Development of a protoplast culture system for carrot (Daucus carota L.)

Abstract

A system was developed for the isolation, culture and transfoffi1ationof protoplasts derived fromcarrot (Daucus carota 1.) roots (var. New Kuroda). Factors such as, protoplaststability, the effect of coconut water concentration on embryoid formation and the effect of PEG concentrationon transformation efficiency were evaluated. Protoplasts isolated enzymatically from carrot roots developed into cell clusters by 10 days in liquid protoplast medium containing 1.0-5.0% (w/v) coconut water and naphthalene acetic acid (NAA). Cells of the resultingcalluses differentiated into embryoids at a higher frequency (16%), on agar medium containing3.0-5.0% (w/v) coconut water. Transient gene expression was observed in carrot rootprotoplasts,usingPEG- mediated direct DNA transformation, with plasmid pCAMBIA 1303containing the green fluorescent protein (gfp) reporter gene, driven by the constitutive 35S promoter from Cauliflower Mosaic Virus (CaMV). Transformation was confirmed by observing under UV light. DNA uptake into protoplasts under different PEG concentrations were evaluated, and a PEG concentration of 30.0% (w/v) proved to be considerably better (with a green fluorescent protein (GFP) expression average of over 30% of transformed I I protoplasts)than a PEG concentration of 20.0% (w/v) based on higher gfp gene expression I frequency, indicating that root - derived protoplasts of carrot are suitable recipients for I transformation