Plant Cell, Tissue and Organ Culture

, Volume 74, Issue 1, pp 73–80 | Cite as

Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus

  • Serap Whitmer
  • Camilo Canel
  • Robert van der Heijden
  • Robert Verpoorte
Article

Abstract

The productivity of several transgenic cell lines of Catharanthus roseus was monitored over a period of 30 months. The transgenic cultures were obtained by Agrobacterium-mediated transformation of leaf explants with constructs containing recombinant versions of the endogenous Str and Tdc genes, which, respectively, encode strictosidine synthase (STR) and tryptophan decarboxylase (TDC). The expression of these transgenes and the β-glucuronidase marker gene were also measured periodically, at the enzymatic level, during this time. Cultures were maintained in selective medium containing either hygromycin or kanamycin and showed GUS activity in the presence of X-gluc, indicating that they carried functional transgenes. The activities of STR and TDC varied greatly over time, occasionally falling to levels not significantly different from those of non-transgenic cultures, and showed susceptibility to the composition of the culture medium. Despite maintaining their transgenic character, the cell lines gradually lost the ability to accumulate terpenoid indole alkaloids (TIAs). The diversity of alkaloids produced was also negatively affected by long-term subculture. We conclude that a strategy of indirect selection, such as the use of antibiotic-resistance genes, is insufficient to maintain the concerted expression of TIA-pathway elements necessary for high productivity.

cell culture genetic engineering secondary metabolism strictosidine synthase terpenoid indole alkaloids transgene expression tryptophan decarboxylase 

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References

  1. Canel C (1999) From genes to phytochemicals: the genomics approach to the characterization and utilization of plant secondary metabolism. Acta Hort. 500: 51-58Google Scholar
  2. Canel C, Lopes Cardoso MI, Whitmer S, van der Fits L, Pasquali G, van der Heijden R, Hoge JHC & Verpoorte R (1998) Effects of over expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta 205: 414-419Google Scholar
  3. Deus-Neumann B & Zenk MH (1984) Instability of indole alkaloid production in Catharanthus roseus cell suspension cultures. Planta Med. 50: 427-431Google Scholar
  4. Galneder E & Zenk MH (1990) Enzymology of alkaloid production in plant cell cultures. In: Nijkamp HJJ, van der Plas LHW & van Aartrijk J (eds) Progress in Plant Cellular and Molecular Biology (pp. 754-762). Kluwer Academic Publishers, Dordrecht, The NetherlandsGoogle Scholar
  5. Grotewold E, Chamberlin M, Snook M, Siame B, Butler L, Swenson J, Maddock S, St Clair G & Bowen B (1998) Engineering secondary metabolism in maize cells by ectopic expression of transcription factors. Plant Cell 10: 721-740Google Scholar
  6. Hirotani M & Furuya T (1977) Restoration of cardenolide synthesis in redifferentiated shoots from callus of Digitalis purpurea. Phytochemistry 16: 610-611Google Scholar
  7. Holden MA, Holden PR & Yeoman MM (1988) Elicitation of secondary product formation in Capsicum frutescens cultures. In: Robins RJ & Rhodes MJC (eds) Manipulating Secondary Metabolism in Culture (pp. 57-65). Cambridge University Press, CambridgeGoogle Scholar
  8. Kim SW, Jung KH, Kwak SS & Liu JR (1994) Relationship between cell morphology and indole alkaloid production in suspension cultures of Catharanthus roseus. Plant Cell Rep. 14: 23-26Google Scholar
  9. Morris P (1986) Long term stability of alkaloid production in cell suspension cultures of Catharanthus roseus. In: Morris P, Scragg AH, Stafford A & Fowler MW (eds) Secondary Metabolism in Plant Cell Cultures (pp. 257-262). Cambridge University Press, CambridgeGoogle Scholar
  10. Morris P, Rudge K, Cresswell R & Fowler MW(1989) Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium. Plant Cell Tiss. Org. Cult. 17: 79-90Google Scholar
  11. Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473-497Google Scholar
  12. Ohta S & Verpoorte R (1992) Some accounts of variation (heterogeneity and/or instability) in secondary metabolites production by plant cell cultures. Ann. Rep. Nat. Sci. Home. Econ. 32: 9-23Google Scholar
  13. Pennings EJM, Hegger I, van der Heijden R, Duine JA & Verpoorte R (1987) Assay of tryptophan decarboxylase from Catharanthus roseus cell cultures by high-performance liquid chromatography. Anal. Biochem. 165: 33-136Google Scholar
  14. Pennings EJM, van den Bosch RA, van der Heijden R, Stevens LH, Duine JA & Verpoorte R (1989) Assay of strictosidine synthase from plant cells by high-performance liquid chromatography. Anal. Biochem. 176: 412-415Google Scholar
  15. Sato F & Yamada Y (1984) High berberine producing cultures of Coptis japonica cells. Phytochemistry 23: 281-285Google Scholar
  16. Schripsema J & Verpoorte R (1992) Search for factors involved in indole alkaloid production in cell suspension cultures of Tabernaemontana divaricata. Planta Med. 58: 245-249Google Scholar
  17. Sierra MI, van der Heijden R, van der Leer T & Verpoorte R (1992) Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during long-term subculture. Plant Cell Tiss. Org. Cult. 28: 59-68Google Scholar
  18. Tabata M & Hiraoka N (1976) Variation in alkaloid production in Nicotiana rustica callus cultures. Physiol. Plant 38: 19-23Google Scholar
  19. Tabata M, Mizukami H, Hiraoka N & Konoshima M (1974) Pigment formation in callus cultures of Lithospermum erythrorhizon. Phytochemistry 13: 927-937Google Scholar
  20. Van der Heijden R, Lamping PJ, Wijnsma R & Verpoorte R (1987) High-performance liquid chromatographic determination of in dole alkaloids in a suspension cell culture of Tabernaemontana divaricata. J. Chromatogr. 396: 287-295Google Scholar
  21. Verpoorte R, van der Heijden R & Memelink J (1998) Plant biotechnology and the production of alkaloids: prospects of metabolic engineering. In: Cordell GA (ed) The Alkaloids Vol. 50 (pp. 453-508). Academic Press, San DiegoGoogle Scholar
  22. 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 (pp. 221-299). Academic Press, San DiegoGoogle Scholar
  23. Von Bodman SB, Domier LL & Farrand SK (1995) Expression of multiple eukaryotic genes from a single promoter in Nicotiana. Biotechnology 13: 587-591Google Scholar
  24. Whitmer S, van der Heijden R & Verpoorte R (2002a) Effect of precursor feeding on alkaloid accumulation by strictosidine synthase over expressing transgenic cell line S1 of Catharanthus roseus. Plant Cell Tiss. Org. Cult. 69: 85-93Google Scholar
  25. Whitmer S, van der Heijden R & Verpoorte R (2002b) Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase overexpressing transgenic cell line T22 of Catharanthus roseus J. Biotechnol. 96: 193-203Google Scholar
  26. Whitmer S, Verpoorte R & Canel C (1998) Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus. Plant Cell Tiss. Org. Cult. 53: 135-141Google Scholar
  27. Yeoman MM & Yeoman CL (1996) Tansley Review No 90 Manipulating secondary metabolism in cultured plant cells. New Phytol. 134: 553-569Google Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • Serap Whitmer
    • 1
  • Camilo Canel
    • 2
  • Robert van der Heijden
    • 1
  • Robert Verpoorte
    • 1
  1. 1.Leiden/Amsterdam Center for Drug Research, Division of Pharmacognosy, Gorlaeus LaboratoriesLeiden UniversityLeidenThe Netherlands
  2. 2.Corning Life Sciences, BF-01CorningUSA

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