Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines

Diploid alfalfa (HG2), capable of plant regeneration from tissue culture, was used to select variant cell lines resistant to growth inhibition due to ethionine (an analog of methionine). Approximately 10(7) suspension-cultured cells were mutagenized with methane sulfonic acid ethylester and then plated in solid media containing ethionine. Callus colonies formed on media with 0.02 mM ethionine. Of the 124 cell lines recovered, 91 regenerated plants. After six months growth on media without ethionine, 15 of 110 cell lines of callus grew significantly better than HG2 on 1 mM ethionine. Several ethionine-resistant callus cultures were also resistant to growth inhibition due to the addition of lysine + threonine to the media. High concentrations, relative to unselected HG2 callus, of methionine, cysteine, cystathionine, and glutathione were found in some, but not all, ethionine-resistant callus cultures. Cell line R32, which had a ca. tenfold increase in soluble methionine, had a 43% increase in total free amino acids and a 40% increase in amino acids in protein as compared to unselected HG2 callus. Relative amounts of each amino acid in protein were the same in both.