Method to Study Gene Expression Patterns During De Novo Root Regeneration from Arabidopsis Leaf Explants.

De novo root regeneration (DNRR) is the process in which adventitious roots are regenerated from damaged plant tissues or organs. We have developed a simple DNRR system in which adventitious roots are formed from detached leaf explants of Arabidopsis (Arabidopsis thaliana) on B5 medium without external hormones. In this chapter, we introduce the methods used to observe gene expression patterns during rooting from leaf explants. Usually, β-glucuronidase (GUS) staining is used to visualize gene expression patterns, since fluorescent proteins are difficult to observe because of the high autofluorescence in leaf explants. Here, we describe the use of the ClearSee technique with Congo red staining for deep imaging to observe fluorescent proteins. This method diminishes autofluorescence in leaf explants and preserves the stability of fluorescent proteins, thus allowing us to investigate the endogenous molecular actions guiding DNRR.