GENE AMPLIFICATION IN GLYPHOSATE TOLERANT TOBACCO CELLS

A series of tobacco (Nicotiana tabacum L.) cell lines have been selected for growth in the presence of normally lethal concentrations of glyphosate, up to 20 mM. Activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), which is inhibited by glyphosate, is elevated in tolerant cells. However, EPSPS isolated from tolerant cells is still inhibited by the herbicide. Increased activity of EPSPS is the result of increased levels of the enzyme in tolerant cells, as determined by immunoblotting, rather than a change in enzyme activity. RNA blots with a petunia EPSPS cDNA demonstrate a correlation between levels of tolerance and steady state level of EPSPS mRNA. Increased expression of EPSPS mRNA results from amplification of at least two genes encoding this enzyme in the tobacco genome. The degree of gene amplification also increases with level of glyphosate tolerance. Selected cells grown in the absence of glyphosate have an elevated EPSPS mRNA abundance and maintain some gene amplification, indicating that selection has resulted in a stable genetic modification. © 1990.