Efficient regeneration from cotyledon protoplasts in Arabidopsis thaliana

An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of alpha-naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.