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Cryopreservation of an endangered Hladnikia pastinacifolia Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

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Abstract

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.

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Funding

The Slovene Ministry of Higher Education, Science, and Technology supported this research within the program “Research to Ensure Food Safety and Health” with the Grant No. P1-0164, led by D. Škorjanc.

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Authors and Affiliations

  1. Faculty of Natural Sciences and Mathematics, University of Maribor, Koroška cesta 160, 2000, Maribor, Slovenia

    Terezija Ciringer, Nina Šajna, Mitja Kaligarič & Jana Ambrožič-Dolinšek

  2. Departemento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040, Madrid, Spain

    Carmen Martín

  3. Faculty of Education, University of Maribor, Koroška cesta 160, 2000, Maribor, Slovenia

    Jana Ambrožič-Dolinšek

Authors
  1. Terezija Ciringer
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  2. Carmen Martín
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  3. Nina Šajna
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  4. Mitja Kaligarič
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  5. Jana Ambrožič-Dolinšek
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Correspondence to Jana Ambrožič-Dolinšek.

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Editor: Barbara Reed

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Ciringer, T., Martín, C., Šajna, N. et al. Cryopreservation of an endangered Hladnikia pastinacifolia Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification. In Vitro Cell.Dev.Biol.-Plant 54, 565–575 (2018). https://doi.org/10.1007/s11627-018-9917-y

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  • DOI: https://doi.org/10.1007/s11627-018-9917-y

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