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In vitro propagation and cryopreservation of the medicinal species Hovenia dulcis Thunb. (Rhamnaceae)

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Abstract

This study describes in vitro propagation and cryopreservation of Hovenia dulcis, a woody species used in traditional medicine. Stem and leaf explants from axenic seedlings were cultivated on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and kinetin (KIN) alone or in combination (0.1, 0.2, 0.5 mg L−1). For in vitro propagation, rates of regeneration (percentage of responsive explants) and proliferation (multiplication capacity of explant-derived shoots) were evaluated after 30 days and five subcultures, respectively. For cryopreservation by V Cryo-plate technique, shoot tips were excised from microcuttings cultured from in vitro-grown stock plants, or excised directly from axillary shoots of stock plants. The shoot tips were precultured in 0.3 M sucrose (24 h), exposed to loading (20 min) and to PVS2 (0–150 min) before storage in liquid nitrogen. The regrowth was assessed by plating of shoot tips on recovery medium (MS with BA + KIN), with or without a sterile filter paper over the culture medium. Cryopreservation was evaluated by survival (4-weeks) and recovery (8-weeks). The highest regeneration by direct organogenesis (100%) were reached on medium with BA + KIN (0.5 mg L−1 each). Shoots maintained multiplication capacity, showing the highest proliferation (87%) in the presence of BA. Shoot elongation and rooting were achieved on growth regulator-free MS. The most efficient cryopreservation protocol (68% survival and 62% recovery) applied exposure to PVS2 (120 min), and recovery on medium containing BA + KIN (0.5 mg L−1 each) with filter paper. The propagation and cryopreservation of H. dulcis may contribute to its conservation and that of other woody species.

Key message

This study aimed to establish an in vitro propagation methodology and the first cryopreservation protocol for Hovenia dulcis, a woody species of commercial, medicinal and nutraceutical values.

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Abbreviations

BA:

6-Benzyladenine

KIN:

Kinetin

PVS2:

Plant Vitrification Solution 2

MS:

Murashige and Skoog (1962) Medium

LN:

Liquid Nitrogen

V Cryo-plate:

Vitrification Cryo-plate technique

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Acknowledgements

This study was supported by the Brazilian Federal Agency for Support and Evaluation of Graduate Education/CAPES (Finance Code 001), the Brazilian Council for Scientific and Technological Development/CNPq and The Carlos Chagas Filho Research Support Foundation/FAPERJ. The authors are grateful to Adriana M. Lanziotti (TCT/FAPERJ) for lab assistance and illustrations of methodology and to Márcio M. Silva for valuable help with graphic design. Special thanks to Dr. Takao Niino (University of Tsukuba, Japan) for the supply of cryo-plates and Dr. Florent Engelmann (Institut de Recherche pour le Développement, France) for introducing the V Cryo-plate methodology to our research group.

Funding

This research is grant aided by the Brazilian Council for Scientific and Technological Development/CNPq (Process 421,538/2016-3) and The Carlos Chagas Filho Research Support Foundation/FAPERJ (E-26/010.001631/2014 and E-26/010.001019/2016).

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Authors and Affiliations

  1. Laboratório de Biotecnologia de Plantas (Labplan), Núcleo de Biotecnologia Vegetal (NBV), Instituto de Biologia Roberto Alcantara Gomes (IBRAG), Universidade do Estado do Rio de Janeiro (UERJ), 524 São Francisco Xavier St., Rio de Janeiro, RJ, 20550-013, Brazil

    Aline Medeiros Saavedra, Tatiana Carvalho de Castro, Lívia da Silva Cordeiro, Thaís Athayde, Norma Albarello & Claudia Simões-Gurgel

Authors
  1. Aline Medeiros Saavedra
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  2. Tatiana Carvalho de Castro
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  3. Lívia da Silva Cordeiro
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  4. Thaís Athayde
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  5. Norma Albarello
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  6. Claudia Simões-Gurgel
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Contributions

AMS, TCC, LSC, NA, CSG conceived and planned the experiments. AMS, TCC, LSC, TA carried out the experiments. LSC, NA, CSG supervised the work. All authors contributed to the interpretation of the results, wrote, read and approved the manuscript.

Corresponding author

Correspondence to Claudia Simões-Gurgel.

Additional information

Communicated by: Qiao-Chun Wang.

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Saavedra, A.M., de Castro, T.C., da Silva Cordeiro, L. et al. In vitro propagation and cryopreservation of the medicinal species Hovenia dulcis Thunb. (Rhamnaceae). Plant Cell Tiss Organ Cult 144, 577–591 (2021). https://doi.org/10.1007/s11240-020-01980-6

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  • DOI: https://doi.org/10.1007/s11240-020-01980-6

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