Meta-topolin Improves In Vitro Morphogenesis, Rhizogenesis and Biochemical Analysis in Pterocarpus marsupium Roxb.: A Potential Drug-Yielding Tree

  • Anees Ahmad
  • Mohammad AnisEmail author


Meta-topolin (mT), a benzyladenine analog [N 6-(3-hydroxybenzylamino) purine] is a highly active cytokinin. The present study evaluates the efficiency of two aromatic cytokinins, mT and BA for inducing in vitro regeneration in a woody legume Pterocarpus marsupium (Roxb.) using cotyledonary node (CN) explants. Of the two cytokinins tested, mT-derived cultures resulted in better shoot multiplication and rhizogenesis than BA. Among the different doses of mT, maximum shoot (9.58 ± 0.30) induction per explant and average shoot length (4.12 ± 0.05 cm) were recorded on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 7.5 µM mT, after 6 weeks of culture. The combined effect of cytokinin and auxin was tested, auxin was mixed with optimum doses of BA or mT separately and the effect of combination was studied. Among the cytokinin–auxin combinations, the highest number of shoots (17.44 ± 0.25) per explant and average shoot length (5.72 ± 0.18 cm) were achieved on MS medium containing 7.5 µM mT with 1.0 µM αnaphthalene acetic acid in 85% of the cultures after 12 weeks. Meta-topolin alone or in combination with auxin has shown an increase in the quality and number of shoots in comparison to BA. In vitro rhizogenesis in individually regenerated microshoots was carried out on half-strength MS medium augmented with 1.0 µM indole-3-butyric acid via a two-step procedure method. After 4 weeks, 7.35 ± 0.11 roots per shootlet with an average root length of 4.54 ± 0.10 cm were recorded in mT-derived microshoots. The well-developed plantlets were acclimatized in a separate batch of single CN explant-derived plantlets. About 80% survival rate was recorded for mT-derived plantlets. Biomass and photosynthetic pigments were also improved in mT-derived plantlets, when compared with the BA derived. Analysis of genetic homogeneity of ten micropropagated plantlets was done through RAPD. Out of 40 RAPD primers, 29 primers produced clearly scorable monomorphic bands, thus exhibiting complete genetic uniformity among in vitro regenerated plantlets.


Acclimatization Cotyledonary node Fabaceae Genetic fidelity Regeneration Woody legume 



We acknowledge the Department of Biotechnology, Government of India, New Delhi for financial support (vide no. BT/PR2189/PBD/17/744/2011 Dated 19.12.2012). We are thankful to the Andhra Pradesh Forest Department, India for providing fruits of Pterocarpus marsupium. Award of UGC-BSR Faculty Fellowship (2017) to MA by the University Grants Commission, New Delhi is gratefully acknowledged.

Author Contributions

Experiment designed and executed by AA. The whole experiment was conducted under the supervision of MA who also read and edited the manuscript.

Compliance with Ethical Standards

Conflict of interest

There is no conflict of interest.


  1. Ahmed MR, Anis M, Alatar AA, Faisal M (2017) In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant. Agroforest Syst 91:637–647CrossRefGoogle Scholar
  2. Amoo SO, Finnie JF, Van Staden J (2011) The role of meta-topolins in alleviating micropropagation problems. Plant Growth Regul 63:197–206CrossRefGoogle Scholar
  3. Anis M, Husain MK, Shahzad A (2005) In vitro plantlet regeneration of Pterocarpus marsupium Roxb., an endangered leguminous tree. Curr Sci 88:861–863Google Scholar
  4. Aremu AO, Bairu MW, Szüčová L, Doležal K, Finnie JF, Van Staden J (2012) Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated ‘Williams’ banana. Acta Physiol Plant 34:2265–2273CrossRefGoogle Scholar
  5. Bairu MW, Stirk WA, Doležal 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 Tissue Organ Cult 90:15–23CrossRefGoogle Scholar
  6. Bairu MW, Stirk WA, Doležal K, Van Staden J (2008) The role of topolins in micropropagation and somaclonal variation of banana cultivars ‘Williams’ and ‘Grand Naine’ (Musa spp. AAA). Plant Cell Tissue Org Cult 95:373–379CrossRefGoogle Scholar
  7. Bogaert I, Van Cauter S, Werbrouck SPO, Doležal K (2006) New aromatic cytokinins can make the difference. Acta Hortic 725:265–270CrossRefGoogle Scholar
  8. Čatský J, Pospíšilová J, Kaminek M, Gaudinová A, Pulkrábek J, Zahradníček J (1996) Seasonal changes in sugar beet photosynthesis as affected by exogenous cytokinin N6-(m-hydroxybenzyl) adenosine. Biol plant 38:511–518CrossRefGoogle Scholar
  9. Chand S, Singh AK (2004) In vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, Pterocarpus marsupium Roxb. Vitro Cell Dev Biol Plant 40:464–466CrossRefGoogle Scholar
  10. Deshmukh VP, Thakare PV, Chaudhari US, Gawande PA (2007) A simple method for isolation of genomic DNA from fresh and dry leaves of Terminalia arjuna (Roxb.) Wight and Argot. Electron J Biotechn 10:468–472Google Scholar
  11. Drewes FE, van Staden J (1989) The effect of 6-benzyladenene derivatives on the rooting of Phaseolus vulgarise L. primary leaf cuttings. Plant Growth Regul 8:289–296CrossRefGoogle Scholar
  12. Ferguson BJ, Beveridge CA (2009) Roles for auxin, cytokinin, and 648 strigolactone in regulating shoot branching. Plant Physiol 149:1929–1944CrossRefGoogle Scholar
  13. Garzuglia M (2006) Threatened, endangered and vulnerable tree species: a comparison between FRA 2005 and the IUCN Red List. Global For Res Assess (2005).
  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 Tissue Org Cult 118:373–381CrossRefGoogle Scholar
  15. Hougee S, Faber J, Sanders A, de Jong RB, van den Berg WB, Garssen J, Hoijer MA, Smit HF (2005) Selective COX-2 inhibition by a Pterocarpus marsupium extract characterized by pterostilbene, and its activity in healthy human volunteers. Planta Med 71:387–392CrossRefGoogle Scholar
  16. Howell SH, Lall S, Che P (2003) Cytokinins and shoot development. Trends Plant Sci 8:453–459CrossRefGoogle Scholar
  17. Husain MK, Anis M, Shahzad A (2007) In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using thidiazuron. In Vitro Cell Dev Biol Plant 43:59–64CrossRefGoogle Scholar
  18. Husain MK, Anis M, Shahzad A (2008) In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants. Acta Physiol Plant 30:353–359CrossRefGoogle Scholar
  19. Husain MK, Anis M, Shahzad A (2010) Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb. Tree 24:781–787CrossRefGoogle Scholar
  20. Javed SB, Anis M, Khan PR, Aref IM (2013) In vitro regeneration and multiplication of Acacia ehrenbergiana Hayne: a potential reclaiment of denude arid lands. Agroforest Syst 87:621–629CrossRefGoogle Scholar
  21. Kubalakova M, Strnad M (1992) The effect of aromatic cytokinins (populins) on micropropagation and regeneration of sugar beet in vitro. Biol Plant 34:578–579Google Scholar
  22. Letham D, Palni L (1983) The biosynthesis and metabolism of cytokinins. Ann Rev Plant Physiol 34:163–197CrossRefGoogle Scholar
  23. Ludwing-Muller J (2000) Indole-3-butyric acid in plant growth and development. Plant Growth Regul 32:219–230CrossRefGoogle Scholar
  24. Mackinney G (1941)) Absorption of light by chlorophyll solution. J Biol Chem 140:315–322Google Scholar
  25. Magyar-Tábori K, Dobránszki J, da Silva JAT, Bulley SM, Hudák I (2010) The role of cytokinins in shoot organogenesis in apple. Plant Cell Tissue Org Cult 101:251–267CrossRefGoogle Scholar
  26. Malá J, Máchová P, Cvrčková H, Karady M, Novák O, Mikulík J, Dostál J, Strnad M, Doležal K (2013) The role of cytokinins during micropropagation of wych elm. Biol Plant 57:174–178CrossRefGoogle Scholar
  27. Manickam M, Ramanathan M, Farboodniay Jahromi MA, Chansouria JPN, Ray AB (1997) Antihyperglycemic activity of phenolics from Pterocarpus marsupium. J Nat Prod 60:915–920CrossRefGoogle Scholar
  28. Martin M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496CrossRefGoogle Scholar
  29. Maurya R, Singh R, Deepak M, Handa SS, Yadav PP, Mishra PK (2004) Constituents of Pterocarpus marsupium: an ayurvedic crude drug. Phytochemistry 65:915–920CrossRefGoogle Scholar
  30. Mohire NC, Salunkhe VR, Bhise SB, Yadav AV (2007) Cardiotonic activity of aqueous extract of heartwood of Pterocarpus marsupium. Indian J Exp Biol 45:532–537Google Scholar
  31. Muller D, Leyser O (2011) Auxin, cytokinin and control of shoot branching. Ann Bot 107:1203–1212CrossRefGoogle Scholar
  32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  33. Nayak SA, Kumar S, Satapathy K, Moharana A, Behera B, Barik DP, Acharya L, Mohapatra PK, Jena PK, Naik SK (2013) In vitro plant regeneration from cotyledonary node of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers. Acta Physiol Plant 35:195–203CrossRefGoogle Scholar
  34. Rahman MH, Rojara OP (2001) Microsatellite DNA somaclonal variation in micropropagation trembling aspen (Populus tremuloides). Plant Cell Rep 20:531–536CrossRefGoogle Scholar
  35. Remsberg CM, Yáñez JA, Ohgami Y, Vega-Villa KR, Rimando AM, Davies NM (2008) Pharmacometrics of pterostilbene: preclinical pharmacokinetics and metabolism, anticancer, antiinflammatory, antioxidant and analgesic activity. Phytother Res 22:169–179CrossRefGoogle Scholar
  36. Sahijaram L, Soonji JR, Ballamma KT (2003) Analyzing somaclonal variation in microropropagated bananas (Muss spp.). In Vitro Cell Dev Biol Plant 35:551–556CrossRefGoogle Scholar
  37. Sakakibara H (2006) Cytokinins: activity, biosynthesis, and translocation. Annu Rev Plant Biol 57:431–449CrossRefGoogle Scholar
  38. Santner A, Estelle M (2009) Recent advances and emerging trends in plant hormone signalling. Nature 459:1071–1078CrossRefGoogle Scholar
  39. Strnad M (1997) The aromatic cytokinins. Physiol Plant 101:674–688CrossRefGoogle Scholar
  40. Strnad M, Hanus J, Vanek T, Kaminek M, Ballantine JA, Fussell B, Hanke DE (1997) Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus × canadensis Moench, cv. Robusta). Phytochemistry 45:213–218CrossRefGoogle Scholar
  41. Valero-Aracama C, Kane M, Wilson S, Philman N (2010) Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy- to acclimatize sea oats (Uniola paniculata L) genotypes. Plant Growth Regul 60:43–49CrossRefGoogle Scholar
  42. Webster CA, Jones OP (1991) Micropropagation of some cold-hardy dwarfing rootstocks for apple. J Hortic Sci 66:1–6CrossRefGoogle Scholar
  43. 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 acclimatisation problems. Plant Cell Rep 14:662–665CrossRefGoogle 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. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic marker. Nucleic Acids Res 18:6531–6535CrossRefGoogle Scholar
  46. Wojtania A (2010) Effect of meta-topolin on in vitro propagation of Pelargonium × hortorum and Pelargonium × hederaefolium cultivars. Acta Soc Bot Pol 79:101–106CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Plant Biotechnology Laboratory, Department of BotanyAligarh Muslim UniversityAligarhIndia

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