Methotrexate-resistance in plants: genetic, molecular and citological analyses.

Carrot cell lines resistant to methotrexate were selected and, upon analysis, shown to overproduce dihydrofolate reductase, the target anzyme of the antifolic agenr, as the main biochemical alteration. This feature behaves as a dominant trait as judged by somatic hybridization. Using this selective marker, asymmetric fertile somatic hybrids between carrot and tobacco were obtained by protoplast fusion using the gammairradiation technique. Dihydrofolate reductase was purified and characterized as regard kinetic and structural properties and shown to be a bifunctional protein associated with thymidylate synthase. The pure protein was injected into a rabbit to obtain polyclonal antibodies which were used for the screning of an expression library in the vector lambda gtll and the subcellular localization of the bifunctional polypeptide.