Tagetes spp. (Marigolds): In Vitro Culture and the Production of Thiophenes

  • H. Breteler
  • D. H. Ketel
Part of the Biotechnology in Agriculture and Forestry book series (AGRICULTURE, volume 21)


Tagetes species were used by ancient civilizations like the Aztecs for various purposes (Neher 1968). The pigments of the flowers were used as a dye and in chicken feed, oil was extracted from the leaves and used as an ingredient of perfumes, and the roots were also assumed to have interesting properties. Field tests in the USA in the 1930s showed that larvae of a root-knot nematode entered the roots of marigolds, but usually failed to develop and neither reached the adult stage nor produced eggs (Steiner 1941). In 1953, a Dutch bulb breeder (Van de Berg-Smit 1953) reported the biological activity of common garden marigolds (Tagetes patula) against root rot in Narcissus caused by free-living nematodes. The latter finding was an incentive for a scientific analysis of the effect of Tagetes plants by the crop protection industry and the academic world. A few years after the initial report by Van de Berg-Smit (1953), Uhlenbroek and Bijloo (1958, 1959, 1960) isolated and described some active principles from Tagetes plants. These chemicals belonged to a group of heterocyclic sulphur-containing compounds, the thiophenes. The thiophene α-terthienyl, which occurs in Tagetes and related species, was first synthesized in 1941 (Steinkopf et al. 1941) and isolated from plants in 1947 (Zechmeister and Sease 1947).


Hairy Root Gibberellic Acid Root Culture Crown Gall Agrobacterium Rhizogenes 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adamse P (1990) Selection of high-yielding cell lines of Tagetes using flow cytometry. In: Nijkamp HJJ, Plas LHW van der, Aartrijk J van (eds) Progress in plant cellular and molecular biology. Kluwer, Dordrecht, pp 726–731CrossRefGoogle Scholar
  2. Arroo RRJ, Kenkel H, Meijers H, Croes AF, Wullems GJ (1990) Cellular aspects of thiophene accumulation in relation to root differentiation. In: Nijkamp HJJ, Plas LHW van dm, Aartrijk J van (eds) Progress in plant cellular and molecular biology. Kluwer, Dordrecht, pp 587–591CrossRefGoogle Scholar
  3. Bakker J, Gommers FJ, Nieuwenhuis J, Wijnberg H (1979) Photoactivation of the nematicidal compound a-terthienyl from roots of marigolds (Tagetes species). J Biol Chem 254: 1841–1844PubMedGoogle Scholar
  4. Berg-Smit J van de (1953) Over het wortelrot bij Narcissen. Weekbl Bollencultuur 93/94: 437Google Scholar
  5. Birot AM, Bouchez D, Casse-Delbart F, Durand-Tardif M, Jouanin L, Pautot V, Robaglia C, Tepfer D, Tepfer M, Tourneur J, Vilaine F (1987) Studies and uses of the Ri plasmids of Agrobacterium rhizogenes. Plant Physiol Biochem 25: 323–335Google Scholar
  6. Bohlmann F, Zdero C (1985) Naturally occurring thiophenes. In: Weissberger A, Taylor EC (eds) Chemistry of heterocyclic compounds, part 1. Gronowitz S (ed) Thiophene and its derivatives. Wiley & Sons, New York, pp 221–323Google Scholar
  7. Bohlmann F, Burkhardt T, Zdero C (1973) Naturally occurring acetylenes. Academic Press, LondonGoogle Scholar
  8. Brown S (1984) Analysis and sorting of plant material by flow cytometry. Physiol Veg 22: 341–349Google Scholar
  9. Burkill JH (1966) A dictionary of the economic products of the Malay peninsula. Ministry of Agriculture and Cooperation, Kuala Lumpur, Malaysia, 2444 ppGoogle Scholar
  10. Cleene M de, Ley J de (1981) The host range of infectious hairy-root. Bot Rev 47: 147–194CrossRefGoogle Scholar
  11. Constabel F (1965) Phenolics in tissue cultures derived from Juniperus communis L.: studies on tannin synthesis. In: White PR, Grove AR (eds) Proc Int Conf Plant tissue Culture, Berkeley McCutcham, Berkeley, pp 183–190Google Scholar
  12. Croes AF, Aarts AM, Bosveld M, Breteler H, Wullems GJ (1989a) Control of thiophene accumulation in calli of two Tagetes species. Physiol Plant 76: 205–210CrossRefGoogle Scholar
  13. Croes AF, Berg AJR van den, Bosveld M, Breteler H, Wullems GJ (1989b) Thiophene accumulation in relation to morphology in roots of Tagetes patula; effects of auxin and transformation by Agrobacterium. Planta 179: 43–50CrossRefGoogle Scholar
  14. Drawert F, Berger RG (1982) Uber die Biogenese von Aromastoffen bei Pflanzen und Früchten. XIX Mitt: Vergleich der Biosynthese von Aromastoffen in Segment-, Kallus- und Suspensionskulturen von Citrusarten. Chem Mikrobiol Technol Lebensm 7: 143–147Google Scholar
  15. Duke J, Ayensu ES (1985) Medicinal plants öf China, vol 1. Reference Publications, MichiganGoogle Scholar
  16. Evans, PK, Cocking, EC (1977) Isolated plant protoplasts. In: Street HE (ed) Plant tissue and cell culture. Botanical monographs, vol 11, 2nd edn. Black well, Oxford, pp 103–135Google Scholar
  17. Flores HE (1987) Use of plant cell and organ cultures in the production of biological chemicals. In: Mumma RO, Lebaron H, Honeycutt RC, Duesing JH (eds) Application of biotechnology to agricultural chemistry. Am Chem Soc Symp Ser Washington DC 334: 67–86Google Scholar
  18. Flores HE, Hoy MW, Pickard J J (1987) Secondary metabolites from root cultures. Trends Biotechnol 5: 64–69CrossRefGoogle Scholar
  19. Flores HE, Pickard JJ, Hoy MW (1988) Production of polyacetylenes and thiophenes in heterotrophic and photosynthetic root cultures of Asteraceae. In: Lam J, Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally-occurring acetylenes and related compounds (NOARC), Elsevier, Amsterdam, pp 233–254Google Scholar
  20. Gommers FJ, Bakker J, Smits L (1980) Effects of singlet oxygen generated by the nematicidal compound a-terthienyl from Tagetes on the nematodeAphelenchus avenae. Nematologica 26: 369–375CrossRefGoogle Scholar
  21. Gommers FJ, Bakker J, Wijnberg H (1982) Dithiophenes as singlet oxygen sensitizers. Photochem Photobiol 35: 615–619CrossRefGoogle Scholar
  22. Groneman AF, Posthumus MA, Tuinstra LGM Th, Traag WA (1984) Identification and determination of metabolites in plant cell biotechnology by gas chromatography and gas chromatography/mass spectrometry: application to nonpolar products ofChrysanthemum cinerariaefolium and Tagetes species. Anal Chim Acta 163: 43–54CrossRefGoogle Scholar
  23. Helsper JPFG, Prins T, Lütke Willink D, Breteler H (1988a) 5-(4-Acetoxy-l-butinyl)-2,2-bithiophene (BBTOAc): acetate esterase in intact plants, calli and cell cultures of Tagetes patula, cv. Nana Furia. Plant Physiol 86 (Suppl): 143CrossRefGoogle Scholar
  24. Helsper JPFG, Ketel DH, Hülst AC, Breteler H (1988b) Production and secretion of thiophenes by differentiated cell cultures of Tagetes. In: Lam J. Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally-occurring acetylenes and related compounds (NOARC). Elsevier, Amsterdam, pp 279–286Google Scholar
  25. Hulst AC, Verlaan P, Breteler H, Ketel DH (1987) Thiophene production by Tagetes patula in a pilot plant airlift-loopreactor (ALR). In: Neyssel OM, Meer RR van der, Luyben KCAM (eds) Proc 4th Eur Congr Biotechnol. Elsevier, Amsterdam, pp 401–404Google Scholar
  26. Hulst AC, Meyer MMT, Breteler H, Tramper J (1989) Effect of aggregate size in cell cultures of T. patula on thiophene production and cell growth. Appl Microbiol Biotechnol 30: 18–25CrossRefGoogle Scholar
  27. Jente R, Olatunji GA, Bosold F (1981) Formation of natural thiophene derivatives from acetylenes by Tagetes patula. Phytochemistry 20: 2169–2175CrossRefGoogle Scholar
  28. Ketel DH (1986) Morphological differentiation and occurrence of thiophenes in leaf callus cultures from Tagetes species: relation to the growth medium of the plants. Physiol Plant 66: 392–396CrossRefGoogle Scholar
  29. Ketel DH (1987a) Distribution and accumulation of thiophenes in plants and calli of different Tagetes species. J Exp Bot 38: 322–330CrossRefGoogle Scholar
  30. Ketel DH (1987b) Callus and cell culture of Tagetes species in relation to production of thiophenes. Thesis, Agric Univ Wageningen, The NetherlandsGoogle Scholar
  31. Ketel DH (1988) Selection of thiophene-producing calli of Tagetes patula in relation to the production of thiophenes in liquid cultures. In: Robins RJ, Rhodes MJC (eds) Manipulating secondary metabolism in culture. Cambridge Univ Press, Cambridge, pp 225–232Google Scholar
  32. Ketel DH, Breteler H, Groot B de (1985) Effect of explant origin on growth and differentiation of calli from Tagetes species. J Plant Physiol 118: 327–333Google Scholar
  33. Ketel DH, Hulst AC, Gruppen H, Breteler H, Tramper J (1987) Effects of immobilization and environmental stress on growth and production of non-polar metabolites ofTagetes minuta cells. Enzyme Microb Technol 9: 303–307CrossRefGoogle Scholar
  34. Ketel DH, Breteler H (1988) Morphogenesis and thiophene production in cell cultures of Tagetes species. In: Lam J, Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally- occurring acetylenes and related compounds (NOARQ. Elsevier, Amsterdam, pp 267–278Google Scholar
  35. Kothari SL, Chandra N (1985) Plant regeneration in callus and suspension cultures of Tagetes erecta L. (African marigold) J Plant Physiol 122: 235–241Google Scholar
  36. Lam J, Thomasen T (1988) Complexing agents for protection of highly conjugated compounds against photodegradation. In: Lam J, Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally-occurring acetylenes and related compounds (NOARC). Elsevier, Amsterdam, pp 47–60Google Scholar
  37. Lam J, Breteler H, Arnason T, Hansen L (1988) Chemistry and biology of naturally occurring acetylenes and related compounds (NOARC). Proc NOARC-Conf Aarhus-Denmark. Elsevier, Amsterdam, 366 ppGoogle Scholar
  38. Metschulat G, Siitfeld R (1988) Acetyl-CoA: 4-hydroxy butinyl bithiophene o-acetyltransferase isoenzymes from Tagetes patula seedlings. Z Naturforsch 42c: 885–890Google Scholar
  39. Mihalik GJ (1978/79) Guyanese ethnomedical botany. A folk pharmacopoeia. Ethnomedicine 1/2: 83–96Google Scholar
  40. Muir WH, Hildebrandt AC, Riker AJ (1954) Plant tissue cultures produced from single isolated cells. Science 119: 877–878CrossRefGoogle Scholar
  41. Mulder-Krieger T, Verpoorte R, Water A de, Gekel M van, Oeveren BCJA van, Baerheim Svendsen A (1982) Identification of the alkaloids and anthraquinones in Cinchona ledgeriana callus cultures. Planta Med 46: 19–24PubMedCrossRefGoogle Scholar
  42. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15: 473–497CrossRefGoogle Scholar
  43. Neher RT (1968) The ethnobotany ofTagetes. Econ Bot 22: 317–325CrossRefGoogle Scholar
  44. Nester EW, Gordon MP, Amasino RM, Yanofsky MF (1984) Crown gall: a molecular and physiological analysis. Annu Rev Plant Physiol 35: 387–413CrossRefGoogle Scholar
  45. Norton RA, Finlayson AJ, Towers GHN (1985) Thiophene production by crown galls and callus tissues of Tagetes patula. Phytochemistry 24: 719–722CrossRefGoogle Scholar
  46. Offringa JA, Melchers LS, Regenburg-Tuink AJG, Constantino P, Schilperoort RA, Hooykaas PJJ (1986) Complementation of Agrobacterium tumefaciens tumor-inducing aux mutants by genes from the Tr-region of the Ri plasmid of Agrobacterium rhizogenes. Proc Natl Acad Sci 83: 6935–6939PubMedCrossRefGoogle Scholar
  47. Otten LABM, Schilperoort RA (1978) A rapid microscale method for the detection of lysopine andnopaline dehydrogenase activities. Biochim Biophys Acta 527: 497–500PubMedGoogle Scholar
  48. Pensl R, Siitfeld R (1985) Occurrence of 3,4-diacetoxybutinylbithiophene in Tagetes patula and its enzymatic conversion. Z Naturforsch 40c: 3–7Google Scholar
  49. Petit A, David C, Dahl GA, Ellis JG, Guyon P, Casse-Delbart F, Tempe J (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190: 204–214CrossRefGoogle Scholar
  50. Philogene BJR, Arnason JT, Berg CW, Duval F, Champagne D, Taylor RG, Leitch LC, Morand P (1985) Synthesis and evaluation of the naturally occurring phototoxin, Alpha-terthienyl as a control agent for larvae ofAedes intrudens, Aedes atropalpus (Diptera: Culicidae) and Simulium verecundum (Diptera: Simuliidae). J Econ Entomol 78: 121–126PubMedGoogle Scholar
  51. Quisumbing E (1951) Medicinal plants of the Philippines. Bureau of printing, Manila, 100 ppGoogle Scholar
  52. Reis Altschul S von (1973) Drugs and foods from little-known plants. Harvard Univ Press, Cambridge, MA, 366 ppGoogle Scholar
  53. Rhodes MJC, Robins RJ, Kamill JD, Parr A J, Walton NJ (1987) Secondary product formation using Agrobacterium rhizogenes transformed “hairy root” cultures. IAPTC Newslett 53: 2–15Google Scholar
  54. Sahai OP, Shuler ML (1984) Environmental parameters influencing phenolics production by batchcultures of Nicotiana tabacum. Biotech Bioeng 26: 111–120CrossRefGoogle Scholar
  55. Sanchez de Jimenez E, Fernandez L (1983) Biochemical parameters to assess cell differentiation of Bouvardia termifolia Schecht callus. Planta 158: 377–383CrossRefGoogle Scholar
  56. Slogteren GMS van, Hoge JHC, Hooykaas PJJ, Schilperoort RA (1983) Clonal analysis of heterogeneous crown gall tumor tissues induced by wild-type and shooter mutant strains of Agrobacterium tumefaciens expression of T-DNA genes. Plant Mol Biol 2: 321–333CrossRefGoogle Scholar
  57. Steiner G (1941) Nematodes parasitic on and associated with roots of marigolds (Tagetes hybrids). Proc Biol Soc Wash 54: 31–34Google Scholar
  58. Steinkopf W, Leitsmann R, Hofmann KH (1941) Studien in der Thiophenreihe. LVII. Uber a-Polythienyle. Liebigs Ann Chem 546: 180–199CrossRefGoogle Scholar
  59. Street HE, Henshaw GG, Buiatti MC (1965) The culture of isolated plant cells. Chem Ind 2 (January): 27–33Google Scholar
  60. Sütfeld R (1982) Distribution of thiophene derivatives in different organs of Tagetes patula seedlings grown under various conditions. Planta 156: 536–540CrossRefGoogle Scholar
  61. Sütfeld R (1987) HPLC of thiophenes for phytochemical and biochemical research. In: Linskens HF, Jackson JF (eds) High performance liquid chromatography in plant sciences. Springer, Berlin Heidelberg New York, pp 104–113Google Scholar
  62. Sütfeld R (1988) Enzymological investigations into the metabolism of bithiophene derivatives. In: Lam J, Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally occurring acetylenes and related compounds (NOARC), vol 7. Elsevier, Amsterdam, pp 201–208Google Scholar
  63. Sütfeld R (1989) Preparative liquid chromatography with analytical separation quality. Interval injection/displacement reversed-phase high performance liquid chromatography. J Chromatogr 464: 103–115CrossRefGoogle Scholar
  64. Sütfeld R, Breteler H (1988) Effects of plant material and extract treatment on the yield of natural products from Tagetes. In: Lam J, Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally occurring acetylenes and related compounds (NOARC). Elesevier, Amsterdam, pp 101–106Google Scholar
  65. Sütfeld R, Towers GHN (1982) 5-(4-Acetoxy-l-butinyl)-2,2’-bithiopheneacetate esterase from Tagetespatula. Phytochemistry 21: 277–279CrossRefGoogle Scholar
  66. Tang CS, Wat CK, Towers GHN (1987) Thiophenes and benzofurans in the undisturbed rhizosphere of Tagetes patula L. Plant Soil 98: 93–97CrossRefGoogle Scholar
  67. Thellier M, Desbiez MO, Champagnat P, Kergosien Y (1982) Do memory processes occur also in plants? Physiol Plant 56: 281–284CrossRefGoogle Scholar
  68. Thorpe A (1978) Regulation of organogenesis in vitro. In: Hughes KW, Henke R, Constantin M (eds) Propagation of higher plants through tissue culture. A bridge between research and application. Univ Tennessee Symp Proc Conf 7804111, 16–19 April, USD of Energy, WashingtonGoogle Scholar
  69. Uhlenbroek JH, Bijloo JD (1958) Investigations on nematicides I. Isolation and structure of a nematicidal principle occurring in Tagetes roots. Rec Trav Chim 77: 1004–1009CrossRefGoogle Scholar
  70. Uhlenbroek JH, Bijloo JD (1959) Investigations on nematicides II. Structure of a second nematicidal rinciple isolated from Tagetes roots. Rec Trav Chim 78: 382–390CrossRefGoogle Scholar
  71. Uhlenbroek JH, Bijloo JD (1960) Investigations on nematicides III. Polythienyls and related compounds. Rec Trav Chim 79: 1181–1198CrossRefGoogle Scholar
  72. Watt G (1889–1896) A dictionary of the economic products of India, 7 Volumes. Periodical Experts, Delhi, IndiaGoogle Scholar
  73. Wiermann R (1981) Secondary plant products and cell and tissue differentiation. In: Stumpf PK, Conn EE (eds) The biochemistry of plants. A comprehensive treatise, vol 7. Academic Press, New York, pp 86–116Google Scholar
  74. Wijnberg H (1988) The chemistry of a-terthienyl. In: Lam J, Breteler H, Arnason T, Hansen L (eds) Chemistry and biology of naturally-occurring acetylenes and related compounds (NOARC). Elsevier, Amsterdam, pp 21–28Google Scholar
  75. Yeoman MM, Miedzybrodzka MB, Lindsey K, McLauchlan WR (1979) The synthetic potential of cultured plant cells. In: Sala F, Parasi B, Cella R, Ciferri O (eds) Plant cell cultures: results and perspectives. Developments in plant biology, vol 5. Elsevier, Amsterdam, pp 327–348Google Scholar
  76. Zechmeister L, Sease JW (1947) A blue-fluorescing compound, terthienyl, isolated from marigolds. J Am Chem Soc 69: 273–275PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1993

Authors and Affiliations

  • H. Breteler
  • D. H. Ketel
    • 1
  1. 1.Centre for Agrobiological Research (CABO)WageningenThe Netherlands

Personalised recommendations