In vitro shoot Proliferation of Hypericum perforatum L. through Indirect and Direct Plant Regeneration

Authors

1 Department of Horticultural Science, Tehran Universitty, Karaj, Iran.

2 Department of Horticultural Science, Tehran University, Karaj, Iran.

3 Department of Horticultural Science, Ferdowsi University of Mashhad, Mashhad, Iran.

4 Central Agriculture and Natural Resource of Fars Province, Shiraz, Iran.

Abstract

Hypericum perforatum L. (St. Johns’ wort) is the most commercially important species of the genus Hypericum and contains a wide range of components including naphthodianthrones, phloroglucinols, tannins, xanthones, phenolic acids and essential oil. In order to establish an efficient protocol for regeneration, the effects of explant type and plant growth regulators on direct and indirect shoot regeneration in H. perforatum were evaluated. According to obtained results the media supplemented with 0.1 mg l-1 Benzyl Adenine (BA) was effective for shoot proliferation from shoot tip explants of H. perforatum that showed the highest shoots number (15.5 shoots per explant) and shoot height (2.07 cm). In second experiment a method for rapid micro propagation of H. perforatum through indirect plant regeneration from calli has been developed. The results demonstrated that a combination of auxin and cytokinin was needed for optimum callus induction and leaf segments were suitable explant for callus induction in H. perforatum. Callus induction was observed in most studied treatments but the highest callus volume (1.43 cm3) was obtained by leaf segments in media supplemented with 0.25 mg l-1 2,4-Dichlorophenoxyacetic acid (2,4-D)+1 mg l-1 Kinetin. Successful shoot regeneration from callus was observed in MS medium containing 0.5 mg l-1 BA, which resulted the highest shoot proliferation rate (61.75%) and maximum number of induced shoots (9 shoots per explant). All of shoots formed root in media with 0.5 mg l-1 Indole-3-Butyric Acid (IBA) on which 100% of the regenerated shoots developed roots with an average number of 5 roots per shoot. The plantlets were acclimatized and transferred to the greenhouse with 80% survival. This in vitro propagation protocol should be useful for conservation as well as mass propagation of H. perforatum plant.

Keywords


1. Crockett SL. Essential oil and volatile components of the genus Hypericum (Hypericaceae). Nat Prod Commun. 2010;5:1493-1506.
2. Karppinen K, Taulavuori E, Hohtola A. Optimization of protein extraction from Hypericum perforatum tissues and immunoblotting detection of hyp-1 at different stages of leaf development. Mol Biotechnol. 2010;46:219-226.
3. Greeson JM, Sanford B, Monti DA. St. John's wort (Hypericum perforatum): A review of the current pharmacological, toxicological, and clinical literature. J Psychopharmacol. 2001;153:402-414.
4. Bilia AR, Gallori S, Vincieri FF. St Johns wort and depression. Efficacy, safety and tolerability-an update. Life Sci. 2002;70:3077-3096. 
5. Barnes J, Anderson LA, Phillipson JD. St John's wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. J Pharmacy Pharmacol. 2001;53:583-600.
6. Gaudin M, Simmonet X, Debrunner N. Breeding for a Hypericum perforatum L. variety both productive and Colletotrichumgloeos poroidestolerant. J Herbs Spices Med Plants. 2002;9:107-120.
7. Moura M. Conservation of Hypericum foliosumaiton, an endemic Azorean species, by micro propagation. In vitro Cell Dev Biol Plant. 1998;34:244-248.
8. Cardoso MA, De Oliveira DE. Tissue culture of Hypericum brasiliense Choisy: shoot multiplication and callus induction. Plant Cell Tissue Organ Cult. 1996;44:91-94.
9. Mederos S, San Andrés L, Luis JG. Rosmanol controls explants browning of Hypericum canariense L. during in vitro establishment of shoots. Acta Societatis Botanic Poloniae. 1997;66:347-349.
10. Cellárová E, Kimákova K, Brutóvska R. Multiple shoot formation in Hypericum perforatum L. and variability of R0. Biol Plant. 1992;34:536-561.
11. Kartnig T, Brantner A. Secondary constituents in cell cultures of Hypericum perforatum and Hypericum maculatum. Planta Med. 1990;56:634-638.
12. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant. 1962;15:473-497.
13. Gamborg OL, Miller PA, Ojima K. Nutrient requirement of suspension cultures of soybean root cells. Exp Cell Res. 1968;50:151-158.
14. Campble MH. Germination emergence and seedling growth of Hypericum perforatum. Weed Res. 1985;25:259-266.
15. Nedkov N. Research on the effect of pre-sowing treatment on seed germination of Hypericum perforatum L. Bulg J Agric Sci. 2007;13:31-37.
16. Clark N. The biology of St. John’s wort (Hypericum perforatum L. var angustifolium DC.) in Ovens Valley, Victoria, with particular references to entomological control. Aust J Bot. 1953;1:95-120.
17. Fenner M. The effects of the parent environment on seed germinability. Seed Sci. Res. 1991;1:75-84.
18. Gutterman Y. Maternal effects on seeds during development. In: Fenner M. (ed.). Seeds: The Ecology of Regeneration in Plant Communities. CAB International, Wallingford. 1992;27-59.
19. Cruz A, Perez B, Velasco A, Moreno JM. Variability in seed germination at the interpopulation and intraindividual levels of shrub Erica Australis in response to fire-related cues. Plant Ecol. 2003;169:93-103.
20. Ayan AK, Cirak C, Kevserolu K, Sokmen A. Effects of explants types and different concentrations of sucrose and phytoharmones on plant regeneration and hypericin content in Hypericum perforatum L. Turk J Agric For. 2005;29:197-204.
21. Murch SJ, Choffe KL, Victor JMR, Slimmon TY, Krishna Raj S, Saxena PK. Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum cv. "Anthos"). Plant Cell Rep. 2000;19:576-581.
22. Gadzovska S, Maury S, Ounnar S, Righezza M, Kascakova S, Refregiers M, Spasenoski M, Joseph C, Hagege D. Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures. Plant Physiol Biochem. 2005;43:591-601.
23. Bezo M, Stefunova V. In direct regeneration of Hypericum perforatum L. under in vitro conditions. Acta Fytotechnica Et Zootechnica. 2001;4:277-279.
24. Santarem ER, Astarita LV. Multiple shoot formation in Hypericum perforatum L. and hypericin production. Braz J Plant Physiol. 2003;15:43-47.
25. Briskin DP. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiol. 2000;124:507-514.
26. Yazaki K, Okuda T. Procyaninins in callus and multiple shoots of Hypericum erectum. Planta Med. 1990;56:490-491.
27. Kartnig T, Gobel I, Heydel B. Production of hypericin, pseudohypericin and flavonoids in cell culture of various Hypericum species and their chemotypes. Planta Med. 1996;62:51-53.
28. Dias AC, Tomas-Barberan FA, Fernandes-Ferreira M, Ferreres F. Unusual flavonoids produced by callus of Hypericum perforatum. Phytochem. 1998;48:1165-1168.
29. Mulinacci N, Giaccherini C, Santamaria AR, Caniato R, Ferrari F,Valletta A, Vincieri FF, Pasqua G. Anthocyanins and xanthones in the calli and regenerated shoots of Hypericum perforatum var. angustifolium Borkh. Plant Physiol Biochem. 2008;46:414-420.
30. Gadzovska S, Maury S, Delaunay A, Spasenoski M, Hagege D, Courtois D, Joseph C. The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L. Plant Cell Tissue Organ Cult. 2013;113:25-39.
31. Pretto FR, Santarem ER. Callus formation and plant regeneration from Hypericum perforatum L. leaves. Plant Cell Tissue Organ Cult. 2000;67:107-113.
32. Blakesley D, Constantine D. Uptake and metabolism of 6-benzyadenine in shoot cultures of a range of species. Plant Cell Tissue Organ Cult. 1992;28:183-186.
33. Kirakosyan A, Hayashi HI, Inoue K, Charchoglyan A, Vardapetyan H. Stimulation of the production of hypericins by mannan in Hypericum perforatum shoot cultures. Phytochemistry. 2000;53:345-348.