Characterization of Carrot and Tobacco Cell Cultures Resistant to p-Fluorophenylalanine

Abstract

This study describes the isolation and characterization of p-fluorophenylalanine-resistant diploid tobacco (Nicotiana tabacum L.) and diploid carrot (Daucus carota L.) cultured cell lines. The p-fluorophenylalanine-resistant tobacco and carrot lines can grow in medium containing p-fluorophenylalanine concentrations 10 to more than 100 times those which inhibit the growth of susceptible cells, respectively. The resistance trait was retained when the cells were grown in a medium lacking the phenylalanine analog for 50 generations. All 14 single cell clones started from the resistant carrot line remained resistant. The resistant lines incorporated much less p-fluorophenylalanine into protein, partially due to a decrease in uptake. In carrots, an increase in the levels of free phenylalanine and tyrosine also apparently contributed to the decreased incorporation of p-fluorophenylalanine into protein by increasing the metabolic pool size which diluted the incoming analog and caused a lowered percentage of incorporation, which was observed. Apparently, phenylalanine and tyrosine synthesis was also increased in resistant tobacco lines, since chorismate mutase was found to have greater activity and to be less sensitive to inhibition by phenylalanine, tyrosine, and p-fluorophenylalanine. It appears, however, that phenylalanine and tyrosine do not accumulate above the normal levels in the resistant tobacco cells, as these amino acids were apparently converted into phenolic compounds which were found in higher levels (6 times). The low frequency of appearance, the stability of the trait, and the biochemical nature of the resistance, indicate that the p-fluorophenylalanine resistance found in the carrot and tobacco lines described here is due to a mutation.