|Year : 2021 | Volume
| Issue : 6 | Page : 225-232
Effect of salinity stress on growth, water content, and guggulsterone production in callus cultures of Commiphora wightii (Arnott.) Bhandari
Soumi Datta1, Jatin Katna1, Dhiraj Shrivastav1, Sarvepalli Badari Narayan1, Shahid Umar2, Jatavallabhula Lakshminarayana Sastry3, Ranjan Mitra1, Sayeed Ahmad4
1 Dabur Research and Development Centre, Ghaziabad, Uttar Pradesh, India
2 Department of Botany, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India
3 National Medicinal Plant Board (NMPB), Ministry of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homeopathy (AYUSH), Government of India, New Delhi, India
4 Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, SPER, Jamia Hamdard, New Delhi, India
|Date of Submission||27-Nov-2020|
|Date of Decision||29-Dec-2020|
|Date of Acceptance||16-Feb-2021|
|Date of Web Publication||15-Sep-2021|
Department of Pharmacognosy and Phytochemistry, Bioactive Natural Product Laboratory, SPER, Jamia Hamdard, New Delhi - 110 062
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objectives: A traditional herb Commiphora wightii (Arnott.) is well known for the management of different ailments, including hypolipidemic and hypocholesterolemic disorders. Overexploitation has resulted in enlisting it under endangered species list; thus, in vitro studies are desirable to circumvent further depletion of the wild population. The objective of our study is to analyze the effect of salinity stress on growth and guggulsterone content in callus cultures. Materials and Methods: The establishment of leaf-derived calli of C. wightii and the effect of salinity stress such as calcium chloride (CaCl2), sodium chloride (NaCl), potassium chloride, and ammonium sulfate at different concentrations and time periods on callus growth and steroid biosynthesis were studied. High-performance liquid chromatography and liquid chromatography-mass spectrometry/mass spectrometry-based spectrometric systems were carried to identify the guggulsterones. Results: The 50 mg/L CaCl2-induced callus was found to have the highest content of guggulsterones after 15 months of incubation as compared to 9 and 12 months and indicated a positive correlation between callus age and guggulsterone biosynthesis. Conclusion: Salinity stress played an important role in callus growth of C. wightii, while calcium-induced calli showed in vitro synthesis of guggulsterones, and this can be further explored for the synthesis of guggulsterones in vitro on large commercial scale using bioreactor, and thus helps to conserve the endangered species in its natural habitat.
Keywords: Callus proliferation, Commiphora wightii, salinity stress, salt tolerance, tissue culture
|How to cite this article:|
Datta S, Katna J, Shrivastav D, Narayan SB, Umar S, Sastry JL, Mitra R, Ahmad S. Effect of salinity stress on growth, water content, and guggulsterone production in callus cultures of Commiphora wightii (Arnott.) Bhandari. Phcog Mag 2021;17, Suppl S2:225-32
|How to cite this URL:|
Datta S, Katna J, Shrivastav D, Narayan SB, Umar S, Sastry JL, Mitra R, Ahmad S. Effect of salinity stress on growth, water content, and guggulsterone production in callus cultures of Commiphora wightii (Arnott.) Bhandari. Phcog Mag [serial online] 2021 [cited 2022 Jun 26];17, Suppl S2:225-32. Available from: http://www.phcog.com/text.asp?2021/17/6/225/326026
- The leaf-derived, 50 mg/L calcium chloride (CaCl2)-induced callus of Commiphora wightii was found to have highest content of guggulsterone steroid in 15 months as compared to 12 and 9, indicating a strong correlation between steroid content and callus age. Further, 100 mg/L sodium chloride and 50 mg/L CaCl2-induced calli were found to have maximum relative growth rate and relative water content response. This study result suggests that salinity stress played an important role in callus growth while calcium may act as a direct elicitor of the steroid production.
Abbreviations used: ANOVA: Analysis of Variance, DW: Dry Weight, FW: Fresh Weight, FWi: Initial Weight, FWf: Final Weigh, HPLC: High Performance Liquid Chromatography, Kn: Kinetin, LC-MS: Liquid Chromatography and Mass Spectrometry, MS: Murashinge and Skoog, RGR: Relative Growth Rate, RWC: Relative Water Content, CaCl2: Calcium Chloride, HCl: Hydrochloric acid, NaCl: Sodium Chloride, KCl: Potassium Chloride, Na2SO4: Ammonium Sulphate, NaOH: Sodium Hydroxide, 2,4-D: 2,4-Dichlorophenoxyacetic acid
| Introduction|| |
Commiphora wightii, commonly called Guggulu, belongs to the Burseraceae family. It is an endangered, slow-growing halophytic dwarf tree with immense therapeutic properties, found in the arid and rocky tracts of Rajasthan and Gujarat (India). C. wightii is a commercially valuable plant primarily because of its tree exudates which comprise true gums, resins, gum resins, oleo-resins, and mucilage. The bioactivities of guggulsterones extracted exudates from Guggulu such as lipid-lowering, antimicrobial,, anti-inflammatory, hypolipidemic, and hypocholesterolemic activities,,,,, are reported which are possibly used in the treatment of a varied range of ailments. Further, Guggulu is reported as a reliever from epilepsy, ulcer, obesity, and rheumatoid arthritis.,,, Slow growth, poor seed set, faulty gum tapping method, indiscriminate wild collection, and inadequate replenishment strategies have led to overexploitation and have resulted it enlisted in the critically endangered status. Therefore, an alternate in vitro guggulsterone biosynthesis protocol and conservation strategy is important for the sustainable utilization of Guggulu. Due to the significance of guggulsterones from C. wightii, several in vitro studies have been reported using morphactin, modified media,, growth retardants, plant gums, and calcium deprivation. Scientists and biotechnologists considered plant cell, tissue, and organ cultures as an alternate to produce secondary metabolites from callus/cell suspension cultures as the chemical synthesis of secondary metabolites was achieved through field cultivation in plants where natural growth was difficult.
Several callus culture studies have reported the production of steroids such as saponins,,,,, saikosaponins, diosgenin,,,, cardenolides,,,,,,, phytosterols,,,, sterols and phenolic compounds, diosgenin and 20E, 20E-3-acetate,,,,, polypodine B,, paclitaxel,, taxoids, ecdysterone, and withanoids.,
The plants remodel their plasticity to abiotic and biotic stresses and enable accumulation of secondary metabolites as a defense response and activated by elicitors., Elicitation is a strategy widely used in in vitro cultures to enhance the production of secondary metabolites by abiotic elicitors such as physical agents (i.e., osmotic pressure, heat, cold, and ultraviolet light) and chemical agents (i.e., heavy metals, ethylene, fungicides, salts, and antibiotics). Elicitation also helps control gene expression in response to the stimulus, induce enzyme synthesis, and thus stimulate the synthesis of various secondary metabolites, such as steroids, phenylpropanoid, polypeptides, and flavonoids. Under stress, two mechanisms are activated, i.e., enzymatic and non-enzymatic. The former mechanism is governed by the enzymes, superoxide dismutase, catalase (CAT), and peroxidase, while the latter is formed by antioxidant molecules within the cell such as phenolic compounds, flavonoids, steroids, and various secondary metabolites. In plants, salinity stress may create both ionic and osmotic stress and result in an increase or decrease of particular secondary metabolites.
The establishment of an in vitro callus induction and guggulsterone biosynthesis technique for C. wightii will highly facilitate the production of the steroid to various industries and accolade conservation strategies. Since C. wightii is listed under rare and endangered category, the current natural population will be insufficient to achieve the raw material demand. Hence, the establishment of a reliable in vitro synthesis of guggulsterone is vital to acquire the steroid demand as raw materials for product development. The effect of plant growth regulators on callus growth was previously studied; however, the effect of salinity stress on callus growth in C. wightii has not yet been researched. Therefore, this investigation was aimed to study the influence of salinity stress on callus growth and to establish an efficient process of in vitro guggulsterone biosynthesis. This study will help as a mode of expediting the guggulsterone production on a bioreactor-based large scale and help to conserve the germplasm of this endangered species in its natural habitat.
| Materials and Methods|| |
Plant material and surface sterilization
The young leaves of C. wightii were collected from the field. Leaves were kept under running water for 15 min to eliminate the foreign particles/microbes. The leaves were then surface sterilized by first dipping in a soap solution (10 mL Labolene® + 5 ml cetrimide [Savlon] + few drops polyethylene glycol sorbitan monolaurate [Tween-20]) for 15 min, after which the leaves were disinfected by soaking in 1.5% (w/v) methyl N-(1H-benzimidazol-2-yl) carbamate (Bavistin) for 20 min. The leaves were rinsed thoroughly with sterile distilled water at least 2–3 times to remove all traces of Bavistin. Finally, the leaves were surface sterilized by 0.15% (w/v) mercuric chloride for 8 min and washed three times with sterile water.
Media and culture condition
The leave explants were cultured in Murashige and Skoog, MS basal medium containing 30 g/L sucrose and 100 mg/L myo-inositol supplemented with plant growth hormones 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.3 mg/L kinetin (Kn). The pH of the medium was adjusted to 5.8–5.9 using 1 N NaOH or HCl before adding 3 g/L Clarigel® (HiMedia, India). Semi-solid MS media prepared were poured in culture bottles, secured with polypropylene caps, and autoclaved at 121°C for 20 min at 15 psi.
Establishment of callus cultures
The surface sterilized leaves were excised (measuring 1 cm × 1 cm) aseptically with a sterile sharp scalpel blade to reduce injury and place onto sterilized media. The cultures were maintained under 16 h/8 h (light/dark) photoperiod at 23°C ± 2°C with a light intensity of 30 μmol/m/s using cool white fluorescent tubes; and the callus was regularly sub-cultured at 21-day intervals by transferring 0.5 g callus to a fresh medium and was maintained for 1 year. The callus cultures were maintained for 1 year 4 months on the mentioned media; and their growth and steroid formation were measured after an interval of every 3 months. The minimum amount of callus required for analysis was obtained at 9 months, followed by 12 and 15 months.
Elicitation experiment of guggulsterones with salt stress
The culture bottles containing 25 mL of standardized MS medium, added with three iso-osmotic concentrations (50, 100, and 150 mg/L) of sodium chloride (NaCl), calcium chloride (CaCl2), potassium chloride (KCl), and ammonium sulfate (Na2SO4), were inoculated with 0.5 g callus, which were fragmented as described in the previous section. Samples were taken at an interval of 9, 12, and 15 months, and the callus growth, water content, and guggulsterone content were monitored. The experiment was carried out in quintuplicate.
Harvesting for measuring responses
For culture growth and water content, the calluses were harvested and weighted on the 90th day of inoculation in all experiments, and the growth and water content were measured. Relative growth rate (RGR) was calculated using the formula (FWf − FWi)/FWi, where FWf is the final weight and FWi is the initial weight (here 500 mg) of the calli,, while relative water content (RWC) was determined using the formula = (FW − DW)/DW, where FW and DW represent fresh weight and dry weight (DW), respectively. DW was determined by drying the callus overnight at 60°C in an oven. For guggulsterone content, the calluses were harvested at intervals of 9, 12, 15 months from the day of inoculation from NaCl- and CaCl2-induced media (as KCl- and Na2SO4-induced media failed to proliferate), and the guggulsterone content was measured.
The extraction of guggulsterone from C. wightii in vitro callus was carried out following the methodology of Mathur et al., 2007. The 0.2 g of dried callus was finely crushed using pestle mortar and extracted overnight with 25 ml methanol. The methanol layer was evaporated, and the residue was extracted thrice with 2 ml of ethyl acetate. Finally, the ethyl acetate was evaporated to dryness in the sample concentrator. The crude extracts were stored at 4°C until analysis. For high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LCMS), the callus extract was dissolved in methanol (HPLC-grade) to yield a final concentration of 200 mg dried tissue per 25 ml. Samples were filtered through syringe filters (0.45 μm, 4 mm nylon filter, Axiva Sichem Pvt. Ltd., India) to remove suspended particles and transferred to autosampler vials and used for HPLC and LCMS analysis.
High-performance liquid chromatography analysis
The extracted callus was analyzed with the Waters HPLC system equipped with a separation module (Model e2695), PDA detector (Model 2998), controlled with “Empower-3” software (Waters Corporation, Milford, MA, USA) and reverse-phase column (LUNA; 250 mm × 4.6 mm C185 μm). The column oven temperature was programmed at 25°C and the column flow rate was 1 mL/min. The mobile phase consisted of 65% (v/v) acetonitrile (A) and 35% (v/v) water (B). The isocratic elution program was used and monitored at 242 nm. Standards of guggulsterone E (T17D025; Natural Remedies) and guggulsterone Z (T17D026; Natural Remedies) 2 mg/L were used as references to identify the presence of guggulsterones. The experiment was carried out in triplicates.
Liquid chromatography-mass spectrometry/mass spectrometry analysis
The extracted callus were analyzed with an Agilent 1260 Liquid Chromatography coupled with Agilent 6460 MS (LC-MS/MS) triple quad with Jetstream instrument equipped with column (2.7 μm × 2.1 EC-C18 column [100 mm; Porashell 120]) and the flow rate was 0.3 mL/min. The mobile phase consisted of 35% (v/v) water (A) and 65% (v/v) acetonitrile (B). The isocratic elution program was used. Under positive ESI mode, pure isomers of guggulsterones produced abundant protonated molecule (M + H)+ at 313.2. Dominant fragments ions at m/z 97, 109.2, and 135.2 were derived from guggulsterone [Supplementary Table 1]. Similar results were inferred by Bhatta et al., 2010. Experiments were conducted in triplets.
Standards of guggulsterone E (T17D025; Natural Remedies) and guggulsterone Z (T17D026; Natural Remedies) 2 mg/L were used as references to identify the presence of guggulsterones in HPLC and LC-MS/MS analysis. The experiment was carried out in triplicates.
The data were expressed as mean ± standard deviation (SD), and P < 0.05 was considered to be statistically significant. The mean and SD were calculated by MS Excel tools. The effect of salinity stress on growth and water content of callus was assessed by one-way ANOVA, while the correlation between (a) RGR and RWC and (b) callus age and guggulsterone content was measured by Pearson's R correlation coefficient. [Supplementary Table 2] shows the mean ± SD of the quintuplicate.
| Results|| |
Callus induction and growth
Friable callus was developed on MS medium fortified with 3 mg/L 2,4-D and 0.3 mg/L Kn (our unpublished previous work). The callus was initiated from young leaves explants after 4 weeks of inoculation; however, profuse and measurable growth was obtained after 90 days. The non-embryogenic callus was white to cream in color but subsequently turned darker with time, which may be due to the presence of phenolic substances within the cells.
Effect of salinity stress on callus growth
To optimize the concentration of salinity stress on the growth of callus, water content, and biosynthesis of guggulsterone in vitro, an experiment was designed to grow C. wightii callus cultures in three iso-osmotic concentrations (50, 100, and 150 mg/L) of NaCl, CaCl2, KCl, and Na2SO4 [Supplementary Table 2] and [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 3]a, [Figure 3]b, [Figure 3]c, and [Figure 4]a, [Figure 4]b, [Figure 4]c. Results showed that MS medium with 100 mg/L NaCl resulted in the highest growth of callus (11.76 ± 0.18 g) [Figure 1]e and, when supplemented with 50 mg/L CaCl2, exhibited second-highest callus growth (9.74 ± 0.17 g); [Figure 2]d. Callus without stress weighed 3.55 ± 0.06 g [Figure 1]a. The elicitation using KCl and Na2SO4 showed inhibition of calli growth. Our results showed that the study for rate of callus growth was significant at P < 0.05 [Supplementary Table 3]. The growth of NaCl-induced calli showed a bell-shaped curve from low to high salinity concentration (50 mg/L <100 mg/L >150 mg/L = 8.68 ± 0.03 g <11.76 ± 0.18 g >6.39 ± 0.12 g, respectively). In contrast to the observation in NaCl-induced calli, CaCl2-induced calli showed decreased growth of callus from low to high salinity concentration (50 mg/L >100 mg/L >150 mg/L = 9.74 ± 0.17 g >5.61 ± 0.36 g >3.56 ± 0.27 g, respectively). From [Supplementary Table 2], it is evident that the growth of the callus was suppressed at all concentrations of KCl and Na2SO4; the latter is more detrimental.
|Figure 1: Callus proliferated from the tender leaves of Commiphora wightii cultured on MS basal with 3 mg/L 2,4-dichlorophenoxyacetic acid + 0.3 mg/L kinetin within 30 days. (a) Untreated (control), (b) sodium chloride (50 mg/L), (c) sodium chloride (100 mg/L), (d) sodium chloride (150 mg/L), and (e) sodium chloride (100 ml/L) within 90 days|
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|Figure 2: Callus proliferated from the tender leaves of Commiphora wightii cultured on MS basal with 3 mg/L 2,4-dichlorophenoxyacetic acid + 0.3 mg/L kinetin within 30 days. (a) Calcium chloride (50 mg/L), (b) calcium chloride (100 mg/L), (c) calcium chloride (150 mg/L), and (d) calcium chloride (50 mg/L) within 90 days|
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|Figure 3: Callus proliferated from the tender leaves of Commiphora wightii cultured on MS basal with 3 mg/L 2,4-dichlorophenoxyacetic acid + 0.3 mg/L kinetin within 90 days. (a) Potassium chloride (50 mg/L), (b) potassium chloride (100 mg/L), and (c) potassium chloride (150 mg/L)|
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|Figure 4: Callus proliferated from the tender leaves of Commiphora wightii cultured on MS basal with 3 mg/L 2,4-dichlorophenoxyacetic acid + 0.3 mg/L kinetin within 90 days. (a) Ammonium sulfate (50 mg/L), (b) ammonium sulfate (100 mg/L), and (c) ammonium sulfate (150 mg/L)|
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Effect of salinity stress on the water content in callus
Contrasting cellular responses, it is to note that the total water content in callus followed a reverse trend to callus growth. Results indicated that MS medium fortified with 50 mg/L Na2SO4 showed the highest water content [11.66 ± 0.52 g; [Figure 4]a], while 100 mg/L NaCl the lowest (9.22 ± 0.01 g) [Figure 1]e and followed by 50 mg/L CaCl2 [9.28 ± 0.09 g; [Figure 2]d]. Our study was significant at P < 0.05 [Supplementary Table 4], and also, Pearson's correlation coefficient R resulted in a strong negative correlation (−0.861) and supported the fact that the growth of callus is indirectly related to its water content.
Effect of salinity stress on guggulsterones production
Column chemistry, solvent type, solvent strength (volume fraction of organic solvent[s] in the mobile phase and pH of the buffer solution), detection wavelength, and flow rate were diverse to regulate the chromatographic conditions giving the best separation. The mobile phase conditions were adjusted to avoid interference from solvent and other compounds. For HPLC analysis, primarily, many mobile phases were tried in attempts to obtain the best separation and resolution between guggulsterones E and Z; however, the optimized chromatographic conditions for an adequate resolution were obtained by using the mobile phase consisting of isocratic elution of acetonitrile (65% [v/v]) and water (35% [v/v]). Under this system, the chromatogram of guggulsterones E and Z from C. wightii extract is shown in [Figure 5]a, [Figure 5]b, [Figure 5]c. The retention times for guggulsterones E and Z were 9.046, 8.76 and 8.75 min and 11.50, 11.077 and 11.08 min at 9, 12, and 15 months, respectively. Our results indicated that MS medium fortified with 50 mg/L CaCl2 showed the presence of guggulsterones by HPLC analysis, when measured (in ppm) at an interval of 9, 12, and 15 months of incubation, and the highest was recorded at 15 months [Supplementary Table 5]. Furthermore, an LC-MS experiment was performed to confirm the study [Figure 6]a, [Figure 6]b, [Figure 6]c.
|Figure 5: High-performance liquid chromatography profile of callus grown on 50 mg/L calcium chloride induced media showing the presence of guggulsterone (E and Z) at (a) 9 months; (b) 12 months, and (c) 15 months|
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|Figure 6: Liquid chromatography-mass spectrometry/mass spectrometry profile of callus grown on 50 mg/L calcium chloride induced media showing the presence of guggulsterone (E and Z) at (a) 9 months; (b) 12 months, and (c) 15 months|
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The increase of basal CaCl2 concentration by 18% promoted the accumulation of guggulsterone (E and Z) when kept for 15 months. The results suggest that 50 mg/L CaCl2 is an effective stress inducer and higher calcium is required for guggulsterone production in in vitro callus cultures of C. wightii. Our data suggest a strong positive correlation (Pearson's R coefficient is 0.962) between the age of the callus and the guggulsterone content.
| Discussion|| |
The occurrence of phenolic substances within the cells, amassed in the cytoplasm, undergoes oxidation and polymerization and oxidized products appear brown in the callus. Our result corroborates with the findings that the osmotic stress tolerance in intact plants is not necessarily matched by tolerance exhibited by callus, as C. wightii in natural habitat preferred arid climate and sandy loam soil with alkaline pH (7.5–9); however, at in vitro level, the pH optimum for growth of the callus was observed to be subacidic (5.8–5.9). Our study also suggested that salinity stress affects the growth of the callus; similar results were reported in Cicer arietinum and suggested that NaCl-stressed media increase callus weight.
It is observed that when the salt concentration is increased beyond a threshold level, the growth of the calli progressively decreases and ultimately affects the size of most plant species; NaCl-induced calli showed declined growth of callus at a higher concentration in rice species, like indica rice cultivars Pusa Basmati 1 and Basmati 370; and also in soybean; while in Populus euphratica, calli reported maximum growth at 50 mM NaCl and reduced at 150 and 250 mM. Similar results were recorded in the present study as, with increase of salt concentration, RGR reduced [Supplementary Table 5] for NaCl and CaCl2 salts. The reduced weight with increased salinity could be attributed to either due to ion toxicity or low exterior osmotic potential or reduced P, K, and Fe and increased Na, Mn, Cu, and Zn uptake, and thus, increased NaCl in the culture media may indicate enhanced sodium uptake and translocation. Thus, our results corroborated previous findings that there is a direct relationship between salt tolerance and callus induction capacity. Our results suggest that the callus growth and water content are indirectly related; and are in agreement with the previous observations recorded in Sugarcane variety cvs. R570 where mannitol-induced calli showed the highest growth of the callus and the lowest water content.
Secondary metabolites are accumulated in low concentration in plant cell cultures but could be enhanced by elicitation. Tissue differentiation, growth, and development in plants determine the site and accumulation of secondary metabolites in plant cell and tissue cultures. Therefore, the reaction to specific stress may vary from plant to plant and different cell lines; thus, it becomes critical to determine suitable concentrations of elicitors for product optimization. The stress inducers/elicitors have different mechanisms of elicitations and, when used in combination, synergistically enhance secondary metabolite production in vitro cultures.
Our study showed that the increase of basal CaCl2 concentration by 18% promoted the accumulation of guggulsterone (E and Z) when kept for 15 months; contrasting cellular responses with the same elicitor, calcium deprivation was found to be potent for guggulsterone production. This result suggests that calcium plays a role in guggulsterone production, but the effect is dose dependent. Our results corroborated with the previous observations of calcium involvement in the production of anthocyanin in Vitis vinifera cells, callose in liquid culture of Glycine max, cholecalciferol in Solanum malacoxylon, risithin in Solanum tuberosum tubers, sanguinarine and chelerythrine in liquid culture of Sanguinaria canadensis, and anthraquinones, chrysophanol, and emodin in Rhubarb cells.
Calcium acts as a secondary messenger in metabolic activities such as cell division and increases accumulation of secondary metabolites. Elicitors of latter biosynthesis result in a transient increase in cytosolic calcium level,,,, which are strongly linked to increased oxygen species (known to act as a second messenger). The calcium transient is controlled by overall downstream mechanisms, especially gene expression.,,,, A change in cytosolic calcium represents a signal which either directly or via calcium-binding proteins regulates the activities of the targeted enzymes; therefore, it may be inferred that instead of calcium channels, calcium-dependent enzymes are affected by calcium enhancement and thus resulted in enhanced guggulsterone synthesis.
It has been reported through in vitro and in vivo studies that the accumulation of secondary metabolites is related to the age of callus/plant as at a later stage of the growth cycle, the growth of the callus slows down or reaches a plateau and results in more production of secondary metabolites, example, in Morinda citrifolia and cryptotanshinone production in Salvia miltiorrhiza. Agarwal and Kamal, 2007 reported maximum flavonoids content in 6-week-old callus (2.90 mg/g DW) while the minimum was 2 weeks old (1.83 mg/g DW). Therefore, our results concur with previous observations and suggest that there exists a direct correlation between the age of the plant/callus and the accumulation of secondary metabolites. Interestingly, our studies indicated that NaCl-induced calli failed to promote guggulsterone production. This effect was also observed by Hagimori et al., 1983, in which Ca2+ but not Cl− was responsible for digitoxin production in Digitalis purpurea L.
The results suggest that salinity stress affects the growth of the callus and production of guggulsterone in vitro callus cultures of C. wightii. The elicitor, CaCl2 at a concentration of 50 mg/L may cause high-stress conditions in callus cultures and triggers differential changes and promotes the biosynthesis of guggulsterone. The present result also suggests a strong positive correlation between the age of the callus and the accumulation of guggulsterone.
| Conclusion|| |
In this study, the effect of salinity stress on the growth of the callus and production of guggulsterone in vitro callus cultures of C. wightii was investigated, which was favored by the calcium elicitation. The results indicated that 100 mg/L NaCl was most suitable for the growth of callus; therefore, it may be concluded that C. wightii is a salt-tolerant medicinal plant that may be intolerant to salinity stress above a concentration of 100 mg/L NaCl in in vitro system. The study also reported that the accumulation of guggulsterone in the callus cultures was enhanced under stress condition, primarily by CaCl2 at a concentration of 50 mg/L incubated for 15 months, thus suggesting a positive correlation between the callus age and guggulsterone produced. The positive effect of calcium on the biosynthesis of guggulsterone in C. wightii may be due to direct elicitation of the steroid production or deviation of the biosynthetic pathway and stimulation of any other metabolic mechanisms ensuing indirectly in the enhancement of guggulsterone production; however, further investigations are required to assess the gamut of this physiological and biological basis of ion effects on stressed cell metabolism, before any irrefutable conclusion regarding the precise nature of salt-tolerant mechanisms.
Due to the increasing demand for therapeutically important plant C. wightii in pharmaceutical and nutraceutical industries, this study will be a beneficial basis for large-scale production of guggulsterone and germplasm conservation and also encouraged studies on other stimuli such as light, injury, and temperature to improve the phytochemical contents.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Dev S. Chemistry of resinous exudates of some Indian trees. Proceedings of the National Academy of Sciences of the United States of America. 49A 1983;(30):359-385.
Kasera PK, Mohammed S. Critically threatened plants of the Thar desert, India – Their utilization and conservation strategies. J Econ Taxon Bot 2007;31:579-83.
Tuchila C, Jianu I, Rujescu CI, Butur M, Ahmadi-Khoie M, Negrea I. Evaluation of the antimicrobial activity of some plant extracts used as food additives. J Food Agric Environ 2008;6:68-70.
Srivastava M, Nityanand S, Kapoor NK. Effect of hypocholesterolaemic agents of plant origin on catecholamine biosynthesis in normal and cholesterol fed rabbits. J Biosci 1984;6:277-82.
Mehta VL, Malhotra CL, Kalrah NS. The effects of various fractions of gum Guggul on experimentally produced hypercholestraemia in chicks. Indian J Physiol Pharmacol 1968;12:91-5.
Satyavati GV, Raghunathan K, Prasad DN, Rathor RS. Commiphora mukul
Engl. and Tinospora cordifolia
Wild – A study of the anti-inflammatory activity. Rheumatism 1969b; 4:141.
Malhotra CL, Agarwal YK, Mehta VL, Prasad S. The effect of various fractions of Gum Guggul one experimentally produced hypercholesterolemia in chicks. Indian J Med Res 1970;58:394-5.
Nityanand S, Kapoor NK. Hypocholesterolemic effect of Commiphora mukul
resin (guggal). Indian J Exp Biol 1971;9:376-7.
Satyavati GV. A promising hypolipidemic agent from gum Guggul (Commiphora wightii
). Econ Med Plant Res 1991;5:47-82.
Nohr LA, Rasmussen LB, Straand J. Resin from the mukul myrrh tree, Guggul, can it be used for treating hypercholesterolemia? A randomized, controlled study. Complement Ther Med 2009;17:16-22.
Arora RB, Kapoor V, Gupta SK, Sharma RC. Isolation of a crystalline steroidal compound from Commiphora mukul
& its anti-inflammatory activity. Indian J Exp Biol 1971;9:403-4.
Satyavati GV, Dwarakanath C, Tripathi SN. Experimental studies on the hypocholesterolaemic effect of Commiphora mukul
Engl. (Guggul). Indian J Med Res 1969a; 57:1950-62.
Santhakumari G, Gujral ML, Sareen K. Further studies on the anti-arthritic and anti-inflammatory activities of gum Guggul. Indian J Physiol Pharmacol 1964;8:36.
Gujral ML, Sareen K, Tangri KK, Amma MK, Roy AK. Antiarthritic and anti-inflammatory activity of gum Guggul (Balsamodendron mukul
Hook). Indian J Physiol Pharmacol 1960;4:267-73.
Tajuddin Agarwal SK, Agarwal SK, Tyagi BR, Ram M, Dwivedi S, Kumar S. Development of cultivar marusudha of Guggul (Commiphora wightii
). J Med Aromat Plant Sci 1997;19:1043-4.
Tanwar YS, Mathur M, Ramawat KG. Morphactin influences guggulusterone production in callus cultures of Commiphora wightii
. Plant Growth Regul 2007;51:93-8.
Mathur M, Jain AK, Dass S, Ramawat KG. Optimization of guggulusterone production in callus cultures of Commiphora wightii
(Arnott.) Bhandari. Indian J Biotechnol 2007;6:525-31.
Mishra SK, Kumar A. Biosynthesis of guggulsterone in the callus culture of Commiphora wightii
. Arnott. Bhandari (Burseraceae
). Res J Pharm Biol Chem Sci 2010;1:35-41.
Suthar S, Ramawat KG. Growth retardants stimulate guggulusterones production in the presence of fungal elicitor in fed-batch cultures of Commiphora wightii
. Plant Biotechnol Rep 2009;4:9-13.
Dass S, Ramawat KG. Elicitation of guggulusterones production in cell cultures of Commiphora wightii
by plant gums. Plant Cell Tissue Org 2009;96:349-53.
Dass S, Ramawat KG. Calcium deprivation markedly enhances guggulusterone accumulation in cell cultures of Commiphora wightii
. Curr Sci 2008;96:1022-4.
Smetanska I. Production of secondary metabolites using plant cell cultures. In: Stahl U, Donalies UE, Nevoigt E, editors. Food Biotechnology. Advances in Biochemical Engineering/Biotechnology. Berlin: Springer, Verlag; 2008. p. 187-228.
Bourgaud F, Gravot A, Milesi S, Gontier E. Production of plant secondary metabolites: A historical perspective. Plant Sci 2001;161:839-51.
Andrijany VS, Indrayanto G, Soehono LD. Simultaneous effect of calcium, magnesium, copper and cobalt on sapogenin steroids content in callus cultures of agave amaniensis. Plant Cell Tissue Org 1999;55:103-8.
Pilgrim H. Sapogenin formation in tissue cultures of Digitalis purpurea L. Pharmaize 1972a; 27:121-2.
Furuya T, Yoshikawa T, Orihara Y, Oda H. Studies of the culture conditions for Panax ginseng
cells in jar fermentors. J Nat Prod 1984;47:70-5.
Desbène S, Hanquet B, Shoyama Y, Wagner H, Lacaille-Dubois MA. Biologically active triterpene saponins from callus tissue of Polygala amarella
. J Nat Prod 1999;62:923-6.
Oncina R, Delrio JA, Gomez P, Ortuno A. Effect of ethylene on diosgenin accumulation in callus culture of Trigonella foenum-graecum
L. Food Chem 2002;76:475-9.
Wang PJ, Huang CI. Production of saikosaponins by callus and re-differentiated organs of Bupleurum falcatum
L. In: Fujiwara A, editor. Plant Tissue Culture. Maruzen. Tokyo: 1982. p. 71-2.
Punyarani K, Sharma JG. Micropropagation of Costus speciosus
(Koen.) Sm. using nodal segment culture. Not Sci Biol 2010;2:58-62.
Singh I, Kumar GY, Vimala Y. Detection and isolation of diosgenin from Costus speciosus
callus raised from non-germinal seeds. IJCLS 2010;2:1240-2.
Solouki M, Hoshyar H, Ramroudi M, Tavassoli A. Comparison and evaluation of steroid alkaloid solasodine on in vivo
and in vitro
cultures of Solanum surattense
Burm L. Afr J Microbiol Res 2011;5:3810-4.
Khatodia S, Biswas K, Bhatotia K. Induction and establishment of hairy root culture of Solanum xanthocarpum
using Agrobacterium rhizogenes
. J Pharm Biosci 2013;1:59-63.
Hagimori M, Matsumotto T, Kisaki T. Determination of digitoxin and digoxin contents in first and second passage calli and organ re-differentiating calli of several Digitalis
species by radioimmunoassay. Plant Cell Physiol 1980;21:1391-404.
Kartnig TP, Kobosil P. On the separation of Digitalis
cardenolides with the aid of high-performance thin-layer chromatography. J Chromatogr 1977;138:238-42.
Lui JH, Staba EJ. Effect of precursor of serially propagated Digitalis lanata
leaf and root cultures. Phytochemistry 1979;18:1913-6.
Rücker W, Jentzsch K, Wichtl M. Root differentiation and glycoside formation in tissues of Digitalis purpurea
L. cultured in vitro
. Z Pflanzenphysiol 1976;80:323-35.
Kartnig T, Russheim U, Maunz B. Incidence and biosynthesis of cardenolides in tissue cultures of Digitalis purpurea
and Digitalis lanata
. 1. Cardenolides in surface-cultures of cotyledones and leaves of Digitalis purpurea
. Planta Med 1976;29:275-82.
Kartnig TH. Cardiac glycosides in cell cultures of Digitalis
. In: Barz W, Reinhard E, Zenk MF, editors. Plant Tissue Culture and Its Bio-Technological Application. Berlin, Heidelberg, New York: Springer; 1977. p. 44-51.
Wichtl M, Jentzsch K, Rücker W. Growth and glycoside formation in callus cultures and tissues of various organs of Digitalis purpurea
L. Pharmazie 1978;33:229-33.
Helmdold H, Voelter W, Reinhard E. Sterols in cell cultures of Digitalis lanata
. Plant Med 1978a; 33:185-7.
Helmdold H, Wahl J, Reinhard E. Attempts to increase the hydroxylation of β-methyldigitoxin by cell cultSterane derivatives in tissue cultures of Digitalis purpurea. Plant Med 1978b; 33:280.
Gurny L, Kapetanidis I. Sterane derivatives in tissue cultures of Digitalis purpurea. Planta Med 1978;33:286-7.
Gurny L, Tabacchi R, Baud C, Kapetanidis I. Callus cultures of Digitalis purprea L. Part 2: Study of growth by analysis of variance. Pharm Acta Helv 1981;56:49-54.
Venkateswara R, Sankara Rao S, Vaidyanathan CS. Phytochemical constituents of cultured cells of Eucalyptus tereticornis
SM. Plant Cell Rep 1986;5:231-3.
Shriram V, Shitole MG. Indirect organogenesis and plant regeneration in Helicteres isora
L. an important medicinal plant. In Vitro
Cell Dev Biol- Plant 2008;44:186-93.
Deshpande HA, Bhalsing SR. Isolation and characterization of diosgenin from in vitro
cultured tissues of Helicteres isora
L. Physiol Mol Biol Plants 2014;20:89-94.
Kumar A. Improvement of fenugreek through breeding approaches and in vitro
applications. The American Journal of Social Issues and Humanities 2014;3:120-7.
Nikam TD, Ebrahimi MA, Patil VA. Embryogenic callus culture of Tribulus terrestris
L. a potential source of harmaline, harmine and diosgenin. Plant Biotechnol Rep 2009;3:243-50.
Sharareh R. Assessment of diosgenin production by Trigonella foenum-graecum
L. in vitro
conditions. Am J Plant Physiol 2011;6:261-8.
Corio-Costet MF, Chapuis L, Mouillet JF, Delbecque JP. Sterol and ecdysteroid profiles of Serratula tinctoria
(L.): Plant and cell cultures producing steroids. Insect Biochem Mol Biol 1993a; 23:175-80.
Corio-Costet MF, Chapuis L, Delbecque JP. Serratula tinctoria
L. (Dyer's Savory): In vitro
culture and the production of ecdysteroids and other secondary metabolites. In: Bajaj YP, editor. Biotechnology in Agriculture and Forestry. Medicinal and Aromatic Plants IX. Vol. 37. Berlin: Springer; 1996. p. 384-401.
Fett-Neto AG, Melanson SJ, Sakata K, DiCosmo F. Improved growth and taxol yield in developing calli of Taxus cuspidata
by medium composition modification. Biotechnology 1993;11:731-4.
Fett-Neto AG, Stewart JM, Nicholson SA, Pennington JJ, DiCosmo F. Improved taxol yield by aromatic carboxylic acid and amino acid feeding to cell cultures of T. cuspida
. Biotechnol Bioeng 1994;(44):967-71.
Parc G, Canaguier A, Landre P, Hocquemiller R, Chriqui D, Meyer M. Production of taxoids with biological activity by plants and callus cultures from selected Taxus genotypes. Phytochemistry 2002;59:725-30.
Ravishankar GA, Mehta AR. Control of ecdysterone biogenesis in tissue cultures of Trianthema portulacastrum
. J Nat Prod 1979;42:152-8.
Sabir F, Sangwan NS, Chaurasiya ND, Misra LN, Sangwan RS. In vitro
withanolide production by Withania somnifera
L. cultures. Z Naturforsch C J Biosci 2008;63:409-12.
Sivanandhan G, Selvaraj N, Ganapathi A, Manickavasagam M. Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera
(L.) Dunal in shake-flask culture and bioreactor. PLoS One 2014;9:e104005.
Badea C, Basu SK. Impact of Drought on Plant Proteome and Metabolome. In: Proceedings of the UGC State Level Seminar on Emerging Trends in Contemporary Education: Implications for 21st
Century; Howrah, India: 2010. p. 104-20.
Zhao J, Davis LC, Verpoorte R. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 2005;23:283-333.
Efferth T. Biotechnology applications of plant callus cultures. Engineering 2019;5:50-9.
Fritz VA, Justen VL, Bode AM, Schuster T, Wang M. Glucosinolate enhancement in cabbage induced by jasmonic acid application. Hort Sci 2010;45:1188-91.
Kumar A. Plant Genetic Transformation and Molecular Markers. Jaipur: Pointer Publishers; 2010.
Alvarado-Orea IA, Paniagua-Vega D, Capataz-Tafur J, Toress-Lopez A, Vera-Reyes I, Garcia-Lopez E, et al
. Photoperiod and elicitors increase steviol glycosides, phenolics and flavonoid contents in root cultures of Stevia rebaudiana
. In Vitro
Cell Dev Biol Plant 2020;56:298-306.
Ramos-Valdivia AC, Huerta-Heredia AA, Trejo Tapia G, Cerda-García-Rojas CM. Secondary metabolites as noenzymatic plant protectors from oxidative stress. In: Anjum NA, Umar S, Ahmad A, editors. Oxidative Stress in Plants Causes, Consequences and Tolerance. Ch. 14. New Delhi, India: I.K. International Publishing House Pvt. Ltd.; 2012. p. 414-33.
Prajapati S, Kumar A, Parmar AK, Kant T. Growth optimization of Commipora wightii
callus – A potential source of in vitro
guggulusterone production. J Phytol Res 2010;23:35-40.
Georgiev MI, Weber J, Maciuk A. Bioprocessing of plant cell cultures for mass production of targeted compounds. Appl Microbiol Biotechnol 2009;83:809-23.
Murashige T, Skoog FA. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 1962;15:473-97.
Lutts S, Almansouri M, Kinet JM. Salinity and water stress have contrasting effects on the relationship between growth and cell viability during and after stress exposure in durum wheat callus. Plant Sci 2004;167:9-18.
Errabbi T, Gandonou CB, Essalmani H, Abrini J, Idaoma RM, Senhaji NS. Effects of NaCl and mannitol induced stress on sugarcane (Saccharum
sp.) callus cultures. Acta Physiol Plant 2007;29:95-102.
Resin G. Guggulu Resin. Indian Pharmacopeia 2018;3:3756-97.
Bhatta RS, Kumar D, Chhonker YS, Jain GK. Simultaneous estimation of E- and Z-isomers of guggulusterone in rabbit plasma using liquid chromatography tandem mass spectrometry and its application to pharmaco kinetic study. Biomed Chromatogr 2011;25:1054-60.
Lukas AM, Christoper DG, Rebecca AS, Virgnia W. AN9-a petunia glutathione s-transferase required for anthocyanin sequestration, is a flavonoid binding protein. Plant Physiol 2000;123:1561-70.
Smith MK, McComb JA. Effect of NaCl on the growth of whole plants and their corresponding callus cultures. Aust J Plant Physiol 1981;8:267-75.
Pandey R, Ganapathy PS. The proline enigma: NaCl-tolerant and NaCl-sensitive callus lines of Cicer arietinum
. Plant Sci 1985;40:13-7.
Garg BK, Gupta IC. Saline Wastelands Environment and Plant Growth. Jodhpur, India: Scientific Publishers; 1997. p. 287.
Reddy PJ, Vaidyanath K. In vitro
characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa
L.). Theor Appl Genet 1986;71:757-60.
Shankhdhar D, Shankhdhar SC, Mani SC, Pant RC. In vitro
selection for salt-tolerance in rice. Biol Plant 2000;43:477-80.
Vaziri A, Ali-Reza A, Hamid F, Franceva B, Hossain S. The effect of different concentrations of NaCl on the callus induction and lipids of soybean. Plant Tissue Cult 2004;14:75-7.
Zhang JZ, Creelman RA, Zhu JK. From laboratory to field. Using information from Arabidopsis to engineer salt, cold, and drought tolerance in crops. Plant Physiol 2004;135:615-21.
Munns R, Termeat M. Whole plant responses to salinity. Aust J Plant Physiol 1986;13:142-60.
Safwan MS, Shibli RA, Mohammad MM. Influence of sodium chloride salt stress on growth and nutrient acquisition of sour orange in vitro
. J Plant Nutr 2003;26:985-96.
Gandi S, Rao K, Chodisetti B, Giri A. Elicitation of andrographolide in the suspension cultures of Andrographis paniculata. Appl Biochem Biotechnol 2012;168:1729-38.
Collin HA. Secondary product formation in tissue culture. Plant Growth Regul 2001;34:119-34.
Shinde AN, Malpathak N, Fulzele DP. Optimised production of isoflavones in cell cultures of Psoralea coryifolia
L. using elicitation and precursor and precursor feeding. Biotech Bioproc Eng 2009;14:612-8.
Zhao J, Hu Q, Zhu WH. Enhanced cartharmine production in Carthamus
roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors. Enzyme Microb Technol 2001;28:673-81.
Vitrac X, Larronde F, Krisa S, Decendit A, Deffieux G, Mérillon JM. Sugar sensing and Ca2+-calmodulin requirement in Vitis vinifera
cells producing anthocyanins. Phytochemistry 2000;53:659-65.
Kohle H, Jeblick W, Poten F, Blaschek W, Kauss H. Chitosan-elicited callose synthesis in Soybean cells as a Ca2+
dependent process. Plant Physiol 1985;77:544-51.
Aburjai T, Bernasconi S, Manzocchi LA, Pelizzoni F. Effect of calcium and cell immobilization on the production of choleocalciferol and its derivatives by Solanum malacoxylon cell cultures. Phytochemistry 1997;46:1015- 8.
Zook MN, Rush JS, Kuć JA. A role for Ca2+
in the elicitation of rishitin and lubimin accumulation in potato tuber tissue. Plant Physiol 1987;84:520-25.
Mahady GB, Beecher CW. Elicitor-stimulated benzophenanthridine alkaloid biosynthesis in bloodroot suspension cultures is mediated by calcium. Phytochemistry 1994;37:415-419.
Kurosaki F, Nagase H, Nishi A. Stimulation of anthraquinone production I in rhubarb tissue culture by an ethylene generating reagent. Phytochemistry 1992;31:2735-38.
Bush DS. Calcium regulation in plant cells and its role in signalling. Ann Rev Plant Physiol 1995;46:95-122.
Ohler U, Niemann H. Identification and analysis of eukaryotic promoters: Recent computational approaches. Trends Genet 2001;17:56-60.
Knight MR, Campbell AK, Smith SM, Trewavas AJ. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 1991;352:524-6.
Knight MR, Smith SM, Trewavas AJ. Wind-induced plant motion immediately increases cytosolic calcium. Proc Natl Acad Sci U S A 1992;89:4967-71.
Sheen J. Ca2+-dependent protein kinases and stress signal transduction in plants. Science 1996;274:1900-2.
Knight H, Trewavas AJ, Knight MR. Cold calcium signalling in Arabidopsis
involved two cellular pools and a change in calcium signature after acclimation. Plant Cell 1996;8:489-503.
Knight H, Trewavas AJ, Knight MR. Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J 1997;12:1067-78.
Polisensky DH, Braam J. Cold-shock regulation of the Arabidopsis TCH genes and the effects of modulating intracellular calcium levels. Plant Physiol 1996;111:1271-9.
Clayton H, Knight MR, Knight H, McAinsh MR, Hetherington AM. Dissection of the ozone-induced calcium signature. Plant J 1999;17:575-9.
Moreno-Valenzuela OA, Minero-García Y, Chan W, Mayer-Geraldo E, Carbajal E, Loyola-Vargas VM. Increase in the indole alkaloid production and its excretion into the culture medium by calcium antagonists in Catharanthus roseus hairy roots. Biotechnol Lett 2003;25:1345-9.
Jan T, Naqvi B, Qadri R, Nisar M. Effect of age of cultures and hormones on the synthesis of secondary metabolites from callus of Salvia santolinifolia
(boiss), a medicinal herb. Eur J Biotechnol Biosci 2015;3:19-22.
Hagendoorn MJ, Jamar DC, Meykamp B, Plas LH. Cell division versus secondary metabolites production in Morinda citrifolia
cell suspension culture. J Plant Physiol 1997;150:325-30.
Tsay HS, Agrawal DC. Tissue culture technology of Chinese medicinal plant resources in Taiwan and their sustainable utilization. Int J Appl Eng Res 2005;3:215-23.
Agarwal M, Kamal R. Studies on flavonoid production using in vitro
cultures of Momordica charantia
L. Indian J Biotechnol 2007;6:277-9.
Hagimori M, Matsumoto T, Obi Y. Effects of mineral salts, initial pH and precursors on digitoxin formation by shoot-forming cultures of Digitalis purpurea
L. grown in liquid media. Agric Biol Chem 1983;47:565-71.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]