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Advances in cryopreservation of vanilla (Vanilla planifolia Jacks.) shoot-tips: assessment of new biotechnological and cryogenic factors

  • Plant Tissue Culture
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Abstract

The effect of dehydration, cryopreservation, and reculture conditions on growth recovery (%) of vanilla (Vanilla planifolia) shoot-tips was evaluated using a D-cryoplate procedure. Tissues were excised from in vitro grown plantlets, preconditioned on MS semisolid medium supplemented with 0.15 M trehalose for 1 d, loaded in a solution of 0.4 M sucrose or trehalose and 2 M glycerol for 30 min, and dehydrated within a laminar flow cabinet for various durations (30, 60, 90, 120, 150, and 180 min). The same preconditioning and loading treatments were compared using dehydration with vitrification solutions (PVS2 or PVS3) for 30 min at room temperature according to droplet-vitrification and V-cryoplate methods. The highest (33%) recovery of cryopreserved shoot-tips was achieved using the D-cryoplate method after 0.15 M trehalose preconditioning, loading with sucrose-glycerol solution and desiccation for 180 min. DSC analyses revealed that the osmotically active water (OAW) content of the shoot-tips was reduced from 77% (fresh weight basis) to 17% after the only effective drying duration (180 min). Melting endotherms indicated that crystallization events accompanied cryopreservation of the tissues. Proliferation of multiple shoots was obtained by indirect organogenesis. Histological analysis of the explants during post-cryopreservation recovery confirmed the organogenic nature of the callus formed after 3–4 mo of reculture in the dark on semisolid multiplication medium. This was followed by a secondary organogenesis on MS medium with kinetin (2 mg L−1) and exposure to a photoperiod. At present, this is the most optimized cryopreservation protocol for shoot-tips of V. planifolia.

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Acknowledgments

The authors are very grateful to Prof. Takao Niino for his friendly and valuable technical assistance in the use of cryoplate procedures and thank to MSc. Fernando Ortega-Escalona for his technical support in the histological analysis. The authors also acknowledge the kind proofreading of the manuscript by Dr. Sershen Naidoo and Dr. Sean Lahmeyer.

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Authors and Affiliations

  1. Facultad de Ciencias Químicas, Universidad Veracruzana, Prolongación Oriente 6, No. 1009, 94340, Orizaba, Veracruz, Mexico

    F. Hernández-Ramírez, O. Flores-Castaños, M.P. Rascón & M.T. González-Arnao

  2. Doctorado en Ciencias Agropecuarias, Facultad de Ciencias Biológicas y Agropecuarias, Universidad Veracruzana, Peñuela, Veracruz, Mexico

    F. Hernández-Ramírez & O. Flores-Castaños

  3. Instituto de Botánica del Nordeste (IBONE), Universidad Nacional del Nordeste (UNNE), Corrientes, Argentina

    N. Dolce

  4. Instituto de Ecología, El Haya, Xalapa, Veracruz, Mexico

    G. Ángeles-Álvarez

  5. Botanical Division, The Huntington Library, Art Collections and Botanical Gardens, San Marino, CA, USA

    R. Folgado

Authors
  1. F. Hernández-Ramírez
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  2. N. Dolce
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  3. O. Flores-Castaños
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  4. M.P. Rascón
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  5. G. Ángeles-Álvarez
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  6. R. Folgado
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  7. M.T. González-Arnao
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Correspondence to M.T. González-Arnao.

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Editor: Yong Eui Choi

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Hernández-Ramírez, F., Dolce, N., Flores-Castaños, O. et al. Advances in cryopreservation of vanilla (Vanilla planifolia Jacks.) shoot-tips: assessment of new biotechnological and cryogenic factors. In Vitro Cell.Dev.Biol.-Plant 56, 236–246 (2020). https://doi.org/10.1007/s11627-020-10069-w

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