MICROPROPAGATION AND MOLECULAR CHARACTERIZATION OF HYPERICUM PERFORATUM L. SUBSP. VERONENSE (SCHRANK) H.LINDB., A VALUABLE MEDICINAL PLANT WITH ORNAMENTAL VALUE
DOI:
https://doi.org/10.51258/RJH.2023.01Keywords:
St John’s wort, in vitro clonal propagation, SNP, PVP, triacontanol, acclimatization, phylogeny, DNA BarcodingAbstract
St. John's wort (Hypericum perforatum) is a perennial species with a worldwide interest due to its pharmaceutical characteristics which is also of ornamental value for gardening with native elements. Given that in vitro culture allows mass multiplication of selected genotypes, we investigated the micropropagation of the ex situ cultivated Greek native H. perforatum subsp. veronense using novel substances and its genetic fingerprinting. Apart from common auxins and cytokinins, a culture medium (BB) was used for the first time for its micropropagation and the effect of betaine, PVP, SNP and triacontanol were explored for the first time. Betaine addition of 200 mg/L produced proliferated shoots and roots without the need of plant growth regulators although a 0.5 mg L-1 BA was the best treatment in proliferation experiments. Shoot number was highest in 0.5 BA + 0.1 IBA (mg L-1), in 1 μΜ sodium nitroprusside (SNP) + 1 mg L-1 IAA+200 mg L-1 betaine and in PVP (0.5-1.5 mg L-1) + 1 mg L-1 IBA + 200 mg L-1. Greater SNP concentration had either no differences or negative effect, while the greatest root number were recorded in Control. All three treatments of triacontanol (1-10 μg L-1) + 0.5 mg L-1 BA + 200 mg L-1 betaine resulted in higher shoot number. However, the statistically greatest root number were recorded in low concentration of SNP, or in absence of PVP and triacontanol. The micro-plants of H. perforatum subsp. veronense obtained from the SunCapTM sealed vessels and transplanted into the soil substrates presented 90.2% success; they were then transplanted to 1 L pots and were successfully established in the field for cultivation. The molecular characterization of H. perforatum subsp. veronense was achieved with DNA barcoding fingerprints deposited in the GenBank regarding four molecular markers of cpDNA (petB/petD, rbcL, trnL/trnF and rpoC1).
Downloads
References
Akula A., Akula C. and Bateson M. (2000). Betaine a novel candidate for rapid induction of somatic embryogenesis in tea (Camellia sinensis (L.) O. Kuntze). Plant Growth Regulation. 30: 241-246.
Alhaithloul H.A., Soliman M.H., Ameta K.L., El-Esawi M.A. and Elkelish A. (2020). Changes in ecophysiology, osmolytes, and secondary metabolites of the medicinal plants of Mentha piperita and Catharanthus roseus subjected to drought and heat stress. Biomolecules. 10: 43.
Annunziata M.G., Ciarmiello L.F., Woodrow P., Dell’Aversana E. and Carillo P. (2019). Spatial and temporal profile of glycine betaine accumulation in plants under abiotic stresses. Front. Plant Sci. 10: 230.
Ashraf M. and Foolad M.R. (2007). Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ. Exp. Bot. 59(2): 206-216.
Auer C.A., Motyka V., Březinová A. and Kamínek M. (1999). Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida. Physiol. Plantarum. 105(1): 141-147.
Aziz N., Roger J. Sauve R.J., Long L. and Cherry M. (2006). Genetic and Phytochemical Diversity Assessment Among Eleven Hypericum Accessions via AFLP and HPLC Analyses. J Herbs Spices Med Plants. 12(1-2): 97-105.
Banerjee A., Bandyopadhyay S. and Raychaudhuri S.S. (2012). In vitro regeneration of Hypericum perforatum
L. using thidiazuron and analysis of genetic stability of regenerants. Indian J. Biotechnol.. 11: 92-98.
Barcaccia G., Arzenton F., Sharbel T., Varotto S., Parrini P. and Lucchin M. (2006). Genetic diversity and reproductive biology in ecotypes of the facultative apomict Hypericum perforatum L. Heredity. 96:322– 334.
Béjaoui A., Boulila A., Messaoud C. and Boussaid M. (2011). Population genetic structure of Tunisian
Hypericum humifusum assessed by RAPD markers. Biologia. 66(6): 1003-1010.
Béjaoui A., Boulila A., Messaoud C., Rejeb M. N. and Boussaid M. (2010). Genetic diversity and population structure of Hypericum humifusum L. (Hypericacae) in Tunisia: Implications for conservation, Plant Biosyst. 144(3): 592-601.
Bi D., Chen D., Khayatnezhad M., Hashjin Z., Li Z. and Ma, Y. (2021). Molecular identification and genetic diversity in Hypericum L.: A high value medicinal plant using RAPD markers markers. Genetika, 53(1), 393–405.
Cao W., Chen X. and Cao Z.W. (2022). Morphometric analysis and genetic diversity in Hypericum L. using sequence related amplified poly morphism. Caryologia 75(2): 23-31.
Carimi F. and Pasquale F. (2003). Micropropagation of Citrus. In: Jain S.M., Ishii K. (eds), Micropropagation of Woody Trees and Fruits. Forestry Sciences, vol 75. Springer, Dordrecht
Chen W. and Xu L. (2014). Growth-regulating activity of cinnamamide and betaine cinnamamide on wheat. Adv. J. Food Sci.Techn.. 7: 584-588.
Correa-Aragunde N., Graziano M., Chevalier C. and Lamattina L. (2006). Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato. J. Exp. Bot.. 57(3): 581– 588.
Costa J., Campos B., Amaral J.S., Nunes M.E., Oliveira M.B.P.P. and Mafra I. (2016). HRM analysis targeting ITS1 and matK loci as potential DNA mini-barcodes for the authentication of Hypericum perforatum and Hypericum androsaemum in herbal infusions. Food Control. 61:105-114.
Council of Europe (2000). Natural sources of flavourings. Report No. 1 Strasbourg: Council of Europe.
Crockett S.L., Douglas A.W., Scheffler B.E. and Khan I.A. (2004). Genetic profiling of Hypericum (St. John's Wort) species by nuclear ribosomal ITS sequence analysis. Planta Med. 70(10):929-35.
Dlugosch, K.M. and Parker, I.M. (2007). Molecular and quantitative trait variation across the native range of the invasive species Hypericum canariense: evidence for ancient patterns of colonization via pre- adaptation. Mol. Ecol. 16: 4269-4283.
EMA (European Medicines Agency) Monograph (2023). European Union herbal monograph on Hypericum perforatum L. herb. EMA/HMPC/7695/2021 Committee on Herbal Medicinal Products (HMPC). Available online: https://www.ema.europa.eu/en/documents/herbal-monograph/final-european- union-herbal-monograph-hypericum-perforatum-l-herba-revision-1_en.pdf (accessed 26 November 2023)
Feito I., Rodriguez A., Centeno M. L., Sánchez-Tamés R. and Fernández B. (1994). Effect of the physical nature of the culture medium on the metabolism of benzyladenine and endogenous cytokinins in Actinida deliciosa tissues cultured in vitro. Physiol. Plantarum. 91(3): 449-453.
Fornasiero R. B., Bianchi A. & Pinetti A. (1998). Anatomical and ultrastructural observations in Hypericum perforatum L. leaves. J. Herbs Spices Med. Plants 5: 21–23.
Gadzovska S., Maury S., Ounnar S., Righezza M., Kascakova S., Refregiers M., Spasenoski M., Joseph C. and Hagège D. (2005). Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures. Plant Physiol. Biochem. 43(6): 591-601.
Gaudeul M. (2006). Disjunct distribution of Hypericum nummularium L. (Hypericaceae): molecular data suggest bidirectional colonization from a single refugium rather than survival in distinct refugia. Biol. J. Linn. Soc. 87(3): 437-447.
George E. F. (1993). Plant Propagation by Tissue Culture, Part 1 The Technology. Exegetics, England.
Goel M. K., Kukreja A. K. and Bisht N. S. (2009). In vitro manipulations in St. John's wort (Hypericum perforatum L.) for incessant and scale up micropropagation using adventitious roots in liquid medium and assessment of clonal fidelity using RADP analysis. Plant Cell Tissue Organ Cult. 96: 1-9.
Grigoriadou K., Krigas N., Lazari N. and Maloupa E. (2020). Sustainable use of mediterranean medicinal- aromatic plants. In Florou-Paneri P., Christaki E., Giannenas I. (eds), Feed Additives, Academic Press, 57- 74.ISBN 9780128147009 (https://doi.org/10.1016/B978-0-12-814700-9.00004-2)
He M. and Wang Z. (2013). Genetic diversity of Hypericum perforatum collected from the Qinling Mountains of China. Biochem. Syst. Ecol. 50: 232-239.
Howard C., Bremner P.D., Fowler M.R., Isodo B., Scott N.W. and Slater A. (2009). Molecular identification of
Hypericum perforatum by PCR amplification of the ITS and 5.8S rDNA region. Planta Med. 75(8): 864-9.
Howard C., Hill E., Kreuzer M., Mali P., Masiero E., Slater A. and Sgamma T. (2019). DNA Authentication of St John’s wort (Hypericum perforatum L.) commercial products targeting the ITS region. Genes 10(4).
Hutchinson J. F. and Zimmerman R. H. (1987). Tissue culture of temperate fruit and nut trees. Horticult. Rev. 9: 273-349.
Isenberg L. (1998). Herbal products are driving supplement industry growth. Herbalgram. 42: 65.
Istikoglou C.I., Mavreas V. and Geroulanos G. (2010). History and therapeutic properties of Hypericum perforatum from antiquity until today. Psychiatriki. 21(4): 332-338.
Ivanova NV, Kuzmina ML, Braukmann TWA, Borisenko AV and Zakharov EV (2016). Correction: Authentication of Herbal Supplements Using Next-Generation Sequencing. PLOS ONE 11(12).
Jayanthi M. and Seeni S. (2004). Assessment of Genetic Diversity and Population Discrimination in
Hypericum Hookerianum of Western Ghats Using RAPD. Plant Cell Biotechnol Mol Biol. 5(1-2):81-84.
Jenfaoui H., Uras M.E., Bahri B.A., Ozyigit I.I. and Souissi T. (2021). Morphological variation, genetic diversity and phylogenetic relationships of Hypericum triquetrifolium Turra populations from Tunisia. Biotechnol. Biotechnol. Equip. 35(1): 1505-1519.
Kakouri E., Trigas P., Daferera D., Skotti E., Tarantilis P. A. and Kanakis C. (2023). Chemical characterization and antioxidant activity of nine Hypericum species from Greece. Antioxidants. 12(4): 899.
Kirakosyan A., Vardapetyan R.R. and Charchoglyan A. G. (2000). The content of hypericin and pseudohypericin in cell cultures of Hypericum perforatum. Russ. J. Plant Physiol. 47: 270-273.
Krishna H., Sairam R. K., Singh S. K., Patel V. B., Sharma R. R., Grover M., and Sachdev A. (2008). Mango explant browning: Effect of ontogenic age, mycorrhization and pre-treatments. Sci. Horticult. 118(2): 132-138.
Kwiecien I., Miceli N., Kedzia E., Cavò E., Taviano M.F., Beerhues L. and Ekiert H. (2023). Different types of Hypericum perforatum cvs. (Elixir, Helos, Topas) in vitro cultures: A rich source of bioactive metabolites and biological activities of biomass extracts. Molecules. 28: 2376.
Liu X., Bai Y., Wang Y., Chen Y., Dong W. and Zhang, Z (2023). Complete Chloroplast Genome of Hypericum perforatum and Dynamic Evolution in Hypericum (Hypericaceae). Int. J. Mol. Sci. 24: 16130.
Mallón R., Rodríguez-Oubiña J. and González M. L. (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: 31-39.
McCoy J.-A. and Camper N. D. (2002). Development of a micropropagation protocol for St. John's wort (Hypericum perforatum L.). HortScience. 37(6): 978-980.
Meseguer A.S., Aldasoro J.J. and Sanmartín I. (2013). Bayesian inference of phylogeny, morphology and range evolution reveals a complex evolutionary history in St. John’s wort (Hypericum). Mol. Phylogenet. Evol. 67(2): 379-403.
Meseguer A.S., Lobo J.M, Ree R., Beerling D.J. and Sanmartín I. (2015). Integrating Fossils, Phylogenies, and Niche Models into Biogeography to Reveal Ancient Evolutionary History: The Case of Hypericum (Hypericaceae). Syst. Biol. Volume 64(2): 215–232.
Meseguer, A. S., Sanmartín, I., Marcussen, T. and Pfeil, B. E. (2014). Utility of low-copy nuclear markers in phylogenetic reconstruction of Hypericum L. (Hypericaceae). Plant Syst. Evol. 300(6): 1503–1514.
Mikhovich Z.E., Echishvili E.E., Portnyagina N.V. and Skrotskaya O.V. (2021). Peculiarities of Hypericum perforatum L. reproduction in vitro culture and development of plants in the open field. Samara J. Sci. 10:79–86
Mohammed N.R., Dalar A., Ozdemir F.A. and Turker M. (2019) In vitro propagation and secondary metabolites investigation of Hypericum perforatum L. Fresenius Environ. Bull. 28: 5569–5576.
Molins M.P., Corral J.M., Aliyu O.M., Koch M.A., Betzin A., Maron J.L. and Sharbel T.F. (2014). Biogeographic variation in genetic variability, apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range. Ann. Bot. 113 (3): 417–427.
Morshedloo M.R., Moghadam M.R.F., Ebadi A. and Yadjani D. (2015). Genetic relationships of Iranian
Hypericum perforatum L. wild populations as evaluated by ISSR markers. Plant Syst. Evol. 301: 657–665.
Moura M. (1998). Conservation of Hypericum foliosum Aiton, an endemic Azorean species, by micropropagation. In Vitro Cell. Devel. Biol.-Plant. 34: 244-248.
Murashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum. 15: 473-497.
Murch S. J., and Saxena P. K. (2006). A melatonin-rich germplasm line of St John's wort (Hypericum perforatum L.). J. Pineal Res. 41(3): 284-287.
Murch S.J., KrishnaRaj S. and Saxena P.K. (2000a). Phytopharmaceuticals: Problems, limitations and solutions. Sci. Rev. Altern. Med. 4: 33–38.
Murch S. J., Choffe K. L., Victor J. M. R., Slimmon T. Y., KrishnaRaj S. and Saxena P. K. (2000b). Thidiazuron- induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos'). Plant Cell Rep. 19(6): 576-581.
National Center for Biotechnology Information (NCBI). Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; [1988] – [accessed on 20 Nov 2023]. Available from: https://www.ncbi.nlm.nih.gov/
Nürk N.M, Madriñán S., Carine M.A., Chase M.W. and Blattner F.R. (2013). Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum; Hypericaceae), Mol. Phylogenet. Evol. Volume 66(1): 1-16.
Nürk N.M., Uribe-Convers S., Gehrke B., Tank B.C. and Blattner F.R. (2015). Oligocene niche shift, Miocene diversification – cold tolerance and accelerated speciation rates in the St. John’s Worts (Hypericum, Hypericaceae). BMC Evol. Biol. 15:80.
Obul Reddy B., Giridhar P. & Ravishankar G. A. (2002). The effect of triacontanol on micropropagation of
Capsicum frutescens and Decalepis hamiltonii W & A. Plant Cell Tissue Organ Cult. 71: 253-258.
Ötvös K., Pasternak T. P., Miskolczi P., Domoki M., Dorjgotov D. and Fehér A. (2005). Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures. Plant J. 43(6): 849-860.
Pagnussat G.C., Simontacchi M., Puntarulo S. and Lamattina L. (2002) Nitric oxide is required for root organogenesis. Plant Physiol. 129: 954–956.
Park S.J. and Kim K.J. Molecular phylogeny of the genus Hypericum (Hypericaceae) from Korea and Japan: evidence from nuclear rDNA ITS sequence data (2004). J. Plant Biol. 47:366–374.
Percifield R.J., Hawkins J.S., McCoy J.A., Widrlechner M.P. and Wendel J.F. (2007). Genetic diversity in Hypericum and AFLP Markers for species-specific identification of H. perforatum L.. Planta Med. 73(15):1614-21.
Piperić K.H., Maleš Z. and Plazibat M. (2008). Genetic structure in Hypericum perforatum L. population. Period. boil. 110(4): 110.
Prasad, M. N. V., Hagemeyer, J. & Greger, M. (1999). Metal availability and bioconcentration in plants. Heavy Metal Stress in Plants: From Molecules to Ecosystems, 1-27.
Pretto F. R. and Santarém E. R. (2000). Callus formation and plant regeneration from Hypericum perforatum
leaves. Plant Cell Tissue Organ Cult. 62: 107-113.
Pyrka I., Stefanaki A. and Vlachonasios K.E. (2021). DNA Barcoding of St.Johnʼs wort (Hypericum spp.) Growing Wild in North-Eastern Greece. Planta Med. 87(07): 528-537
Raclariu A.C., Paltinean R., Vlase L., Labarre A., Manzanilla V., Ichim M.C., Crisan G., Brysting A.K. and de Boer
H. (2017). Comparative authentication of Hypericum perforatum herbal products using DNA metabarcoding, TLC and HPLC-MS. Scientific Reports, 7(1), 1-12.
Rahnavard A. (2017). Genetic and biochemical diversity of Hypericum perforatum L. Grown in the Caspian climate of Iran. Appl Ecol Environ Res. 15: 665-675.
Ravindran B.M., Rizzo P., Franke K., Fuchs J. and D’Auria J. (2022). Simple and robust multiple shoot regeneration and root induction cycle from different explants of Hypericum perforatum L. genotypes. Plant Cell Tissue Organ Cult. 152(1):1–15
Reustle G. and Natter I. (1994). Effect of polyvinylpyrrolidone and activated charcoal on formation of microcallus from grapevine protoplasts (Vitis sp.). Vitis. 33: 117-121.
Reustle G., Harst M. and Alleweldt, G. (1995). Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue. Plant Cell Rep. 15: 238-241.
Rezaei L., Qaderi A., Naghavi M.R., Ebrahimi, M.A., Riazi A.S. Mehrafarin A. and Naghdi Badi H. (2012). Assessment of genetic diversity in the populations of Hypericum perforatum L. Using AFLP Markers. J. Med. Plant Res. 11(44): 62-69.
Rico-Lemus M. and Rodrìguez-Garay B. (2014) SNP as an effective donor of nitric oxide for in vitro plant cell and tissue culture. J. Plant Biochem. Physiol. 2:127–128
Robson N. K. B. (2016). And then came molecular phylogenetics - Reactions to a monographic study of
Hypericum (Hypericaceae). Phytotaxa 255(3): 181-198.
Saddiqe Z., Naeem I., and Maimoona A. (2010). A review of the antibacterial activity of Hypericum perforatum L. J. Ethnopharmacol. 131(3): 511-521.
Saini U., Kaur D., Chanda S., Bhattacharya A. and Ahuja P. S. (2012). Application of betaine improves solution uptake and in vitro shoot multiplication in tea. Plant Growth Regul. 67: 65-72.
Santarém E. R. and Astarita L. V. (2003). Multiple shoot formation in Hypericum perforatum L. and hypericin production. Braz. J. Plant Physiol. 15(1): 43-47.
Sarropoulou V. and Maloupa E. (2019). Effect of glycine betaine, polyvinylpyrrolidone and D-mannitol on micropropagation of Sideritis raeseri Boiss and Heldr. subsp. raeseri. GSC Biological and Pharmaceutical Sciences. 7(2).
Savio L. E. B., Astarita L. V. and Santarém E. R. (2012). Secondary metabolism in micropropagated Hypericum perforatum L. grown in non-aerated liquid medium. Plant Cell Tissue Organ Cult. 108: 465- 472.
Schempp C. M., Kirkin V., Simon-Haarhaus B., Kersten A., Kiss J., Termeer C. C. and Simon J. C. (2002). Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St. John's wort that acts by induction of apoptosis. Oncogene. 21(8): 1242-1250.
Shiji P.C. and Siril E.A. (2018). An improved micropropagation and ex vitro rooting of a commercially important crop Henna (Lawsonia inermis L.). Physiol. Mol. Biol. Plants. 24(6): 1273-1284.
Smelcerovic A., Verma V., Spiteller M., Ahmad S.M., Puri S.C. and Qazi G.N. (2005). Phytochemical analysis and genetic characterization of six Hypericum species from Serbia. Phytochemistry. 67(2):171-7.
Southwell I. A., and Bourke C. A. (2001). Seasonal variation in hypericin content of Hypericum perforatum L. (St. John’s wort). Phytochemistry. 56(5): 437-441.
Tamura K., Stecher G., and Kumar S. (2021). MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol. Biol. Evol. 38: 3022-3027.
Tan, B.C., Chin, C.F. & Alderson, P. (2013). Effects of sodium nitroprusside on shoot multiplication and regeneration of Vanilla planifolia Andrews. In Vitro Cell. Dev. Biol.-Plant 49, 626–630. https://doi.org/10.1007/s11627-013-9526-8
Tantos A., Mésáros A., Farkas T., Szalai J. and Horváth G. (2001). Triacontanol-supported micropropagation of woody plants. Plant Cell Rep. 20: 16–21.
Tavakoli F., Rafeiolhossaini M., Ravash R. and Ebrahimi M. (2020). UV-B radiation and low temperature promoted hypericin biosynthesis in adventitious root culture of Hypericum perforatum. Plant Signal Behav. 15: 1559–2324.
Touchell D., Smith J. and Ranney T. G. (2008). Organogenesis from Hypericum frondosum leaves. Proc. SNA Res. Conf. Annu. Rpt. 53: 222-225.
Tsoktouridis G., Krigas N., Sarropoulou V., Kampouropoulou S., Papanastasi K., Grigoriadou K., Menexes C.G. and Maloupa, E. (2019). Micropropagation and molecular characterization of Thymus sibthorpii Benth. (Lamiaceae), an aromatic-medicinal thyme with ornamental value and conservation concern. In Vitro Cell. Devel. Biol.-Plant. 55(6): 647-658.
Tzatzani T.T., MichailI., Boutsika A., Sarrou E. and Ganopoulos I. (2023). Micropropagation of guava (Psidium guajava) seedlings, a plant with interest in cool subtropics, using an innovative culture medium. Biotechn. Biotechn. Equip. 37(1): 139-150.
Verma A., Malik C.P., Gupta V.K. and Bajaj B.K. (2011). Effects of in vitro triacontanol on growth, antioxidant enzymes, and photosynthetic characteristics in Arachis hypogaea (L.) Braz. J. Plant Physiol. 23: 271–277.
Zeliou K., Koui E-M., Papaioannou C., Koulakiotis N.S., Iatrou G., Tsarbopoulos A., Papasotiropoulos V. and Lamari F.N. (2020). Metabolomic fingerprinting and genetic discrimination of four Hypericum taxa from Greece. Phytochemistry. 174(112290).
Downloads
Published
Issue
Section
License
Copyright (c) 2023 ROMANIAN JOURNAL OF HORTICULTURE
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
