In vitro propagation of Nyactanthes arbor-tristis L. was achieved by culturing N-phenyl-N′-benzothiazol-6-yl-urea (PBU)-pretreated nodal explants in Murashige and Skoog (MS) medium without any phytohormones. Pretreatment of nodal explants in liquid MS medium with 100 μM N-phenyl-N′-benzothiazol-6-yl-urea for 4 d showed the highest shoot proliferation by producing maximum number of shoots (17.40 ± 1.02) per explant, with average shoot length of 5.96 ± 0.08 cm at the end of 8 wk. Effective rooting was accomplished by preincubating the cut-end of shoots with half-strength MS medium containing 6 μM indole-3-butyric acid for 1 wk, followed by implantation into half-strength MS medium; an average of 6.20 ± 0.049 roots per shoot were produced. Seventy-eight percent of the plantlets regenerated in vitro were successfully acclimatized and transferred to soil. These plantlets appeared to be morphologically similar to the donor plants. The genetic fidelity of these in vitro-regenerated plantlets was confirmed by start codon targeted polymorphism (SCoT) marker analysis, followed by comparative evaluations of the bioactive metabolites (ursolic acid, rengyolone, arbortristoside-A, and nyctanthoside), antioxidant-rich phytochemicals, and radical scavenging activities. This optimized in vitro propagation protocol should be an aid for the conservation of N. arbor-tristis germplasm, as well as cater to the needs of herbal industries for the production of therapeutic molecules.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Adeniran AA, Sonibare MA, Rajacharya GH, Kumar S (2018) Assessment of genetic fidelity of Dioscorea bulbifera L. and Dioscorea hirtiflora Benth. and medicinal bioactivity produced from the induced tuberous roots. Plant Cell Tissue Organ Cult 132:343–357
Adeyemi O, Aremu AO, Bairu MW, Szucova L, Dolezal K, Finnie JF, Van Staden J (2012) Assessment of the role of metatopolins on in vitro produced phenolics and acclimatization competence of micro-propagated ‘Williams’ banana. Acta Physiol Plant 34:2265–2273
Agrawal J, Pal A (2013). Nyctanthes arbor-tristis Linn—a critical ethnopharmacological review. J Ethnopharmacol 146:645–658.
Agarwal J, Shanker K, Chanda D, Pal A (2013) Nyctanthes arbor-tristis positively affects immunopathology of malaria-infected mice prolonging its survival. Parasitol Res 112:2601–2609
Agarwal T, Gupta AK, Patel AK, Shekhawat NS (2015) Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers. Plant Cell Tissue Organ Cult 120:313–323
Amoo SO, Aremu AO, Van Staden J (2013) Shoot proliferation and rooting treatments influence secondary metabolite production and antioxidant activity in tissue culture derived Aloe arborescens grown ex vitro. Plant Growth Regul 70:115–122
Amoo SO, Van Staden J (2013) Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix. Plant Cell Tissue Organ Cult 112:249–256
Bantawa P, Roy OS, Ghosh P, Mondal TK (2009) Effect of bavistin and adenine sulphate on in vitro shoot multiplication of Picrorhiza scrophulariiflora. Plant Tissue Cult Biotechnol 19:237–245
Bhattacharyya P, Kumaria S, Diengdoh R, Tandon P (2014) Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid. Meta Gene 2:489–504
Braca A, Sortino C, Politi M, Morelli I, Mendez J (2002) Antioxidant activity of flavonoids from Licania licaniaeflora. J Ethnopharmacol 79:379–381
Carra A, Del Signore MB, Sottile F, Ricci A, Carimi F (2012) Potential use of new diphenylurea derivatives in micropropagation of Capparis spinosa L. Plant Growth Regul 66:229–237
Cheruvathur MK, Kumar GK, Thomas TD (2012) Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz. Plant Cell Tissue Organ Cult 113:63–71
Collard BCY, Mackill DJ (2009) Start Codon Targeted (SCoT) Polymorphism: a simple, novel DNA marker technique for generating gene targeted markers in plants. Plant Mol Biol Report 27:86–93
Coste A, Vlase L, Halmagyi A, Deliu C, Coldea G (2011) Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and H. maculatum. Plant Cell Tissue Organ Cult 106:279–288
Dasgupta N, De B (2007) Antioxidant activity of some leafy vegetables of India: a comparative study. Food Chem 101:471–474
Dörnenburg H, Knorr D (1995) Strategies for the improvement of secondary metabolite production in plant cell cultures. Enzym Microb Technol 17:674–684
Ghaderi N, Jafari M (2014) Efficient plant regeneration, genetic fidelity and high-level accumulation of two pharmaceutical compounds in regenerated plants of Valeriana officinalis L. S Afr J Bot 92:19–27
Gul MZ, Bhakshu LM, Ahmad F, Kondapi AK, Qureshi IA, Ghazi IA (2011) Evaluation of Abelmoschus moschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using in vitro assays. BMC Complement Altern Med 11:64. https://doi.org/10.1186/1472-6882-11-64
Guo B, Abbasi BH, Zeb A, Xu LL, Wei YH (2011) Thidiazuron: a multi-dimensional plant growth regulator. Afr J Biotechnol 10:8984–9000
Harter HL (1960) Critical values for Duncan’s new multiple range test. Biometrics 16:671–685
Huang ZC, Zeng FH, Lu XY (2010) Efficient regeneration of Eucalyptus urophylla from seedling-derived hypocotyls. Biol Plant 54:131–134
Huetteman CA, Preece JE (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult 33:105–119
Jahan AA, Anis M, Aref IM (2011) Preconditioning of axillary buds in thidiazuron-supplemented liquid media improves in vitro shoot multiplication in Nyctanthes arbor-tristis L. Appl Biochem Biotechnol 163:851–859
Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188
Kakkar P, Das B, Viswanathan PN (1984) A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 21:130–132
Khanapur M, Avadhanula RK, Setty OH (2014) In vitro antioxidant, antiproliferative, and phytochemical study in different extracts of Nyctanthes arbor-tristis flowers. BioMed Res Intl 2014:291–271.
Kumari A, Baskarana P, Plačková L, Němčáková H, Nisler J, Doležal K, Van Staden J (2018) Plant growth regulator interactions in physiological processes for controlling plant regeneration and in vitro development of Tulbaghia simmleri. J Plant Physiol 223:65–71
Larkin PJ, Scowcroft WR (1981) Somaclonal variation-a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214
Li HB, Wong CC, Cheng KW, Chen F (2008) Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. Food Sci Technol-LEB 41:385–390
Li LM, Ouyang LJ, Gan SM (2015) Towards an efficient regeneration protocol for Eucalyptus urophylla. J Trop For Sci 27:289–297
Li S, Chen G, Zhang C, Wu M, Wu S, Liu Q (2014) Research progress of natural antioxidants in food for the treatment of diseases. Food Sci Hum Wellness J 3:110–116
Ludwig-Müller J, Vertocnik A, Town CD (2005) Analysis of indole-3-butyric acid induced adventitious root formation on Arabidopsis stem segments. J Exp Bot 56:2095–2105
Luximon-Ramma A, Bahorun T, Soobrattee MA, Aruoma OI (2002) Antioxidant activities of phenolic, proanthocyanidin, and flavonoid components in extracts of Cassia fistula. J Agric Food Chem 50:5042–5047
Mahesh A, Jeyachandran R (2013) Influence of plant growth regulators on micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br. Plant Biosyst 147:493–499
Malik S, Sharma S, Sharma M, Ahuja PS (2010) Direct shoot regeneration from intact leaves of Arnebia euchroma (Royle) Johnston using thidiazuron. Cell Biol Int 34:537–542
Marquez G, Alarcon MV, Salguero J (2016) Differential responses of primary and lateral roots to IAA, IBA and NAA in maize seedlings. Biol Plant 60:367–375
Masondo NA, Aremu AO, Finnie JF, Van Staden J (2015) Growth and Phytochemical levels in micropropagated Eucomis autumnalis subspecies autumnalis using different gelling agents, explant sources, and plant growth regulator. In vitro Cell Dev Biol–Plant 51:102–110
Mendham J, Dennet RC, Bernes JD, eds TMJK (2003) Vogel’s text book of quantitative chemical analysis. Pearson Education Pvt. Ltd., New Delhi, India
Mishra AK, Upadhyay R, Chaurasia JK, Tiwari KN (2016) Comparative antioxidant study in different flower extracts of Nyctanthes arbor-tristis (L.) (Oleaceae): an important medicinal plant. Brazillian J Bot 39:813–820
Mishra RR, Sahu AR, Rath SC, Behera B, Panigrahi J (2013) Molecular mapping of locus controlling resistance to Helicoverpa armigera (Hubner) in Cajanus cajan L. (Millspaugh) using interspecifc F2 mapping population. Nucleus 56:91–97
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays for tobacco tissue cultures. Physiol Plant 15:473–497
Muthukumar M, Kumar TS, Rao MV (2016) Organogenesis and evaluation of genetic homogeneity through SCoT and ISSR markers in Helicteres isora L., a medicinally important tree. S Afr J Bot 106:204–210
Pawar B, Kale P, Bahurupe J, Jadhav A, Kale A, Pawar S (2015) Proline and glutamine improve in vitro callus induction and subsequent shooting in rice. Rice Sci 22:283–289
Phillips GC (2004) In vitro morphogenesis in plants-recent advances. In vitro Cell Dev Biol–Plant 40:342–345.
Podwyszyńska M, Novák O, Doležal K, Strnad M (2014) Endogenous cytokinin dynamics in micropropagated tulips during bulb formation process influenced by TDZ and iP pretreatment. Plant Cell Tissue Organ Cult 119:331–346
Ramirez-Mosqueda MA, Iglesia-Andreau LG (2015) Indirect organogenesis and assessment of somaclonal variation in plantlets of Vanilla planifolia Jacks. Plant Cell Tissue Organ Cult 123:657–664
Rani C, Chawla S, Mangal M, Mangal AK, Kajla S, Dhawan AK (2012) Nyctanthes arbor-tristis Linn. (Night Jasmine): A sacred ornamental plant with immense medicinal potentials. Indian J Tradit Knowl 11:427–435
Rathee JS, Hassarajani SA, Chattopadhyay S (2007) Antioxidant activity of Nyctanthes arbor-tristis leaf extract. Food Chem 103:1350–1357
Riaz UR, Chaudhary MF, Khawar KM, Lu G, Manan M, Zia M (2014) In vitro propagation of Caralluma tuberculate and evaluation of antioxidant potential. Biol Plant 69:341–349
Ricci A, Bertoletti C (2008) Urea derivatives on the move: cytokinin-like activity and adventitious rooting enhancement depend on chemical structure. Plant Biol 11:262–272
Rolli E, Ricci A, Bianchi A, Bruni R (2011) Optimisation of in vitro propagation of Hyssopus officinalis L. using two-node explants and N-phenyl-N′-benzothiazol-6-yl-urea (PBU), a new urea-type cytokinin. J Hortic Sci Biotechnol 86:141–145
Rout GR, Mahato A, Senapati SK (2008) In vitro clonal propagation of Nyctanthes arbor-tristis. Biol Plant 52:521–524
Sahu RC, Sahu A, Padhi S, Pattanaik L, Mishra RR, Panigrahi J (2012) In vitro regeneration of plantlets from nodal explants of Nyctanthes arbor-tristis linn. and evaluation of genetic fidelity through RAPD analysis. Bioscan 7:583–589
Saini P, Gayen P, Kumar D, Nayak A, Mukherjee N, Mukherjee S, Pal BC, Babu SPS (2014) Antifilarial effect of ursolic acid from Nyctanthes arbor-tristis: Molecular and biochemical evidences. Parasitol Int 63:717–728
Seth S, Panigrahi J (2018) In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers. J Hortic Sci Biotechnol 94:70–79
Seth S, Rath SC, Rout GR, Panigrahi J (2017) Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers. Plant Biosyst 151:704–714
Shahsavari E (2011) Impact of tryptophan and glutamine on the tissue culture of upland rice. Plant Soil Environ 57:7–10
Siragusa M, Carra A, Salvia L, Puglia AM, De Pasquale F, Carimi F (2007) Genetic instability in calamondin (Citrus madurensis Lour.) plants derived from somatic embryogenesis induced by diphenylurea derivatives. Plant Cell Rep 26:1289–1296
Sivanesan I, Jeong BR (2007) Direct shoot regeneration from nodal explants of Sida cordifolia Linn. In vitro Cell Dev Biol–Plant 43:436–441
Sugiyama M (1999) Organogenesis in vitro. Curr Opin Plant Biol 2:61–64
Torelli A, Borinato M, Francia S, Carra A, Ricci A, Branca C (2006) Adeninic and ureidic cytokinins: Primary response events in in vitro tomato caulogenesis. Plant Sci 171:60–73
Tuntiwachwuttikul P, Rayanil K, Taylor WC (2003) Chemical constituents from the flowers of Nyctanthes arbor-tristis. Sci Asia 29:21–30
Van Staden J, Zazimalova E, George EF (2008) Plant growth regulators II: Cytokinins, their analogues and antagonist. Plant Propagation by Tissue Culture (vol-1) The Background. Springer, Dordrecht, The Netherlands, p 2008
Vargas MH (2000) ED50 plus v1.0 programs. Instituto Nacional de Enfermedades Respiratorias (www. sciencegateway.org/protocols/cellbio/drug/data/ ed50v10.xls).
Zatloukal M, Gemrotova M, Dolezal K, Havlicek L, Spichal L, Strnad M (2008) Novel potent inhibitors of Arabidopsis thaliana cytokinin oxidase/dehydrogenase. Bioorg Med Chem 16:9268–9275
Zhao XY, Su YH, Cheng ZJ, Zhang XS (2008) Cell fate switch during in vitro plant organogenesis. J Integr Plant Biol 50:816–824
Zielinska S, Piatczak E, Kalemba D, Matkowski A (2011) Influence of plant growth regulators on volatiles produced by in vitro grown shoots of Agastache rugosa (Fischer and C. A. Meyer) O. Kuntze. Plant Cell Tissue Organ Cult 107:161–167
This study is supported by the UGC, Govt. of India, and the Vice Chancellor, Sambalpur University, Odisha, India, through financial support and the facilities to carry out this work.
Conflict of interest
The authors declare that they have no conflict of interest.
The authors declare that the study was carried out following scientific ethics and conduct. However, this study did not involve any use of animals; hence, no ethical approval has been obtained from the concerned committee.
Editor: Pamela Weathers
About this article
Cite this article
Rath, S.C., Seth, S., Mishra, S.K. et al. Genetic homogeneity assessment of in vitro-regenerated plantlets of Nyctanthes arbor-tristis L. and comparative evaluation of bioactive metabolites and antioxidant activity. In Vitro Cell.Dev.Biol.-Plant 56, 72–87 (2020). https://doi.org/10.1007/s11627-019-10004-8
- Antioxidant activity
- Bioactive metabolites (arbortristoside-A, nyctanthoside, rengyolone, and ursolic acid)
- N-Phenyl-N′-benzothiazol-6-yl-urea (PBU)
- Start codon targeted polymorphism (SCoT) marker analysis