Molecular and General Genetics MGG

, Volume 248, Issue 6, pp 649–656 | Cite as

Transgene inactivation inPetunia hybrida is influenced by the properties of the foreign gene

  • Paula Elomaa
  • Yrjö Helariutta
  • Mika Kotilainen
  • Teemu H. Teeri
  • Robert J. Griesbach
  • Pauli Seppänen
Original Paper


Petunia mutant RL01 was transformed with maizeA1 and gerberagdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The sameAgrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, theA1 andgdfr transformants showed clearly different phenotypes. The flowers of the primaryA1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of thegdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in theA1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In thegdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while thegdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonousgdfr cDNA with a lower GC content and fewer possible methylation sites is more ‘compatible’ the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.

Key words

Transgenic plants Gene silencing Methylation GC content 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors forAgrobacterium-mediated gene transfer to plants. Nucleic Acids Res 13:4777–4788Google Scholar
  2. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  3. Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back! Bio/Technology 12:883–888Google Scholar
  4. Griesbach RJ (1993) Characterization of the flavonoids fromPetunia ×hybrida flowers expressing theA1 gene ofZea mays. Hort Sci 28:659–660Google Scholar
  5. Hain R, Stahel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Uptake, integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Mol Gen Genet 199:161–168Google Scholar
  6. Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids ofAgrobacterium tumefaciens. EMBO J 2:411–417Google Scholar
  7. Helariutta Y, Elomaa P, Kotilainen M, Seppänen P, Teeri TH (1993) Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization ofdfr expression in the corollas ofGerbera hybrida var. Regina (Compositae). Plant Mol Biol 22:183–193Google Scholar
  8. Hobbs SLA, Kpodar P, DeLong CMO (1990) The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol 15:851–864Google Scholar
  9. Hobbs SLA, Warkentin TD, DeLong CMO (1993) Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21:17–26Google Scholar
  10. 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
  11. Huits HSM, Gerats AGM, Kreike MM, Mol JNM, Koes RE (1994) Genetic control of dihydroflavonol 4-reductase gene expression inPetunia hybrida. Plant J 6:295–310Google Scholar
  12. Jones JDG, Duinsmuir P, Bedbrook J (1985) High level expression of introduced chimeric genes in regenerated transformed plants. EMBO J 4:2411–2418Google Scholar
  13. Kilby NJ, Leyser HMO, Furner IJ (1992) Promoter methylation and progressive transgene inactivation in Arabidopsis. Plant Mol Biol 20:103–112Google Scholar
  14. Van Larebeke N, Engler G, Holsters M, Van den Esacker S, Schilperoort RA, Schell J (1974) Large plasmid inAgrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252:169–170Google Scholar
  15. Linn F, Heidmann I, Saedler H, Meyer P (1990) Epigenetic changes in the expression of the maizeA1 gene inPetunia hybrida: role of numbers of integrated gene copies and state of methylation. Mol Gen Genet 222:329–336Google Scholar
  16. Matassi G, Montero LM, Salinas J, Bernardi G (1989) The isochore organization and the compositional distribution of homologous coding sequences in the nuclear genome of plants. Nucleic Acids Res 17:5273–5290Google Scholar
  17. Matzke MA, Matzke AJM (1991) Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol 16:821–830Google Scholar
  18. Matzke AJM, Neuhuber F, Park Y-D, Ambros PF, Matzke MA (1994) Homology-dependent gene silencing in transgenic plants: epistatic silencing loci containing multiple copies of methylated transgenes. Mol Gen Genet 244:219–229Google Scholar
  19. Matzke MA, Primig M, Trnovsky J, Matzke AJM (1989) Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J 8:643–649Google Scholar
  20. Meyer P, Heidmann I (1994) Epigenetic variants of a transgenic petunia line show hypermethylation in transgene DNA: an indication for specific recognition of foreign DNA in transgenic plants. Mol Gen Genet 243:390–399Google Scholar
  21. Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower color generated by transformation of a mutant with a maize gene. Nature 330:677–678Google Scholar
  22. Meyer P, Linn F, Heidmann I, Meyer ZAH, Niedenhof I, Saedler H (1992) Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Mol Gen Genet 231:345–352Google Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plantarum 15:473–498Google Scholar
  24. Pröls F, Meyer P (1992) The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA inPetunia hybrida. Plant J 2:465–475Google Scholar
  25. Salinas J, Matassi G, Montero LM, Bernardi G (1988) Compositional compartmentalization and compositional patterns in the nuclear genomes of plants. Nucleic Acids Res 16:4269–4285Google Scholar
  26. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  27. Zoubak S, Richardson JH, Rynditch A, Höllsberg P, Haller DA, Boeri E, Lever AML, Bernardi G (1994) Regional specificity of HTLV-I proviral integration in the human genome. Gene 143:155–163Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Paula Elomaa
    • 1
  • Yrjö Helariutta
    • 1
  • Mika Kotilainen
    • 1
  • Teemu H. Teeri
    • 1
  • Robert J. Griesbach
    • 2
  • Pauli Seppänen
    • 3
  1. 1.Institute of BiotechnologyUniversity of HelsinkiFinland
  2. 2.U.S. Department of AgricultureFloral/Nursery Plants Research, U.S. National ArboretumBeltsvilleUSA
  3. 3.Kemira Agro Oy, Espoo Research CentreEspooFinland

Personalised recommendations