Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 106, Issue 2, pp 215–223 | Cite as

In vitro propagation of four saponin producing Maesa species

  • Ahmad Faizal
  • Ellen Lambert
  • Kenn Foubert
  • Sandra Apers
  • Danny Geelen
Original Paper


A successful micropropagation system was developed for four different medicinal Maesa species. Multiple shoots were induced through both axillary bud formation and adventitious shoot regeneration from leaf explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), thidiazuron (TDZ) and/or α-naphthalene acetic acid (NAA). The success of regeneration varied for different species and depended on the type and concentration of plant growth regulators. Regenerated shoots spontaneously developed roots within 6 weeks on MS hormone-free medium. The rooted shoots were transferred to the greenhouse with a 100% success rate. Furthermore, flow cytometry analysis indicated that there were no changes in ploidy level of those regenerated shoots as compared with wild type adult plants. Thin layer chromatography (TLC) analysis revealed that common and distinguishing spot of saponins were similarly observed in regenerated shoots compared to the control plants. Therefore, the protocol also provides an effective means for the in vitro conservation of Maesa spp. that produce pharmaceutically interesting saponins.


Adventitious shoot Axillary shoot Maesa species Conservation Flow cytometry Saponin 



This research was funded by FWO-Flanders (project No. G.0014.08). A.F. is supported by the Directorate General of Higher Education, Ministry of National Education, Republic of Indonesia. We also acknowledge the help from Nathalie Vanden Bleeken for improving the saponin detection protocol.


  1. Ataei-Azimi A, Hashemloian BD, Ebrahimzadeh H, Majd A (2008) High in vitro production of anti canceric indole alkaloids from periwinkle (Catharanthus roseus) tissue culture. Afr J Biotechnol 7:2834–2839Google Scholar
  2. Bairu M, Stirk W, 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 Tissue Organ Cult 90:15–23CrossRefGoogle Scholar
  3. Bopana N, Saxena S (2008) In vitro propagation of a high value medicinal plant: Asparagus racemosus Willd. In Vitro Cell Dev Biol Plant 44:525–532CrossRefGoogle Scholar
  4. Carimi F, Terzi M, De Michele R, Zottini M, Lo Schiavo F (2004) High levels of the cytokinin BAP induce PCD by accelerating senescence. Plant Sci 166:963–969CrossRefGoogle Scholar
  5. Corredoira E, Ballester A, Vieitez AM (2008) Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees. Plant Cell Tissue Organ Cult 95:197–208CrossRefGoogle Scholar
  6. Cousin A, Heel K, Cowling WA, Nelson MN (2009) An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants. Cytometry Part A 75A:1015–1019CrossRefGoogle Scholar
  7. De Gyves EM, Sparks CA, Fieldsend AF, Lazzeri PA, Jones HD (2001) High frequency of adventitious shoot regeneration from commercial cultivars of evening primrose (Oenothera spp.) using thidiazuron. Ann App Biol 138:329–332CrossRefGoogle Scholar
  8. Debnath M, Malik CP, Bisen PS (2006) Micropropagation: a tool for the production of high quality plant-based medicines. Curr Pharm Biotechno 7:33–49CrossRefGoogle Scholar
  9. Espinosa AC, Pijut PM, Michler CH (2006) Adventitious shoot regeneration and rooting of Prunus serotina in vitro cultures. HortScience 41:193–201Google Scholar
  10. Feng J-C, Yu X, Shang X, Li J, Wu Y (2010) Factors influencing efficiency of shoot regeneration in Ziziphus jujuba Mill. ‘Huizao’. Plant Cell Tissue Organ Cult 101:111–117CrossRefGoogle Scholar
  11. Foubert K, Vermeersch M, Apers S, Pieters L, Maes L (2008) LC-MS analysis of 13, 28-epoxy-oleanane saponins in Maesa spp. extracts with antileishmanial activity. Planta Med 74:1086CrossRefGoogle Scholar
  12. Foubert K, Vermeersch M, Theunis M, Apers S, Cos P, Claeys M, Van Puyvelde L, Pieters L, Maes L (2009) LC-MS analysis of 13, 28-epoxy-oleanane saponins in Maesa spp. extracts with antileishmanial activity. Phytochem Anal 20:159–167PubMedCrossRefGoogle Scholar
  13. Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues. Science 220:1049–1051PubMedCrossRefGoogle Scholar
  14. Germonprez N, Puyvelde LV, Maes L, Tri MV, Kimpe ND (2004) New pentacyclic triterpene saponins with strong anti-leishmanial activity from the leaves of Maesa balansae. Tetrahedron 60:219–228CrossRefGoogle Scholar
  15. Ghimire BK, Seong ES, Goh EJ, Kim NY, Kang WH, Kim EH, Yu CY, Chung IM (2010) High-frequency direct shoot regeneration from Drymaria cordata Willd. leaves. Plant Cell Tissue Organ Cult 100:209–217CrossRefGoogle Scholar
  16. Gonçalves S, Fernandes L, Romano A (2010) High-frequency in vitro propagation of the endangered species Tuberaria major. Plant Cell Tissue Organ Cult 101:359–363CrossRefGoogle Scholar
  17. Huetteman CA, Preece JE (1993) Thidiazuron—a potent cytokinin for woody plant-tissue culture. Plant Cell Tissue Organ Cult 33:105–119CrossRefGoogle Scholar
  18. Jones MPA, Yi ZJ, Murch SJ, Saxena PK (2007) Thidiazuron-induced regeneration of Echinacea purpurea L.: micropropagation in solid and liquid culture systems. Plant Cell Rep 26:13–19PubMedCrossRefGoogle Scholar
  19. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188PubMedCrossRefGoogle Scholar
  20. Kanchanapoom K, Boonvanno K (2000) A protocol towards micropropagation of the piscicidal plant, Maesa ramentacea A. DC. ScienceAsia 26:201–205CrossRefGoogle Scholar
  21. Karuppusamy S, Kiranmai C, Aruna V, Pullaiah T (2009) In vitro conservation of Ceropegia intermedia—an endemic plant of south India. Afr J Biotechnol 8:4052–4057Google Scholar
  22. Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell-cultures for plant improvement. Theor Appl Genet 60:197–214CrossRefGoogle Scholar
  23. Loureiro J, Rodriguez E, Dolezel J, Santos C (2006) Comparison of four nuclear isolation buffers for plant DNA flow cytometry. Ann Bot 98:679–689PubMedCrossRefGoogle Scholar
  24. Lu C-Y (1993) The use of thidiazuron in tissue culture. In Vitro Cell Dev Biol Plant 29:92–96CrossRefGoogle Scholar
  25. Maes L, Berghe DV, Germonprez N, Quirijnen L, Cos P, De Kimpe N, Van Puyvelde L (2004a) In vitro and in vivo activities of a triterpenoid saponin extract (PX-6518) from the plant Maesa balansae against visceral Leishmania species. Antimicrob Agents Ch 48:130–136CrossRefGoogle Scholar
  26. Maes L, Germonprez N, Quirijnen L, Van Puyvelde L, Cos P, Berghe DV (2004b) Comparative activities of the triterpene saponin maesabalide III and liposomal amphotericin B (AmBisome) against Leishmania donovani in hamsters. Antimicrob Agents Ch 48:2056–2060CrossRefGoogle Scholar
  27. Mallón R, Rodríguez-Oubiña J, González M (2010) In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tissue Organ Cult 101:31–39CrossRefGoogle Scholar
  28. Mukherjee P, Husain N, Misra SC, Rao VS (2010) In vitro propagation of a grape rootstock, deGrasset (Vitis champinii Planch.): effects of medium compositions and plant growth regulators. Sci Hort 126:13–19CrossRefGoogle Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  30. Murovec J, Eler K, Bohanec B (2010) Adventitious shoot regeneration from leaf and internodal explants of Mimulus Aurantiacus Curtis. Propag Ornam Plants 10:18–23Google Scholar
  31. Nicasio-Torres MD, Erazo-Gomez JC, Cruz-Sosa F (2009) In vitro propagation of two antidiabetic species known as guarumbo: Cecropia obtusifolia and Cecropia peltata. Acta Physiol Plant 31:905–914CrossRefGoogle Scholar
  32. Obae S, West T (2010) Nuclear DNA content of Hydrastis canadensis L. and genome size stability of in vitro regenerated plantlets. Plant Cell Tissue Organ Cult 102:259–263CrossRefGoogle Scholar
  33. Prakash S, Van Staden J (2008) Micropropagation of Searsia dentata. In Vitro Cell Dev Biol Plant 44:338–341CrossRefGoogle Scholar
  34. Radić S, Prolić M, Pavlica M, Pevalek-Kozlina B (2005) Cytogenetic stability of Centaurea ragusina long-term culture. Plant Cell Tissue Organ Cult 82:343–348CrossRefGoogle Scholar
  35. Rout GR, Samantaray S, Das P (2000) In vitro manipulation and propagation of medicinal plants. Biotech Adv 18:91–120CrossRefGoogle Scholar
  36. Sindambiwe JB, Balde AM, De Bruyne T, Pieters L, Van den Heuvel H, Claeys M, Berghe VD, Vlietinck AJ (1996) Triterpenoid saponins from Maesa lanceolata. Phytochemistry 41:269–277PubMedCrossRefGoogle Scholar
  37. Singh S, Rathod Z, Saxena OP (2009) In vitro plant regeneration and production of saponins of Bacopa monnieri. Natl Acad Sci Lett 32:77–82Google Scholar
  38. Vongpaseuth K, Roberts SC (2007) Advancements in the understanding of paclitaxel metabolism in tissue culture. Curr Pharm Biotechno 8:219–236CrossRefGoogle Scholar
  39. Vujovic T, Ruzic D, Cerovic R, Momirovic GS (2010) Adventitious regeneration in blackberry (Rubus fruticosus L.) and assessment of genetic stability in regenerants. Plant Growth Regul 61:265–275CrossRefGoogle Scholar
  40. Wiart C (2006) Medicinal plants of Asia and the Pacific. In: Wiart C (ed) Medicinal plants of Asia and the Pacific. CRC press, Boca Raton, FL, pp 9–14Google Scholar
  41. Wu J, Zhong J-J (1999) Production of ginseng and its bioactive components in plant cell culture: current technological and applied aspects. J Biotechnol 68:89–99PubMedCrossRefGoogle Scholar
  42. Zheng W, Xu XD, Dai H, Chen LQ (2009) Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species. Sci Hort 122:138–141CrossRefGoogle Scholar
  43. Zhou HC, Li M, Zhao X, Fan XC, Guo AG (2010) Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ (Prunus persica x P. davidiana). Plant Cell Tissue Organ Cult 101:79–87CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Ahmad Faizal
    • 1
  • Ellen Lambert
    • 1
  • Kenn Foubert
    • 2
  • Sandra Apers
    • 2
  • Danny Geelen
    • 1
  1. 1.Department of Plant Production, Faculty of Bioscience EngineeringGhent UniversityGhentBelgium
  2. 2.Laboratory of Pharmacognosy and Pharmaceutical Analysis, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary SciencesUniversity of AntwerpAntwerpBelgium

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