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Optimizing protocols for arabidopsis shoot and root protoplast cultivation

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  • Taras Pasternak, University of Freiburg
  • ,
  • Ivan A. Paponov
  • Serhii Kondratenko, National Academy of Sciences of Ukraine

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

Original languageEnglish
Article number375
Pages (from-to)1-17
Number of pages17
Publication statusPublished - Feb 2021

Bibliographical note

Funding Information:
Funding: This research was supported by the National Academy of Agrarian Sciences of Ukraine in the course of the second level assignment (type of assignment F) of scientific research program No. 18 "Vegetable and melon growing". This work was supported by Bundesministerium für Bildung und Forschung (BMBF 349 Microsystems).

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Copyright 2021 Elsevier B.V., All rights reserved.

    Research areas

  • Arabidopsis thaliana, Auxin, Protoplasts, Reprograming

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