Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 134, Issue 2, pp 241–250 | Cite as

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of Allamanda cathartica L. using TDZ and ultrasound assisted extraction of quercetin

  • Mehrun Nisha Khanam
  • Mohammad AnisEmail author
Original Article


The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00 ± 1.30) and shoot length (7.50 ± 0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50 ± 0.16 mean roots/shoot with 4.05 ± 0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.


Micropropagation Cytokinins Colorimetry Flavonoid Ultrasonication Mass spectrometry 



Award of UGC-BSR Faculty fellowship (2017) by the University Grants Commission, New Delhi to MA is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abdel-Kader MS, Wisse J, Evans R, Vander WH, Kingston DG (1997) Bioactive iridoids and a new lignan from Allamanda cathartica and Himatanthus fallax from the suriname rainforest. J Nat Prod 6:1294–1297CrossRefGoogle Scholar
  2. Agbo MO, Uzor PF, Akazie-Nneji UN, Eze-Odurukwe CU, Ogbatue UB, Mbaoji EC (2015) Antioxidant, total phenolic and flavonoid content of selected nigerian medicinal plants. Dhaka Univ J Pharm Sci 14(1):00–00CrossRefGoogle Scholar
  3. Ahmad N, Anis M (2007) Rapid clonal multiplication of a woody tree, Vitex negundo L. through axillary shoots proliferation. Agrofor Syst 71:195–200CrossRefGoogle Scholar
  4. Ahmed MR, Anis M (2012) Role of TDZ in the quick regeneration of multiple shoots from nodal explant of Vitex trifolia L.: an important medicinal plant. Appl Biochem Biotechnol 168:957–966CrossRefPubMedGoogle Scholar
  5. Ahmed MR, Anis M, Al-Atta HA (2014) An efficient in vitro process for cyclic clonal production of shoots from adult tree of Cassia alata L. and evaluation of genetic stability using DNA-based markers. Appl Biochem Biotechnol 174(8):2886–2896CrossRefPubMedGoogle Scholar
  6. Anis M, Ahmad N, 2016. Plant tissue culture: a journey from research to commercialization. Anis M, Ahmad N (eds), Plant tissue culture: propagation, conservation & crop improvement. Springer Nature, New York 3–13CrossRefGoogle Scholar
  7. Bonomini TJ, Holzmann I, Thiesen LC, Fratoni E, Muller AFF, Lucinda-Silva RM et al (2017) Neuropharmacological and acute toxicological evaluation of ethanolic extract of Allamanda cathartica L. flowers and plumieride. Regulatory Toxicol Pharmacol 91:9–19CrossRefGoogle Scholar
  8. Capelle SC, Mok DWS, Kirchner SC, Mok MC (1983) Effects of thidiazuron on cytokinin autonomy and the metabolism of N6-(D2-isopentenyl)[8-14C]adenosine in callus tissues of Phaseolus lunatus L. Plant Physiol 73:796–802CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cook NC, Samman S (1996) Flavonoids-chemistry, metabolism, cardioprotective effects and dietary sources. J Nutr Biochem 7(2):66–76CrossRefGoogle Scholar
  10. Deb CR, Deb MS, Jamir NS (2014) Effect of different factors on in vitro axillary shoot proliferation and plant regeneration of Cinnamomum tamala Nees: a spice yielding plant. Indian J Biotechnol 13:520–526Google Scholar
  11. Essiett AU, Udo ES (2015) Comparative phytochemical screening and nutritional potentials of the stems, leaves and flowers of Allamanda cathartica (Apocynaceae). Int J Sci Technol 4:248–253Google Scholar
  12. Faisal M, Ahmad N, Anis M (2005) Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron. Plant Cell Tiss Org Cult 80:187–190CrossRefGoogle Scholar
  13. Faisal M, Ahmad N, Anis M (2007) An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant. Plant Biotechnol Report 1(3):155–161CrossRefGoogle Scholar
  14. Faisal M, Alatar AA, Ahmad N, Anis M, Hegazy AK (2012) An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers. Appl Biochem Biotechnol 168(7):1739–1752CrossRefPubMedGoogle Scholar
  15. Fartyal M (2016) Allamanda cathartica L.: extraction and pharmaceutical evaluation of various extracts of leaves and flowers. Int J Curr Pharm Res 8(4):28–32CrossRefGoogle Scholar
  16. Fatima N, Anis M (2011) Thidiazuron induced high frequency axillary shoot multiplication in Withania somnifera L. (Dunal.). J Medicinal Plants Res 5:6681–6687CrossRefGoogle Scholar
  17. Fiola JA, Hassan MA, Swartz HJ, Bors RH, McNicols R (1990) Effect of thidiazuron, light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves. Plant Cell Tiss Org Cult 20:223–228Google Scholar
  18. Huetteman A, Preece EJ (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Org Cult 33:105–119CrossRefGoogle Scholar
  19. Hutchinson MJ, Krishnaraj S, Saxena PK (1996) Morphological and physiological changes during thidiazuron-induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) hypocotyl cultures. Int J Plant Sci 157:440–446CrossRefGoogle Scholar
  20. Jahan AA, Anis M (2009) In vitro rapid multiplication and propagation of Cardiospermum halicacabum L. through axillary bud culture. Acta Physiologia Plantarum 31:133–138CrossRefGoogle Scholar
  21. Kardono LB, Tsauri S, Padmawinata K, Pezzuto JM, Kinghorn AD (1990) Cytotoxic constituents of the bark of Plumeria rubra collected in Indonesia. J Nat Prod 53:1447–1455CrossRefPubMedGoogle Scholar
  22. Khan MI, Anis M (2012) Modulation of in vitro morphogenesis in nodal segments of Salix tetrasperma Roxb. through the use of TDZ, different media types and culture regimes. Agrofor Syst 86(1):95–103CrossRefGoogle Scholar
  23. Kostic DA, Dimitrijevic DS, Mitic SS, Mitic MN, Stojanovic GS, Zivanovic AV (2013) Phenolic content and antioxidant activities of fruit extracts of Morus nigra L (Moraceae) from Southeast Serbia. Trop J Pharmac Res 12:105–110Google Scholar
  24. Kozai T, Kubota C, Jeong BR (1997) Environmental control for the large-scale production of plants through in vitro techniques. Plant Cell Tiss Org Cult 51:49CrossRefGoogle Scholar
  25. Kumari P, Singh S, Yadav S, Tran LSP (2018) Pretreatment of seeds with thidiazuron delimits its negative effects on explants and promotes regeneration in chickpea (Cicer arietinum L.). Plant Cell Tiss Org Cult 133(1):103–114CrossRefGoogle Scholar
  26. Kupchan SM, Dessertme AL, Blaylock BT, Bryan RF (1974). Isolation and structural elucidation of allamandin, and antileukemic iridoid lactone from Allamanda cathartica. J Org Chem 39(17):2477–2482CrossRefPubMedGoogle Scholar
  27. Liu Y, Lu J, Zhu H, Li L, Shi Y, Yin X (2016) Efficient culture protocol for plant regeneration from cotyledonary petiole explants of Jatropha curcas L. Biotechnol. Biotechnol Equipment 30(5):907–914CrossRefGoogle Scholar
  28. Mok MC, Mok DWS, Armstrong DJ, Shudo K, Isogai Y, Okamoto T (1982) Cytokinin activity of N-phenyl-N’-1,2,3-thiadiazol-5-ylurea (thidiazuron). Phytochemistry 21:1509–1511CrossRefGoogle Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  30. Oliveira JPS, Hakimi O, Murgu M, Koblitz MGB, Ferreira MSL, Cameron LC, Macedo AF (2018) Tissue culture and metabolome investigation of a wild endangered medicinal plant using high definition mass spectrometry. Plant Cell Tiss Org Cult. CrossRefGoogle Scholar
  31. Parveen S, Shahzad A (2010) TDZ–induced high frequency shoot regeneration in Cassia sophera Linn. via cotyledonary node explants. Physiol Mol Biol Plants 16(2):201–206CrossRefPubMedPubMedCentralGoogle Scholar
  32. Perveen S, Khanam MN, Anis M, El Atta HA (2015) In vitro mass propagation of Murraya koenigii L. J Appl Res Med Aromatic Plants 2:60–68CrossRefGoogle Scholar
  33. Rupasinghe HPV, Kathirvel P, Huber GM (2011) Ultrasonication-assisted solvent extraction of quercetin glycosides from ‘Idared’ apple peels. Molecules 16:9783–9791CrossRefGoogle Scholar
  34. Saeed N, Khan MR, Shabbir M (2012) Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Comp Altern Med 12:221CrossRefGoogle Scholar
  35. Sambandam B, Thiyagarajan D, Ayyaswamy A, Raman P (2016) Extraction and isolation of flavonoid quercetin from the leaves of trigonella foenum-graecum and their anti-oxidant activity. Int J Pharm Sci 8(6):120–124Google Scholar
  36. Savithramma N, Linga Rao M, Suhrulatha D (2013) Qualitative and quantification analysis of phytochemicals from leaf aqueous extract of Allamanda cathartica L. and Terminalia paniculata Roth An Int J 1(8):821–825Google Scholar
  37. Tiwari TN, Pandey VB, Dubey NK (2002) Plumieride from Allamanda cathartica as an antidermatophytic agent. Phytother Res 16(4):393–394CrossRefPubMedGoogle Scholar
  38. Wong KF, Taha RM, 2012. The effect of 2,4-dichlorophenoxyacetic Acid and 6-benzylaminopurine on callus induction and plant regeneration of Allamanda cathartica—a valuable medicinal plant. Res J Biotech 7(3):75–80Google Scholar
  39. Xu W, Chu K, Li H, Zhang Y, Zheng H, Chen R, Chen L (2012) Ionic liquid-based microwave-assisted extraction of flavonoids from Bauhinia championii (Benth.). Molecules 17:14323–14335CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

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

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