New Tools for Engineering the Arabidopsis Plastid Genome

Plastid transformation in the spectinomycin-hypersensitive Col-0 acc2-1 and Sav-0 backgrounds was found to be 100-fold more efficient than the wild type (Yu et al., 2017). As a follow up to this study, we developed new tools to expand plastid transformation biotechnology in Arabidopsis. Newly constructed pMEK vectors harbor a left targeting region truncated at the HindIII site in the plastid rrn16 gene to enable utilization of the EcoRI-HindIII multiple cloning site. A one-step selective SPEED medium permits rapid evaluation of plastid transformation efficiency in 35 to 42 days (5 to 6 weeks) and transplastomic cells can be readily identified by GFP fluorescence. Mutations in the ACC2 N-terminal chloroplast transit peptide of RLD and Ws produced ACC2-knockout lines that are spectinomycin-hypersensitive and pliable for plastid transformation. We have advanced efforts to develop plastid transformation in Arabidopsis by developing a system for rapid evaluation of plastid transformation efficacy, complete with new vectors, culture media, and new ACC2-knockout lines that expand the range of accessions available for experimentation.