A rapid in vivo shikimate accumulation assay with excised leaf discs

An in vivo shikimate accumulation assay with excised leaf tissue was developed to provide a fast and reliable method for identifying glyphosate-resistant plants. The assay is based on glyphosate-induced accumulation of shikimate. There was a linear accumulation of shikimate in excised leaf discs of soybean and canola treated with 250 mu M glyphosate for 48 h. The IC50 for the accumulation of shikimate in soybean and corn leaf discs was 34 and 87 mu M, respectively. Leaf discs excised from glyphosate-resistant corn or soybean did not accumulate shikimate when treated with 500 mu M glyphosate. Leaf discs taken from a number of field-grown plants accumulated shikimate in a glyphosate dose-dependent manner. The accumulation of shikimate was dependent on light and the age of the leaf from which the disc was taken. The assay worked either in 96-well microtiter plates or in vials, and it clearly differentiated between glyphosate-resistant and -susceptible crops in which the resistance is due to an alteration of the target site for glyphosate. The assay was simple and robust and has the potential to be used as a high throughput assay to detect glyphosate resistance in weeds.