Abstract
We have employed Populus as a model system to investigate the stability and expression of transgenes at the morphological, physiological, and molecular levels. Four clones from European aspen (Populus tremuld) and hybrid aspen (P. tremula x P. tremuloides) clones were genetically transformed, using an Agrobacterium binary vector system, with different gene constructs, in particular roIC. Two types of promoters, namely 35S from cauliflower mosaic virus, and rbcS from potato, were employed for control of roIC gene expression. An improved transformation method employing leaf discs was developed, and putative transformants were regenerated on a kanamycin-containing medium. More than 1000 transgenic aspens have been raised in the greenhouse during the last two years. The morphology and physiology of the transgenic plants seems to be influenced by the type of promoter employed. Transgenic aspens carrying the 35S-ro/C gene construct exhibited much smaller leaves, as compared to the untransformed controls. On the other hand, rbcS-ro/C carrying transgenic aspens showed only slightly smaller leaves, as compared to the controls. However, transgenic plants carrying the chimeric gene 35S-ro/C or rbcS-ro/C exhibited yellowish-green leaves, as compared to dark green leaf color of the untransformed controls. A number of leaf abnormalities, chimeras, revertants to normal state have been observed in the transgenic aspens. In order to silence the roIC gene expression, two constructs carrying the transposable element Ac from maize, along with roIC, were employed for genetic transformation in aspen. Transgenic aspens carrying the transgenes 35S-Ac-rolC and rbcS-Ac-rolC were morphologically similar to the untransformed aspens, in terms of leaf size, shape and color, indicating that at least the phenotypic expression of the roIC gene has been repressed by Ac. The Ac excision events were monitored by variegated spots on the leaves. There were significantly more Ac excisions in the transgenic plants carrying 35S-Ac-rolC than in rbcS-Ac-rolC transgenes. These observations suggest that the type of promoter and/or gene construct seems to influence the phenotype and physiology in the transgenic plants. Transformation was confirmed by PCR amplifications of the roIC and Ac coding regions, and Southern blot analysis was carried out to determine the copy number of the transgenes and their relationship to the morphogenetic response, as well as genetic stability in the transgenic aspens.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Ahuja, M.R. (1986) Aspen, in Evans, D.E., Sharp, E.R. and Anomirato, P.J. (eds.), Handbook of Plant Cell Culture, vol. 4. Macmillan Publishing Company, New York, pp. 626–651.
Ahuja, M.R. (1987) In vitro propagation of poplar and aspen, in Bonga, IM and Durzan, D.J. (eds.), Cell and Tissue Culture in Forestry, vol. 3, Martinus Nrjhoff Publishers, Dordrecht, pp. 207–223.
Ahuja, M.R. (1988a) Gene transfer in forest trees, in Hanover, IE. and Keathley, D.E. (eds.), Genetic Manipulation of Woody Plants, Plenum Press, New York, pp. 25–41.
Ahuja, M.R. (1988b) Gene transfer in woody plants: problems and perspectives, in Ahuja, M.R. (ed.), Somatic Cell Genetics of Woody Plants, Kluwer Academic Publishers, Dordrecht, pp. 83–101.
Ahuja, M.R. (1988c) Molecular genetics of transgenic plants, in Hällgren, J.E. (ed.),Molecular Genetics of Forest Trees, Swedish University of Agricultural Sciences, Umeå, pp. 127–145.
Ahuja, M.R. (1993) Regeneration and germplasm preservation in aspen, in Ahuja, M.R. (ed.), Micropropagation of Woody Plants, Kluwer Academic Publishers, Dordrecht, pp. 187–194.
Chun, Y.W. (1993) Clonal propagation in non-poplar aspen poplar hybrids, in Ahuja, M.R. (ed.), Micropropagation of Woody Plants, Kluwer Academic Publishers, Dordrecht, pp. 209–222.
Confalonieri, M, Balestrazzi, A. and Bisoffi, S. (1994) Genetic transformation of Populus nigra by Agrobacterium tumefaciens, Plant Cell Reports 13, 256–261.
Doyle, J.J. and Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue, Phytochemical Bull. 19, 11–15
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.
Ernst, S.G. (1993) In vitro culture of pure species non-aspen poplars, in Ahuja, M.R. (ed.), Micropropagation of Woody Plants, Kluwer Academic Publishers, Dordrecht, pp. 195–207.
Fillatti, J.J., McCown, B.H., Selmer, I, Haissig, B.E. and Cornai, L. (1987) Agrobacterium-mediated transformation and regeneration of poplar,Molec. Gen. Genetics 206, 192–199.
Finnegan, I and McElroy, D. (1994) Transgene inactivation: plants fight back, Bio/Technology, 12, 127–145.
Fladung, M. (1990) Transformation of diploid and tetraploid potato clones with the rolC gene of Agrobacterium rhizogenes and characterization of transgenic plants, Plant Breeding 104, 295–304.
Fladung, M and Ahuja, M.R. (1995) ’sandwich’ method for non-radioactive hybridization, Biotechniques 18, 3–5.
Fladung, M and Ballvora, A. (1992) Further characterization of rolC transgenic tetraploid potato clones, and influence of daylength and level ofrolC expression on yield parameters, Plant Breeding 109, 18–27.
Fladung, M., Ballvora, A. and Schmülling, T. (1993) Constitutive or light regulated expression of the rolC gene in transgenic potato plant has different effects on yield attributes and tuber carbohydrate composition, Plant Mol. Biol. 23, 749–757.
Fladung, M., Kumar, S. and Ahuja, M.R. (1996) Genetic transformation of Populus genotypes with different chimeric gene constructs: Transformation efficiency and molecular analysis, Transgenic Res. 5, 1–11.
Howe, G.T., Strauss, S.H. and Goldfarb, B. (1991) Insertion of the maize transposable element Ac into poplar, in Ahuja, M.R. (ed), Woody Plant Biotechnology, Plenum Press, New York, pp. 283–294.
Howe, G.T., Goldfarb, B. and Strauss, S.H. (1994) Agrobacterium-mediated transformation of hybrid poplar suspension cultures and regeneration of transformed plants, Plant Cell Tissue & Organ Culture 36, 59–71.
Jones, J.B.G., Bishop, G., Carrol, B., Dickson, M., English, J., Harrison, K., Jones, D., Scofield, S. and Thomas, CM. (1992) Prospects of establishing a tomato gene tagging system using the maize transposon activator (Ac). Proc. Royal Soc. Edinburgh, 99B, 107–119.
Klopfenstein, N.B., Shi, N.Q., Keman, A., McNabb, H.S., Hall, R.B., Hart, E.R. and Thomburg, R.W. (1991) Transgenic Populus hybrid expresses a wound inducible potato proteinase inhibitor JJ-CAT gene fusion, Can. J. For. Res. 21, 1321–1328.
Klopfenstein, N.B., McNabb, H.S., Hart, E.R., Hall, R.B., Hanna, R.D., Heuchelin, S.A., Allen, K, Shi, N.Q. and Thomburg, R.W. (1993) Transformation of Populus hybrids to study and improvepest resistance, Silvae Genetica 42, 86–90.
Logemann, J., Schell, I and Wilhnitzer, L. (1987) Improved method for the isolation of RNA from plant tissues, Anal. Biochemistry 163, 16–20.
Meyer, P., Heidemann, I. and Niedenhof, I. (1993) Differences in DNA methylation are associated with paramutation phenomenon in transgenic petunia, The Plant J. 4, 89–100.
Nilsson, O., Moritz, T., Imbault, N, Sandberg, G. and Olsson, O. (1993) Hormonal charaterization of transgenic tobacco plants expression the rolC gene of Agrobacterium rhizogenes TL-DNA, Plant Physiol. 102, 363–371.
Olsson, O., Nilsson, O., Sundberg, B., Little, C.H.A and Sandberg, G. (1992) RolC biosynthesizing transgenic Populus plants goes bonzai, Fifth Workshop of the IUFRO WP Molecular Genetics, Carcans, France, p. 3.6.
Schmülling, T., Schell, J. and Spena, A (1988) Single genes from Agrobacterium rhizogenes influence plant development, EMBO J 9, 2621–2639.
Schmülüng, T., Fladung, M., Grossmann, K. and Schell, J. (1993) Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes, The Plant J. 3, 587–598.
Spena, A, Schtnülling, T., Koncz, C. and Schell, J. (1987) Independent and synergistic activity of rol A,B and C loci in stimulating abnormal growth in plants, EMBO J. 6, 3891–3899.
Spena, A, Aalen, R.B. and Schulze, S.C. (1989) Cell autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay of transposon excision in transgenic plants, The Plant J. 1, 1157–1164.
Van Bokland, R., Van der Geest, N., Mol, J.N.M and Kooster, J.M. (1994) Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from increase inn RNA turnover, The Plant J. 6, 861–877.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Ahuja, M.R., Fladung, M. (1996). Stability and Expression of Chimeric Genes in Populus . In: Ahuja, M.R., Boerjan, W., Neale, D.B. (eds) Somatic Cell Genetics and Molecular Genetics of Trees. Forestry Sciences, vol 49. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-3983-0_12
Download citation
DOI: https://doi.org/10.1007/978-94-011-3983-0_12
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-5761-5
Online ISBN: 978-94-011-3983-0
eBook Packages: Springer Book Archive