Plant Cell Reports (2000) 19 : 315–320 Q Springer-Verlag 2000 Communicated by S. Merkle K.-H. Han 1 7 R. Meilan 7 C. Ma 7 S.H. Strauss (Y) Department of Forest Science, Oregon State University, Corvallis, OR 97331–7501, USA e-mail: strauss6FSL.orst.edu Fax: c541–737–1393 Present address: 1 Department of Forestry, 126 Natural Resources, Michigan State University, East Lansing, MI 48824-1222 K.-H. Han 7 R. Meilan 7 C. Ma 7 S.H. Strauss An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus) Received: 16 June 1998 / Revision received: 5 February 1999 / Accepted: 14 April 1999 Abstract We describe a protocol for Agrobacterium tumefaciens-mediated transformation of hybrid cotton- woods (Populus sections Tacamahaca Spach. and Aigeiros Duby). The protocol has allowed routine transformation of several economically important cottonwood hybrids (Populus trichocarpa Torr. & Gray!P. deltoides Bartr. ex. Marsh. and P. deltoides!P. nigra L.) that were previously difficult to transform. The procedure was applied to 11 different hybrid cottonwood genotypes and one P. deltoides genotype using kanamycin as the selection agent. Addi- tional experiments showed a very strong interaction between auxin preculture and the effectiveness of various cytokinins for induction of shoot organogen- esis. The data also demonstrated the superiority of Agrobacterium strain EHA105 over C58 and LBA4404 for T-DNA transfer based on transient assays with a reporter gene. Key words Poplar 7 Populus 7 Aspen 7 Cottonwood 7 Transformation 7 Agrobacterium Abbreviations AS: Acetosyringone 7 BA: 6-Benzyladenine 7 CIM: Callus induction media 7 GUS: b-Glucuronidase 7 IBA: Indole-3-butyric acid 7 MES: 2-[N-morpholinol]ethanesulfonic acid 7 MS: Murashige and Skoog (1962) 7 NAA: Naphthalene-acetic acid 7 NPTII: Neomycin phosphotransferase gene 7 RIM: Root induction media 7 SIM: Shoot induction media 7 TDZ: Thidiazuron 7 2,4-D: 2,4-Dichloro-phenoxyacetic acid 7 2iP: N 6 –(2-isopentenyl) adenine Introduction Asexual transformation methods provide a means for introducing new traits that are difficult to obtain via traditional breeding and allow modification of valuable clones without the genetic recombination that occurs during sexual reproduction. Transformation requires that DNA be inserted into plant cells, incorporated into chromosomes, and expressed in cells that can be induced to regenerate into plants. Although the transfer of DNA into plant cells via Agrobacterium, biolistics, and other physical methods is now routine (reviews in Han et al. 1996; Jouanin et al. 1993; Kim et al. 1997), coupling transformation with the selection of transgenic cells and plant regeneration is still difficult and costly in many economically important species, thereby limiting the use of transgenic crops (Birch 1997). Hybrid cottonwoods, aspens, and other poplar species (genus Populus) are highly valued by the pulp and paper industry for their fast growth and high quality fiber (Stettler et al. 1996). Although poplars have been transformed for research purposes far more than any other species of forest tree, many economi- cally important genotypes and species remain difficult to transform (Han et al. 1996). Highly efficient trans- formation systems for poplars have been reported by several laboratories (e.g., Leple et al. 1992; Tsai et al. 1994; Tzifra et al. 1996, 1997; Fladung et al. 1997), however much of the work has been restricted to a few model hybrids and species of section Leuce (aspens and white poplars), many of which had been selected for ease of transformation. Transformation of cottonwood species (sections Tacamahaca Spach and Aigeiros Duby) has been uncommon by comparison and has usually involved one or a few genotypes (e.g., DeBlock