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

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

Abstract

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 

Notes

Acknowledgments

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).

References

  1. 1.
    Liu J et al (2014) WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis. Plant Cell 26:1081–1093CrossRefGoogle Scholar
  2. 2.
    Xu L, Huang H (2014) Genetic and epigenetic controls of plant regeneration. Curr Top Dev Biol 108:1–33CrossRefGoogle Scholar
  3. 3.
    Ikeuchi M, Ogawa Y, Iwase A, Sugimoto K (2016) Plant regeneration: cellular origins and molecular mechanisms. Development 143:1442–1451CrossRefGoogle Scholar
  4. 4.
    Kareem A et al (2016) De novo assembly of plant body plan: a step ahead of Deadpool. Regeneration 3:182–197CrossRefGoogle Scholar
  5. 5.
    Chen L et al (2016) YUCCA-mediated auxin biogenesis is required for cell fate transition occurring during de novo root organogenesis in Arabidopsis. J Exp Bot 67:4273–4284CrossRefGoogle Scholar
  6. 6.
    Chen X et al (2016) Auxin-independent NAC pathway acts in response to explant-specific wounding and promotes root tip emergence during de novo root organogenesis in arabidopsis. Plant Physiol 170:2136–2145CrossRefGoogle Scholar
  7. 7.
    Chen X et al (2014) A simple method suitable to study de novo root organogenesis. Front Plant Sci 5:208CrossRefGoogle Scholar
  8. 8.
    Hu X, Xu L (2016) Transcription factors WOX11/12 directly activate WOX5/7 to promote root primordia initiation and organogenesis. Plant Physiol 172:2363–2373CrossRefGoogle Scholar
  9. 9.
    Yu J, Liu W, Liu J, Qin P, Xu L (2017) Auxin control of root organogenesis from callus in tissue culture. Front Plant Sci 8:1385CrossRefGoogle Scholar
  10. 10.
    Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci U S A 94:2122–2127CrossRefGoogle Scholar
  11. 11.
    Kim MK et al (2002) Specimen block counter-staining for localization of GUS expression in transgenic arabidopsis and tobacco. Plant Cell Rep 21:35–39CrossRefGoogle Scholar
  12. 12.
    Kurihara D, Mizuta Y, Sato Y, Higashiyama T (2015) ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142:4168–4179CrossRefGoogle Scholar
  13. 13.
    Muller SM, Galliardt H, Schneider J, Barisas BG, Seidel T (2013) Quantification of Forster resonance energy transfer by monitoring sensitized emission in living plant cells. Front Plant Sci 4:413PubMedCentralGoogle Scholar
  14. 14.
    Truernit E et al (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20:1494–1503CrossRefGoogle Scholar
  15. 15.
    Haseloff J (2003) Old botanical techniques for new microscopes. BioTechniques 34:1174–1178, 1180, 1182CrossRefGoogle Scholar
  16. 16.
    Kumar R, Silva L (1973) Light ray tracing through a leaf cross section. Appl Opt 12:2950–2954CrossRefGoogle Scholar
  17. 17.
    He C, Chen X, Huang H, Xu L (2012) Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS Genet 8:e1002911CrossRefGoogle Scholar
  18. 18.
    Bennett T, van den Toorn A, Willemsen V, Scheres B (2014) Precise control of plant stem cell activity through parallel regulatory inputs. Development 141:4055–4064CrossRefGoogle Scholar
  19. 19.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  20. 20.
    Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefGoogle Scholar
  21. 21.
    Tsuge T, Tsukaya H, Uchimiya H (1996) Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 122:1589–1600PubMedGoogle Scholar

Copyright information

© 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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