Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 138, Issue 1, pp 167–180 | Cite as

Establishment of callus-cultures of the Argentinean mistletoe, Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae) and screening of their polyphenolic content

  • M. V. Ricco
  • M. L. Bari
  • F. Bagnato
  • C. Cornacchioli
  • M. Laguia-Becher
  • L. U. Spairani
  • A. Posadaz
  • C. Dobrecky
  • R. A. Ricco
  • M. L. Wagner
  • M. A. ÁlvarezEmail author
Original Article


Ligaria cuneifolia (R. et P.) Tiegh (Loranthaceae), known as liga, muérdago criollo, or Argentinean mistletoe, is a hemiparasitic plant with a broad distribution in central and northern Argentina. Pharmacological studies showed that L. cuneifolia extracts have hypolipemic, antioxidant, antibacterial, and immunomodulatory effects. We have established callus cultures from embryo and haustoria fragments. The highest frequency of callus formation from embryos (85%) was obtained on White medium with 4% (w/v) sucrose and 2.5 µM 1-naphtalene acetic acid and 9.2 µM kinetin as plant growth regulators (PGRs). From haustoria, the best result (35%) was obtained on Gamborg medium with 3% (w/v) sucrose and 0.45 µM 2,4-dichlorephenoxyacetic acid and 0.47 µM zeatin as PGRs. Thin layer chromatography showed that callus methanolic extract (2.5% w/v) had a lower content of flavonoids and proanthocyanins as compared to the wild plant (5% w/v for leaves, stems, and flowers), but a higher content of hydroxycinnamic acids. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) showed the presence of quercetin glycosides and phenolic acids in the methanolic extracts both from the parent plant and the callus obtained from embryo.

Key message

Callus cultures were established from embryo and haustorium explants of Ligaria cuneifolia. Leaves, stems, and meristems were recalcitrant to in vitro culture. Callus tissues contained quercetin glycosides and phenolic acids.


Medicinal plants Liga Hemiparasitic plant Callus culture Flavonoids 



We wish to thank Dr. Sabrina Flor for her assistance in the mass spectrometry analysis (Pharmaceutical Technology Department of the Faculty of Pharmacy and Biochemistry from the University of Buenos Aires), Dr. Javier Calcagno (CONICET/CEBBAD) for his advice regarding the statistical analysis, Dr. Chana Pilberg (Universidad Maimónides) for kindly providing us from plant material from Merlo, and M. Julian Schecter for his advice and careful revision of English. This work was supported by Fondo Nacional de Ciencia y Tecnología (FONCyT), Ministerio de Ciencia, Tecnología e Innovación Productiva from Argentina (PICT2015-2024), Universidad de Buenos Aires, and Universidad Maimónides. M A Alvarez and M Laguia-Becher are researchers from CONICET, MVR has a scholarship from CONICET-Universidad Maimónides, and MLB has a scholarship from FONCyT.

Author’s contributions

MVR and MLB carried out the experiments and participated in drafting the manuscript; CC and FB carried out experiments; ML-B and CD participated in the analysis of the results; AP participated in selecting, collecting, and classifying the plant material; LUS performed statistical analysis; MLW, RAR and MAA initiated the project and supervised the work throughout, MAA also drafted the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2019_1615_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb). On line resource 1: Antioxidant treatments used for avoiding browning in Ligaria cuneifolia leaves in vitro cultures. Explants (n = 10) were washed with the antioxidants during 5 min, 15 min and 30 min. Then, explants were transferred to Petri dishes with White medium plus 2,4-d (4.5 µM).The treatments were performed in dark or light, with similar results in both cases. l-CysHCl: l-Cysteine hydrochloride monohydrate


  1. Abiatti D (1946) Las Lorantáceas Argentinas. Rev Mus La Plata NS Sec Bot 7:1–110Google Scholar
  2. Ahmad I, Hussain T, Ashraf I, Nafees M, Maryam RM, Iqbal M (2013) Lethal effects of secondary metabolites on plant tissue culture. Am Eurasian J Agric Environ Sci 13:539–547Google Scholar
  3. Ahmad I, Jaskani MJ, Nafees M, Ashraf I, Qureshi R (2016) Control of Media Browning in Micropropagation of Guava (Psidium guajava L.). Pak J Bot 48(2):713–716Google Scholar
  4. Amico GC, Nickrent DL (2007) Phylogeography of the Argentine mistletoe, Ligaria cuneifolia (Loranthaceae). Darwiniana 45:55–131Google Scholar
  5. Amuchástegui A, Petryna L, Cantero JJ, Núñez C (2003) Plantas parásitas del centro de Argentina. Acta Botánica Malacitana 28:37–46Google Scholar
  6. Bajaj YPS (1967) In vitro studies on the embryos of two Mistletoes, Amyema pendula and Amyema miquelli. NZ J Bot 5(1):49–56CrossRefGoogle Scholar
  7. Batchbaroiva RB, Slavov SB, Bossolova SN (1999) In vitro culture of Orobanche ramosa. Weed Res 39:191–197CrossRefGoogle Scholar
  8. Cerdá Zolezzi P, Fernández T, Aulicino PC, Cavaliere V, Greczanik S, Caldas Lopez E, Wagner ML, RIcco RA, Gurni A, Hajos S, Alvarez E (2005) Ligaria cuneifolia flavonoid fractions modulate cell growth of normal lymphocytes and tumor cells as well as multidrug resistant cells. Immunobiology 209(10):737–749CrossRefGoogle Scholar
  9. Debergh PC, Read PE (1991) Micropropagation. In: Debergh PC, Zimmerman RH (eds) Micropropagation technology and application. Kluwer Academic Publishers, Dordrecht, pp 1–14CrossRefGoogle Scholar
  10. Dobrecky CB, Flor SA, López PG, Wagner ML, Lucangioli SE (2017) Development of a novel dual CD-MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh- Loranthaceae- extracts. Electrophoresis 38(9–10):1292–1300CrossRefGoogle Scholar
  11. Espinosa-Leal CA, Puente-Garza CA, García-Lara S (2018) In vitro plant tissue culture: means for production of biological active compounds. Planta 248:1–8CrossRefGoogle Scholar
  12. Fukui M, Azuma JI, Okamura K (1990) Induction of Callus from mistletoe and interaction with its host cells. Bull Kyoto Univ Forests 62:261–269Google Scholar
  13. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefGoogle Scholar
  14. Gonzálvez J, García G, Galliano S, Dominghini A, Urli L, Monti J, Ronco MT, Frances D, Wagner M, Carnovale C, Luquita A (2017) The enriched proanthocyanidin extract of Ligaria cuneifolia shows a marked hypocholesterolemic effect in rats fed with cholesterol-enriched diet. Recent Pat Endocr Metab Immune Drug Discov 11(1):47–53CrossRefGoogle Scholar
  15. Hall PJ, Letham DS, Barlow BA (1987) The influence of hormones on development of Amyema seedlings cultured in vitro. In: Weber HC, Forstreuter W (eds) Proceedings of the 4th International Symposium on Parasitic Flowering Plants. Philips University, Marburg, pp 285–291Google Scholar
  16. Ishrad M, Rizwan HM, Debnath B, Anwar M, Li M, Liu S, He B, Qiu D (2018) Ascorbic acid controls lethal browning and pluronic F-68 promotes high-frequency multiple shoot regeneration from coltyldonary node explant of okra (Abelmoschus esculentus L.). HortScience 53(2):183–190CrossRefGoogle Scholar
  17. Johri BM, Bajaj YPS (1962) Behaviour of Mature Embryos of Dendrophthoe falcata (l.F) Ettingsh in vitro. Nature 193:194–195CrossRefGoogle Scholar
  18. Johri BM, Bajaj YPS (1964) Growth of embryos of Amyema, Anylotheca, and Scurrula on synthetic media. Nature 204:1220–1221CrossRefGoogle Scholar
  19. Karunaratne MLWOM, Peries SE, Egodawatta WCP (2014) Callus induction and organogenesis from leaf explants of Tectona grandis. Ann Biol Res 5(4):74–82Google Scholar
  20. Khana P, Staba J (1968) Antimicrobials from plant tissue cultures. Lloydia 31:180–189Google Scholar
  21. Kim SW, Ko SM, Liu JR (2008) In vitro seed germination and callus formation on flower bud of Korean mistletoe [Viscum album L. var. cololatum (Kom.) Ohwi]. J Plant Biotechnol 35:47–53CrossRefGoogle Scholar
  22. Lee KP, Lee DW (2013) The Identification of in Vitro Production of Lectin from Callus Cultures of Korean Mistletoe (Viscum album L. var. coloratum). Biosci Biotechnol Biochem 77(4):884–887CrossRefGoogle Scholar
  23. Majid I, Muhammad J, Rizwan R, Syed ZU, Muhammad SI, Misbah R, Salman M (2014) Effect of plant growth regulators on callus formation in potato. J Agri Food Appl Sci 2:77–81Google Scholar
  24. Martínez GJ (2010) Las plantas en la medicina tradicional de las Sierras de Córdoba. Un recorrido por la cultura campesina de Paravachasca y Calamuchita, Ediciones del Copista, p 212Google Scholar
  25. McHugh ML (2013) The chi square test of independence. Biochemia Medica 23(2):143–149CrossRefGoogle Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  27. Nag KK, Johri BM (1976) Experimental morphogenesis of the embryo of Dendrophthoe, Taxillus, and Nuytsia. Bot Gaz 37:378–390CrossRefGoogle Scholar
  28. Ndakidemi CF, Mneney E, Ndakidemi PA (2014) Effects of ascorbic acid in controlling lethal browning in in vitro culture of Brachylaena huillensis using nodal segments. Am J Plant Sci 5(1):5CrossRefGoogle Scholar
  29. Nigra HM, Caso O, Giulietti AM (1987) Production of solasodine by calli from different parts of Solanum eleagnifolium Cav. plants. Plant Cell Rep 6:135–137Google Scholar
  30. Ohofeghara FA (1971) The effects of growth substances on the growth of Tapinanthus bauguensis (Loranthaceae) in vitro. Am Bot 36:563–570CrossRefGoogle Scholar
  31. Payne G, Bringi V, Prince C, Shuler M (1991) Quantifying growth and product synthesis: kinetics and stoichiometry. In: Michael S (ed) Plant cell and tissue culture in liquid systems. Hanser/Oxford University Press, Oxford, pp 47–70Google Scholar
  32. R Core Team (2018). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.
  33. RStudio Team (2018). RStudio: Integrated Development for R. RStudio, Inc., Boston
  34. Ram RL, Singh MPN (1991) In vitro haustoria regeneration from embryo and in vitro-formed leaf callus cultures in Dendrophthoe falcata (L.f.) Ettings. Adv Plant Sci 4:48–53Google Scholar
  35. Rousset A, Simier P, Fer A (2003) Characterization of simple in vitro cultures of Striga hermonthica suitable for metabolic studies. Plant Biol 5:265–273CrossRefGoogle Scholar
  36. Rustán JJ, Balseiro D, Degrange FJ, Halpern K, Sferco ME, Luna ML, Giudice GE (2003) Características estructurales de los haustorios de Ligaria cuneifolia (Loranthaceae) de Argentina. Bol Soc Arg Bot 38:107–108Google Scholar
  37. Scarpa GF, Montani MC (2011) Etnobotánica médica de las “ligas” (Loranthaceae sensu latu) entre indígenas y criollos de Argentina. Dominguezia 27(2):5–19Google Scholar
  38. Sharpe D (2015) Your chi square test is statistically significant: now what? Pract Assess Res Eval 20(8):1–10Google Scholar
  39. Soberón JR, Sgariglia MA, Dip Maderuelo MR, Andina ML, Sampietro DA, Vattuone MA (2014) Antibacterial activities of Ligaria cuneifolia and Jodina rhombifolia leaf extracts against phytopathogenic and clinical bacteria. J Biosci Bioeng 118(5):599–605CrossRefGoogle Scholar
  40. Srivastava S, Dwivedi UN (2001) Plant regeneration from callus of Cuscuta reflexa—an angiospermic parasite– and modulation of catalase and peroxidase activity by salicylic acid and naphthalene acetic acid. Plant Physiol Biochem 39:529–538CrossRefGoogle Scholar
  41. Stat-Ease, Inc. (2018). Design-Expert Trial (version 64-bit)Google Scholar
  42. Trigiano RN (2011) Chapter 12-Propagation of shoot culture. In: Trigiano RN, Gray DJ (eds) Plant tissue culture, development and biotechnology. CRC Press Taylor & Francis Group, Boca Raton, pp 181–192Google Scholar
  43. Varela BG, Fernández T, Taira C, Cerdá Zolezzi P, Ricco RA, Caldas López E, Álvarez E, Gurni AA, Hajos S, Wagner ML (2001) El “muérdago criollo”, Ligaria cuneifolia (R. et P.) Tiegh -Loranthaceae- Desde el uso popular hacia el estudio de los efectos farmacológico. Dominguezia 17(1):31–50Google Scholar
  44. Vázquez y Novo SP, Wagner ML, Gurni AA, Rondina RVD (1989) Importancia Toxicológica de la presencia de sustancias aminadas en ejemplares de Ligaria cuneifolia var. cuneifolia colectados en diferentes áreas de la República Argentina. Acta Farmacéutica Bonaerense 8(1):23–29Google Scholar
  45. White PR (1963) The cultivation of animal and plant cells. Ronald Press, New YorkGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • M. V. Ricco
    • 1
    • 2
  • M. L. Bari
    • 1
  • F. Bagnato
    • 1
  • C. Cornacchioli
    • 1
  • M. Laguia-Becher
    • 2
    • 5
  • L. U. Spairani
    • 1
    • 6
  • A. Posadaz
    • 4
  • C. Dobrecky
    • 3
    • 7
  • R. A. Ricco
    • 3
  • M. L. Wagner
    • 3
  • M. A. Álvarez
    • 1
    • 2
    Email author
  1. 1.Carreras de Farmacia y Bioquímica, Cátedra de Farmacobotánica y Farmacognosia, Facultad de Ciencias de la SaludUniversidad MaimónidesCiudad de Buenos AiresArgentina
  2. 2.Consejo Nacional de Investigaciones Científicas y TecnicasCiudad de Buenos AiresArgentina
  3. 3.Departamento de Farmacología, Cátedra de Farmacobotánica, Facultad de Farmacia y BioquímicaUniversidad de Buenos AiresCiudad de Buenos AiresArgentina
  4. 4.Facultad de Turismo y UrbanismoUniversidad Nacional de San LuisVilla de Merlo, San LuisArgentina
  5. 5.Centro de Estudios Biotecnológicos, Biológicos, Ambientales y Diagnóstico (CEBBAD)Universidad MaimónidesCiudad de Buenos AiresArgentina
  6. 6.Instituto Antártico Argentino, Dirección Nacional del AntárticoSan Martín, Buenos AiresArgentina
  7. 7.Cátedra de Tecnología Farmacéutica I, Facultad de Farmacia y BioquímicaUniversidad de Buenos AiresCiudad de Buenos AiresArgentina

Personalised recommendations