High efficiency poplar transformation.
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 procedures 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 transformed 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 beta-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.
Cseke
Leland J
LJ
Department of Biological Sciences, The University of Alabama in Huntsville, 142 Wilson Hall, Huntsville, AL 35899, USA.
Cseke
Sarah Beth
SB
Podila
Gopi K
GK
D016960:Agrobacterium tumefaciens / Q000502:physiology; D015152:Blotting, Northern; D002934:Cinnamates / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000187:drug effects; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D018517:Plant Roots / Q000187:drug effects / Q000382:microbiology; D030821:Plants, Genetically Modified; D032107:Populus / Q000187:drug effects / Q000235:genetics*; D013997:Time Factors; D014170:Transformation, Genetic / Q000187:drug effects
D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A