Characterization of the glyphosate selection of carrot suspension cultures resulting in gene amplification

When Daucus carota L. (carrot) suspension cultures, which had been under gradual selection for increased glyphosate resistance, were plated in glyphosate-containing medium, the populations became gradually more resistant with I50 values about equal to the final selection concentration used in the liquid medium. Wild type cells did not grow on plates with 2 mM glyphosate, but some colonies formed after 90 days. Cells in these colonies, after growth away for the inhibitor to obtain sufficient cells, had increased glyphosate resistance and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity and usually had amplified EPSPS genes. One step selection in liquid or solidified medium with 10 mM glyphosate was not successful. However, in liquid medium with 2 mM glyphosate, the cell viability decreased to about 30% by 30 days and then increased as growth became evident. Kinetic analysis indicates that at least 0.8% of the cells grow. The growth rate seen when the cells are subjected to a gradual selection scheme, where glyphosate was about doubled at each step, also indicates that a high proportion of the original population survives to form the new population. These results indicate that the gene amplification which causes the glyphosate tolerance in carrot cell cultures is a gradual and relatively stable process that occurs in a relatively high proportion of the cell population.