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Plant Methods. 2009; 5: 3.
Published online 2009 February 27. doi: 10.1186/1746-4811-5-3.
PMCID: PMC2660325
Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana
Amanda M Davis,#1 Anthony Hall,#2 Andrew J Millar,3 Chiarina Darrah,#1 and Seth J Daviscorresponding author1
1Max Planck Institute, Cologne, Germany
2University of Liverpool, Liverpool, UK
3University of Edinburgh, Edinburgh, UK
corresponding authorCorresponding author.
#Contributed equally.
Amanda M Davis: amdavis/at/mpiz-koeln.mpg.de; Anthony Hall: anthony.hall/at/liverpool.ac.uk; Andrew J Millar: andrew.millar/at/ed.ac.uk; Chiarina Darrah: Chiarina.Darrah/at/UGent.be; Seth J Davis: davis/at/mpiz-koeln.mpg.de
Received December 9, 2008; Accepted February 27, 2009.
Abstract
Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore, where multiple transgenes are to be introduced, achieving this by sequential transformations over multiple generations adds significantly to the time required. To circumvent these bottlenecks, we have developed three streamlined sub-protocols. First, we find that A. thaliana can be transformed by dipping directly into an A. tumefaciens culture supplemented with surfactant, eliminating the need for media exchange to a buffered solution. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. These sub-protocols, which can be used separately or in combination, save time and money, and reduce the possibility of contamination.