M2WISH: An easy and efficient protocol for whole‐mount mRNA in situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana

SUMMARY Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co‐expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems. Significance Statement Gene expression analysis is crucial for understanding plant development mechanisms. We developed a protocol utilizing MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis that offers high‐yield, homogeneous hybridization without cellular damage. Combined with histochemical staining, it provides cellular resolution for gene expression. Demonstrating its robustness with 10 genes across various plant organs, this protocol generates extensive gene expression localization data for modeling complex systems.