Efficient Regeneration-based Agrobacterium-Mediated Transformation of an Asexual Amphibious Brassicaceae Species,...

Stable genetic transformation of plants mediated by Agrobacterium tumefaciens has been a powerful tool for fundamental plant biology research as well as plant biotechnologies that directly impact crop improvement. Simple and effective transformation pipelines, such as the floral-dipping method for Arabidopsis, have drastically advanced large-scale analyses of gene function. However, such a pipeline cannot be applied to plant species that do not undergo sexual reproduction. Rorippa aquatica is an emerging amphibious Brassicaceae model species exhibiting heterophylly, drastic changes of leaf and plant form in terrestrial or underwater environments. R. aquatica exclusively propagates vegetatively. The optimized pipeline presented here provides efficient and reliable Agrobacterium-mediated R. aquatica transformation that does not require sterile tissue culture. Leaf cuttings are briefly immersed in Agrobacterium suspended in transformation buffer containing a binary vector that expresses a visual marker, RUBY, under the constitutive promoter. Subsequently, the inoculants are grown on a water-soaked filter paper in a growth chamber. Then, regenerating plantlets with RUBY red pigmentation are separated and potted in soil. Those initial T1 regenerants (R1-T1) express red sectors. Through cutting out the red sector and repeating the regeneration steps, a large number of transgenic siblings are produced in the next R2-T1 generation, some of which are nearly uniformly red; indicative of RUBY expression. This simple transformation pipeline can be adopted for R. aquatica and possibly other vegetatively propagating plant species to introduce genes of interest to investigate their functions in the adaptation to terrestrial vs. aquatic environments.