Molecular and General Genetics MGG

, Volume 220, Issue 2, pp 245–250 | Cite as

Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation

  • G. Vancanneyt
  • R. Schmidt
  • A. O'Connor-Sanchez
  • L. Willmitzer
  • M. Rocha-Sosa


Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the β-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.

Key words

Agrobacterium tumefaciens β-glucuronidase Portable intron Splicing Transformation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Amasino RM (1986) Acceleration of nucleic hybridisation rate by polyethylene glycol. Anal Biochem 152:304–307Google Scholar
  2. Bartha A, Sommergruber K, Thompson D, Hartmuth K, Matzke MA, Matzke AJM (1986) The expression of a nopaline synthase — human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Mol Biol 6:347–357Google Scholar
  3. Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721Google Scholar
  4. Bradford MM (1979) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  5. Brown JWS (1986) A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acids Res 14:9549–9559Google Scholar
  6. Brown JWS, Feix G, Frendewey D (1986) Accurate in vitro splicing of two pre-mRNA plant introns in a Hela cell nuclear extract. EMBO J 5:2749–2758Google Scholar
  7. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montag M, Leemans J (1985) Efficient octopine Ti plasmidderived vectors for Agrobacterium-mediated gene transfer. Nucleic Acids Res 13:4777–4788Google Scholar
  8. Eckes P, Rosahl S, Schell J, Willmitzer L (1986) Isolation and characterisation of a light-inducible, organ-specific gene from potato and the analysis of its expression after tagging and transfer into tobacco and potato shoots. Mol Gen Genet 199:216–224Google Scholar
  9. Harmuth K, Bartha A (1986) In vitro processing of a plant premRNA in a Hela cell nuclear extract. Nucleic Acids Res 14:7513–7529Google Scholar
  10. Hawkins JD (1988) A survey on intron and exon lengths. Nucleic Acids Res 16:9893–9908Google Scholar
  11. Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877Google Scholar
  12. Horsch RB, Klee HJ (1986) Rapid assay of foreign gene expression in leaf disc transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process. Proc Natl Acad Sci USA 83:4428–4432Google Scholar
  13. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231Google Scholar
  14. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
  15. Jefferson RA, Kavanagh TA, Bevan M (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907Google Scholar
  16. Kingston RE (1987) In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology, pp 4.8.1–4.8.3Google Scholar
  17. Klee H, Horsch R, Rogers S (1987) Agrobacterium-mediated plant transformation and its further applications to plant biology. Annu Rev Plant Physiol 38:467–486Google Scholar
  18. Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:21–26Google Scholar
  19. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning — a laboratory manual. Cold ptring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  20. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase. Science 239:487–491Google Scholar
  21. Schell J (1987) Transgenic plants as tools to study the molecular organisation of plant genes. Science 237:1176–1183Google Scholar
  22. Schmidt R, Willmitzer L (1988) High efficienty Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants. Plant Cell Rep 7:583–586Google Scholar
  23. Shapiro MB, Senepathy P (1987) RNA splice junctions of different classes of eucaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174Google Scholar
  24. Töpfer R, Pröls M, Schell J, Steinbiss H-H (1987) A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res 15:5890Google Scholar
  25. Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540Google Scholar
  26. Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterisation of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48Google Scholar
  27. Wiebauer K, Herrero J-J, Filipowicz W (1988) Nuclear pre-mRNA processing in plants: Distinct modes of 3′-splice-site selection in plants and animals. Mol Cell Biol 8:2042–2051Google Scholar
  28. Zambrisky P, Tempé J, Schell J (1989) Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell 56:193–201Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • G. Vancanneyt
    • 1
  • R. Schmidt
    • 1
  • A. O'Connor-Sanchez
    • 1
  • L. Willmitzer
    • 1
  • M. Rocha-Sosa
    • 1
  1. 1.Institut für Genbiologische ForschungBerlin 33

Personalised recommendations