IMPROVED APPROACHES OF IN SITU HYBRIDIZATION AND IN SITU ENZYME HISTOCHEMISTRY FOR GENE EXPRESSION ANALYSES IN PLANTS

Improved methods for both in situ hybridization and in situ enzyme histochemistry were described. The procedures for both methods have been significantly simplified by omitting some unnecessary treatments and substituting the cumbersome and laborious techniques, and the reliability of in situ histochemistry was increased by reversing the operations of Block and Debrouwer's procedure. The improved steps are: Instead of the conventional fixation, a simplified FAA procedure by adding liquid nitrogen onto the embedded tissues during sectioning to ensure high quality of the sections;labeled DNA by randompriming, other than labeled RNA by transcription, was used as probes in hybridization, which was conducted in a moisturesaturated plastic chamber other than emerging in mineral oil. The improved procedure for tissue in situ histochemical study was that the GUS coloration was carried out before fixation, embedding and sectioning, which was different from the procedure as described by Block and Debrouwer.