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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High-performance liquid chromatography
Murashige and Skoog
Plant growth regulator
Specific peroxidase activity
Total protein content
Akram M, Aftab F (2015) Effect of cytokinins on in vitro seed germination and changes in chlorophyll and soluble protein contents of teak (Tectona grandis L.). Biochem Physiol 4:166. doi:10.4172/2168-9652.1000166
Alali FQ, Amrine CSM, El-Elimat T, Alkofahi A, Tawaha K, Gharaibah M, Swanson SM, Falkinham JO, Cabeza M, Sánchez A, Figueroa M (2014) Bioactive withanolides from Withania obtusifolia. Phytochem Lett 9:96–101
Albayrak S, Aksoy A, Sagdic O, Hamzaoglu E (2010) Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from Turkey. Food Chem 119(1):114–122
Allen DE, Hatfield G (2004) Medicinal plants in folk tradition. Timber Press, Portland
Barba-Espin G, Diaz-Vivancos P, Clemente-Moreno MJ, Albacete A, Faize L, Faize M, Pérez-Alfocea F, Hernández JA (2010) Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant Cell Environ 33(6):981–994
Beck GM, Maly K, Bijelćić Ž (1974) Knautia. In: Čović B (ed) Flora Bodnae et Herzegovinae, IV Sympetalae, vol 3. Zemaljski muzej Bosne i Hercegovine, Sarajevo, p 75
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254
Gadzovska S, Maury S, Delaunay A, Spasenoski M, Joseph C, Hagege D (2007) Jasmonic acid elicitation of Hypericum perforatum L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones. Plant Cell Tissue Organ Cult 89(1):1–13
González LF, Rojas MC, Perez FJ (1999) Diferulate and lignin formation is related tobiochemical differences of wall-bound peroxidases. Phytochemistry 50(5):711–717
Grzegorczyk-Karolak I, Rytczak P, Bielecki S, Wysokińska H (2015) The influence of liquid systems for shoot multiplication, secondary metabolite production and plant regeneration of Scutellaria alpina. Plant Cell Tissue Organ Cult 122(3):699–708
Grzegorczyk-Karolak I, Rytczak P, Bielecki S, Wysokińska H (2017) The influence of liquid systems for shoot multiplication, secondary metabolite production and plant regeneration of Scutellaria alpina. Plant Cell Tissue Organ Cult 128(2):479–486
Hendrawati O, Hille J, Woerdenbag HJ, Quax WJ, Kayser O (2012) In vitro regeneration of wild chervil (Anthriscus sylvestris L.). In Vitro Cell Dev Biol Plant 48(3):355–361
Karalija E, Ćavar Zeljković S, Tarkowski P, Muratović E, Parić A (2017) Media composition effects seed dormancy, apical dominance and phenolic profile of Knautia sarajevensis (Dipsacaceae), Bosnian endemic. Acta Bot Croat. doi:10.1515/botcro-2017-0011
Kasparavičienė G, Ramanauskienė K, Savickas A, Velžienė S, Kalvėnienė Z, Kazlauskienė D, Ragažinskienė O Ivanauskas K (2013) Evaluation of total phenolic content and antioxidant activity of different Rosmarinus officinalis L. ethanolic extracts. Biologija 59(1):39–44
Kikowska M, Kędziora I, Krawczyk A, Thiem B (2015) Methyl jasmonate, yeast extract and sucrose stimulate phenolic acids accumulation in Eryngium planum L. shoot cultures. Acta Biochim Pol 62(2):197–2000
Kuhlmann A, Röhl C (2006) Phenolic antioxidant compounds produced by in vitro. Cultures of Rosemary (Rosmarinus officinalis.) and their anti-inflammatory effect on lipopolysaccharide-activated microglia. Pharm Biol 44(6):401–410
Lou Z, Wang H, Li J, Zhu S, Lu W, Ma C (2011) Effect of simultaneous ultrasonic/microwave assisted extraction on the antioxidant and antibacterial activities of burdock leaves. J Med Plant Res 5(22):5370–5377
Matkowski A (2008) Plant in vitro culture for the production of antioxidants: a review. Biotechnol Adv 26(6):548–560
Mattalia G, Quave CL, Pieroni A (2013) Traditional uses of wild food and medicinal plants among Brigasc, Kyé, and Provençal communities on the Western Italian Alps. Genet Resour Crop Evol 60(2):587–603
McFarland J (1907) The nephelometer: an instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. JAMA 49(14):1176–1178
Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG (2005) Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem 91(3):571–577
Moore DM (1980) Dipsacaceae. In: Tutin TG, Heywood VH, Burges NA, Moore DM, Valentine DH, Walters SM, Webb DA (eds) Flora Europea, volume 4: plantaginaceae to compositae. University Press, Cambridge, p 56.
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497
Murthy HN, Lee EJ, Paek KY (2014) Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult 118(1):1–16
Ordonez AAL, Gomez JD, Vattuone MA (2006) Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food Chem 97(3):452–458
Ramirez-Estrada K, Vidal-Limon H, Hidalgo D, Moyano E, Golenioswki M, Cusidó RM, Palazon J (2016) Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 21(2):182
Raomai S, Kumaria S, Kehie M, Tandon P (2015) Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitors. Plant Growth Regul 75(1):341–353
Saher S, Piqueras A, Hellin E, Olmos E (2004) Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress. Physiol Plant 120(1):152–161
Sakamoto K, Iida K, Sawamura K, Hajiro K, Asada Y, Yoshikawa T, Furuya T (1994) Anthocyanin production in cultured cells of Aralia cordata Thunb. Plant Cell Tissue Organ Cult 36(1):21–26
Savio, L. E. B., Astarita LV, Santarém ER (2012) Secondary metabolism in micropropagated Hypericum perforatum L. grown in non-aerated liquid medium. Plant Cell Tissue Organ Cult 108(3):465–472
Shani E, Ben-Gera H, Shleizer-Burko S, Burko Y, Weiss D, Ori N (2010) Cytokinin regulates compound leaf development in tomato. Plant Cell 22(10):3206–3217
Takayama S, Akita M (2005) Practical aspects of bioreactor application in mass propagation of plants. In: Liquid culture systems for in vitro plant propagation. Springer, Dordrecht, pp 61–78
Tawaha K, Alali FQ, Gharaibeh M, Mohammad M, El-Elimat T (2007) Antioxidant activity and total phenolic content of selected Jordanian plant species. Food Chem 104(4):1372–1378
Tosun M, Ercisli S, Sengul M, Ozer H, Polat T, Ozturk E (2009) Antioxidant properties and total phenolic content of eight Salvia species from Turkey. Biol Res 42(2):175–181
Von Wettstein D, Jende-Strid B, Ahrenst-Larsen B, Sørensen JA (1977) Biochemical mutant in barley renders chemical stabilization of beer superfluous. Carlsberg Res Commun 42(5):341–351
Wawrosch C, Benda E, Kopp B (2009) An improved 2-step liquid culture system for efficient in vitro shoot proliferation of Sundew (Drosera rotundifolia L.). Sci Pharm 77(4):827–836
Wolfe K, Wu X, Liu RH (2003) Antioxidant activity of apple peels. J Agric Food Chem 51(3):609–614
Xia D, Wu X, Shi J, Yang Q, Zhang Y (2011) Phenolic compounds from the edible seeds extract of Chinese Mei (Prunus mume Sieb. et Zucc) and their antimicrobial activity. LWT Food Sci Technol 44(1):347–349
Zaheer M, Giri CC (2015) Multiple shoot induction and jasmonic versus salicylic acid driven elicitation for enhanced andrographolide production in Andrographis paniculata. Plant Cell Tissue Organ Cult 122(3):553–563
Zaheer M, Reddy VD, Giri CC (2016) Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L.(Fabaceae). Nat Prod Res 30(13):1542–1547
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.
Conflict of interest
The authors declare that they have no conflict of interest.
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). https://doi.org/10.1007/s11240-017-1288-2
- Antibacterial activity
- Antioxidant activity
- In vitro cultures