Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

Abstract

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.

Development of Protoporphyrinogen Oxidase as an Efficient Selection Marker forAgrobacterium tumefaciens-Mediated Transformation of Maize

“…Selection based on herbicide tolerance could potentially lead to an increase in gene copy number as a result of strong selection for high gene expression (Shyr et al, 1992). The stability of transgenes has been shown to be related to the copy number of insertions (Sidorenko and Peterson, 2001).…”

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

“…Glyphosate inhibits the conversion of shikimate 3-phosphate to EPSP, which is catalyzed by EPSP synthase (Reinbothe et al, 1991). The mRNA levels of EPSP synthase in two lines of resistant carrot cells, CAR and CI, subjected to stepwise selection were 59 and 32 times the level of their respective wild-type cells (Shyr et al, 1992). Moreover, the resistant suspension-cultured cells of Corydalis sempervirens (Holländer-Czytko et al, 1992) and petunia (Shah et al, 1986) contained several-times higher levels of EPSP synthase mRNA than wild-type cells.…”

Resistance to Protoporphyrinogen Oxidase-inhibiting Compound S23142 from Overproduction of Mitochondrial Protoporphyrinogen Oxidase by Gene Amplification in Photomixotrophic Tobacco Cells

“…For example, many plant cell cultures selected for glyphosate resistance have higher EPSPS activity due to overproduction by EPSPS gene ampliˆcation, [34][35][36] and alfalfa cells selected for phosphinothricin resistance have a 4-11-fold higher copy number of the glutamine synthetase genes, resulting in a roughly 8-fold higher mRNA level and a 3-to 7-fold higher enzyme activity. 37) Chlorsulfuron resistance in carrot cells is also associated with the increased activity of the target enzyme, acetohydroxyacid synthase, which is due to overproduction by gene ampliˆcation.…”