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.
Highlights
The generation of transgenic plants has allowed for new insights into gene function
Agrobacterium tumefaciens cultures and culturing methods A. tumefaciens strains ABI and GV3101 were both obtained from the Amasino group (University of Wisconsin-Madison, WI, USA)
Whereas the rate of transformation of A. thaliana transgenics was significantly lower from A. tumefaciens grown in LB media supplemented with 25% sucrose (w/v) and 0.02% Silwet L77, this preparation was found to support the generation of transgenic plants (Figure 1)
Summary
The generation of transgenic plants has allowed for new insights into gene function. The creation of the floral-dip protocol [1] markedly advanced the ease of creating transformants in Arabidopsis thaliana. We found that after saturated growth in YEBS media of A. tumefaciens cell line ABI harboring the CCR2:LUC transgene [11], the addition of both 25 g per L of sucrose and 200 μL Silwet L77 per L of culture supported robust transfomation (Figure 1).
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