Targeted C-to-T Base Editing in the Arabidopsis Plastid Genome

Arabidopsis thaliana, particularly the ecotype Columbia-0 (Col-0), has been extensively employed in the study of genetics of the nuclear genome. However, the difficulty of modifying the plastid genome of Col-0, the most widely used ecotype, has hindered investigation of the functional interactions between nuclear-encoded and plastid-encoded genes in this ecotype. Recently, we achieved targeted base editing, substituting a specific C:G pair with a T:A pair in the plastid genome of Col-0 through the application of genome-editing technology. This article introduces the method employed to accomplish this targeted base editing. The process involves four steps: (i) designing and constructing a binary vector encoding the genome-editing enzyme, (ii) introducing the binary vector into the nuclear genome of Col-0 via floral dipping, (iii) identifying base-edited plants, and (iv) verifying inheritance of the edited base(s) by the next generation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design and construction of a binary vector encoding ptpTALECD or ptpTALECD_v2 Basic Protocol 2: Agrobacterium-mediated introduction of a binary vector into the Arabidopsis nuclear genome Basic Protocol 3: Selection of plants harboring T-DNA in the nucleus and detection of base editing in the plastid genome.