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

  • Jie Yu
  • Ning Zhai
  • Lin Xu
  • Wu LiuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2094)


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.

Key words

De novo root regeneration Gene expression pattern GUS staining ClearSee Congo red staining Plant regeneration Adventitious root 



We thank B. Scheres for Arabidopsis lines. This work was supported by grants from the Strategic Priority Research Program “Molecular Mechanism of Plant Growth and Development” of CAS (XDPB0403) and China Postdoctoral Science Foundation (2017M611627).


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant SciencesInstitute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of SciencesShanghaiChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

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