Applied Biochemistry and Biotechnology

, Volume 168, Issue 3, pp 681–696 | Cite as

Optimization of Elicitation Conditions with Methyl Jasmonate and Salicylic Acid to Improve the Productivity of Withanolides in the Adventitious Root Culture of Withania somnifera (L.) Dunal

  • Ganeshan Sivanandhan
  • Muthukrishnan Arun
  • Subramanian Mayavan
  • Manoharan Rajesh
  • Murugaraj Jeyaraj
  • Gnanajothi Kapil Dev
  • Markandan Manickavasagam
  • Natesan Selvaraj
  • Andy GanapathiEmail author


Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70 g FW) on 30-day-old adventitious root cultures with 150 μM SA for 4 h elicitor exposure period resulted in the production of 64.65 mg g−l dry weight (DW) withanolide A (48-fold), 33.74 mg g−l DW withanolide B (29-fold), 17.47 mg g−l DW withaferin A (20-fold), 42.88 mg g−l DW withanone (37-fold), 5.34 mg g−l DW 12-deoxy withastramonolide (nine fold), 7.23 mg g−l DW withanoside V (seven fold), and 9.45 mg g−l DW withanoside IV (nine fold) after 10 days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.


Adventitious root culture Methyl jasmonate Salicylic acid Solanaceae Withania somnifera Withanolides 



The first author gratefully acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India, for the award of CSIR-SRF. The authors wish to thank Council of Scientific and Industrial Research [CSIR] (sanction no. 38 (1180/08/EMR-II)), Government of India for financial support.

Supplementary material

12010_2012_9809_MOESM1_ESM.pdf (181 kb)
ESM 1 PDF 181 kb
12010_2012_9809_MOESM2_ESM.pdf (180 kb)
ESM 2 PDF 180 kb
12010_2012_9809_MOESM3_ESM.doc (42 kb)
Table S1 Effect of MeJ and SA elicitation and elicitation time on fresh weight, dry weight, and growth index from adventitious root culture of Kolli hill and Cumbum varieties of W. somnifera (DOC 42 kb)


  1. 1.
    Gupta, G. L., & Rana, A. C. (2007). Withania somnifera (Ashwagandha): a review. Pharmacognosy Reviews, 1, 129–136.Google Scholar
  2. 2.
    Jayaprakasam, B., Zhang, Y., Seeram, N. P., & Nair, M. G. (2003). Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves. Life Sciences, 74, 125–132.CrossRefGoogle Scholar
  3. 3.
    Choudhary, M. I., Hussain, S., Yousuf, S., Dar, A., & Mudassar, A.-R. (2005). Chlorinated and diepoxy withanolides from Withania somnifera and their cytotoxic effects against human lung cancer cell line. Phytochemistry, 71, 2205–2209.CrossRefGoogle Scholar
  4. 4.
    Kaileh, M., Berghe, W. V., Heyerick, A., Horion, J., Piette, J., Libert, C., De Keukeleire, D., Essawi, T., & Haegeman, G. (2007). Withaferin A strongly elicits IKKβ hyperphosphorylation, concomitant with potent inhibition of its kinase activity. The Journal of Biological Chemistry, 282, 4253–4264.CrossRefGoogle Scholar
  5. 5.
    Mishra, L. C., Singh, B. B., & Dagenais, S. (2000). Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Alternative Medicine Review, 5, 334–346.Google Scholar
  6. 6.
    Chaurasiya, N. D., Uniyal, G. C., Lal, P., Misra, L., Sangwan, N. S., Tuli, R., & Sangwan, R. S. (2008). Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection. Phytochemical Analysis, 19, 148–154.CrossRefGoogle Scholar
  7. 7.
    Dhar, R. S., Verma, V., Suri, K. A., Sangwan, R. S., Satti, N. K., Kumar, A., Tuli, R., & Qazi, G. N. (2006). Phytochemical and genetic analysis in selected chemotypes of Withania somnifera. Phytochemistry, 67, 2269–2276.CrossRefGoogle Scholar
  8. 8.
    Praveen, N., & Murthy, H. N. (2010). Production of withanolide A from adventitious root cultures of Withania somnifera. Acta Physiologiae Plantarum, 5, 1017–1022.CrossRefGoogle Scholar
  9. 9.
    Sivanandhan, G., Mariashibu, T. S., Arun, M., Rajesh, M., Kasthurirengan, S., Selvaraj, N., & Ganapathi, A. (2011). The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants. Acta Physiologiae Plantarum, 33, 2279–2288.CrossRefGoogle Scholar
  10. 10.
    Sudha, C. G., & Seeni, S. (2001). Establishment and analysis of fast growing normal root culture of Decalepis arayalpathra, a rare endemic medicinal plant. Current Science, 81, 371–374.Google Scholar
  11. 11.
    Rani, G., Arora, S., & Nagpal, A. (2003). Direct rhizogenesis from in vitro leaves of Withania somnifera (L.) Dunal. Journal of Herbs, Spices and Medicinal Plants, 10, 47–54.CrossRefGoogle Scholar
  12. 12.
    Wadegaonkar, P. A., Bhagwat, K. A., & Rai, M. K. (2006). Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal. Plant Cell Tissue Organ Culture, 84, 223–225.CrossRefGoogle Scholar
  13. 13.
    Singh, A., Duggal, S., Singh, H., Singh, J., & Katekhaye, S. (2011). Withanolides: phytoconstituents with significant pharmacological activities. International Journal of Green Pharmacy, 4, 229–237.CrossRefGoogle Scholar
  14. 14.
    Kim, S. G., Soh, W. Y., & Cho, D. Y. (1995). Saikosaponin content in adventitious root formed from callus of Bupleurum falcatum. Korean Journal of Plant Tissue Culture, 22, 29–33.Google Scholar
  15. 15.
    Choi, S. M., Son, S. H., Yun, S. R., Kwon, O. W., Seon, J. H., & Paek, K. Y. (2000). Pilot-scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell Tissue Organ Culture, 62, 187–193.CrossRefGoogle Scholar
  16. 16.
    Chatterjee, S., Srivastava, S., Khalid, A., Singh, N., Sangwan, R. S., Sidhu, O. P., Roy, R., Khetrapal, C. L., & Tuli, R. (2010). Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry, 71, 1085–1094.CrossRefGoogle Scholar
  17. 17.
    Saenz-Carbonell, L., & Loyola-Vargas, V. M. (1997). Datura stramonium hairy roots tropane alkaloid content as a response to changes in Gamborg’s B5 medium. Applied Biochemistry and Biotechnology, 61, 321–337.CrossRefGoogle Scholar
  18. 18.
    Mizukami, H., Tavira, Y., & Ellis, B. E. (1993). Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures. Plant Cell Reports, 12, 706–709.CrossRefGoogle Scholar
  19. 19.
    Draper, J. (1997). Salicyliate × superoxide synthesis and cell suicide in plant defense. Trends in Plant Science, 2, 162–165.CrossRefGoogle Scholar
  20. 20.
    Mehmetoglu, U., & Curtis, W. R. (1997). Effects of abiotic inducers on sesquiterpene synthesis in hairy root and cell-suspension cultures of Hyoscyamus muticus. Applied Biochemistry and Biotechnology, 67, 71–77.CrossRefGoogle Scholar
  21. 21.
    Ciddi, V. (2006). Withaferin A from cell cultures of Withania somnifera. Indian Journal of Pharmaceutical Sciences, 68, 490–492.CrossRefGoogle Scholar
  22. 22.
    Baldi, A., Singh, D., & Dixit, V. K. (2008). Dual elicitation for improved production of withaferin A by cell suspension cultures of Withania somnifera. Applied Biochemistry and Biotechnology, 151, 556–564.CrossRefGoogle Scholar
  23. 23.
    Chaudhury, K., Das, S., Bandhyopadhyay, M., Zalar, A., Kollmann, A., Jha, S., & Tepfer, D. (2009). Transgenic mimicry of pathogen attack stimulates growth and secondary metabolite accumulation. Transgenic Research, 18, 121–134.CrossRefGoogle Scholar
  24. 24.
    Sivanandhan, G., Arun, M., Mayavan, S., Rajesh, M., Mariashibu, T. S., Manickavasagam, M., Selvaraj, N., & Ganapathi, A. (2012). Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Industrial Crops and Products, 1, 124–129.CrossRefGoogle Scholar
  25. 25.
    Mathew, K. M. (1982). The flora of the Tamil Nadu carnatic, vol II (1st ed.). Tiruchirappalli: The Rapinat Herbarium.Google Scholar
  26. 26.
    Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, 15, 473–497.CrossRefGoogle Scholar
  27. 27.
    Rani, G., & Grover, I. S. (1999). In vitro callus induction and regeneration studies in Withania somnifera. Plant Cell Tissue Organ Culture, 57, 23–27.CrossRefGoogle Scholar
  28. 28.
    Rani, G., Virk, G. S., & Nagpal, A. (2003). Callus induction and plantlet regeneration in Withania somnifera (L.) Dunal. In Vitro Cellular and Developmental Biology—Plant, 39, 468–474.CrossRefGoogle Scholar
  29. 29.
    Chong, T. M., Abdullah, M. A., Lai, O. M., Nor’ Aini, F. M., & Lajis, N. H. (2005). Effective elicitation factors in Morinda elliptica cell suspension culture. Process Biochemistry, 40, 3397–3405.CrossRefGoogle Scholar
  30. 30.
    Kim, Y. S., Hahn, E. J., Murthy, H. N., & Paek, K. Y. (2004). Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate. Biotechnology Letters, 26, 1619–1622.CrossRefGoogle Scholar
  31. 31.
    Sakunphueak, A., & Panichayupakaranant, P. (2010). Increased production of naphthoquinones in Impatiens balsamina root cultures by elicitation with methyl jasmonate. Bioresource Technology, 101, 8777–8783.CrossRefGoogle Scholar
  32. 32.
    Lu, M. B., Wong, H. L., & Teng, W. L. (2000). Effects of elicitation on the production of saponin in cell culture of Panax ginseng. Plant Cell Reports, 120, 674–677.Google Scholar
  33. 33.
    Zhao, J., Zhu, W. H., & Hu, Q. (2001). Selection of fungal elicitors to increase indole alkaloid accumulation in Catharanthus roseus suspension cell culture. Enzyme and Microbial Technology, 28, 666–672.CrossRefGoogle Scholar
  34. 34.
    Shabani, L., Ehsanpour, A. A., Asghari, G., & Emami, J. (2009). Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid. Russian Journal of Plant Physiology, 56, 621–626.CrossRefGoogle Scholar
  35. 35.
    Kang, S. M., Jung, H. Y., Kang, Y. M., Yun, D. J., Bahk, J. D., Yang, J., & Choi, M. S. (2004). Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora. Plant Science, 166, 745–751.CrossRefGoogle Scholar
  36. 36.
    Ali, M. B., Yu, K. W., Hahn, E. J., & Paek, K. Y. (2006). Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors. Plant Cell Reports, 25, 613–620.CrossRefGoogle Scholar
  37. 37.
    Yu, Z. Z., Fu, C. X., Han, Y. S., Li, Y. X., & Zhao, D. X. (2006). Salicylic acid enhances jaceosidin and syringin production in cell cultures of Saussurea medusa. Biotechnology Letters, 28, 1027–1031.CrossRefGoogle Scholar
  38. 38.
    Satdive, R. K., Fulzele, D. P., & Eapen, S. (2007). Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization. Journal of Biotechnology, 128, 281–289.CrossRefGoogle Scholar
  39. 39.
    Zhao, J. L., Zhou, L. G., & Wu, J. Y. (2010). Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures. Applied Microbiology and Biotechnology, 87, 137–144.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ganeshan Sivanandhan
    • 1
  • Muthukrishnan Arun
    • 1
  • Subramanian Mayavan
    • 1
  • Manoharan Rajesh
    • 1
  • Murugaraj Jeyaraj
    • 1
  • Gnanajothi Kapil Dev
    • 1
  • Markandan Manickavasagam
    • 1
  • Natesan Selvaraj
    • 2
  • Andy Ganapathi
    • 1
    Email author
  1. 1.Department of Biotechnology and Genetic Engineering, School of BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  2. 2.Periyar E. V. R College (Autonomous)TiruchirappalliIndia

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