Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues

Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, sec ondary cell walls non-specifically bind probes used for in situ hybridizati on thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techn iques are used. Here we describe an in situ hybridization technique which u ses fast freezing and freeze substitution to cryoimmobilize the mRNA follow ed by embedding in a methacrylate resin for high-resolution analysis of gen e expression. By using a transgenic poplar line harbouring rolC::uidA, rolC ::iaaM, the gene expression pattern could be compared with histochemical GU S staining. This in situ hybridization technique results in superior preser vation of cellular contents, retention of mRNA in all cell types in the pop lar stem, a significant reduction of non-specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of gene s involved in xylogenesis and wood formation.