Skip to main content
SpringerLink
Log in

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large- scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene β-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co- cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

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

Similar content being viewed by others

References

  1. An GM: High efficiency transformation of cultured tobacco cells. Plant Physiol 79: 568–570(1985).

    Google Scholar 

  2. An G, Ebert PR, Mitra A, Ha SB: Binary vectors. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, pp. A3/1–A3/19. Kluwer Academic Publishers, Dordrecht, Netherlands (1987).

    Google Scholar 

  3. Atherly AG: Agrobacterium, Rhizobium and other gramnegative soil bacteria. In: Wang K, Herrera-Estrella A, Van Montagu M (eds) Transformation of Plants and Soil Microorganisms, pp. 23–33. Cambridge University Press, Cambridge, UK (1995).

    Google Scholar 

  4. Bergmann BA, Stomp A-M: Effect of host plant genotype and growth rate on Agrobacterium tumefaciens-mediated gall formation in Pinus radiata. Phytopathology 82: 1457–1462 (1992).

    Google Scholar 

  5. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Coner TW, Wan Y: Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971–980 (1997).

    PubMed  Google Scholar 

  6. Curtis IS, Power JB, Blackhall NW, de Laat AMM, Davey MR: Genotype-independent transformation of lettuce using Agrobacterium tumefaciens. J Exp Bot 45: 1441–1449(1994).

    Google Scholar 

  7. Davis ME, Lineberger RD, Miller AR: Effects of tomato cultivar, leaf age and bacterial strain on transformation by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 24: 115–121(1991).

    Google Scholar 

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

    Google Scholar 

  9. Drake PMW, John A, Power JB, Davey MR: Expression of the gusA gene in embryogenic cell lines of sitka spruce following Agrobacterium-mediated transformation. J Exp Bot 48: 151–155 (1997).

    Google Scholar 

  10. Ellis D:Transformation in spruce (Picea species) In: Bajaj YPS (ed), Biotechnology in Agriculture and Forestry, vol. 23, pp. 315–330. Springer-Verlag, Berlin/Heidelberg (1993).

    Google Scholar 

  11. Ellis D, Roberts D, Sutton B, Lazaroff W, Webb D, Flinn B: Transformation of white spruce and other conifer species by Agrobacterium tumefaciens. Plant Cell Rep 8: 16–20(1989).

    Google Scholar 

  12. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments for high specific activity. Anal Biochem 132: 6–13(1983).

    PubMed  Google Scholar 

  13. Fullner KJ, Lara JC, Nester EW: Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273: 1107–1109 (1996).

    PubMed  Google Scholar 

  14. Gelvin SB, Liu C-N: Genetic manipulation of Agrobacterium tumefaciens strains to improve transformation of recalcitrant species. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, 2nd ed., pp. B4/1–B4/13. Kluwer Academic Publishers, Dordrecht, Netherlands (1994).

    Google Scholar 

  15. Gupta PK: Method for reproducing conifers by somatic embryogenesis using a maltose enriched maintenance medium. US Patent 5,563,061, October 8, 1996.

  16. Gupta PK, Durzan DJ: Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos of Picea abies (Norway Spruce). In Vitro Cell Dev Biol 22: 685–688(1986).

    Google Scholar 

  17. Hajdukiewicz P, Svab Z, Maliga P: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25: 989–994(1994).

    PubMed  Google Scholar 

  18. Hansen G, Das A, Chilton M-D: Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium. Proc Natl Acad Sci USA 91: 7603–7607(1994).

    PubMed  Google Scholar 

  19. Hiei Y, Ohta S, Komari T, Kumashiro T: Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282(1994).

    Article  PubMed  Google Scholar 

  20. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir-and Tregion of the Agrobacterium tumefaciens Ti plasmid. Nature 303: 179–180(1983).

    Google Scholar 

  21. Hood EE, Gelvin SB, Melchers LS, Hoekema A: New Agrobacterium helper plasmids for gene transfer to plants. Trans Res 2: 208–218(1993).

    Google Scholar 

  22. Huang Y, Diner AM, Karnosky DF: Agrobacterium rhizogenes-mediated genetic transformation and regeneration of a conifer: Larix decidua. In Vitro Cell Dev Biol 27P: 201–207(1991).

    Google Scholar 

  23. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T: High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnol 14: 745–750(1996).

    Google Scholar 

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

    Google Scholar 

  25. Jin S, Komari T, Gordon MP, Nester EW: Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J Bact 169: 4417–4425(1987).

    PubMed  Google Scholar 

  26. Komari T: Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep 9: 303–306 (1990).

    Google Scholar 

  27. Koncz Cs, Schell J: The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383–396(1986).

    Article  Google Scholar 

  28. Levee V, Garin E, Seguin A: Genetic transformation of white pine (Pinus strobus) using Agrobacterium tumefaciens. Joint meeting of UIFRO working parties 2.04–07 and 2.04–06: Somatic Cell Genetics and Molecular Genetics of Trees, Aug. 12- 16, Quebec City, Quebec, Abstract 26 (1997).

  29. Li H-Q, Sautter C, Potrykus I, Pounti-Kaerlas J: Genetic transformation of cassava (Manihot esculenta Crantz). Nature Biotechnol 14: 736–740(1996).

    Google Scholar 

  30. Liu C-N, Li X-Q, Gelvin SB: Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens. Plant Mol Biol 20: 1071–1087 (1992).

    PubMed  Google Scholar 

  31. Ni M, Cui D, Einstein J, Narasimhulu S, Vergara SE, Gelvin SB: Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. Plant J 7: 661–676(1995).

    Article  Google Scholar 

  32. Pullman GS, Webb DT: An embryo staging system for comparison of zygotic and somatic embryo development. TAPPI R & D Division, Biological Sciences Symposium, October 3- 6, Minneapolis, MN, pp. 31–34(1994).

  33. Robertson D, Weissinger AK, Ackley R, Glover S, Sederoff RR: Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment. Plant Mol Biol 19: 925–935(1992).

    PubMed  Google Scholar 

  34. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  35. Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan-Norreel B: Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta 188: 439–456(1992).

    Google Scholar 

  36. Sederoff RR: Forest trees. In: Wang K, Herrera-Estrella A, Van Montagu M (eds), Transformation of Plants and Soil Microorganisms, pp. 150–163. Cambridge University Press, Cambridge, UK (1995).

    Google Scholar 

  37. Sederoff RR, Stomp AM: DNA transfer to conifers. In Ahuja MR, Libby WJ (eds) Clonal Forestry. I. Genetics and Biotechnology, pp. 241–254.Springer-Verlag, Berlin/Heidelberg (1993).

    Google Scholar 

  38. Sederoff R, Stomp A-M, Chilton WS, Moore LW: Gene transfer into loblolly pine by Agrobacterium tumefaciens. Bio/technology 4: 647–649(1986).

    Google Scholar 

  39. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA: An improved PCR method for walking in uncloned genomic DNA. Nucl Acids Res 23: 1087–1088 (1995).

    PubMed  Google Scholar 

  40. Stomp A-M, Loopstra C, Chilton WS, Sederoff RR, Moore LW: Extended host range of Agrobacterium tumefaciens in the genus Pinus. Plant Physiol 92: 1226–1232(1990).

    Google Scholar 

  41. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517(1975).

    PubMed  Google Scholar 

  42. Tautorus TE, Fowke LC, Dunstan DI: Somatic embryogenesis in conifers. Can J Bot 69: 1873–1899(1991).

    Google Scholar 

  43. Tremblay L, Tremblay FM: Somatic embryogenesis in black spruce [Picea mariana (Mill.) B.S.P.] and red spruce (P. rubens Sarg.). In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, vol 30, pp. 431–445. Springer-Verlag, Berlin/Heidelberg (1995).

    Google Scholar 

  44. Verhagen SA, Wann SR: Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryoss. Plant Cell Organ Cult 16: 103–111(1989).

    Google Scholar 

  45. Villemont E, Dubois F, Sangwan RS, Vasseur G, Bourgeois Y, Sangwan-Norreel B: Role of the host cell cycle in the Agrobacterium-mediated genetic transformation of Petunia: evidence of an S-phase control mechanism for T-DNA transfer. Planta 201: 160–172(1997).

    Google Scholar 

  46. Von Arnold S, Clapham D, Egertsdotter U, Mo LH, Somatic embryogenesis in conifers: a case study of induction and development of somatic embryos in Picea abies. Plant Growth Regul 20: 3–9(1996).

    Google Scholar 

  47. Walkerpeach CR, Velten J: Agrobacterium-mediated gene transfer to plant cells: cointegrate and binary vector systems. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, 2nd ed. pp. B1/1–B1/19. Kluwer Academic Publishers, Dordrecht, Netherlands (1994).

    Google Scholar 

  48. Yibrah HS, Manders G, Clapham DH, Von Arnold S: Biological factors affecting transient transformation in embryogenic suspension culture of Picea abies. J Plant Physiol 144: 472–478(1994).

    Google Scholar 

  49. Zheng Z, Haykashimoto A, Li Z, Murai N: Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts. Plant Physiol 97: 832–835 (1991).

    Google Scholar 

  50. Zupan J, Zambryski P: The Agrobacterium DNA transfer complex. Crit Rev Plant Scis 16: 279–295(1997).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Forest Biotechnology Group, North Carolina State University, Raleigh, NC, 27695, USA

    Allan Richard Wenck, Michelle Quinn, Ross W. Whetten & Ronald Sederoff

  2. Institute of Paper Science and Technology, Atlanta, GA, 30318, USA

    Gerald Pullman

Authors
  1. Allan Richard Wenck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle Quinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ross W. Whetten
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerald Pullman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ronald Sederoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Richard Wenck, A., Quinn, M., Whetten, R.W. et al. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol Biol 39, 407–416 (1999). https://doi.org/10.1023/A:1006126609534

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006126609534

Search

Navigation