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
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