, Volume 74, Issue 5, pp 543–553 | Cite as

Effect of abiotic elicitation and pathway precursors feeding over terpenoid indole alkaloids production in multiple shoot and callus cultures of Catharanthus roseus

  • Abhishek SharmaEmail author
  • Ajay Kumar Mathur
  • Jawahar Ganpathy
  • Bhrugesh Joshi
  • Prittesh Patel
Original Article


Catharanthus roseus is a well-known herb with great pharmaceutical value for being the only source of natural antineoplastic drugs vinblastine and vincristine. The terpenoid indole alkaloids (TIAs) biosynthetic pathway responsible for the production of these antineoplastic drug molecules is under a strict spatiotemporal regulation that requires five different cellular as well as at least four intracellular compartmentations for its completion. Therefore, several efforts were made to investigate the cell and tissue cultures of Catharanthus roseus to establish as an alternative source of TIAs production. Cell suspension, hairy roots and callus cultures of Catharanthus roseus do not provide the required cellular complexity for the completion of entire TIAs pathway, therefore, failed to produce vinblastine and vincristine. However, the multiple shoot cultures do provide the required cellular complexity to complete the entire TIAs pathway. Therefore, in the present study, the multiple shoot cultures along with callus cultures were subjected to the abiotic elicitors and TIAs pathway precursors feeding for the first time to chase the effect of these abiotic elicitors and pathway precursors on the production of major TIAs of Catharanthus roseus. The multiple shoot cultures treated with TIAs pathway precursor tryptamine 300 mg/L accumulated highest vinblastine content (0.0277% dry wt) followed by tryptophan feeding at 300 mg/L (0.0180% dry wt) and 500 mg/L (0.0175% dry wt) whereas, callus cultures failed to produce vinblastine.


Catharanthus roseus Terpenoid indole alkaloids Elicitation 



Terpenoid indole alkaloids






geraniol 8-hydroxylase

AVLB a-3,4’



peroxidase a-3’,4’-anhydrovinblastine synthase


Growth Index



AS is highly thankful to Department of Science and Technology (DST-INSPIRE), Gov. of India for providing an INSPIRE fellowship. AS also gratefully acknowledge Director, CSIR-CIMAP, Lucknow and Director, CGBIBT-UTU.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11756_2019_202_MOESM1_ESM.docx (13 kb)
ESM 1 (DOCX 12 kb)


  1. Arora R, Malhotra P, Mathur AK, Mathur A, Govil CM, Ahuja PS (2010) Anticancer alkaloids of Catharanthus roseus: transition from traditional to modern medicine. In: Arora R (ed) Herbal medicine: a cancer chemopreventive and therapeutic perspective. Jaypee Brothers Medical Publishers Pvt. Ltd, New Delhi, pp 292–310CrossRefGoogle Scholar
  2. Baenas N, García-Viguera C, Moreno DA (2014) Elicitation: a tool for enriching the bioactive composition of foods. Molecules 19:13541–11356. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Caarls L, Pieterse CMJ, Van Wees SCM (2015) How salicylic acid takes transcriptional control over jasmonic acid signaling. Front Plant Sci.
  4. Caputi L, Franke J, Farrow SC, Chung K, Payne RME, Nguyen TD, Dang TTT et al (2018) Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science 360:1235–1239. CrossRefPubMedGoogle Scholar
  5. Duarte JD, Cooper-DeHoff RM (2010) Mechanisms for blood pressure lowering and metabolic effects of thiazide and thiazide-like diuretics. Expert Rev Cardiovasc Ther 8:793–802. CrossRefPubMedPubMedCentralGoogle Scholar
  6. El-Sayed M, Verpoorte R (2007) Catharanthus terpenoid indole alkaloids: biosynthesis and regulation. Phytochem Rev 6:277–305. CrossRefGoogle Scholar
  7. Giri CC, Zaheer M (2016) Chemical elicitors versus secondary metabolite production in vitro using plant cell, tissue and organ cultures: recent trends and a sky eye view appraisal. Plant Cell Tissue Organ Cult 126:1–18. CrossRefGoogle Scholar
  8. Godoy–Hernandez G, Loyola–Vargas VM (1991) Effect of fungal homogenate, enzyme inhibitors and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures. Plant Cell Rep 10:537–540. CrossRefPubMedGoogle Scholar
  9. Hughes RH, Shanks JV (2002) Metabolic engineering of plants for alkaloid production. Metab Eng 4:41–48. CrossRefPubMedGoogle Scholar
  10. Karuppusamy S (2009) A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell culture. J Med Plant Res 3:1222–1239Google Scholar
  11. Kumar S, Bhatia S (2016) A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don. Scientific Reports 6 (1)Google Scholar
  12. Li CY, Leopold AL, Sander GW et al (2013) The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway. BMC Plant Biol 13:155–159. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Liu J, Cai J, Wang R, Yang S (2017) Transcriptional regulation and transport of terpenoid indole alkaloid in Catharanthus roseus: exploration of new research directions. Int J Mol Sci 18:53–59. CrossRefGoogle Scholar
  14. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  15. Murthy HM, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult 118:1–16. CrossRefGoogle Scholar
  16. Morgan JA, Shanks JV (2000) Determination of metabolic rate limitations by precursor feeding in Catharanthus roseus hairy root cultures. J Biotechnol 79:137–145Google Scholar
  17. Neuss N, Neuss MN (1990) The therapeutic use of bisindole alkaloids from Catharanthus. In: Brossi A, Suffness M (eds) The alkaloids, vol 37. Academic Press, San Diego, pp 228–240. CrossRefGoogle Scholar
  18. Noe W, Mollenschott C, Berlin J (1984) Tryptophan decarboxylase from Catharanthus roseus cell suspension cultures: purification, molecular and kinetic data of the homogenous protein. Plant Mol Biol 3:281–288. CrossRefPubMedGoogle Scholar
  19. Pandey SS, Singh S, Babu CSV, Shanker K, Srivastava NK, Shukla AK, Kalra A (2016) Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis. Scientific Reports 6 (1)Google Scholar
  20. Peebles CA, Hughes EH, Shanks JV, San KY (2009) Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. Metab Eng 11:76–86. CrossRefPubMedGoogle Scholar
  21. Qu Y, Michael LAEE, Jordan F, Razvan S, Tomas H, Luca VD (2015) Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast. PNAS 112:6224–6229. CrossRefPubMedGoogle Scholar
  22. Qu Y, Easson M, Simionescu R, Hajicek J, Thamm AMK, Salim V, De Luca V (2018) Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidospermamonoterpenoid indole alkaloids from 19E-geissoschizine. PNAS 114:3180–3185. CrossRefGoogle Scholar
  23. Rijhwani SK, Shanks JV (1998) Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures. Biotechnol Prog 14:442–449. CrossRefPubMedGoogle Scholar
  24. Rodriguez S, Compagnon V, Crouch NP, St-Pierre B, De Luca V (2003) Jasmonate- induced epoxidation of tabersonine by a cytochrome P-450 in hairy root cultures of Catharanthus roseus. Phytochemistry 64:401–409. CrossRefPubMedGoogle Scholar
  25. Sharma A, Verma P, Mathur A, Mathur AK (2017) Genetic engineering approach using early Vinca alkaloid biosynthesisgenes led to increased tryptamine and terpenoid indole alkaloids biosynthesis in differentiating cultures of Catharanthus roseus. Protoplasma 255:425–435. CrossRefPubMedGoogle Scholar
  26. Sharma A, Verma P, Mathur A, Mathur AK (2018) Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced terpenoid indole alkaloids pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus. Protoplasma.
  27. Smith JI, Smart NJ, Kurz WGW, Misawa M (1987) Stimulation of indole alkaloid production in cell suspension cultures of Catharanthusroseus by abscisic acid. Planta Med 53:470–474. CrossRefPubMedGoogle Scholar
  28. Tatsis EC, Carqueijeiro I, Bernonville TDD, Oudin A et al (2017) A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate. Nat Commun 8:316. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Thakore D, Srivastava AK, Sinha AK (2015) Model based fed batch cultivation and elicitation for the overproduction of ajmalicine from hairy roots of Catharanthus roseus. Biochem Eng J 97:73–80. CrossRefGoogle Scholar
  30. Vázquez-Flota FA, Loyola-Vargas VM (1994) A Catharanthus roseus salt tolerant line II. Alkaloid production. J Plant Physiol 144:613–616. CrossRefGoogle Scholar
  31. Vázquez-Flota F, Hernández-Domínguez E, De Lourdes Miranda-Ham M, Monforte-González M (2009) A differential response to chemical elicitors in Catharanthus roseus in vitro cultures. Biotechnol Lett 31:591–595. CrossRefPubMedGoogle Scholar
  32. Verma P, Mathur AK, Srivastava A, Mathur A (2012) Emerging trends in research on spatial and temporal organization of terpenoid indole alkaloid pathway in Catharanthus roseus: a literature update. Protoplasma 249:255–268. CrossRefPubMedGoogle Scholar
  33. Verma P, Khan SA, Mathur AK, Ghosh S, Shankar K, Kalra A (2014) Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor. Protoplasma 251:1359–1371. CrossRefPubMedGoogle Scholar
  34. Verma P, Sharma A, Khan SA, Shanker K, Mathur AK (2015) Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor. Protoplasma 252:373–381. CrossRefPubMedGoogle Scholar
  35. Verpoorte R, Memelink J (2002) Engineering secondary metabolite production in plants. CurrOpin Biotechnol 13:181–187. CrossRefGoogle Scholar
  36. Verpoorte R, van der Heijden R, Moreno PRH (1997) Biosynthesis of terpenoid indole alkaloids in Catharanthus roseus cells. In: Cordell GA (ed) The alkaloids, vol 49. Academic, San Diego, pp 221–299. CrossRefGoogle Scholar
  37. Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary metabolites. Phytochem Rev 1:13–25. CrossRefGoogle Scholar
  38. Wang Q, Xing S, Pan Q et al (2012) Development of efficient Catharanthus roseus regeneration and transformation system using Agrobacteriumtumefaciens and hypocotyls as explants. BMC Biotechnol 12:34–46. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Wang X, Pan YJ, Chang BW, Hu YB, Guo XR, Tang ZH (2016) Ethylene-induced vinblastine accumulation is related to activated expression of downstream TIA pathway genes in Catharanthus roseus. Biomed Res Int.
  40. Whitmer S, Canel C, Hallard D, Goncalves C, Verpoorte R (1998) Influence of precursor availability on alkaloid accumulation by transgenic cell line of Catharanthus roseus. Plant Physiol 116:853–857. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Whitmer S, van der Heijden R, Verpoorte R (2002) Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus. J Biotechnol 96:193–203. CrossRefGoogle Scholar
  42. Zaheer M, Giri CC (2015) Multiple shoot induction and jasmonic versus salicylic acid driven elicitation for enhanced andrographolide production in Andrographispaniculata. Plant Cell Tissue Organ Cult 122:553–563. CrossRefGoogle Scholar
  43. Zhao J, Verpoorte R (2007) Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering. Phytochem Rev 6:435–457. CrossRefGoogle Scholar
  44. Zhao J, Zhu W, Hu Q, He XW (2000) Improved indole alkaloid production in Catharanthus roseus suspension cultures by various chemicals. Biotechnol Lett 22:1221–1226. CrossRefGoogle Scholar
  45. Zhao J, Zhu WH, Hu Q, Guo YQ (2001) Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shocks. Biotechnol Lett 22:1227–1231. CrossRefGoogle Scholar
  46. Zhou ML, Zhu XM, Shao JR, Wu YM, Tang YX (2010) Transcriptional response of the catharanthine biosynthesis pathway to methyl jasmonate/nitric oxide elicitation in Catharanthus roseus hairy root culture. App Microbiol Biotechnol 88:737–750. CrossRefGoogle Scholar

Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019

Authors and Affiliations

  1. 1.C. G. Bhakta Institute of BiotechnologyUka Tarsadia UniversitySuratIndia
  2. 2.Department of Plant BiotechnologyCentral Institute of Medicinal and Aromatic Plants (CIMAP), Council of Scientific and Industrial ResearchLucknowIndia

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