Skip to main content
SpringerLink
Log in

Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the β-glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.

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. 1A–C
Fig. 2
Fig. 3A, B
Fig. 4A, B
Fig. 5
Fig. 6A, B

Similar content being viewed by others

References

  • Aitken-Christie J, Gough K, Maddocks D, Sigley M, Burger F, Carter PCS (1994) Towards commercialisation of conifer embryogenesis. In: Proc 2nd Int Symp Applic Biotechnol Tree Cult Protect Utilization. USDA-For Serv Gen Tech Rep NC-175:181–190

  • Aitken-Christie J, Singh AP, Davies H (1988) Multiplication of meristematic tissue: A new tissue culture system for radiata pine. In: Hanover JW, Keathley DE (eds) Genetic manipulation of woody plants. Plenum, New York, pp 413–432

  • Aronen TS (1997) Interactions between Agrobacterium tumefaciens and coniferous defence compounds α-pinene and trans-stilbene. Eur J For Pathol 27:55–67

    Google Scholar 

  • Birch RG (1997) Plant transformation: problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol 48:297–326

    CAS  Google Scholar 

  • Brukhin V, Clapham D, Elfstrand M, von Arnold S (2000) Basta tolerance as a selectable and screening marker for transgenic plants of Norway spruce. Plant Cell Rep 19:899–903

    Article  CAS  Google Scholar 

  • Campbell MA, Kinlaw CS, Neale DB (1992) Expression of luciferase and β-glucuronidase in Pinus radiata suspension cells using electroporation and particle bombardment. Can J For Sci 22:2014–2018

    CAS  Google Scholar 

  • Charest PA, Devantier Y, Lachance D (1996) Stable genetic transformation of Picea mariana (black spruce) via particle bombardment. In Vitro Cell Dev Biol Plant 32:91–99

    Google Scholar 

  • Charity JA, Holland L, Donaldson SS, Grace L, Walter C (2002) Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration. Plant Cell Tissus Organ Cult 70:51–60

    Article  CAS  Google Scholar 

  • Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33

    Article  CAS  Google Scholar 

  • De Buck S, Jacobs A, Van Montagu M, Depicker A (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J 20:295–304

    PubMed  Google Scholar 

  • Deroles S, Gardner RC (1988) Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11:365–377

    CAS  Google Scholar 

  • Doyle J, Doyle J (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Ellis DD, McCabe DE, McInnis S, Ramachandran R, Russell DR, Wallace KM, Martinell BJ, Roberts DR, Raffa KF, McGown BH (1993) Stable transformation of Picea glauca by particle acceleration. Biotechnology 11:84–89

    Google Scholar 

  • Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51:223–256

    CAS  Google Scholar 

  • Gould JH, Zhou Y, Padmanabhan V, Magallanes-Cedeno ME, Newton RJ (2002) Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium. Mol Breed 10:131–141

    Article  CAS  Google Scholar 

  • Holland L, Gemmell JE, Charity JA, Walter C (1997) Foreign gene transfer into Pinus radiata cotyledons by Agrobacterium tumefaciens. N Z J For Sci 27:289–304

    CAS  Google Scholar 

  • Horgan K, Aitken J (1981) Reliable plantlet formation from embryos and seedling shoot tips of radiata pine. Physiol Plant 53:170–175

    CAS  Google Scholar 

  • Humara JM, Lopez M, Ordas RJ (1999) Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer. Plant Cell Rep 19:51–58

    Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • Klimaszewska K, Devantier Y, Lachance D, Lelu MA, Charest PJ (1997) Larix laricina (tamarack): somatic embryogenesis and genetic transformation. Can J For Res 27:538–550

    Article  Google Scholar 

  • Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95:7203–7208

    CAS  PubMed  Google Scholar 

  • Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, 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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9:963–967

    CAS  PubMed  Google Scholar 

  • Levée V, Lelu M-A, Jouanin L, Cornu D, Pilate G (1997) Agrobacterium tumefaciens-mediated transformation of hybrid larch (Larix kaempferi x L. decidua) and transgenic plant regeneration. Plant Cell Rep 16:680–685

    Google Scholar 

  • Levée V, Garin E, Klimaszewska K, Séguin A (1999) Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. Mol Breed 5:429–440

    Article  CAS  Google Scholar 

  • Liew OW (1994) Expression of atrial naturiuretic factor in transgenic potatoes. PhD thesis, Lincoln University, Canterbury

  • McCullagh P, Nelder JA (1989) Generalised linear models. Chapman and Hall, London

  • Pawlowski WP, Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc Natl Acad Sci USA 95:12106–12110

    CAS  PubMed  Google Scholar 

  • Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207–7221

    CAS  PubMed  Google Scholar 

  • Reilly K, Washer J (1977) Vegetative propagation of radiata pine by tissue culture: plantlet formation from embryonic tissue. N Z J For Sci 7:199–206

    CAS  Google Scholar 

  • Smith DR (1986) Forest and nut trees. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 1. Trees. Springer, Berlin Heidelberg New York, pp 274–289

  • Smith DR, Walter C, Warr A, Hargreaves CL, Grace LJ (1994) Somatic embryogenesis joins the plantation forestry revolution in New Zealand. In: Proc Tappi Biol Sci Symp. TAPPI, Minneapolis, pp 19–29

  • Tang W (2003) Additional virulence genes and sonication enhance Agrobacterium tumefaciens-mediated loblolly pine transformation. Plant Cell Rep 21:555–562

    CAS  PubMed  Google Scholar 

  • Tang W, Sederoff R, Whetton R (2001) Regeneration of transgenic loblolly pine (Pinus taeda L.) from zygotic embryos transformed with Agrobacterium tumefaciens. Planta 213:981–989

    CAS  PubMed  Google Scholar 

  • Tinland B (1996) The integration of T-DNA into plant genomes. Trends Plant Sci 1:178–184

    Article  Google Scholar 

  • Vergunst AC, Hooykaas PJJ (1999) Recombination in the plant genome and its application in biotechnology. Crit Rev Plant Sci 18:1–31

    Article  CAS  Google Scholar 

  • Walter C, Grace LJ, Wagner A, White DWR, Walden AR, Donaldson SS, Hinton H, Gardner RC, Smith DR (1998) Stable transformation and regeneration of transgenic plants of Pinus radiata D. Don. Plant Cell Rep 17:460–468

    CAS  Google Scholar 

  • Walter C, Grace LJ, Donaldson J, Moody J, Gemmell JE, van der Maas S, Kvaalen H, Lőnneborg A (1999) An efficient Biolistic transformation protocol for Picea abies embryogenic tissue and regeneration of transgenic plants. Can J For Res 29:1539–1546

    Article  Google Scholar 

  • Wenck AR, Quinn M, Whetton RW, Pullman G, Sederoff R (1999) High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol Biol 39:407–416

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was supported by GEENZ Ltd, Rayonier NZ Ltd, Ernslaw One, Wenita Forest Products, the NZ Foundation for Research, Science and Technology and a Bright Futures Enterprise Scholarship to TMD. We thank Lynn Thomson for technical support, Ruth Butler for statistical analysis, Tracy Williams for helpful suggestions on the manuscript, Dr Dieter Adam and Ms Tania Smith for ongoing encouragement.

Author information

Authors and Affiliations

  1. New Zealand Institute for Crop and Food Research Limited, Private Bag 4704, Christchurch, New Zealand

    J. E. Grant, P. A. Cooper & T. M. Dale

Authors
  1. J. E. Grant
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. A. Cooper
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. M. Dale
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. E. Grant.

Additional information

Communicated by P. Debergh

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grant, J.E., Cooper, P.A. & Dale, T.M. Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons. Plant Cell Rep 22, 894–902 (2004). https://doi.org/10.1007/s00299-004-0769-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-004-0769-z

Keywords

Search

Navigation