Stability and culture medium limitations of gene amplification in glyphosate resistant carrot cell lines

Abstract

The stability of resistance to the herbicide glyphosate caused by amplification of the 5-enolpyruvylshiki-mate-3-phosphate synthase (EPSPS) gene was followed in six cloned Daucus carota (carrot) cell lines and other carrot lines grown in the absence of the selection agent for periods up to about four years. The lines originated from three different carrot suspension cultures with high levels of glyphosate resistance. The cloned lines lost resistance with a half-time of about two years. The resistance was correlated with decreased levels of EPSPS enzyme activity and EPSPS gene copy number. In some, but not all cases, glyphosate resistance was correlated with increased DNA content as measured by flow cytometry. The C1 wild type cell line, which had 2.3-times as much nuclear DNA as carrot plants, also had a correspondingly higher chromosome number, about 42 versus 18. The medium containing high glyphosate (70 mmol · L−1) was much less toxic if the K+ level was decreased, indicating that growth inhibition of the most resistant lines was caused in part by the high K+ concentration of the medium. These studies show that the selected glyphosate resistance was unstable and was lost slowly when grown away from the selection agent and that high K+ concentrations limited growth of resistant lines at high glyphosate concentrations.