohain, 2005). So far, biotechnological attempts to over produce asiaticoside through cell or tissue culture have encountered limited success (Kim et al., 2002).

Figure 3. Chromatogram of (a) 1 mg/ml ASD standard (b) control shoot culture (MS +1 mg/l BAP) (c) elicitor treated shoot cultures (1 mg/l BAP + 100µM MeJA) (d) control callus cultures (1 mg/l BAP + 1.5 mg/l NAA) (e) elicitor elicited callus cultures (1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA) (f) control cell suspension culture (1 mg/l BAP + 1.5 mg/l NAA) (g) elicited treated cell suspension culture (1 mg/l BAP + 1.5 mg/l NAA+100 µM MeJA). [Click here to view]

Table 2. Asiaticoside estimation in different cultures of Centella asiatica treated with 1 mg/l BAP + 100 µM MeJA in case of shoot culture and 1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA in case of callus and cell suspension culture. In control cultures MeJA was not present. [Click here to view]

Figure 4. Chromatogram of (a) 1 mg/ml ATA standard (b) control shoot culture (MS + 1 mg/l BAP) (c) elicitor treated shoot cultures (1 mg/l BAP + 100 µM MeJA) (d) control callus cultures (1 mg/l BAP + 1.5 mg/l NAA) (e) elicitor elicited callus cultures (1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA) (f) control cell suspension culture (1 mg/l BAP + 1.5 mg/l NAA) (g) elicited treated cell suspension culture (1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA). [Click here to view]

Table 3. ATA estimation in different cultures of Centella asiatica treated with 1 mg/l BAP + 100 µM MeJA in case of shoot culture, and 1 mg/l BAP + 1.5 mg/l NAA + 100 µM MeJA in case of callus and cell suspension culture. In control cultures MeJA was not present. There was no evidence of presence of ATA in both the treated and untreated cell suspension culture. [Click here to view]

The retention time for standard ATA was found to be approximately at 22.6 minutes (Fig. 4a). Treatment with 100 μM MeJA resulted to 1.9-fold increase in ATA in callus extract, whereas in shoot extract ATA was reduced after elicitation (Table 3). ATA was found absent in both control and elicited culture of cell suspension culture. Chromatogram of ATA content before and after elicitation in shoot and callus culture are shown in Figure 4b–e. ATA was not detected in cell suspension culture even after the treatment with MeJA, because plant secondary metabolites are synthesized by specialized cells at particular stage of its development and certain compounds are not synthesized if cells remain undifferentiated as in cell suspension culture (Kim et al., 2002). Another important factor for regulation of secondary metabolic processes is the distribution between mRNA transcripts, enzymes, and biosynthetic products within and between cells (James et al., 2008). The callus is undifferentiated tissue which has the ability to develop into any plant organ such as root, shoot, or leaf under correct hormone concentrations and this might be the reason for presence of all the triterpenoids in callus and not in cell suspension culture (James et al., 2008). Previous studies showed that four triterpenoid, asiaticoside, ATA, madecassic acid, and madecassoside were found in the undifferentiated cultures cells, with higher concentrations in calli as compared to the cell suspensions of C. asiatica belonging to two different phenotypes from South Africa (James et al., 2008). Another problem is the instability of cell cultures for continued production of secondary products as some cell lines loss their ability to synthesize desired compound after prolonged culture (James et al., 2008). Comparative estimation of asiaticoside and ATA are shown in Tables 2 and 3 for shoot, callus, and cell suspension culture, respectively. The highest asiaticoside content was found in MeJA-elicited callus culture, whereas the highest ATA content was found in the control shoot culture.