Skip to main content
SpringerLink
Log in

Genomic stability and long-term transgene expression in poplar

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula × Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Ahuja MR (2009) Transgene stability and dispersal in forest trees. Trees 23:1125–1135

    Article  CAS  Google Scholar 

  • Ahuja MR (2011) Fate of transgenes in the forest tree genome. Tree Gen Genomes 7:221–230

    Article  Google Scholar 

  • Altpeter F, Varshney A, Abderhalden O, Douchkov D, Sautter C, Kumlehn J, Dudler R, Schweizer P (2005) Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance. Plant Mol Biol 57:271–283

    Article  PubMed  CAS  Google Scholar 

  • Alvarez Viveros MF, Inostroza-Blancheteau C, Timmermann T, González M, Arce-Johnson P (2013) Overexpression of GlyI and GlyII genes in transgenic tomato (Solanum lycopersicum Mill.) plants confers salt tolerance by decreasing oxidative stress. Mol Biol Rep 40:3281–3290

    Article  PubMed  CAS  Google Scholar 

  • Anand A, Trick HN, Gill BS, Muthukrishnan S (2003) Stable transgene expression and random gene silencing in wheat. Plant Biotech J 1:241–251

    Google Scholar 

  • Arruda P (2012) Genetically modified sugarcane for bioenergy generation. Curr Opin Biotechnol 23:315–322

    Article  PubMed  CAS  Google Scholar 

  • Bonadei M, Zelasco S, Giorcelli A, Gennaro M, Calligari P, Quattrini E, Balestrazzi A (2012) Transgene stability and agronomical performance of two transgenic Basta (R)-tolerant lines of Populus alba L. Plant Biosyst 146:33–40

    Article  Google Scholar 

  • Borejsza-Wysocka E, Norelli JL, Aldwinckle HS, Malnoy M (2010) Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period. BMC Biotech 10:41

    Article  Google Scholar 

  • Brunner AM, Li JY, DiFazio SP, Shevchenko O, Montgomery BE, Mohamed R, Wei H, Ma C, Elias AA, VanWormer K, Strauss SH (2007) Genetic containment of forest plantations. Tree Gen Genomes 3:75–100

    Article  Google Scholar 

  • Dalakouras A, Moser M, Boonrod K, Krczal G, Wassenegger M (2011) Diverse spontaneous silencing of a transgene among two Nicotiana species. Planta 234:699–707

    Article  PubMed  CAS  Google Scholar 

  • Fladung M (1999) Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure. Mol Gen Genet 260:574–581

    Article  PubMed  CAS  Google Scholar 

  • Fladung M, Ahuja MR (1995) Sandwich method for non-radioactive hybridization. Biotechniques 18:800–802

    PubMed  CAS  Google Scholar 

  • Fladung M, Hoenicka H (2012) Fifteen years of forest tree biosafety research in Germany. Iforest 5:126–130

    Article  Google Scholar 

  • Fladung M, Kumar S (2002) Gene stability in transgenic aspen-Populus. III. T-DNA repeats influence transgene expression differentially among different transgenic lines. Plant Biol 4:329–338

    Article  CAS  Google Scholar 

  • Fladung M, Muhs HJ, Ahuja MR (1996) Morphological changes in transgenic Populus carrying the rolC gene from Agrobacterium rhizogenes. Silv Genet 45:349–354

    Google Scholar 

  • Fladung M, Kumar S, Ahuja MR (1997) Genetic transformation of Populus genotypes with different chimeric gene constructs: transfromation efficiency and molecular analysis. Transgen Res 6:111–121

    Article  CAS  Google Scholar 

  • Hawkins S, Leple JC, Cornu D, Jouanin L, Pilate G (2003) Stability of transgene expression in poplar: a model forest tree species. Ann For Sci 60:427–438

    Article  Google Scholar 

  • Hoenicka H, Fladung M (2006) Biosafety in Populus spp. and other forest trees: from non-native species to taxa derived from traditional breeding and genetic engineering. Trees 20:131–144

    Article  Google Scholar 

  • Kumar S, Fladung M (2000) Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Mol Gen Genet 264:20–28

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Fladung M (2001) Gene stability in transgenic aspen (Populus). II. Molecular characterization of variable expression of transgene in wild and hybrid aspen. Planta 213:731–740

    Article  PubMed  CAS  Google Scholar 

  • Kumar S, Fladung M (2002) Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration. Plant J 31:543–551

    Article  PubMed  CAS  Google Scholar 

  • Lachance D, Hamel LP, Pelletier F, Valero J, Bernier-Cardou M, Chapman K, Seguin A (2007) Expression of a Bacillus thuringiensis cry1Ab gene in transgenic white spruce and its efficacy against the spruce budworm (Choristoneura fumiferana). Tree Gen Genomes 3:153–167

    Article  Google Scholar 

  • Lardet L, Leclercq J, Benistan E, Dessailly F, Oliver G, Martin F, Montoro P (2011) Variation in GUS activity in vegetatively propagated Hevea brasiliensis transgenic plants. Plant Cell Rep 30:1847–1856

    Article  PubMed  CAS  Google Scholar 

  • Larkin PJ, Scowcroft WR (1981) Somaclonal variation: a novel source of variability from cell-cultures for plant improvement. Theor Appl Genet 60:197–214

    Article  Google Scholar 

  • Latham JR, Wilson AK, Steinbrecher RA (2006) The mutational consequences of plant transformation. J Biomed Biotech 2006:25376

    Article  Google Scholar 

  • Li J, Brunner AM, Meilan R, Strauss SH (2008a) Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons. Tree Physiol 29:299–312

    Article  PubMed  Google Scholar 

  • Li J, Meilan R, Ma C, Barish M, Strauss SH (2008b) Stability of herbicide resistance over 8 years of coppice in field-grown, genetically engineered poplars. West J Appl For 23:89–93

    Google Scholar 

  • Maghuly F, AdC Machado, Leopold S, Khan MA, Katinger H, Laimer M (2007) Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species. J Biotech 127:310–321

    Article  CAS  Google Scholar 

  • Markussen T, Tusch A, Stephan BR, Fladung M (2004) Identification of molecular markers for selected wood properties of Norway spruce Picea abies L. (Karst.) I. Wood density. Silv Genet 53:45–50

    Google Scholar 

  • Masclaux FG, Pont-Lezica R, Galaud JP (2005) Relationship between allelic state of T-DNA and DNA methylation of chromosomal integration region in transformed Arabidopsis thaliana plants. Plant Mol Biol 58:295–303

    Article  PubMed  CAS  Google Scholar 

  • Meilan R, Auerbach DJ, Ma C, DiFazio SP, Strauss SH (2002) Stability of herbicide resistance and GUS expression in transgenic hybrid poplars (Populus sp.) during four years of field trials and vegetative propagation. HortScience 37:277–280

    CAS  Google Scholar 

  • Meng L, Ziv M, Lemaux PG (2006) Nature of stress and transgene locus influences transgene expression stability in barley. Plant Mol Biol 62(1–2):15–28

    Article  PubMed  CAS  Google Scholar 

  • Ming R, Hou SB, Feng Y, Yu QY, Dionne-Laporte A, Saw JH, Alam M (2008) The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature 452:991–997

    Article  PubMed  CAS  Google Scholar 

  • Mirza B (2005) Influence of the nature of the T-DNA insertion region on transgene expression in Arabidopsis thaliana. Genetika 411601–411607

  • Pons E, Peris JE, Peña L (2012) Field performance of transgenic citrus trees: assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics. BMC Biotechnol 12–41

  • Saika H, Nonaka S, Osakabe K, Toki S (2012) Sequential monitoring of transgene expression following Agrobacterium-mediated transformation of rice. Plant Cell Physiol 53:1974–1983

    Article  PubMed  CAS  Google Scholar 

  • Schmulling T, Fladung M, Grossmann K, Schell J (1993) Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single Rol genes of Agrobacterium rhizogenes T-DNA. Plant J 3:371–382

    Article  Google Scholar 

  • Spena A, Schmulling T, Koncz C, Schell JS (1987) Independent and synergistic activity of Rol-A, Rol-B and Rol-C loci in stimulating abnormal growth in plants. EMBO J 6:3891–3899

    PubMed  CAS  Google Scholar 

  • Srivastava V (2013) Site-specific gene integration in rice. Methods Mol Biol 956:83–93

    Article  PubMed  Google Scholar 

  • Vanyushin BF, Ashapkin VV (2011) DNA methylation in higher plants: past, present and future. Biochim Biophys Acta 1809:360–368

    Article  PubMed  CAS  Google Scholar 

  • Vos P, Hogers R, Bleeker M, Reijans M, Vandelee T, Hornes M, Zabeau M (1995) AFLP: a new technique for DNA-fingerprinting. Nucl Acid Res 23:4407–4414

    Article  CAS  Google Scholar 

  • Walter C, Fladung M, Boerjan W (2010) The 20-year environmental safety record of GM trees. Nature Biotech 28:656–658

    Article  CAS  Google Scholar 

  • Wei A, He C, Li B, Li N, Zhang J (2011) The pyramid of transgenes TsVP and BetA effectively enhances the drought tolerance of maize plants. Plant Biotechnol J 9:216–229

    Article  PubMed  CAS  Google Scholar 

  • Wilson A, Latham J, Steinbrecher R (2005) Regulatory regimes for transgenic crops. Nature Biotech 23:785

    Article  CAS  Google Scholar 

  • Yao Q, Cong L, Chang JL, Li KX, Yang GX, He GY (2006) Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. J Exp Bot 57:3737–3746

    Article  PubMed  CAS  Google Scholar 

  • Zeng FS, Zhan YG, Zhao HC, Xin Y, Qi FH, Yang CP (2010a) Molecular characterization of T-DNA integration sites in transgenic birch. Trees 24:753–762

    Article  CAS  Google Scholar 

  • Zeng FS, Qian J, Luo W, Zhan YG, Xin Y, Yang CP (2010b) Stability of transgenes in long-term micropropagation of plants of transgenic birch (Betula platyphylla). Biotech Lett 32:151–156

    Article  CAS  Google Scholar 

  • Zeng FS, Xin Y, Li B, Zhan YG, Yang CP (2011) The stability of transgene expression and effect of DNA methylation on post transcriptional gene silencing (PTGS) in birch. Afr J Biotech 10:8188–8193

    CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by the Federal Ministry for Education and Research (BMBF). We deeply thank Dr. Trevor Fenning (Northern Research Station of Forest Research, Roslin, UK) for critically reading the manuscript. The technical assistance of Olaf Polak, Doris Ebbinghaus and Anke Schellhorn is gratefully acknowledged.

Author information

Author notes

  1. M. Raj Ahuja

    Present address: , 60 Shivertown Road, New Paltz, NY, 12561, USA

Authors and Affiliations

  1. Thuenen-Institute (TI) of Forest Genetics, Sieker Landstrasse 2, 22927, Grosshansdorf, Germany

    Matthias Fladung, Hans Hoenicka & M. Raj Ahuja

Authors
  1. Matthias Fladung
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hans Hoenicka
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Raj Ahuja
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthias Fladung.

Additional information

The experiments reported comply with the current law of Germany.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fladung, M., Hoenicka, H. & Raj Ahuja, M. Genomic stability and long-term transgene expression in poplar. Transgenic Res 22, 1167–1178 (2013). https://doi.org/10.1007/s11248-013-9719-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-013-9719-2

Keywords

Search

Navigation