Selection of methotrexate-resistant cell lines in Daucus carota: biochemical analysis and genetic characterization by protoplast fusion.

In this work we report the isolation and characterization of carrot cell lines resistant to methotrexate (Mtx). Selection of spontaneous variants was accomplished by one step and two step exposure to Mtx. Among lines that showed normal Mtx uptake, no difference from the parental line was found in inactivation of the inhibitor or affinity of dihydrofolate reductase (DHFR) for Mtx.
One step resistant lines, when maintained in a medium lacking Mtx for at least 75 days, showed a DHFR specific activity and capacity to bind Mtx 3 -4 times that of the parental one and these values remained stable up to 6 months. DHFR activity appeared to be very low in cell lines grown with Mtx, due to the presence of an inhibitor, possibly Mtx itself, in the cell extract.
Nevertheless, measurement of the amount of DHFR using antibodies to the pure enzyme indicated that the level of the enzyme is about the same in lines maintained either in the presence or in the absence of Mtx. Genetic analysis by protoplast fusion has shown that resistance is a dominant or semidominant trait.