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Does somaclonal variation play advantageous role in conservation practice of endangered species?: comprehensive genetic studies of in vitro propagated plantlets of Viola stagnina Kit. (Violaceae)

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Abstract

In vitro regeneration of Viola stagnina Kit., endangered in most part of its European distribution range, was successfully obtained based on the newly developed protocol. Adventitious shoots via direct and indirect organogenesis were induced on leaf blade and petiole fragments on solidified Murashige and Skoog (MS) medium supplemented with 0.5 or 1 mg l−1 thidiazuron, respectively. Shoots were rooted on half-strength MS medium with 2% sucrose and 0.5 mg l−1 indole-3-acetic acid (IAA), and plantlets were successfully acclimatized. Sixty-five of the regenerated plants (72% of isolated shoots cultured on rooting medium) survived at the experimental plot conditions. Among recovered via organogenesis plants, individuals ‘true-to-type’ derived from initial plant G1 and also plants genetically distant from initial plants were detected by ISSR markers. In all groups of clones genetic indices (number of genotypes, polymorphic markers, gene diversity, total gene diversity, mean gene diversity) were lower than in natural populations. Regenerated plantlets had the same genome size estimated by flow cytometry as initial material and plants from natural populations. They developed chasmogamous flowers with highly viable pollen grains (over 90%), cleistogamous flowers, and set seeds from both flower types in the first and second seasons cultivated at experimental plots. This is the first report of a successfully developed micropropagation protocol of V. stagnina, and the first detailed genetic analysis of recovered plants with the use of ISSR markers and genome size measurements allowing to discuss the advantageous role of somaclonal variation in ex situ plant conservation with the use of in vitro micropropagation.

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Acknowledgements

Authors would like to thank dr. Artur Pliszko from Institute of Botany, Jagiellonian University and Drs. Ryszard Marecki and Tomasz S. Olszewski from Gdansk University for their help in collecting of plant material. This work was funded in part by the Dean of the Faculty of Biology and Earth Sciences of Jagiellonian University (Project DS/MND/WBiNoZ/IB/25/2012 for P. Żabicki).

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Authors and Affiliations

  1. Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, 9 Gronostajowa St., 30-387, Cracow, Poland

    Piotr Żabicki, Monika Tuleja, Justyna Żabicka, Aneta Słomka, Monika Kwiatkowska & Elżbieta Kuta

  2. Laboratory of Molecular Biology and Cytometry, Department of Agricultural Biotechnology, UTP University of Science and Technology, 7 Prof. S. Kaliskiego Ave., 85-789, Bydgoszcz, Poland

    Elwira Sliwinska

  3. Botanical Garden, Jagiellonian University, 27 Kopernika St., 31-501, Cracow, Poland

    Józef Mitka

  4. Department of Plant Breeding and Seed Science, University of Agriculture, 24 Łobzowska St., 31-140, Cracow, Poland

    Agnieszka Sutkowska

  5. Institute of Biology, Pedagogical University of Cracow, 2 Podchorążych St., 30-084, Cracow, Poland

    Grzegorz Migdałek

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  1. Piotr Żabicki
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  2. Elwira Sliwinska
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Contributions

All Authors made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, participated in drafting the article or revising it critically. PŻ study conception and design, acquisition of data, analysis and interpretation of data, drafting of manuscript; EŚ performed and evaluated flow cytometric analyses and participated in writing the manuscript; JM conducted calculations of genetic indices; AS acquisition, analysis and interpretation of data; MT acquisition of data, participated in drafting the article; GM analysis and interpretation of data, participated in drafting the manuscript; JŻ analysis and interpretation of data, participated in revising the manuscript; AS acquisition of data, participated in revising the manuscript; MK participated in revising the manuscript and formatting it according to instructions for authors; EK contribution to interpretation of data, supervising experiments, critical revision of the manuscript.

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Correspondence to Piotr Żabicki.

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Communicated by Sergio J. Ochatt.

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Żabicki, P., Sliwinska, E., Mitka, J. et al. Does somaclonal variation play advantageous role in conservation practice of endangered species?: comprehensive genetic studies of in vitro propagated plantlets of Viola stagnina Kit. (Violaceae). Plant Cell Tiss Organ Cult 136, 339–352 (2019). https://doi.org/10.1007/s11240-018-1519-1

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