AN IMPROVED PROTOCOL FOR ELIMINATING ENDOGENOUS BETA-GLUCURONIDASE BACKGROUND IN BARLEY

Scutella of immature barley embryos were found to exhibit high activity of endogenous beta-glucuronidase which interferes with the expression of bacterial beta-glucuronidase that was transferred into the scutella by biolistic transformation, The amount of intrinsic activity was strongly genotype dependent and could be detected with both the histochemical and fluorimetrical assay. This interfering activity could not be eliminated by performing the assay at pH 7.0 nor using additional methanol in the incubation buffer. Only pretreatment of the bombarded scutella for 60 min at 55 degrees C followed by staining at the same temperature in a modified buffer was found to completely suppress barley-endogenous beta-glucuronidase, In addition to the spots of strong GUS activity, these improved conditions made it possible to observe high numbers of smaller GUS-spots that became visible on the white scutellum, being no longer masked by a previous non-specific blue staining pattern. Further, this protocol can be used for unequivocally differentiating between transgenic and non-transgenic barley plants.