Length of time in tissue culture can affect the selected glyphosate resistance mechanism. 
Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene that leads to resistance by increasing EPSPS mRNA and enzyme activity.  We show that glyphosate selection with newly initiated suspension cultures can produce resistant lines with resistance mechanisms other than gene amplification and that usually as the cultures age gene amplification becomes the predominant mechanism.  Gene amplification did not occur in 3 lines selected from 5-month-old Datura innoxia Mill.  cultures but did occur in all 10 lines selected after 52 months.  Selection with Nicotiana tabacum L. (tobacco) less than 5 months old produced 2 lines out of 24 with no EPSPS amplification while all 17 lines selected from older cultures contained amplified genes.  Lines selected from the oldest culture (35 years) also exhibited amplification of several different genes, indicating the expression of different EPSPS genes or an enhanced gene amplification incidence.  None of the 15 lines selected from 2 different 5-month-old Daucus carota L. (carrot) lines exhibited amplification while amplification led to the resistance of all 7 lines selected from one of the original carrot lines (DHL) after 3 years.  However, the other line (Car4) was exceptional and produced only non-amplified lines (9 of 9) after 8 years in culture.  These results show that plant tissue cultures change with time in culture and that several different new mechanisms can result in glyphosate resistance.