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Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots

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Abstract

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22–71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39–45 %) was already observed after the sucrose pretreatment; it increased to 54–68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

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Acknowledgments

This work has received partial financial support from ARCAD, a flagship programme of Agropolis Fondation (Montpellier, France) (I. Engelmann-Sylvestre), and from the PHC 2010–11 STAR project no. 23200UJ. Montpellier Rio Imaging (MRI) is gratefully acknowledged for providing access to its facilities.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. UMR DIADE, IRD, 911 Avenue Agropolis, BP 64501, 34394, Montpellier Cedex 5, France

    Mohammad Salma, Isabelle Engelmann-Sylvestre, Myriam Collin & Florent Engelmann

  2. Plateforme d’Histologie et d’Imagerie Cellulaire Végétale, UMR AGAP, CIRAD, Avenue Agropolis, TAA-108/02, 34398, Montpellier Cedex 5, France

    Jacques Escoute, Marc Lartaud & Jean-Luc Verdeil

  3. National Academy of Agricultural Science, Suwon, 441-853, Republic of Korea

    Jung-Yoon Yi & Haeng-Hoon Kim

  4. Department of Well-Being Resources, Sunchon National University, 225 Jungang-ro, Suncheon, Jeonnam, 540-742, Republic of Korea

    Haeng-Hoon Kim

Authors
  1. Mohammad Salma
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  2. Isabelle Engelmann-Sylvestre
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  3. Myriam Collin
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  4. Jacques Escoute
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  5. Marc Lartaud
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  6. Jung-Yoon Yi
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  7. Haeng-Hoon Kim
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  8. Jean-Luc Verdeil
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  9. Florent Engelmann
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Corresponding author

Correspondence to Florent Engelmann.

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Handling Editor: Peter Nick

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Salma, M., Engelmann-Sylvestre, I., Collin, M. et al. Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots. Protoplasma 251, 649–659 (2014). https://doi.org/10.1007/s00709-013-0565-0

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