Abstract
Regeneration problems are one of the main limitations preventing the wider application of genetic engineering strategies to the genus Eucalyptus. Seedlings from Eucalyptus grandis × Eucalyptus urophylla were selected according to their regeneration (adventitious organogenesis) and transformation capacity. After in vitro cloning, the best genotype of 250 tested was transformed via Agrobacterium tumefaciens. A cinnamyl alcohol dehydrogenase (CAD) antisense cDNA from Eucalyptus gunnii was transferred, under the control of the 35S CaMV promoter with a double enhancer sequence, into a selected genotype. According to kanamycin resistance and PCR verification, 120 transformants were generated. 58% were significantly inhibited for CAD activity, and nine exhibited the highest down-regulation, ranging from 69 to 78% (22% residual activity). Southern blot hybridisation showed a low transgene copy number, ranging from 1 to 4, depending on the transgenic line. Northern analyses on the 5–16 and 3–23 lines (respectively one and two insertion sites) demonstrated the antisense origin of CAD gene inhibition. With respectively 26 and 22% of residual CAD activity, these two lines were considered as the most interesting and transferred to the greenhouse for further analyses.
Similar content being viewed by others
References
Azmi A (1999) Bases cellulaires et physiologiques de la régénération et de la transformation génétique chez Eucalyptus globulus Labill. PhD Thesis, Pierre et Marie Curie University, Paris, France.
Bernard Vailhé MA, Cornu A, Robert D, Maillot MP and Besle JM (1996) Cell wall degradability of transgenic tobacco stems in relation to their chemical extraction and lignin quality. J Agric Food Chem 44: 1164–1169.
Baucher M, Chabbert B, Pilate G, Van Doorsselaere J, Tollier M-T, Petit-Conil M et al. (1996) Red xylem and higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenase in poplar (Populus tremula×Populus alba). Plant Physiol 112: 1479–1490.
Boudet A-M (2000) Lignins and lignification: selected issues. Plant Physiol Biochem 38(1): 1–16.
FAO (2001) Global Forest Resources Assessment 2000 - FRA 2000 Main Report. FAO Forestry Paper 140, Rome, 479 pp.
Gawel NJ and Jarret RL (1991) A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Mol Biol Rep 9: 262–266.
Goffner D, Joffroy I, Grima-Pettenati J, Halpin C, Knight M, Schuch W et al. (1992) Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem. Planta 188: 48–53.
Grima-Pettenati J, Feuillet C, Goffner D, Borderies G and Boudet A-M (1993) Molecular cloning and expression of a Eucalyptus gunnii cDNA clone encoding cinnamyl alcohol dehydrogenase. Plant Mol Biol 21: 1085–1095.
Halpin C, Knight ME, Foxon GA, Campbell MM, Boudet A-M, Boon JJ et al. (1994) Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase. Plant J 6(3): 339–350.
Jefferson RA, Kavanagh TA and Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13): 3901–3907.
Machado LO, de Andrade GM, Cid LPB, Penchel RM and Brasileiro ACM (1997) Agrobacterium strain specificity and shooty tumour formation in eucalypt (Eucalyptus grandis×E. urophylla). Plant Cell Rep 16: 299–303.
MacRae S and van Staden J (1999) Transgenic eucalyptus. In: Bajaj YPS (ed.), Biotechnology in Agriculture and Forestry. Vol. 44 (pp. 88–114) Springer, Heidelberg.
Moralejo M, Rochange F, Boudet A-M and Teulières C (1998) Generation of transgenic Eucalyptus globulus plantlets through Agrobacterium tumefaciens mediated transformation. Aust J Plant Physiol 25: 207–212.
Mullins KV, Hartney VJ, Llewellyn DJL, Strauss S and Dennis ES (1995) Regeneration and transformation of Eucalyptus camaldulensis. In: CRCTHF-IUFRO Conference Eucalypt Plantations: Improving Fibre Yield and Quality. (pp. 413–415) Hobart, Australia.
Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A laboratory Manual. Cold Spring Harbor, New York.
Serrano L, Rochange F, Semblat JP, Marque C, Teulières C and Boudet AM (1996) Genetic transformation of Eucalyptus globulus through biolistics: parallel development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue. J Exp Bot 47(295): 285–290.
Tepfer M and Casse-Delbart F (1987) Agrobacterium rhizogenes as a vector for transforming higher plants.Microbiol Sci 4: 24–28.
Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L and Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220: 245–250.
Yahiaoui N, Marque C, Myton KE, Negrel J and Boudet A-M (1998) Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants. Planta 204: 8–15.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Tournier, V., Grat, S., Marque, C. et al. An Efficient Procedure to Stably Introduce Genes into an Economically Important Pulp Tree (Eucalyptus grandis × Eucalyptus urophylla). Transgenic Res 12, 403–411 (2003). https://doi.org/10.1023/A:1024217910354
Issue Date:
DOI: https://doi.org/10.1023/A:1024217910354