Plant Growth Regulation

, Volume 82, Issue 1, pp 103–110 | Cite as

Differential responses to isoprenoid, N 6-substituted aromatic cytokinins and indole-3-butyric acid in direct plant regeneration of Eriocephalus africanus

  • Olwethu Madzikane-Mlungwana
  • Mack Moyo
  • Adeyemi O. Aremu
  • Lucie Plíhalová
  • Karel Doležal
  • Johannes Van Staden
  • Jeffrey F. Finnie
Original paper


Eriocephalus africanus is a medicinal and aromatic plant species that is part of South Africa’s remarkable diversity. As a result of illegal and over-harvesting, most plant communities have become unsustainable and as such, effective and efficient conservation strategies have to be implemented. In the present study, an isoprenoid cytokinin (CK): isopentenyladenine (iP) and four aromatic CKs namely benzyladenine (BA), meta-topolin (mT), meta-topolin riboside (mTR) and 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine (mTTHP) at 1, 5 or 10 µM were evaluated for in vitro plant regeneration in E. africanus. Different concentrations of indole-3-butyric acid (IBA) were also evaluated for shoot and root organogenesis. The highest number of shoots was produced by mT (1 and 5 µM) treatment, longest shoots were stimulated by iP (1 µM) and the highest fresh mass was obtained in BA (5 and 10 µM), mT (5 and 10 µM) and mTTHP (5 µM)-treated plants. During acclimatization, all the in vitro plants obtained from the lowest concentration of CKs survived and 1 µM iP regenerants recorded a 100% survival rate. For the rooting experiment, more vigorous adventitious roots were observed in the 1 µM IBA treatment. All IBA treatments had 100% survival rate after 6 weeks of acclimatization. Overall, the concentration and type of plant growth regulators had a remarkable influence on the growth and development of in vitro-derived E. africanus.


Auxins Cytokinins Flavonoids Plant regeneration Phenolics 



N 6-Benzyladenine


Catechin equivalents




Dry weight


Gallic acid equivalents


Indole-3-butyric acid


N 6-Isopentenyladenine




meta-Topolin riboside




Murashige and Skoog (1962) medium


Plant growth regulator


Photosynthetic photon flux



The University of KwaZulu-Natal and National Research Foundation, South Africa provided financial support. LP and KD thank the Ministry of Education, Youth and Sports, Czech Republic (Grant LO1204 from the National Program of Sustainability I.) for financial support. We thank Mrs Alison Young (UKZN Botanical Garden, Pietermaritzburg, South Africa) and her Staff for their assistance in maintaining the greenhouse facilities.

Author contributions

OMM conducted the experiments, collected and analysed data and wrote the draft paper with the help of MM and AOA. LP and KD synthesized meta-topolin, meta-topolin riboside and 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine. JFF and JVS provided funding and research facilities, respectively. All authors edited the paper.


  1. Amoo SO, Van Staden J (2012) Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix. Plant Cell Tiss Organ Cult 112:249–256CrossRefGoogle Scholar
  2. Amoo SO, Aremu AO, Van Staden J (2012) In vitro plant regeneration, secondary metabolite production and antioxidant activity of micropropagated Aloe arborescens Mill. Plant Cell Tiss Organ Cult 111:345–358CrossRefGoogle Scholar
  3. Aremu AO, Bairu MW, Doležal K, Finnie JF, Van Staden J (2012) Topolins: a panacea to plant tissue culture challenges? Plant Cell Tiss Organ Cult 108:1–16CrossRefGoogle Scholar
  4. Bairu MW, Stirk WA, Dolezal K, Van Staden J (2007) Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell Tiss Organ Cult 90:15–23CrossRefGoogle Scholar
  5. Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA, Taiz L, Muday GK (2001) Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis. Plant Physiol 126:524–535CrossRefPubMedPubMedCentralGoogle Scholar
  6. Buer CS, Muday GK (2004) The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. Plant Cell 16:1191–1205CrossRefPubMedPubMedCentralGoogle Scholar
  7. Catarino MD, Silva AMS, Saraiva SC, Sobral AJFN, Cardoso SM (2015) Characterization of phenolic constituents and evaluation of antioxidant properties of leaves and stems of Eriocephalus africanus. Arab J Chem.  10.1016/j.arabjc.2015.04.018 Google Scholar
  8. Chang HS, Charkabarty D, Hahn EJ, Paek KY (2003) Micropropagation of calla lily (Zantedeschia albomaculata) via in vitro shoot tip proliferation. In Vitro Cell Dev Biol-Plant 39:129–134CrossRefGoogle Scholar
  9. de Klerk G-J, van der Krieken W, de Jong JC (1999) Review the formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Biol-Plant 35:189–199CrossRefGoogle Scholar
  10. Doležal K, Popa I, Kryštof V, Spíchal L, Fojtíková M, Holub J, Lenobel R, Schmülling T, Strnad M (2006) Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells. Bioorg Med Chem 14:875–884CrossRefPubMedGoogle Scholar
  11. Doležal K, Malá J, Máchová P, Cvrcková H, Karady M, Novák O, Szucova L, Mikulik J, Spichal L, Strnad M. 2011. New cytokinin derivatives—their discovery, development and use for micropropagation of endangered tree species. BMC Proceedings. BioMed Central Ltd, p O46Google Scholar
  12. Escalona M, Cejas I, González-Olmedo J, Capote I, Roels S, Cañal M, Rodríguez R, Sandoval J, Debergh P (2003) The effect of meta-topolin on plantain propagation using a temporary immersion bioreactor. InfoMusa 12:28–30Google Scholar
  13. Frébort I, Kowalska M, Hluska T, Frébortová J, Galuszka P (2011) Evolution of cytokinin biosynthesis and degradation. J Exp Bot 62:2431–2452CrossRefPubMedGoogle Scholar
  14. Gentile A, Gutiérrez MJ, Martinez J, Frattarelli A, Nota P, Caboni E (2014) Effect of meta-topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell Tiss Organ Cult 118:373–381CrossRefGoogle Scholar
  15. Kamínek M, Motyka V, Vaňková R (1997) Regulation of cytokinin content in plant cells. Physiol Plant 101:689–700CrossRefGoogle Scholar
  16. Khan T, Abbasi BH, Khan MA, Shinwari ZK (2016) Differential effects of thidiazuron on production of anticancer phenolic compounds in callus cultures of Fagonia indica. Appl Biochem Biotechnol 179:46–58CrossRefPubMedGoogle Scholar
  17. Lall N, Kishore N (2014) Are plants used for skin care in South Africa fully explored? J Ethnopharmacol 153:61–84CrossRefPubMedGoogle Scholar
  18. Ljung K, Hull AK, Celenza J, Yamada M, Estelle M, Normanly J, Sandberg G (2005) Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17:1090–1104CrossRefPubMedPubMedCentralGoogle Scholar
  19. Ludwig-Müller J (2000) Indole-3-butyric acid in plant growth and development. Plant Growth Regul 32:219–230CrossRefGoogle Scholar
  20. Makunga N, Philander L, Smith M (2008) Current perspectives on an emerging formal natural products sector in South Africa. J Ethnopharmacol 119:365–375CrossRefPubMedGoogle Scholar
  21. Malá J, Máchová P, Cvrčková H, Karady M, Novák O, Mikulík J, Hauserová E, Greplová J, Strnad M, Doležal K (2009) Micropropagation of wild service tree (Sorbus torminalis [L.] Crantz): the regulative role of different aromatic cytokinins during organogenesis. J Plant Growth Regul 28:341–348CrossRefGoogle Scholar
  22. Moyo M, Finnie JF, Van Staden J (2012) Topolins in Pelargonium sidoides micropropagation: do the new brooms really sweep cleaner? Plant Cell Tiss Organ Cult 110:319–327CrossRefGoogle Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. Murphy A, Peer AW, Taiz L (2000) Regulation of auxin transport by aminopeptidases and endogenous flavonoids. Planta 211:315–324CrossRefPubMedGoogle Scholar
  25. Niedz RP, Evens TJ (2010) The effects of benzyladenine and meta-topolin on in vitro shoot regeneration of a Citrus citrandarin rootstock. Res J Agric Biol Sci 6:45–53Google Scholar
  26. Njenga E, Viljoen A (2006) In vitro 5-lipoxygenase inhibition and anti-oxidant activity of Eriocephalus L.(Asteraceae) species. S Afr J Bot 72:637–641CrossRefGoogle Scholar
  27. Njenga E, Van Vuuren S, Viljoen A, Eloff J (2005) Antimicrobial activity of Eriocephalus L. species. S Afr J Bot 71:81–87CrossRefGoogle Scholar
  28. Palacio L, Cantero JJ, Cusidó RM, Goleniowski ME (2012) Phenolic compound production in relation to differentiation in cell and tissue cultures of Larrea divaricata (Cav.). Plant Sci 193–194:1–7CrossRefPubMedGoogle Scholar
  29. Peer WA, Murphy AS (2007) Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci 12:556–563CrossRefPubMedGoogle Scholar
  30. Philander LA (2011) An ethnobotany of Western Cape Rasta bush medicine. J Ethnopharmacol 138:578–594CrossRefGoogle Scholar
  31. Plíhalová L, Vylíčilová H, Doležal K, Zahajská L, Zatloukal M, Strnad M (2016) Synthesis of aromatic cytokinins for plant biotechnology. New Biotechnol 33:614–624CrossRefGoogle Scholar
  32. Salie F, Eagles P, Leng H (1996) Preliminary antimicrobial screening of four South African Asteraceae species. J Ethnopharmacol 52:27–33CrossRefPubMedGoogle Scholar
  33. SANBI (2015). Red list of South African plants.
  34. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
  35. Štefančič M, Štampar F, Osterc G (2005) Influence of IAA and IBA on root development and quality of Prunus ‘GiSelA 5’ leafy cuttings. HortScience 40:2052–2055Google Scholar
  36. Szopa A, Ekiert H (2012) In vitro cultures of Schisandra chinensis (Turcz.) Baill.(Chinese Magnolia Vine)—a potential biotechnological rich source of therapeutically important phenolic acids. Appl Biochem Biotechnol 166:1941–1948CrossRefPubMedPubMedCentralGoogle Scholar
  37. Szopa A, Ekiert H (2014) Production of biologically active phenolic acids in Aronia melanocarpa (Michx.) Elliott in vitro cultures cultivated on different variants of the Murashige and Skoog medium. Plant Growth Regul 72:51–58CrossRefGoogle Scholar
  38. Szüčová L, Spíchal L, Doležal K, Zatloukal M, Greplová J, Galuszka P, Kryštof V, Voller J, Popa I, Massino FJ, Jørgensen J-E, Strnad M (2009) Synthesis, characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives. Bioorg Med Chem 17:1938–1947CrossRefPubMedGoogle Scholar
  39. Taylor LP, Grotewold E (2005) Flavonoids as developmental regulators. Curr Opin Plant Biol 8:317–323CrossRefPubMedGoogle Scholar
  40. Tubić L, Savić J, Mitić N, Milojević J, Janošević D, Budimir S, Zdravković-Korać S (2015) Cytokinins differentially affect regeneration, plant growth and antioxidative enzymes activity in chive (Allium schoenoprasum L.). Plant Cell Tiss Organ Cult 124:1–14Google Scholar
  41. Verdeguer M, Blázquez MA, Boira H (2009) Phytotoxic effects of Lantana camara, Eucalyptus camaldulensis and Eriocephalus africanus essential oils in weeds of Mediterranean summer crops. Biochem Syst Ecol 37:362–369CrossRefGoogle Scholar
  42. Verstraeten I, Geelen D (2015) Adventitious rooting and browning are differentially controlled by auxin in rooting-recalcitrant Elegia capensis (Burm. f.) Schelpe. J Plant Growth Regul 34:475–484CrossRefGoogle Scholar
  43. Viljoen AM, Njenga EW, Van Vuuren SF, Bicchi C, Rubiolo P, Sgorbini B (2006) Essential oil composition and in vitro biological activities of seven Namibian species of Eriocephalus L.(Asteraceae). J Essent Oil Res 18:124–128Google Scholar
  44. Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC (1996) Meta-topolin, an alternative to benzyladenine in tissue culture? Physiol Plant 98:291–297CrossRefGoogle Scholar
  45. Wiesman Z, Riov J, Epstein E (1989) Characterization and rooting ability of indole-3-butyric acid conjugates formed during rooting of mung bean cuttings. Plant Physiol 91:1080–1084CrossRefPubMedPubMedCentralGoogle Scholar
  46. Zhishen J, Mengcheng T, Jianming W (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 64:555–559CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Olwethu Madzikane-Mlungwana
    • 1
  • Mack Moyo
    • 1
    • 2
  • Adeyemi O. Aremu
    • 1
  • Lucie Plíhalová
    • 3
  • Karel Doležal
    • 3
  • Johannes Van Staden
    • 1
  • Jeffrey F. Finnie
    • 1
  1. 1.Research Centre for Plant Growth and Development, School of Life SciencesUniversity of KwaZulu-NatalScottsvilleSouth Africa
  2. 2.Department of Horticultural Sciences, Faculty of Applied SciencesCape Peninsula University of TechnologyCape TownSouth Africa
  3. 3.Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of SciencePalacký University and Institute of Experimental Botany AS CROlomoucCzech Republic

Personalised recommendations