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Article

Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation

1
Institute of Biology II/Molecular Plant Physiology, Centre for BioSystems Analysis, BIOSS Centre for Biological Signalling Studies University of Freiburg, 79104 Freiburg, Germany
2
Department of Food Science, Aarhus University, 8200 Aarhus, Denmark
3
Institute of Vegetables and Melon Growing of National Academy of Agricultural Sciences of Ukraine, 62478 Kharkiv, Ukraine
*
Authors to whom correspondence should be addressed.
Academic Editor: Yoselin Benitez-Alfonso
Plants 2021, 10(2), 375; https://doi.org/10.3390/plants10020375
Received: 13 December 2020 / Revised: 26 January 2021 / Accepted: 10 February 2021 / Published: 15 February 2021
(This article belongs to the Special Issue Stars in Plant Cell Biology)
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. View Full-Text
Keywords: Arabidopsis thaliana; protoplasts; reprograming; auxin Arabidopsis thaliana; protoplasts; reprograming; auxin
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MDPI and ACS Style

Pasternak, T.; Paponov, I.A.; Kondratenko, S. Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation. Plants 2021, 10, 375. https://doi.org/10.3390/plants10020375

AMA Style

Pasternak T, Paponov IA, Kondratenko S. Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation. Plants. 2021; 10(2):375. https://doi.org/10.3390/plants10020375

Chicago/Turabian Style

Pasternak, Taras; Paponov, Ivan A.; Kondratenko, Serhii. 2021. "Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation" Plants 10, no. 2: 375. https://doi.org/10.3390/plants10020375

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