Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 123, Issue 3, pp 443–453 | Cite as

Elicitation of furanocoumarins in poison hemlock (Conium maculatum L.) cell culture

  • Philipp Meier
  • Hannu Hotti
  • Heiko RischerEmail author
Original Article


Furanocoumarins, such as psoralen, xanthotoxin and bergapten, serve as protectants against phytopathogens and are used in pharmaceutical applications e.g. as DNA-crosslinking agents against non-melanoma skin cancers. Poison hemlock plants (Conium maculatum L.) are a known source of furanocoumarins and toxic alkaloids, but systematic research on callus and suspension cultures with the aim of eliciting secondary metabolites is lacking. Therefore callus cultures of poison hemlock were induced with 0.186 mg L−1 6-benzylaminopurine and 2 or 4 mg L−1 naphthalene acetic acid on McCown’s Woody plant medium. A broad variety of elicitors (alginic acid, cellulase, chitosan, ethylene, methyl jasmonate, salicylic acid, copper(II) sulphate and silver nitrate) were tested with an established cell suspension culture for their capacity to trigger differential metabolite accumulation. Samples were extracted and analysed by gas chromatography-mass spectrometry. Elicitation with alginic acid, cellulase, chitosan, silver nitrate and copper(II) sulphate induced furanocoumarins. Plant hormones (ethylene, methyl jasmonate and salicylic acid) were not able to induce furanocoumarins. Extracts contained bergapten, columbianetin, isopimpinellin, marmesin, oroselone, psoralen and xanthotoxin but not piperidine alkaloids. The relative amount of furanocoumarins was generally higher in the medium than in the cells. The report describes the angular furanocoumarins oroselone and columbianetin together with the linear furanocoumarin marmesin, elicited for the first time in poison hemlock.


Callus Cell culture Elicitation Furanocoumarins Poison hemlock (Conium maculatum L.) 





Dry cell weight


Fresh cell weight


Fluorescein diacetic acid


Gas chromatography mass spectrometry


Naphthalene acetic acid


Retention time


Thin layer chromatography


McCown’s Woody plant medium (Lloyd and McCown 1981)



We thank Anna Ruskeepää and Tuulikki Seppänen-Laakso for GC-MS analyses. Prof. Dr. Regine Eibl-Schindler, Zurich University of Applied Sciences, is acknowledged for enabling P.M.’s bachelor thesis at VTT. This work was supported by ERASMUS funding (to P.M.), the Finnish Doctoral Program in Plant Science, Societas pro Fauna et Flora Fennica, Eteläsuomalaisten ylioppilaiden säätiö, Oskar Öflunds Stiftelse, the Otto A. Malm Foundation (all to H.H.), VTT and the Academy of Finland (grant 138808 to H.R.). The authors acknowledge the support of COST Action FA1006, PlantEngine.

Author contributions

P.M., H.H., H.R. planned the experiments; P.M., H.H. performed the experiments; P.M., H.H., H.R. analysed the data; P.M., H.H., H.R. wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2015_847_MOESM1_ESM.doc (71 kb)
Supplementary material 1 (DOC 71 kb)


  1. Al-Barwani FM, Eltayeb EA (2004) Antifungal compounds from induced Conium maculatum L. plants. Biochem Syst Ecol 32:1097–1108CrossRefGoogle Scholar
  2. Baldi A, Srivastava AK, Bisaria VS (2009) Fungal elicitors for enhanced production of secondary metabolites in plant cell suspension cultures. In: Varma A, Kharkwal AC (eds) Soil biology 18: symbiotic fungi. Springer, Berlin, pp 373–380CrossRefGoogle Scholar
  3. Bulgakov VP, Tchernoded GK, Mischenko NP, Khodakovskaya MV, Glazunov VP, Radchenko SV, Zvereva EV, Fedoreyev SA, Zhuravlev YN (2002) Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes. J Biotechnol 97:213–221CrossRefPubMedGoogle Scholar
  4. Carew DP, Bainbridge T (1976) Biotransformations with plant tissue cultures. Lloydia 39:147–149PubMedGoogle Scholar
  5. Corsi G, Biasci D (1998) Secretory structures and localization of alkaloids in Conium maculatum L. (Apiaceae). Ann Bot 81:157–162CrossRefGoogle Scholar
  6. Diwan R, Malpathak N (2011) Bioprocess optimazation of furanocoumarin elicitation by medium renewal and re-elicitation: a prefusion-based approach. Appl Biochem Biotehcnol 163:756–764CrossRefGoogle Scholar
  7. Ekiert H (2000) Medicinal plant biotechnology: the Apiaceae family as the example of rapid development. Pharmazie 55:561–567PubMedGoogle Scholar
  8. Ellialtıoğlu Ş, Üstün AS, Mehmetoğlu Ü (2001) Using AgNO3 and CuSO4 as elicitors in the callus suspension cultures of different pepper genotypes. In: XIth Eucarpia meeting on genetics and breeding of capsicum and eggplant. Accessed 11 Feb 2015
  9. Enan MR (2009) Genotoxicity of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D): higher plants as monitoring systems. J For Environ Sci 25:147–155Google Scholar
  10. Fairbairn JW, Challen SB (1959) The alkaloids of hemlock (Conium maculatum L.): distribution in relation to the development of the fruit. Biochem J 72:556–561PubMedCentralCrossRefPubMedGoogle Scholar
  11. Fairbairn JW, Suwal PN (1961) The alkaloids of hemlock (Conium maculatum L.)—II: evidence for a rapid turnover of the major alkaloids. Phytochemistry 1:38–46CrossRefGoogle Scholar
  12. Gadzovska S, Maury S, Delaunay A, Spasenoski M, Hagège D, Courtois D, Joseph C (2013) The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L. Plant Cell Tissue Organ Cult 113:25–39CrossRefGoogle Scholar
  13. Gangopadhyay M, Dewanjee S, Bhattacharya S (2011) Enhanced plumbagin production in elicited Plumbago indica hairy root cultures. J Biosci Bioeng 111:706–710CrossRefPubMedGoogle Scholar
  14. Häkkinen ST, Moyano E, Cusidó RM, Palazón J, Piñol MT, Oksman-Caldentey K-M (2005) Enhanced secretion of tropane alkaloids in Nicotiana tabacum hairy roots expressing heterologous hyoscyamine-6β-hydroxylase. J Exp Bot 56:2611–2618CrossRefPubMedGoogle Scholar
  15. Holm L, Doll J, Holm E, Pancho J, Herberger J (1997) World weeds: natural histories and distribution. Wiley, New YorkGoogle Scholar
  16. Ishikawa A, Kuma T, Sasaki H, Sasaki N, Ozeki Y, Kobayashi N, Kitamura Y (2009) Constitutive expression of bergaptol O-methyltransferase in Glehnia littoralis cell cultures. Plant Cell Rep 28:257–265CrossRefPubMedGoogle Scholar
  17. Jonsell B, Karlsson T (2010) Flora Nordica 6, Thymelaeaceae-Apiaceae. The Swedish Museum of Natural History, StockholmGoogle Scholar
  18. Karamat F, Olry A, Munakata R, Koeduka T, Sugiyama A, Paris C, Hehn A, Bourgaud F, Yazaki K (2014) A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley. Plant J 77:627–638CrossRefPubMedGoogle Scholar
  19. Lee YR (1995) A concise new synthesis of angular furanocoumarins: angelicin, oroselone and oroselol. Tetrahedron 51:3087–3094CrossRefGoogle Scholar
  20. Leete E (1971) Biosynthesis of the hemlock and related piperidine alkaloids. Acc Chem Res 4:100–107CrossRefGoogle Scholar
  21. Lloyd G, McCown B (1981) Commercially-feasible micropropagation of Mountain laurel, Kalmia latifolia, by use of shoot tip culture. Int Plant Propag Soc Proc 30:421–427Google Scholar
  22. Lopez T, de la Torre M, Cid M (2004) An efficient TLC method for analysis of gamma-coniceine and coniine in Conium maculatum L. foliage. J Planar Chromatogr Mod TLC 17:218–223CrossRefGoogle Scholar
  23. Ma CJ (2008) Cellulase elicitor induced accumulation of capsidiol in Capsicum annuum L. suspension cultures. Biotechnol Lett 30:961–965CrossRefPubMedGoogle Scholar
  24. Madrid E, Corchete P (2010) Silymarin secretion and its elicitation by methyl jasmonate in cell cultures of Silybum marianum is mediated by phospholipase D-phosphatidic acid. J Exp Bot 61:747–754PubMedCentralCrossRefPubMedGoogle Scholar
  25. NIST Mass Spec Data Center, Stein SE (2015) Mass spectra. In: Linstrom PJ, Mallard WG (eds) NIST Chemistry WebBook, NIST Standard Reference Database Number 69. National Institute of Standards and Technology, Gaithersburg MD, 20899. Accessed 7 May 2015
  26. Namdeo AG (2007) Plant cell elicitation for production of secondary metabolites: a review. Pharmacogn Rev 1:69–79Google Scholar
  27. Nétien G, Combet J (1971) Etude comparative dans la composition chimique des cultures de tissus de Conium maculatum cultivées in vitro—I: variation des substances azotees. Paris Soc Biol Compt Rend 165:103–107Google Scholar
  28. Ojala T, Remes S, Haansuu P, Vuorela H, Hiltunen R, Haahtela K, Vuorela P (2000) Antimicrobial activity of some coumarin containing herbal plants growing in Finland. J Ethnopharmacol 73:299–305CrossRefPubMedGoogle Scholar
  29. Patel H, Krishnamurthy R (2013) Elicitors in plant tissue culture. J Pharmacogn Phytochem 2:60–65Google Scholar
  30. Pathak MA, Fitzpatrick TB (1992) The evolution of photochemotherapy with psoralens and UVA (PUVA): 2000 BC to 1992 AD. J Photochem Photobiol B 14:3–22CrossRefPubMedGoogle Scholar
  31. Prince M, Campbell CT, Robertson TA, Wells AJ, Kleiner HE (2006) Naturally occurring coumarins inhibit 7,12-dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland. Carcinogenesis 27:1204–1213CrossRefPubMedGoogle Scholar
  32. Repka V, Fischerová I, Šilhárová K (2004) Methyl jasmonate is a potent elicitor of multiple defense responses in grapevine leaves and cell-suspension cultures. Biol Plant 48:273–283CrossRefGoogle Scholar
  33. Roberts MF (1981) Enzymatic synthesis of γ-coniceine in Conium maculatum chloroplasts and mitochondria. Plant Cell Rep 1:10–13CrossRefPubMedGoogle Scholar
  34. Schoofs G, Teichmann S, Hartmann T, Wink M (1983) Lysine decarboxylase in plants and its integration in quinolizidine alkaloid biosynthesis. Phytochemistry 22:65–69CrossRefGoogle Scholar
  35. Sévon N, Varjonen T, Hiltunen R, Oksman-Caldentey K-M (1992) Effect of sucrose, nitrogen and copper on the growth and alkaloid production of transformed root cultures of Hyoscyamus muticus. Planta Med 58:609–610CrossRefGoogle Scholar
  36. Shul’ts EE, Ganbaatar Z, Petrova TN, Shakirov MM, Bagryanskaya IY, Taraskin VV, Radnaeva LD, Otgonsuren D, Pokrovskii AG, Tolstikov GA (2012) Plant coumarins—IX: phenolic compounds of Ferulopsis hystrix growing in Mongolia: cytotoxic activity of 8,9-dihydrofurocoumarins. Chem Nat Compd 48:211–217CrossRefGoogle Scholar
  37. Suri R, Radzali M, Marziah M, Aspollah SM (2002) Effect of elicitors on the production of naringin and rutin in leech lime (Citrus hystrix) callus. J Trop Agric Food Sci 30:89–94Google Scholar
  38. Udomsuk L, Jarukamjorn K, Tanaka H, Putalun W (2011) Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation. Biotechnol Lett 33:369–374CrossRefPubMedGoogle Scholar
  39. Üstün AS, Ellialtioğlu S, Mehmetoğlu Ü (1998) The effects of biotic and abiotic elicitors on the capsidiol synthesis in cell suspension cultures of pepper (Capsicum annuum L.) Scientific and Technical Research Council of Turkey. Accessed 11 Feb 2015
  40. Vetter J (2004) Poison hemlock (Conium maculatum L.). Food Chem Toxicol 42:1373–1382CrossRefPubMedGoogle Scholar
  41. Wang BY (2007) Environmental biodegradation research focus. Nova Science Publishers, Hauppauge,NYGoogle Scholar
  42. Windholm J (1972) The use of fluorescein diacetate and phenosaphranine for determining viability of cultured plant cells. Stain Technol 47:189–194Google Scholar
  43. Wink M, Witte L (1983) Evidence for a wide-spread occurrence of the genes of quinolizidine alkaloid biosynthesis: induction of alkaloid accumulation in cell suspension cultures of alkaloid-‘free’ species. FEBS Lett 159:196–200CrossRefGoogle Scholar
  44. Wink M, Witte L, Schiebel H-M, Hartmann T (1980) Alkaloid pattern of cell suspension cultures and differentiated plants of Lupinus polyphyllus. Planta Med 38:238–245CrossRefGoogle Scholar
  45. Wink M, Witte L, Hartmann T (1981) Quinilizidine alkaloid composition of plants and of photomixotrophic cell suspension cultures of Sarothamnus scoparius and Orobanche rapum-genistae. Planta Med 43:342–352CrossRefPubMedGoogle Scholar
  46. Wu Q, Christensen LA, Legerski RJ, Vasquez KM (2005) Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells. EMBO Rep 6:551–557PubMedCentralCrossRefPubMedGoogle Scholar
  47. Yoo SH, Kim JS, Kang SS, Son KH, Chang HW, Kim HP, Bae KH, Lee C-O (2002) Constituents of the fruits and leaves of Euodia danielii. Arch Pharm Res 25:824–830CrossRefPubMedGoogle Scholar
  48. Young-Woon J, Kim J, Byun S (1993) Phytohormone effects with elicitation on cell growth and alkaloid production in suspension cell cultures of Eschscholtzia california. J Microbiol Biotechnol 3:238–243Google Scholar
  49. Zobel AM, Brown SA (1993) Furanocoumarins on the surface of callus cultures from species of Rutaceae and Umbelliferae. Can J Bot 71:966–969Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Life Sciences und Facility ManagementZHAW Zurich University of Applied SciencesWaedenswilSwitzerland
  2. 2.VTT Technical Research Centre of Finland LtdEspooFinland

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