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Plant Biol (Stuttg) 2002; 4(3): 329-338
DOI: 10.1055/s-2002-32329
Original Paper
Georg Thieme Verlag Stuttgart ·New York

Gene Stability in Transgenic Aspen-Populus - III. T-DNA Repeats Influence Transgene Expression Differentially among Different Transgenic Lines

M. Fladung, S. Kumar
  • Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics and Forest Tree Breeding, Grosshansdorf, Germany
Further Information

Publication History

December 21, 2001

February 6, 2002

Publication Date:
20 June 2002 (online)

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Abstract

Transgene silencing mechanisms have been confirmed in many crop plants, however, few reports are currently available for long-lived tree species. Three transgenic aspen lines carrying the rolC gene of Agrobacterium rhizogenes, under control of the 35S promoter of the cauliflower mosaic virus, but showing very different types of reversion from rolC-phenotype to wild type are investigated in detail. A complete suppression of rolC phenotype was observed after one year of in vitro culturing in one transgenic line, whereas the second transgenic line showed gradual decrease in rolC expression, and the plants were comparable to control plants after three to four years in the greenhouse. In the third transgenic line, however, transgene inactivation was observed only in some shoots of the greenhouse-grown plants which reverted to the rolC phenotype in subsequent years. Southern blotting experiments using a rolC probe revealed the integration of one or two tandem copies of the foreign genes into the host genome. All three transgenic lines contain in/complete T-DNA repeats, as indicated by reverse primer (rp) PCR. In the reverted tissues of two transgenic lines loss of rolC protein was observed despite the presence of the rolC transcript. The third transgenic line, showing a completely suppressed rolC phenotype, was found positive for the rolC transcript and protein. On the basis of the molecular data presented for the three unstable lines, including Southern, Northern and Western blotting experiments, as well as a survey of transgenic methylation patterns, new insights were obtained with respect to structure and inactivation of transgenes following their transfer in a host tree species.

Key words

Instability - methylation - reversion - silencing - transgene insertion

References

  • 01 Chuang,  C. H., and Meyerowitz,  E. M.. (2000);  Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. .  Proc. Natl. Acad. Sci. USA. 97 4985-4990
    Search in Google Scholar
  • 02 Cogoni,  C., and Macino,  G.. (1999);  Posttranscriptional gene silencing in Neurospora by RecQ DNA helicase.  Science. 286 342-344
    Search in Google Scholar
  • 03 De Neve,  M.,, De Buck,  S.,, Jacobs,  A.,, Van Montagu,  M.,, and Depicker,  A.. (1997);  T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs.  Plant J.. 11 15-29
    Search in Google Scholar
  • 04 Filipowicz,  W., and Hohn,  T.. (1996) Post-transcriptional control of gene expression in plants. Dordrecht, Boston, London; Kluwer Academic Publishers
    Search in Google Scholar
  • 05 Fladung,  M.. (1990);  Transformation of diploid and tetraploid potato clones with the rolC gene of Agrobacterium rhizogenes and characterization of transgenic plants.  Plant Breeding. 104 295-304
    Search in Google Scholar
  • 06 Fladung,  M.. (1996);  Transgenic potato plants can be used to evaluate the stability of foreign genes: reversions and chimeras in multiple copies of rolC harboring clones.  Agronomie. 16 113-121
    Search in Google Scholar
  • 07 Fladung,  M.. (1999 a);  Gene stability in transgenic aspen-Populus. I. Flanking DNA sequences and T-DNA structure.  Mol. Gen. Genet.. 260 574-581
    Search in Google Scholar
  • 08 Fladung,  M.. (1999 b) Prospects for the isolation of genes controlling tree-specific traits by using transposon tagging approach. Plant Biotechnology and In Vitro Biology in the 21st century. Altman, A., Ziv, M., and Izhar, S., eds. Netherlands; Kluwer Academic Publishers pp. 139-142
    Search in Google Scholar
  • 09 Fladung,  M., and Ahuja,  M. R.. (1995);  “Sandwich” method for non-radioactive hybridizations.  Biotechniques. 18 3-5
    Search in Google Scholar
  • 10 Fladung,  M., and Ahuja,  M. R.. (1997);  Excision of the maize transposable element Ac in periclinal leaves of 35S-Ac-rolC transgenic aspen-Populus. .  Plant Mol. Biol.. 33 1097-1103
    CrossrefPubMedSearch in Google Scholar
  • 11 Fladung,  M., and Ballvora,  A.. (1992);  Further characterization of rolC transgenic tetraploid potato clones, and influence of daylength and level of rolC expression on yield parameters.  Plant Breeding. 109 18-27
    PubMedSearch in Google Scholar
  • 12 Fladung,  M.,, Muhs,  H. J.,, and Ahuja,  M. R.. (1996);  Morphological changes observed in transgenic Populus carrying the rolC gene from Agrobacterium rhizogenes. .  Silvae Genetica. 45 349-354
    PubMedSearch in Google Scholar
  • 13 Fladung,  M.,, Großmann,  K.,, and Ahuja,  M. R.. (1997 a);  Alterations in hormonal and developmental characteristics in transgenic Populus conditioned by the rolC gene from Agrobacterium rhizogenes. .  J. Plant Physiol.. 150 420-427
    PubMedSearch in Google Scholar
  • 14 Fladung,  M.,, Kumar,  S.,, and Ahuja,  M. R.. (1997 b);  Genetic transformation of Populus genotypes with different chimeric gene constructs: Transformation efficiency and molecular analysis.  Trans. Res.. 6 111-121
    PubMedSearch in Google Scholar
  • 15 Flavell,  R. B.. (1994);  Inactivation of gene expression in plants as a consequence of specific sequence duplication.  Proc. Nat. Acad. Sci. USA. 91 3490-3496
    PubMedSearch in Google Scholar
  • 16 Hobbs,  S. L. A.,, Kpodar,  P.,, and DeLong,  C. M. O.. (1990);  The effect of T- DNA copy number, position and methylation on reporter gene expression in tobacco transformants.  Plant Mol. Biol.. 15 851-864
    CrossrefPubMedSearch in Google Scholar
  • 17 Hobbs,  S. L. A.,, Warkentin,  T. D.,, and DeLong,  C. M. O.. (1993);  Transgene copy number can be positively or negatively associated with transgene expression.  Plant Mol. Biol.. 21 17-26
    CrossrefPubMedSearch in Google Scholar
  • 18 Kaendler,  C.,, Fladung,  M.,, and Uhrig,  H.. (1996);  Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as morphological marker.  Theor. Appl. Genet.. 92 455-462
    CrossrefPubMedSearch in Google Scholar
  • 19 Kilby,  N. J.,, Leyser,  H. M. O.,, and Furner,  I. J.. (1992);  Promoter methylation and progressive transgene inactivation in Arabidopsis. .  Plant Mol. Biol.. 20 103-112
    CrossrefPubMedSearch in Google Scholar
  • 20 Kohli,  A.,, Griffiths,  S.,, Palacios,  N.,, Twyman,  R. M.,, Vain,  P.,, Laurie,  D. A.,, and Christou,  P.. (1999);  Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination.  Plant J.. 17 591-601
    CrossrefPubMedSearch in Google Scholar
  • 21 Kooter,  J. M.,, Matzke,  M. A.,, and Meyer,  P.. (1999);  Listening to the silent genes: transgene silencing, gene regulation and pathogen control.  Trends Plant Sci.. 4 340-347
    CrossrefPubMedSearch in Google Scholar
  • 22 Krizkova,  L., and Hrouda,  M.. (1998);  Direct repeats of T-DNA integrated in tobacco chromosome: characterisation of junctions region.  Plant J.. 16 673-680
    CrossrefPubMedSearch in Google Scholar
  • 23 Kumar,  S., and Fladung,  M.. (2000 a);  Determination of transgene repeat formation and promoter methylation in transgenic plants.  BioTechniques. 28 1128-1137
    PubMedSearch in Google Scholar
  • 24 Kumar,  S., and Fladung,  M.. (2000 b);  Transgene repeats in aspen: molecular characterization suggests simultaneous integration of independent T-DNAs into a receiptive spot of host genome.  Mol. Gen. Genet.. 264 20-28
    PubMedSearch in Google Scholar
  • 25 Kumar,  S., and Fladung,  M.. (2001);  Gene stability in transgenic aspen (Populus). II. Molecular chracterization of variable expression of transgene in wild and hybrid aspen.  Planta. 213 731-740
    CrossrefPubMedSearch in Google Scholar
  • 26 Linn,  F.,, Heidmann,  I.,, Saedler,  H.,, and Meyer,  P.. (1990);  Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation.  Mol. Gen. Genet.. 222 329-336
    PubMedSearch in Google Scholar
  • 27 Maniatis,  T.,, Fritsch,  E. F.,, Sambrook,  J.. (1989) Molecular Cloning, a Laboratory Manual. New York; Cold Spring Harbor Laboratory Press
    Search in Google Scholar
  • 28 Matzke,  M. A., and Matzke,  A. J. M.. (1993);  Genomic imprinting in plants - parental effects and trans-inactivation phenomena.  Ann. Rev. Plant Physiol.. 44 53-76
    PubMedSearch in Google Scholar
  • 29 Matzke,  M. A., and Matzke,  A. J. M.. (1995);  How and why do plants inactivate homologous (trans)genes?.  Plant Physiol.. 107 679-685
    PubMedSearch in Google Scholar
  • 30 Matzke,  M. A.,, Priming,  M.,, Trnovshy,  J.,, and Matzke,  A. J. M.. (1989);  Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants.  EMBO J.. 8 643-649
    PubMedSearch in Google Scholar
  • 31 Matzke,  A. J. M.,, Neuhuber,  F.,, Park,  Y. D.,, Ambros,  P. F.,, and Matzke,  M. A.. (1994);  Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes.  Mol. Gen. Genet.. 244 219-229
    PubMedSearch in Google Scholar
  • 32 Meins,  F.. (2000);  RNA degradation and models for posttranscriptional gene silencing.  Plant Mol. Biol.. 43 261-273
    CrossrefPubMedSearch in Google Scholar
  • 33 Mette,  M. F.,, Winden,  J. V. D.,, Matzke,  M. A.,, and Matzke,  A. J. M.. (1999);  Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans. .  EMBO J.. 18 241-248
    CrossrefPubMedSearch in Google Scholar
  • 34 Mette,  M. F.,, Aufsatz,  W.,, Van der Winden,  J.,, Matzke,  M. A.,, and Matzke,  A. J. M.. (2000);  Transcriptional silencing and promoter methylation triggered by double-stranded RNA.  EMBO J.. 19 5194-5201
    CrossrefPubMedSearch in Google Scholar
  • 35 Meyer,  P.,, Heidmann,  I.,, and Niedenhof,  I.. (1993);  Differences in DNA-methylation are associated with a paramutation phenomenon in transgenic petunia.  Plant J.. 13 2084-2088
    PubMedSearch in Google Scholar
  • 36 Mittelsten Scheid,  O.,, Afsar,  K.,, and Paszkowski,  J.. (1998);  Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis. .  Proc. Nat. Acad. Sci. USA. 95 632-637
    CrossrefPubMedSearch in Google Scholar
  • 37 Morino,  K.,, Olsen,  O.-A.,, and Shimamoto,  K.. (1999);  Silencing of an aleurone-specific gene in transgenic rice is caused by rearranged transgene.  Plant J.. 17 275-285
    CrossrefPubMedSearch in Google Scholar
  • 38 Park,  Y.-D.,, Papp,  I.,, Moscone,  E. A.,, Iglesias,  V. A.,, Vaucheret,  H.,, Matzke,  A. J. M.,, and Matzke,  M. A.. (1996);  Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity.  Plant J.. 9 183-194
    CrossrefPubMedSearch in Google Scholar
  • 39 Plasterk,  R. H. A., and Ketting,  R. F.. (2000);  The silence of the genes.  Curr. Opin. Genet. Dev.. 10 562-567
    CrossrefPubMedSearch in Google Scholar
  • 40 Schmülling,  T., and Röhrig,  H.. (1995);  Gene silencing in transgenic tobacco hybrids: frequency of the event and visualisation of somatic inactivation pattern.  Mol. Gen. Genet.. 249 375-390
    PubMedSearch in Google Scholar
  • 41 Schmülling,  T., and Schell,  J.. (1993);  Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras.  Plant Mol. Biol.. 21 705-708
    CrossrefPubMedSearch in Google Scholar
  • 42 Spena,  A.,, Schmülling,  T.,, Koncz,  C.,, and Schell,  J.. (1987);  Independent and synergistic activity of rol A, B and C loci in stimulating abnormal growth in plants.  EMBO J.. 6 3891-3899
    PubMedSearch in Google Scholar
  • 43 Spena,  A.,, Aalen,  R. B.,, and Schulze,  S. C.. (1989);  Cell autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants.  Plant Cell. 1 1157-1164
    CrossrefPubMedSearch in Google Scholar
  • 44 Stam,  M.,, Mol,  J. N. M.,, and Kooter,  J. M.. (1997 a);  The silence of genes in transgenic plants.  Ann. Bot.. 79 3-12
    CrossrefPubMedSearch in Google Scholar
  • 45 Stam,  M.,, de Bruin,  R.,, Kenter,  S.,, van der Hoorn,  R. A. L.,, Blokland,  R.,, Mol,  J. N. M.,, and Kooter,  J. M.. (1997 b);  Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats.  Plant J.. 12 63-82
    CrossrefPubMedSearch in Google Scholar
  • 46 Van Houdt,  H.,, Ingelbrecht,  I.,, Van Montagu,  M.,, and Depicker,  A.. (1997);  Post-transcriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3′ flanking regions.  Plant J.. 12 379-392
    CrossrefPubMedSearch in Google Scholar
  • 47 Vaucheret,  H.. (1993);  Identification of a general silencer for 19S and 35S promoters in a transgenic plant: 90 bp of homology in the promoter sequence are sufficient for inactivation.  CR Acad. Sci. Paris, Life Sci.. 316 1471-1483
    PubMedSearch in Google Scholar
  • 48 Vaucheret,  H., and Fagarad,  M.. (2001);  Transcriptional gene silencing in plants: targets, inducers and regulators.  Trends in Genetics. 17 29-35
    CrossrefPubMedSearch in Google Scholar

M. Fladung

Federal Research Centre for Forestry and Forest Products
Institute for Forest Genetics and Forest Tree Breeding

Sieker Landstraße 2
22927 Grosshansdorf
Germany

Email: mfladung@uni-hamburg.de

Section Editor: H. Rennenberg