GENETIC TRANSFORMATION AND HYBRIDIZATION High efficiency poplar transformation Leland J. Cseke Æ Sarah Beth Cseke Æ Gopi K. Podila Received: 10 January 2007 / Revised: 31 March 2007 / Accepted: 12 April 2007 / Published online: 11 May 2007 Ó Springer-Verlag 2007 Abstract With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide- spread tree species in North America, and its rapid growth generates the most abundant wood-based biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation pro- cedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce trans- formed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media. Transformation was verified through b-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes. The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3–4 months, rather than the 6–12 months required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes them better suited for large-scale functional genomics studies in poplars. Keywords Agrobacterium Á Aspen Á Poplar Á Populus Á Transformation Abbreviations AS Acetosyringone Cb Carbenicillin Gm Gentamycin GUS b-glucuronidase HEPT High efficiency poplar transformation HPT Hygromycin phosphotransferase gene Kan Kanamycin LB Lauria Broth MES 2-[N-morpholinil]ethanesulfonic acid NPTII Neomycin phosphotransferase II gene PCR Polymerase chain reaction TDZ N-phenyl-N¢-1,2,3-thiadiazol-5-yl-urea (thidiazuron) WPM Wood plant medium (Lloyd and McCown 1980) X-gluc 5-bromo-4-chloro-3-indolyl-b-glucuronic acid Introduction Apart from pines, Populus species are the most widely distributed trees in the world, and they are the most widely distributed tree species in the North America (Barret 1980; Isebrands and Karnosky 2002). Poplar trees, such as trembling aspen (Populus tremuloides), are a major wood crop in the Great Lakes region, and they are abundantly Communicated by P.P. Kumar. L. J. Cseke Á S. B. Cseke Á G. K. Podila (&) Department of Biological Sciences, The University of Alabama in Huntsville, 142 Wilson Hall, Huntsville, AL 35899, USA e-mail: podilag@uah.edu 123 Plant Cell Rep (2007) 26:1529–1538 DOI 10.1007/s00299-007-0365-0