Methotrexate-resistant Somatic Cells of Glycine max (L.) Merr.: Selection and Characterization

A total of 70 methotrexate-resistant cell lines were isolated from UV-irradiated fast growing suspensions of Glycine max(L.) Merr. The resistant cells grew in 4 X 10(-4) M MTX without significant reduction in growth rate, whereas 100 % of wild type cells died in 2.2 x 10 (-7) M within 24 h. The frequency of resistant cell clones was 2 x 10(-8) per cell generation. Two different selection strategies, a single-step and a multiple-step protocol yielded phenotypically identical resistant lines. All resistant lines have retained their resistance over more than 500 cell generations in the absence of selection. Resistant cells plated on MTX could be selected from an excess of wild type cells. Ten percent of the resistant lines internalized MTX at a reduced rate. DHFR activity in wild type and resistant cells was equally sensitive to inhibition by MTX. Resistant cells did not display an increased activity of DHFR. Likewise MTX binding to cell extracts was the same for all cell lines. In the wild type MTX-blockage of cell division could be partially released by 2 x 10(-4) M thymidine. In the presence of MTX thymidine had no influence on the growth of resistant cells.