Advancement in protocol for in vitro seed germination, plant regeneration and cryopreservation of Viola cornuta
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.
KeywordsHorned 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.
- Alimohammadi M, Bagherieh-Najjar MB (2009) Agrobacterium-mediated transformation of plants: basic principles and influencing factors. Afr J Biotechnol 8:5142–5148Google Scholar
- Antonić D, Trajković M, Cingel A, Subotić A, Jevremović S (2017) Plant regeneration from in vitro-derived leaf and petiole explants of Viola cornuta L.’Lutea Splendens’. Propag Ornam Plants 17(3):95–102Google Scholar
- Babber S, Sharma K (1991) Study of anatomy of vitrified structure in Viola tricolor L. Ann Biol Hissar 7:93–96Google Scholar
- Chalageri G, Babu UV (2012) In vitro plant regeneration via petiole callus of Viola patrinii and genetic fidelity assessment using RAPD markers. Turk J Bot 36:358–368Google Scholar
- El Aou-ouad H, Medrano H, Lamarti A, Gulías J (2014) Seed germination at different temperatures and seedling emergence at different depths of Rhamnus spp. Cent Eur J Biol 9(5):569–578Google Scholar
- George F (1993) Plant propagation by tissue culture, part I: the technology. Exegenetics, Edington, 1–574Google Scholar
- Mitchell WC, Cornetto TM, Nunez M, Patel M (2000) Analysis of horned violet, Viola cornuta seed germination. Bull N J Acad Sci 45:1–5Google Scholar
- Mokhtari A, Zarei M, Samsamzadeh B, Moradi K (2016) Interactive effects of plant growth regulators and explants on direct shoot regeneration of Viola odorata. Biol Tech 97(1):33–39Google Scholar
- Muktadir MA, Habib MA, Mian MAK, Akhond MAY (2016) Regeneration efficiency based on genotype, culture conditions and growth regulators of eggplant (Solanum melongena L.). Agric Natl Res 50:38–42Google Scholar
- Naeem M, Naveed I, Naqvi SMS, Mahmood T (2013) Standardization of tissue culture conditions and estimation of free scavenging activity in Viola odorata L. Pak J Bot 45:197–202Google Scholar
- Reed BM (1990) Survival of in vitro-grown apical meristem of Pyrus following cryopreservation. Hort Sci 25:111–113Google Scholar
- Sakai A, Engelman F (2007) Vitrifiction, encapsulation-vitrification and droplet-vitrification: a review. Cryo Lett 28:151–172Google Scholar
- Vishwakarma UR, Garav AM, Sharma PC (2013) Regeneration of multiple shoots from petiole callus of Viola serpens Wall. Pharm Res 5:86–92Google Scholar
- Wijowska M, Kota E, Przywara L (1999) In vitro culture of unfertilized ovules of Viola odorata L. Acta Biol Cracov Bot 41:95–101Google Scholar