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CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.

ILHigher plant DNA is extensively methylated, but the two methylated sequences (CpG and CpNpG) show different characteristics. Using sequence analysis techniques, we demonstrate that while CpG methylation follows the existing models for cytosine methylation in animals, CpNpG methylation does not. Although there is evidence to support the suggestion that the low CpG frequency has arisen from deaminational conversion of 5-methylcytosine to thymidine, there appears to be no comparable conversion of 5-methylcytosine in the CpNpG configuration. It therefore appears that between the evolution of CpG and CpNpG cytosine methylation systems, a mechanism evolved for the correction of C + T conversion, probably using the methylated strand to direct the repair in the correct direction.