Carrot protoplasts as a suitable method for protein subcellular localization

Carrot (Daucus carota) is a useful plant model for the study of carotenoid biosynthesis, specifically in roots which are enriched in carotenoids. Carrot genome and transcriptome sequences, complemented by optimized methods for carrot transformation, contribute to a comprehensive toolbox for exploring pathway regulation. To expand the repertoire of tools available for the study of D. carota, we present protocols for the isolation of protoplasts from D. carota cell suspension cultures and polyethylene glycol (PEG)-mediated transformation. To obtain carrot protoplasts, in vitro somatic embryogenesis from epicotyls is induced. The somatic embryogenic tissue that develops is transferred to liquid medium to obtain a suspension of cells which are homogenized and incubated with cell-wall degrading enzymes to release protoplasts. For transfection, protoplasts are incubated with a plasmid encoding a protein of interest prior to examination of protein localization by light microscopy. As an example, we demonstrate nuclear localization of a carrot transcription factor, DcAREB3.