Chapter 12 In situ Analysis of Gene Expression in Plants Sinéad Drea, Paul Derbyshire, Rachil Koumproglou, Liam Dolan, John H. Doonan, and Peter Shaw Summary In the post-genomic era, it is necessary to adapt methods for gene expression and functional analyses to more high-throughput levels of processing. mRNA in situ hybridization (ISH) remains a powerful tool for obtaining information regarding a gene’s temporal and spatial expression pattern and can therefore be used as a starting point to define the function of a gene or a whole set of genes. We have decon- structed ‘traditional’ ISH techniques described for a range of organisms and developed protocols for ISH that adapt and integrate a degree of automation to standardized and shortened protocols. We have adapted this technique as a high-throughput means of gene expression analysis on wax-embedded plant tissues and also on whole-mount tissues. We have used wax-embedded wheat grains and Arabidopsis floral meristems and whole-mount Arabidopsis roots as test systems and show that it is capable of highly parallel processing. Key words: High-throughput, Spatial patterns of gene expression, In situ hybridization. In situ hybridization (ISH) is one of the methods of choice for determining the spatial expression pattern of a given gene. High resolution protocols provide cellular and even subcellular resolu- tion. One of the most significant advantages inherent in the tech- nique is that it is applicable to any species whether or not these species are amenable to other methods of functional analyses such as stable transformation. For this reason it has proved to be invaluable in the evo-devo (evolution of development) field for instance where, in the absence of direct functional data in diverse species, it can provide detailed gene expression patterns across 1. Introduction Daryl J. Somers et al. (eds.), Methods in Molecular Biology, Plant Genomics, vol. 513 © Humana Press, a part of Springer Science + Business Media, LLC 2009 DOI: 10.1007/978-1-59745-427-8_12 229