Abstract
Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.
Highlights
Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants
Agrobacterium is routinely used as a plant gene transformation vehicle as it naturally possesses the ability to transfer a segment of its plasmid DNA (T-DNA) into its host nucleus, which leads to integration of the T-DNA into the nuclear genome (Tzfira et al, 2004)
The vacuum infiltration step was replaced by floral dipping where the developing floral tissues are dipped into a solution containing Agrobacterium, sucrose and the surfactant Silwet L-77 (Clough & Bent, 1998)
Summary
Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. The updated version of this manuscript includes additional references on floral dip transformation, clarification of data in Figure 1 in the legend, and addition of the OD600 value of the Agrobacterium cell density at the time of harvest in the Method section. In Agrobacterium-mediated transformation protocols, the concentration of bacterial inoculum has been considered crucial to the success of plant transformation.
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