STABILITY AND CULTURE-MEDIUM LIMITATIONS OF GENE AMPLIFICATION IN GLYPHOSATE RESISTANT CARROT CELL-LINES

The stability of resistance to the herbicide glyphosate caused by ampl ification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) g ene was followed in six cloned Daurcus carota (carrot) cell lines and other carrot lines grown in the absence of the selection agent for per iods 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 a ctivity and EPSPS gene copy number. In some, but not al cases, glyphos ate resistance was correlated with increased DNA content as measured b y flow cytometry; The C1 wild type cell line, which had 2.3-times as m uch nuclear DNA as carrot plants, also had a correspondingly higher ch romosome number, about 42 versus 18. The medium containing high glypho sate (70 mmol . L-1) was much less toxic if the K+ level was decreased , indicating chat growth inhibition of the most resistant lines was ca used in part by the high K+ concentration of the medium. These studies show that the selected glyphosate resistance was unstable and was los t slowly when grown away from the selection agent and that high K+ con centrations limited growth of resistant lines at high glyphosate conce ntrations.