Different strategies have been developed and implemented during the last decades aiming to decipher the function of particular genes. Among the different techniques, in situ hybridization of mRNA remains an essential experiment to fully understand gene function. Here, we describe a protocol for the in situ localization of gene transcripts in plants. It is optimized for use of paraffin-embedded tissues and DIG-labeled probes and has successfully applied to floral bud tissues from Medicago truncatula. Using this protocol, we have analyzed the expression of MADS-box transcription factors where some of them have been preserved as duplicates in the genome. When duplicated genes are analyzed, the tissue and cellular location of the transcripts is the only technique that accounts for small variations in the pattern of gene expression that occurred after duplication and diversification. The use of a well-standardized in situ hybridization protocol is vital for the systematic analysis of the function of genes in Medicago truncatula.
Keywords: Digoxigenin; In situ hybridization; M. truncatula; Riboprobe; Transcript; mRNA.