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Long-term conservation of Tarenaya rosea (Cleomaceae) root cultures: histological and histochemical analyses during cryopreservation using the encapsulation-vitrification technique


Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0–120 min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90 min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

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The authors are grateful to Jeanne A. T. Glória for the valuable technical assistance with histochemical analyses, to Thaís J. Vasconcellos for help in using the Image-Pro Plus software, and Adriana M. Lanziotti for lab assistance.


This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - (CAPES/Brazil) - Finance Code 001 - and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil) through an international collaborative research project (Program Science without Borders - Project no. A054/2013) between the Núcleo de Biotecnologia Vegetal of the Universidade do Estado do Rio de Janeiro (UERJ/Brazil) and the Institut de Recherche pour le Développement (IRD/Montpellier, France). The work was also supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ/Brazil).

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Correspondence to Lívia da Silva Cordeiro.

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da Silva Cordeiro, L., Collin, M., Callado, C.H. et al. Long-term conservation of Tarenaya rosea (Cleomaceae) root cultures: histological and histochemical analyses during cryopreservation using the encapsulation-vitrification technique. Protoplasma (2020).

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  • Adventitious root cultures
  • Cleome rosea
  • Intercellular content
  • Medicinal plant
  • Plasmolysis