Skip to main content

Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin?


Benzyladenine (BA) is the most widely used cytokinin in the micropropagation industry due to its effectiveness and affordability. It, however, has disadvantages such as genetic alteration and abnormal growth in some plants. Naturally occurring zeatin on the other hand is not as widely used as BA and is far more expensive. The use of meta-topolin and its derivatives as alternatives to BA and zeatin, both of which frequently have negative effects in tissue culture was investigated. In vitro grown Aloe polyphylla (an endangered medicinal and ornamental aloe) were cultured on full strength Murashige and Skoog basal medium with different concentrations of cytokinins and solidified with 1% Bacteriological Agar (Oxoid No. 1). mT was the preferred cytokinin both in terms of multiplication rate and rooting. The optimum concentration that induced regeneration and rooting is 5.0 μM. The problem of hyperhydricity was totally controlled. Plants rooted spontaneously in multiplication medium, thus avoiding the extra rooting step of the protocol. More than 91% of the plants transferred to ex vitro conditions were successfully acclimatized.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4



[6-benzylamino-9- β-d-glucopyranosylpurine]




indole butyric acid


Murashige and Skoog (1962) medium












plant growth regulators




  • Abrie AL, Van Staden J (2001) Micropropagation of the endangered Aloe polyphylla. Plant Growth Regul 33:19–23

    Article  CAS  Google Scholar 

  • Aubrey A (2002) Aloe polyphylla. Available online at Cited 11 Sept 2005

  • Baroja-Fernandez E, Aguirreolea J, Martínková H, Hanus J, Strnad M (2002) Aromatic cytokinins in micropropagated potato plants. Plant Physiol Biochem 40:217–224

    Article  CAS  Google Scholar 

  • Chukwujechwu JC, Fennell CW, van Staden J (2002) Optimisation of the tissue culture protocol for the endangered Aloe polyphylla. S Afr J Bot 68:424–429

    Google Scholar 

  • Horgan R, Hewett EW, Horgan JM, Purse JG, Wareing PF (1975) A new cytokinin from Populus × robusta. Phytochemistry 19:1005–1008

    Article  Google Scholar 

  • Iwamura H, Fujiita T, Koyama S, Koshimizu K, Kumazawa Z (1980) Quantitative structure-activity relationship of cytokinin-active adenine and urea derivatives. Phytochemistry 19:1309–1319

    CAS  Google Scholar 

  • Jones LH, Martinková H, Strnad M, Hanke DE (1996) Occurrence of aromatic cytokinins in oil palm (Elaeis guineensis Jacq.). J Plant Growth Regul 15:39–49

    Article  CAS  Google Scholar 

  • Kamínek M, Paces V, Corse J, Challice JS (1979) Effect of stereospecific hydroxylation of N6-(Δ2-Isopentenyl)adenosine on cytokinin activity. Planta 145:239–243

    Article  Google Scholar 

  • Kamínek M, Vanék T, Kalendova A, Pilar J (1987a) Effect of two cytokinins on production of stem cuttings by stock plants of Euphorbia pulcherrima Wild. and Gerbera jamesonii Hook. Sci Hortic 33:281–289

    Article  Google Scholar 

  • Kamínek M, Vanék T, Motyka V (1987b) Cytokinin activities of N6-benzyladenosine derivatives hydroxylated on the side-chain phenyl ring. J Plant Growth Regul 6:113–120

    Article  Google Scholar 

  • Krikorian AD (1995) Hormones in tissue culture and micropropagation. In: Davies PJ (eds) Plant hormones: physiology, biochemistry and molecular biology. Academic Publishers, The Netherlands, pp 774–796

    Google Scholar 

  • Kubalakova M, Strnad M (1992) The effect of aromatic cytokinins (populins) on micropropagation and regeneration in vitro. Biol Plant 34:578–579

    Google Scholar 

  • Leshem B, Sachs T (1985) ‘Vitrified’ Dianthus- teratomata in vitro due to growth factor imbalance. Ann Bot 56:613–617

    Google Scholar 

  • Leshem B, Shaley DP, Izhar S (1988) Cytokinin as an inducer of hyperhydricity in melon. Ann Bot 61:255–260

    CAS  Google Scholar 

  • Miller CO (1965) Evidence for the natural occurrence of zeatin and derivatives: compounds from maize which promote cell division. Proc Natl Acad Sci USA 54:1052–1058

    PubMed  Article  CAS  Google Scholar 

  • Mok MC, Martin RC, Dobrev PI, Vancovà R, Ho PS, Yonekura-Sakakibara K, Sakakibara H, Mok DWS (2005) Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin o-glucosyltransferase with position specificity related to receptor recognition. Plant Physiol 137:1057–1066

    PubMed  Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for a rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Strnad M (1997) The aromatic cytokinins. Physiol Plant 101:674–688

    Article  CAS  Google Scholar 

  • Strnad M, Hanuš J, Vanĕk T, Kamínek M, Ballantine JA, Fussell B, Hanke DE (1997) Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus × canadensis Moench, cv. Robusta). Phytochemistry 45:213–218

    Article  CAS  Google Scholar 

  • Tarkowská D, Doležal K, Tarkowski P, Åstot C, Holub J, Fuksová K, Schmülling T, Sandberg G, Strnad M (2003) Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus × canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography frit-fast atom bombardment mass spectrometry. Physiol Plant 117(4):579–590

    PubMed  Article  Google Scholar 

  • Teramoto H, Momotani E, Tsuji H (1993) Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons. Physiol Plant 87:584–591

    Article  CAS  Google Scholar 

  • Van Staden J, Drewes FE (1991) The biological activity of cytokinin derivatives in soybean callus bioassay. Plant Growth Regul 10:109–115

    Article  Google Scholar 

  • Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC (1996) Meta-topolin, an alternative to benzyladenine in tissue culture. Physiol Plant 98:291–297

    Article  CAS  Google Scholar 

  • Werbrouck SPO, Van der Jeugt B, Dewitte W, Prinsen E, Van Onckelen HA, Debergh PC (1995) The metabolism of benzyladenine in Spathiphyllum floribundum schott ‘petite’ in relation to acclimatization problems. Plant Cell Rep 14:662–665

    Article  CAS  Google Scholar 

Download references


We acknowledge the financial support of the National Research Foundation (NRF), Pretoria. This work was also supported by the Grant Agency of the Czech Republic (GA 522/06/0108).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Johannes Van Staden.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bairu, M.W., Stirk, W.A., Dolezal, K. et al. 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, 15–23 (2007).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Aromatic cytokinins
  • Conservation
  • Hyperhydricity
  • Metabolites
  • Multiplication rate
  • Plant growth regulators
  • Root and shoot growth