Transgenic Research

, Volume 6, Issue 2, pp 111–121 | Cite as

Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis

  • Matthias Fladung
  • Sandeep Kumar
  • M. Raj Ahuja


Aspen (Populus tremula) and hybrid aspen (P. tremula × P. tremuloides) were transformed with different gene constructs using two types of promoter. The aim was to determine the influence of the reporter gene rolC, controlled by promoters of viral or plant origin, on genetic and morphologic expression of different transgenic aspen clones. An improved transformation method using leaf discs was developed, by which putative transgenic plantlets were regenerated at high efficiencies (up to 34%) on kanamycin-containing medium. Transgenic aspen carrying the rolC gene from Agrobacterium rhizogenes under control of the cauliflower-35S-promoter are reduced in size with smaller leaves, whereas aspen transgenic for the same rolC gene, but under control of the light inducible rbcS promoter from potato, are only slightly reduced in size compared to untransformed controls. However, all clones carrying 35S-rolC and rbcS-rolC genes revealed light-green colouration of leaves when compared to untransformed aspen. Owing to this special feature, constructs were used in which expression of the rolC gene was inhibited by insertion of a transposable element, Ac, from maize. Transgenic aspen transformed with the 35S-Ac-rolC and rbcS-Ac-rolC genes were morphologically similar to untransformed aspen, but out of 54 independently regenerated 35S-Ac-rolC transgenic aspen clones, 30 clones showed light-green/dark green variegated leaves. In contrast, out of 19 independently transformed rbcS-Ac-rolC aspen clones, only two clones revealed light-green/dark green variegated leaves. The role of bacterial strains in transformation, and molecular genetics of transgenic aspen plants (including the function of the transposable element, Ac, in the aspen genome) are discussed

Ac Agrobacterium aspen Populus transformation transposition 


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  1. Ahuja, M.R. (1986) Aspen. In: Evans, D.A., Sharp, W.R. and Ammirato, P.J. eds. Handbook of Plant Cell Culture 4, Techniques and Applications, pp. 626-51. New York: Macmillan Publishing Company.Google Scholar
  2. Ahuja, M.R. (1987) In-vitropropagation of poplar and aspen. In: Bonga, J.M. and Durzan, D.J. eds. Cell and Tissue Culture in Forestry, Vol. 3, pp. 621-51. Dordrecht: Martinus Nijhoff.Google Scholar
  3. Ahuja, M.R. (1988) Gene transfer in forest trees. In: Hanover, J.W. and Keathley, D.E. eds. Genetic Manipulation of Woody Plants. pp. 25-41. New York: Plenum Press.Google Scholar
  4. Chung, K.H., Park, Y.G., Noh, E.R. and Chun, Y.W. (1989) Transformation of Populus alba 00D7 P. glandulosaby Agrobacterium rhizogenes. J. Korean For. Soc. 78, 372-80.Google Scholar
  5. Confalonieri, M., Balestrazzi, A, and Bisoffi, S. (1994) Genetic transformation of Populus nigraby Agrobacterium tumefaciens. Plant Cell Reports 13, 256-61.CrossRefGoogle Scholar
  6. DeVerno, L. and Cheliak, W.M. (1988) In vivotransformation of hybrid poplars: clonal variation - a preliminary investigation. In Cheliak, W.M. and Yapa, A.C. eds. Molecular Genetics of Forest Trees. pp. 48-53. Petawawa Chalk Rivers: Petawawa National Forestry Institute Information Report PI-X-80.Google Scholar
  7. Devillard, C. (1992) Genetic transformation of aspen (Populus tremula 00D7 Populus alba) by Agrobacterium rhizogenesand regeneration of plants tolerant to herbicide. C.R. Acad. Sci. Paris 314, 291-8.Google Scholar
  8. Döring, H.P. and Starlinger, P. (1986) Molecular genetics of transposable elements in plants. Annu. Rev. Genet. 20, 175- 200.PubMedGoogle Scholar
  9. Doyle, J.J. and Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bull. 19, 11-5.Google Scholar
  10. Edwards, K., Johnstone, C. and Thompson, C. (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl. Acids Res. 19, 1349.PubMedGoogle Scholar
  11. Fillatti, J.J., Sellmer, J., McCown, B., Haissig, B. and Comai, L. (1987) Agrobacteriummediated transformation and regeneration of Populus. Mol. Gen. Genet. 206, 192-9.CrossRefGoogle Scholar
  12. Finnegan, E.J., Taylor, B.H., Craig, S. and Dennis, E.S. (1989) Transposable elements can be used to study cell lineages in transgenic plants. Plant Cell 1, 757-64.CrossRefPubMedGoogle Scholar
  13. Fladung, M. (1990) Transformation of diploid and tetraploid potato clones with the rolCgene of Agrobacterium rhizogenesand characterization of transgenic plants. Plant Breeding 104, 295-304.Google Scholar
  14. Fladung, M. and Ahuja, M.R. (1995) "sandwich’ method for nonradioactive hybridizations. Biotechniques 18, 3-5.Google Scholar
  15. Fladung, M. and Ballvora, A. (1992) Further characterization of rolCtransgenic tetraploid potato clones, and influence of daylength and level of rolCexpression on yield parameters. Plant Breeding 109, 18-27.Google Scholar
  16. Fladung, M. and Gieffers, W. (1993) Resistance reactions of leaves and tubers of rolCtransgenic tetraploid potato to bacterial and fungal pathogens. Correlation with sugar, and starch and chlorophyll content. Physiol. Mol. Plant Pathol. 42, 123-32.CrossRefGoogle Scholar
  17. Fladung, M., Ballvora, A. and Schmülling, T. (1993) Constitutive or light regulated expression of the rolCgene in transgenic potato plants has different effects on yield attributes and tuber carbohydrate composition. Plant Mol. Biol. 23, 749-57.PubMedGoogle Scholar
  18. Hamilton, R.H. and Fall. M.Z. (1971) The loss of tumor initiating ability in Agrobacterium tumefaciensby inoculation at high temperatures. Experientia 27, 229-30.PubMedGoogle Scholar
  19. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983) A binary plant vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciensTiplasmid. Nature 303, 179-80.Google Scholar
  20. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. (1985) A simple and general method for transferring genes into plants. Science 227, 1229-31.Google Scholar
  21. Howe, G.T., Strauss, S.H. and Goldfarb, B. (1991) Insertion of the maize transposable element Acinto poplar. In: Ahuja, M.R. ed. Woody Plant Biotechnology, pp. 283-94. New York: Plenum Press.Google Scholar
  22. Howe, G.T., Goldfarb, B. and Strauss, S.H. (1994) Agrobacteriummediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants. Plant Cell, Tissue Organ Culture 36, 59-71.Google Scholar
  23. Jones, J.D.G., Bishop, G., Carroll, B., Dickinson, M., English, J., Harrison, K., Jones, D., Scofield, S. and Thomas, C.M. (1992) Prospects for establishing a tomato gene tagging system using the maize transposon Activator (Ac). Proc. Royal Soc. Edinburgh 99B, 107-19.Google Scholar
  24. Klopfenstein, N.B., Shi, N.Q., Kernan, A., McNabb, H.S., Jr, Hall, R.B., Hart, E.R. and Thornburg, R.W. (1991) Transgenic Populushybrid expresses a wound-inducible potato proteinase inhibitor II - CAT gene fusion. Can. J. For. Res. 21, 1321-8.Google Scholar
  25. Klopfenstein, N.B., McNabb, H.S., Jr, Hart, E.R., Hall, R.B., Hanna, R.D., Heuchelin, S.A., Allen, K., Shi, N.Q. and Thornburg, R.W. (1993) Transformation of Populushybrids to study and improve pest resistance. Silvae Genetica 42, 86-90.Google Scholar
  26. Knapp, S., Coupland, G., Uhrig, H., Starlinger, P. and Salamini, F. (1988) Transposition of the maize transposable element Acin Solanum tuberosum . Mol. Gen. Genet. 213, 285-90.Google Scholar
  27. Koncz, C. and Schell, J. (1986) The promoter of the TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacteriumvector. Mol. Gen. Genet. 204, 383-96.CrossRefGoogle Scholar
  28. Leple, J.C., Brasileiro, A.C.M., Michel, M.F., Delmotte, F. and Jouanin, L. (1992) Transgenic poplars: expression of chimeric genes using for different constructs. Plant Cell Rep. 11, 137- 41.CrossRefGoogle Scholar
  29. Lloyd, G. and McCown, B. (1981) Commercially feasible micropropation of mountain laurel (Kalmia latiflora) by use of shoot tip culture. Proc. Int. Plant Propagators Soc. 30, 421-7.Google Scholar
  30. Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved method for the isolation of RNA from plant tissues. Anal. Biochemistry 163, 16-20.Google Scholar
  31. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.Google Scholar
  32. Neuhaus-Url, G. and Neuhaus, G. (1993) The use of nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: a comparison with radioactivity. Transgenic Res. 2, 115-20.Google Scholar
  33. Nilsson, O., Alden, T., Sitbon, F., Little, C.H.A., Chalupa, V., Sandberg, G. and Olsson, O. (1992) Spatial pattern of cauliflower mosaic virus 35S promoter-luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging. Trangenic Res. 1, 209-20.Google Scholar
  34. Nilsson, O., Moritz, T., Imbault, N., Sandberg, G. and Olsson, O. (1993) Hormonal characterization of transgenic tobacco plants expressing the rolCgene of Agrobacterium rhizogenesTL-DNA. Plant Physiol. 102, 363-71.PubMedGoogle Scholar
  35. Olsson, O., Nilsson, O., Sundberg, B., Little, C.H.A. and Sandberg, G. (1992) rolCbiosynthesizing transgenic Populusplants goes bonzai. Fifth workshop of the IUFRO Working Party-S20406 in Carcans, Manbuisson, France, Abstract 3.6.Google Scholar
  36. Owens, L.D. and Cress, D.E. (1985) Genotypic variability of soybean responses to Agrobacteriumstrains harboring Ti or Ri plasmids. Plant Physiol. 77, 87-94.Google Scholar
  37. Porsch, P. (1995) Transformation von Solanum tuberosummit mikrobiellen Genen zur Beeinflussung des CO2-Stoffwechsels. Ph.D. Thesis, University Köln, pp. 1-98.Google Scholar
  38. Schmülling, T., Schell, J. and Spena, A. (1988) Single genes from Agrobacterium rhizogenesinfluence plant development. EMBO J. 9, 2621-39.Google Scholar
  39. Schmülling, T., Fladung, M., Grossmann, K. and Schell, J. (1993) Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rolgenes. Plant J. 3, 587-98.CrossRefGoogle Scholar
  40. Spena, A., Schmülling, T., Koncz, C. and Schell, J. (1987) Independent and synergistic activity of rol A, Band Cloci in stimulating abnormal growth in plants. EMBO J. 6, 3891-9.Google Scholar
  41. Spena, A., Aalen, R.B. and Schulze, S.C. (1989) Cell autonomous behavior of the rolCgene of Agrobacterium rhizogenesduring leaf development: a visual assay for transposon excision in transgenic plants. Plant Cell 1, 1157-64.CrossRefPubMedGoogle Scholar
  42. Sundberg, B., Tuominen, H., Lundberg, A.K., Jacobson, L., Sitbon, F., Little, C.H.A., Olsson, O. and Sandberg, G. (1992) Transgenic Populusplant expressing the T-DNA IAA biosynthesis genes display altered growth. Fifth Workshop of the IUFRO Working Party-S20406 in Carcans, Manbuisson, France, Abstract 3.7.Google Scholar
  43. Tsai, C.J., Podila, G.K. and Chiang, V.L. (1994) Agrobacteriummediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants. Plant Cell Reports 14, 94-7.CrossRefGoogle Scholar
  44. Wordragen, M.F. von, De Jong, J., Huiteman, H.B.M. and Dons, H.J.M. (1991) Genetic transformation of chrysanthemum using wild type Agrobacteriumstrains; strain and cultivar specifity. Plant Cell Reports 9, 505-9.CrossRefGoogle Scholar

Copyright information

© Chapman and Hall 1997

Authors and Affiliations

  • Matthias Fladung
    • 1
  • Sandeep Kumar
    • 2
  • M. Raj Ahuja
    • 1
  1. 1.Federal Research Centre for Forestry and Forest ProductsInstitute of Forest GeneticsGrosshansdorfGermany
  2. 2.Genetics and Plant Propagation DivisionTropical Forest Research InstituteJabalpur (M.P.)India

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