The effect of cytokinins on growth, phenolics, antioxidant and antimicrobial potential in liquid agitated shoot cultures of Knautia sarajevensis


Endemic plants are under constant threat of extinction and micropropagation protocols that can provide not only mechanisms for their revitalisation, but also a possibility for potential sources of new biologically active substances. This study describes the very first use of the liquid culture system for in vitro Knautia sarajevensis shoot multiplication, production of biomass, and an increase in biological activities of the extracts. Murashige and Skoog media containing various concentrations of cytokinins (6-benzyladenine, zeatin, and kinetin) were used to establish agitated shoot cultures of this endemic plant species and to evaluate their effect on shoot morphology. HPLC analysis of phenolic compounds show that the main metabolite in all extracts was salicylic acid with accumulation of rosmarinic and 4-hydroxybenzoic acid for some treatments. The richest sources of phenolics were shoots cultivated in media containing zeatin, which also had high influence on biomass production. Analysis of antioxidant and antimicrobial potential suggests that this plant could have beneficial biological activities. Methanol extracts of shoots cultivated in media containing 2.0 mg/L 6-benzyladenine were moderately active against Staphylococcus aureus and Bacillus spizizeni. DPPH assay showed radical scavenging activity of shoot cultures with IC50 10–90 µg/mL.

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4-Hydroxybenzoic acid


Antioxidant potential




Caffeic acid


Chlorogenic acid




2,2-Diphenyl-1-picrylhydrazyl radical


Standard deviation


Dry weight


Ferulic acid


Gallic acid




High-performance liquid chromatography




Murashige and Skoog




Plant growth regulator






Rosmarinic acid


Salicylic acid


Sinapic acid


Specific peroxidase activity


Syringic acid


Total protein content


Vanillic acid




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This research was supported by Grant No. LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS.

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EK performed all the in vitro culture and analysis of growth, total phenolics, peroxidase activity and DPPH, antimicrobial potential, statistical analysis as well as the writing of the manuscript. SCZ and PT developed HPLC method for separation and quantification of phenolic compounds. They performed construction of calibration curves, analysis of plant extracts, and calculation of the content of each particular phenolic compound in extracts. EM participated in field work by collecting the seeds and identification of plant species used for this research and in statistical analysis of the data. AP supervised the in vitro culture work.

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Correspondence to Erna Karalija.

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The authors declare that they have no conflict of interest.

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Communicated by Nokwanda Pearl Makunga.

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Karalija, E., Ćavar Zeljković, S., Tarkowski, P. et al. The effect of cytokinins on growth, phenolics, antioxidant and antimicrobial potential in liquid agitated shoot cultures of Knautia sarajevensis . Plant Cell Tiss Organ Cult 131, 347–357 (2017).

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  • Antibacterial activity
  • Antioxidant activity
  • In vitro cultures
  • Phenolics
  • HPLC