Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 136, Issue 2, pp 339–352 | Cite as

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)

  • Piotr ŻabickiEmail author
  • Elwira Sliwinska
  • Józef Mitka
  • Agnieszka Sutkowska
  • Monika Tuleja
  • Grzegorz Migdałek
  • Justyna Żabicka
  • Aneta Słomka
  • Monika Kwiatkowska
  • Elżbieta Kuta
Original Article


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.


In vitro organogenesis Plant regeneration Genome size Somaclonal variation ISSR markers Viola 



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).

Author 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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2018_1519_MOESM1_ESM.docx (33 kb)
Supplementary Figure 1 (DOCX 32 KB)
11240_2018_1519_MOESM2_ESM.docx (18 kb)
Supplementary Table 1 (DOCX 17 KB)
11240_2018_1519_MOESM3_ESM.docx (16 kb)
Supplementary Table 2 (DOCX 15 KB)


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Piotr Żabicki
    • 1
    Email author
  • Elwira Sliwinska
    • 2
  • Józef Mitka
    • 3
  • Agnieszka Sutkowska
    • 4
  • Monika Tuleja
    • 1
  • Grzegorz Migdałek
    • 5
  • Justyna Żabicka
    • 1
  • Aneta Słomka
    • 1
  • Monika Kwiatkowska
    • 1
  • Elżbieta Kuta
    • 1
  1. 1.Department of Plant Cytology and Embryology, Institute of BotanyJagiellonian UniversityCracowPoland
  2. 2.Laboratory of Molecular Biology and Cytometry, Department of Agricultural BiotechnologyUTP University of Science and TechnologyBydgoszczPoland
  3. 3.Botanical GardenJagiellonian UniversityCracowPoland
  4. 4.Department of Plant Breeding and Seed ScienceUniversity of AgricultureCracowPoland
  5. 5.Institute of BiologyPedagogical University of CracowCracowPoland

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