3 Biotech

, 9:17 | Cite as

Advancement in protocol for in vitro seed germination, plant regeneration and cryopreservation of Viola cornuta

  • Milena Trajković
  • Dragana Antonić
  • Aleksandar Cingel
  • Nabil Ghalawenji
  • Angelina Subotić
  • Slađana JevremovićEmail author
Original Article


The aim of this study was to develop a fast, reliable and true-to-type protocol for in vitro plant regeneration and long-term storage of horned pansy (Viola cornuta L). Seed germination over 60% was recorded after 12 weeks of growth at 10 °C or 4 °C. Calli formation and shoot induction were obtained in petiole and hypocotyl culture on half-strength MS mineral salts with full concentration of Na–FeEDTA and vitamins (½MS medium) with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1 mg/L) and 6-benzylaminopurine (BAP, 2.0 mg/L) and leaf culture on ½MS medium with thidiazuron (TDZ,1.0 mg/L). The highest frequency of adventitious shoot induction (50%) with six shoots/explant was achieved in hypocotyl culture from top hypocotyl segments, close to epicotyl which was grown 8 weeks at 16 h light/8 h dark photoperiod. Subsequent shoot multiplication was achieved on ½MS medium with α-naphthaleneacetic acid (NAA, 0.1 or 0.5 mg/L) and BAP (1.0 mg/L). Rooting of shoots was obtained on ½MS medium with low concentration (0.1 mg/L) of auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA, or without growth regulators. In vitro-derived plantlets were acclimatized under greenhouse conditions. All plants developed normally, bloomed and set seeds. Shoot tips were cryopreserved succssefully using modified plant vitrification 3 (PVS3-based vitrification procedure). Cold acclimation for 2 weeks significantly improved shoot regrowth (64%) after thawing in comparison to non-acclimated shoots (39%). Clonal fidelity of regenerated plantlets at ploidy level was confirmed by chromosome counting. The presented protocol can be useful for mass propagation, genetic transformation studies and long-term storage of valuable Viola spp.


Horned pansy Seed germination Micropropagation Hypocotyl culture Cryopreservation 



We thank Dr. Zoran Jeknić (Oregon State University, USA) for helpful discussions and critical reading of the manuscript. This research was sponsored by the Ministry for Education, Science and Technological Research, Serbia (Project TR31019).

Compliance with ethical standards

Conflict of interest

The authors declare that there was no conflict of interest.


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”University of BelgradeBelgradeSerbia

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