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
