Plant Cell Reports

, Volume 23, Issue 9, pp 606–616 | Cite as

Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures

  • J. A. Charity
  • L. Holland
  • L. J. Grace
  • C. Walter
Genetic Transformation and Hybridization


An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.


Genetic engineering Conifers Stable transformation Nurse cultures Plant regeneration 



Gene coding for phosphinothricin acetyl transferase


Embryo development medium


Enzyme linked immuno sorbent assay


Genetically modified organism


4-Methylumbelliferyl β-d-glucuronide


Neomycin phosphotransferase gene or protein


Plant growth regulator




β-Glucuronidase gene or protein


Standard error of the mean


  1. Bishop-Hurley SL, Zabkiewicz RJ, Grace LJ, Gardner RC, Wagner A, Walter C (2001) Conifer genetic engineering: transgenic Pinus radiata (D. Don) and Picea abies (Karst) plants are resistant to the herbicide Buster. Plant Cell Rep 20:235–243CrossRefGoogle Scholar
  2. Cerda F, Aquea F, Gebauer M, Medina C, Arce-Johnson P (2002) Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 70:251–257CrossRefGoogle Scholar
  3. Charity JA, Holland L, Donaldson SS, Grace LJ, Walter C (2002) Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum infiltration. Plant Cell Tissue Organ Cult 70:51–60CrossRefGoogle Scholar
  4. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995PubMedGoogle Scholar
  5. Côté C, Rutledge R (2003) An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants. Plant Cell Rep 21:619–624PubMedGoogle Scholar
  6. Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beuchy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33CrossRefGoogle Scholar
  7. Dean C, Jones J, Favreau M, Dunsmuir P, Bedbrook J (1988) Influence of flanking sequences on variability in expression levels of an introduced gene in transgenic tobacco plants. Nucleic Acids Res 16:9267–9283PubMedGoogle Scholar
  8. Ellis DD, McCabe DE, McInnis S, Ramachandran R, Russell DR, Wallace KM, Martinell BJ, Roberts DR, Raffa KF, McCown BH (1993) Stable transformation of Picea glauca by particle acceleration. Biotechnology 11:84–89CrossRefGoogle Scholar
  9. Gallagher SR (1992) GUS protocols: using the GUS gene as a reporter of gene expression. Academic, San Diego, Calif.Google Scholar
  10. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versitile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994PubMedGoogle Scholar
  11. Hargreaves CL, Grace LJ, Holden GD (2002) Nurse culture for efficient recovery of cryopreserved P. radiata D. Don embryogenic cell lines. Plant Cell Rep 21:40–45CrossRefGoogle Scholar
  12. Hobbs SLA, Warkentin TD, DeLong CMO (1993) Transgene copy number can be positively or negatively associated with transgene expression. Plant Mol Biol 21:17–26PubMedGoogle Scholar
  13. Holland L, Gemmell JE, Charity JA, Walter C (1997) Foreign gene transfer into Pinus radiata cotyledons by Agrobacterium tumefaciens. NZ J For Sci 27(3):289–304Google Scholar
  14. Hood EE, Jen G, Kayes L, Kramer J, Fraley RT, Chilton MD (1987) Restriction endonuclease map of pTiB0542, a potential Ti-plasmid vector for genetic engineering of plants. Biotechnology 2:702–709Google Scholar
  15. Horsch TB, Jones GE (1980) A double filter paper technique for plating cultured cells. In Vitro 16:103–108Google Scholar
  16. Jefferson A, Kavanagh A, Bevan W (1987) GUS fusions: glucuronidase as sensitive marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  17. Klimaszewska K, Lachance D, Pelletier G, Lelu M-A, Séguin A (2001) Regeneration of transgenic Picea glauca, P. mariana and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens. In Vitro Cell Dev Biol Plant 37:748–755Google Scholar
  18. Klimaszewska K, Lachance D, Bernier-Cardou M, Rutldege RG (2003) Transgene integration patterns and expression levels in transgenic lines of Picea mariana, P. glauca and P. abies. Plant Cell Rep 21:1080–1087CrossRefPubMedGoogle Scholar
  19. Le VQ, Belles-Isles J, Dusabenyagasani M, Tremblay FM (2001) An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens. J Exp Bot 364:2089–2095Google Scholar
  20. Levée V, Lelu M-A, Jouanin L, Cornu D, Pilate G (1997) Agrobacterium tumefaciens-mediated transformation of hybrid larch (Larix kaempferi × L. decidua) and transgenic plant regeneration. Plant Cell Rep 16:680–685CrossRefGoogle Scholar
  21. 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 5:429–440CrossRefGoogle Scholar
  22. Makarevitch I, Svitashev SK, Somers DA (2003) Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment. Plant Mol Biol 52:421–432CrossRefPubMedGoogle Scholar
  23. Matzke MA, Matzke AJM (1995) How and why do plants inactivate homologous (trans)genes? Plant Physiol 107:679–685PubMedGoogle Scholar
  24. Niu X, Li X, Veronese P, Bressan RA, Weller SC, Hasegawa PM (2000) Factors affecting Agrobacterium tumefaciens-mediated transformation of Peppermint. Plant Cell Rep 9:304–310CrossRefGoogle Scholar
  25. Shelbourne CJA, Carson MJ, Wilcox MD (1989) New technologies in the genetic improvement of radiata pine. Commonw For Rev 68:3Google Scholar
  26. Smith D (1996) Growth medium. US patent no 5,565,355Google Scholar
  27. Tang W, Newton RJ (2003) Genetic transformation of conifers and its application in forest biotechnology. Plant Cell Rep 22:1–15CrossRefPubMedGoogle Scholar
  28. Trontin J-F, Harvengt L, Garin E, Lopez-Bernaza M, Arancia L, Hoebeke J, Canlet F, Pâques M (2002) Towards genetic engineering of maritime pine (Pinus pinaster Ait.). Ann For Sci 59:687–697CrossRefGoogle Scholar
  29. Vaucheret H, Béclin C, Elmayan T, Feuerbach F, Gordon C, Morel JB, Mourrain P, Palauqui JC, Vernhettes S (1998) Transgene-induced silencing in plants. Plant J 16:651–659CrossRefPubMedGoogle Scholar
  30. Veluthambi K, Jayaswal RK, Gelvin SB (1987) Virulence genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proc Natl Acad Sci USA 84:1881–1885Google Scholar
  31. Walter C, Grace LJ (2000) Genetic engineering of conifers for plantation forestry: Pinus radiata transformation. In: Jain SM, Minocha SC (eds) Molecular biology of woody plants, vol 2. Kluwer, Dordecht, pp 79–104Google Scholar
  32. 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 17:460–468CrossRefGoogle Scholar
  33. Walter C, Grace LJ, Donaldson SS, Moody J, Gemmell JE, van der Maas S, Kvaalen H, Lonneborg A (1999) An efficient Biolistic transformation protocol for Picea abies embryogenic tissue and regeneration of transgenic plants. Can J For Res 29:1539–1546CrossRefGoogle Scholar
  34. Walter C, Bishop-Hurley S, Charity JA, Find J, Grace LJ, Hoefig K, Holland L, Moeller R, Moody J, Wagner A, Walden A (2001) Genetic engineering of Pinus radiata and Picea abies, production of transgenic plants and gene expression studies. International Wood Biotechnology Symposium, Narita, Japan, March 2001Google Scholar
  35. Wenck AR, Quinn M, Whetten RW, Pullman G, Sederoff R (1999) High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol Biol 39:407–416CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • J. A. Charity
    • 1
  • L. Holland
    • 1
  • L. J. Grace
    • 1
  • C. Walter
    • 1
  1. 1.Cellwall Biotechnology CentreForest ResearchRotoruaNew Zealand

Personalised recommendations