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

Scutella of immature barley embryos were found to exhibit high activit y of endogenous beta-glucuronidase which interferes with the expressio n of bacterial beta-glucuronidase that was transferred into the scutel la by biolistic transformation, The amount of intrinsic activity was s trongly genotype dependent and could be detected with both the histoch emical and fluorimetrical assay. This interfering activity could not b e eliminated by performing the assay at pH 7.0 nor using additional me thanol in the incubation buffer. Only pretreatment of the bombarded sc utella for 60 min at 55 degrees C followed by staining at the same tem perature 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 n umbers of smaller GUS-spots that became visible on the white scutellum , being no longer masked by a previous non-specific blue staining patt ern. Further, this protocol can be used for unequivocally differentiat ing between transgenic and non-transgenic barley plants.