In vitro propagation of medicinal and aromatic plants: the case of selected Greek species with conservation priority

  • K. Grigoriadou
  • N. KrigasEmail author
  • V. Sarropoulou
  • K. Papanastasi
  • G. Tsoktouridis
  • E. Maloupa
Invited Review


Worldwide, many medicinal and aromatic plants (MAPs) are still collected from the wild and only a small fraction of them are exclusively sourced from cultivation. This practice when performed non-sustainably threatens species and populations. Micropropagation of MAPs is a powerful tool to conserve rare, threatened, and valuable MAPs, and to massively produce high-value plant material for cultivation without seasonal constraints. In this study, the in vitro propagation protocols of 22 Greek native MAPs assigned with conservation priority were assessed (herbaceous perennials, bulbous, subshrubs, and trees), including 17 range-restricted plants and 5 taxa of Orchidaceae. For the latter, current micropropagation efforts include seed germination, callus induction, and protocorm formation for successful plantlet development; however, these propagation protocols are still fragmentary. For the rest (n = 17), a five-stage detailed procedure is outlined (plant material, establishment, proliferation, rooting, and acclimatization), while materials, treatments, and data per stage are shown comparatively and discussed. Emphasis is given on the selection and preparation of plant material obtained from nature for research, sustainable use, and ex situ conservation actions, and on their effectiveness for conservation purposes or mass production needs. The protocol effectiveness was calculated using a specific equation to estimate the potential number of acclimatized plants raised from a single explant within a year. All protocols can facilitate conservation, and almost half of them could be used for commercialization with high cost (five cases), intermediate cost (eight), or low cost (four), which enables their possible sustainable use.


Greek flora Ex situ conservation Sustainable exploitation Propagation protocol effectiveness Plant growth regulators 



The authors would like to thank the anonymous reviewers for their constructive comments. The work of Nikos Krigas, Katerina Grigoriadou, and George Tsoktouridis was supported by the project “Multifaceted Valorisation of single-country endemic plants of Crete, Greece, Tunisia and Rif, Morocco for sustainable exploitation in the agro-alimentary, horticultural-ornamental and medicinal-cosmetic sectors” (MULTI-VAL-END) which is funded through the ARIMNet2 2017 Joint Call by the following funding agencies: HAO-Demeter (Greece), MHESRT (Tunisia), and MESRSFC (Morocco); ARIMNet2 (ERA-NET) has received funding from the European Union’s Seventh Framework Program for research, technological development and demonstration under grant agreement no. 618127.


  1. Antonidaki-Giatromanolaki A, Dragassaki M, Vlahos I, Papadimitriou M (2006) Vegetative propagation in vivo and in vitro of Staehelina petiolata (L.) Hilliard & Burtt. Prop Orn Plant 6:187–193Google Scholar
  2. Bernáth J, Németh É (2000) Ecological diversity of Hungarian medicinal and aromatic plant flora and its regional consequences. Int J Hort Sci 7:10–19Google Scholar
  3. De Paoli G, Rossi V, Scozzoli A (1994) Micropropagazione Delle Piante Ortoflorofrutticole, 1a edizone. Edagricole, Cesena, ItalyGoogle Scholar
  4. Debergh PC, Maene L (1981) A scheme for commercial propagation of ornamental plants by tissue culture. Sci Hortic 14:335–345CrossRefGoogle Scholar
  5. Dias MC, Almeida R, Romano A (2002) Rapid clonal multiplication of Lavandula viridis L’Hér through in vitro axillary shoot proliferation. Plant Cell Tissue Organ Cult 68:99–102CrossRefGoogle Scholar
  6. Dimopoulos P, Raus T, Bergmeier E, Constantinidis T, Iatrou G, Kokkini S, Strid A, Tzanoudakis D (2016) Vascular plants of Greece: an annotated checklist. Supplement Willdenowia 46:301–347CrossRefGoogle Scholar
  7. Dimopoulos P, Raus Th, Bergmeier E, Constantinidis Th, Iatrou G, Kokkini S, Strid A, Tzanoudakis D (2013) Vascular plants of Greece: an annotated checklist –Englera 31, Ser. Publ. Botanischer Garten und Botanisches Museum Berlin-Dahlem, Berlin and by Hellenic Botanical Society, Athens, GreeceGoogle Scholar
  8. Firoozabady E, Gutterson N (2003) Cost-effective in vitro propagation methods for pineapple. Plant Cell Rep 21:844–850Google Scholar
  9. George EF, Debergh PC (2008) Micropropagation: uses and methods. In: George EF, Hall MA, De Klerk GJ (eds) Plant propagation by tissue culture. Springer, Dordrecht, The NetherlandsGoogle Scholar
  10. Georghiou K, Delipetrou P (2010) Patterns and traits of the endemic plants of Greece. Bot J Linn Soc 162:130–422CrossRefGoogle Scholar
  11. Gonçalves S, Romano A (2013) In vitro culture of lavenders (Lavandula spp.) and the production of secondary metabolites. Biotechnol Adv 31:166–174CrossRefGoogle Scholar
  12. Grigoriadou K, Krigas N, Maloupa E (2011) GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta. Plant Cell Tissue Organ Cult 107:531–540CrossRefGoogle Scholar
  13. Grigoriadou K, Krigas N, Maloupa E (2014) GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: seed germination requirements and effect of growth regulators on in vitro proliferation and rooting. Plant Biosyst 148:1169–1177CrossRefGoogle Scholar
  14. Hanlidou E, Lazari D (2013) Essential oils of Thymus leucospermus Hartvig, a Greek endemic rich in phenolic monoterpenes. Nat Prod Res 27:1800–1803CrossRefGoogle Scholar
  15. Hanlidou E, Karousou R, Lazari D (2012) Essential oils of three taxa of the Nepeta argolica aggregate from Greece. Chem Biodivers 9:1559–1566CrossRefGoogle Scholar
  16. Hartmann HT, Kester DE, Davies FT, Geneve RL (2002) Plant propagation principles and practices, 7th edn. Prentice Hall, Upper Saddle River, NJ, pp 367–374Google Scholar
  17. Karioti A, Kitsaki C, Zygouraki S, Ziobora M, Samah D, Skaltsa H, Liakopoulos G (2008) Occurrence of flavonoids in Ophrys (Orchidaceae) flower parts. Flora 203:602–609CrossRefGoogle Scholar
  18. Kartsonas E, Papafotiou M (2007) Mother plant age and seasonal influence on in vitro propagation of Quercus euboica Pap., an endemic, rare and endangered oak species of Greece. Plant Cell Tissue Organ Cult 90:111–116CrossRefGoogle Scholar
  19. Katsalirou E, Gerakis A, Haldas X, Deconninck G (2017) Optimal disinfection times for seeds of Mediterranean orchids propagated on nutrient media. Eur J Environ Sci 7:119–124Google Scholar
  20. Kaur A, Sandhu JS (2015) High throughput in vitro micropropagation of sugarcane (Saccharum officinarum L.) from spindle leaf roll segments: cost analysis for agri-business industry. Plant Cell Tissue Organ Cult 120:339–350CrossRefGoogle Scholar
  21. Kitsaki CK, Zygouraki S, Ziobora M, Kintzios S (2004) In vitro germination, protocorm formation and plantlet development of mature versus immature seeds from several Ophrys species (Orchidaceae). Plant Cell Rep 23:284–290CrossRefGoogle Scholar
  22. Korakis G, Vidakis K (2016) The aromatic and medicinal flora of Samothraki: a list of flora and description of the most important aromatic and medicinal plants on the island of Samothraki. Democritus University of Thrace, Faculty of Agriculture and Forestry Sciences, Department of Forestry and Management of Environment and Natural Resources, Laboratory of Forestry Botany (In Greek) Accessed 13 Aug 2019
  23. Kreziou A, de Boer H, Gravendeel B (2015) Harvesting of salep orchids in north-western Greece continues to threaten natural populations. Oryx 1:1–4Google Scholar
  24. Krigas N, Papadimitriou K, Mazaris AD (2012) GIS and ex-situ plant conservation. In: Alam BM (ed) Application of geographic information systems, In:, Rijeka, pp 153–174 Accessed 13 Aug 2019
  25. Krigas N, Menteli V, Chrysanthou P, Vokou D (2017) The electronic trade in endemic plants of Cyprus through the Internet. Plant Biosyst 151:387–393CrossRefGoogle Scholar
  26. Krigas N, Menteli V, Vokou D (2014a) Analysis of the ex situ conservation of the Greek endemic flora at national, European and global scales and of its effectiveness in meeting GSPC Target 8. Plant Biosyst 150:573–582CrossRefGoogle Scholar
  27. Krigas N, Menteli V, Vokou D (2014b) The electronic trade in Greek endemic plants: biodiversity, commercial and legal aspects. Econ Bot 68:85–95CrossRefGoogle Scholar
  28. Krigas N, Mouflis G, Grigoriadou K, Maloupa E (2010) Conservation of important plants from the Ionian Islands at the Balkan Botanic Garden of Kroussia, N Greece: using GIS to link the in situ collection data with plant propagation and ex-situ cultivation. Biodivers Conserv 19:3583–3603CrossRefGoogle Scholar
  29. Kupke J, Schwierz A, Niefind B (2000) Arznei- und gewürzpflanzen in osteuropa. anbau, verarbeitung und handel in 18 ausgewählten moe-ländern. Materialien zur Marktberichterstattung ZMP Bonn 34:1–95Google Scholar
  30. Lange D (1996) Untersuchungen zum Heilpflanzenhandel in Deutschland. Ein Beitrag zum internationalen Artenschutz. German Federal Agency for Nature Conservation, Bonn-Bad Godesberg, GermanyGoogle Scholar
  31. Lange D (1998) Europe’s medicinal and aromatic plants: their use, trade and conservation. TRAFFIC International, 219c Huntingdon Rd, Cambridge CB3 0DL, UKGoogle Scholar
  32. Litz RE, Moon PA, Chavez Avila VM (2008) Somatic embryogenesis and regeneration of endangered cycad species. Acta Hortic 692:75–80Google Scholar
  33. Lloyd G, McCown B (1980) Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Proc Int Plant Prop Soc 30:421–427Google Scholar
  34. Malmgren S (1996) Orchid propagation: theory and practice. In: Allen C (ed) North American native terrestrial orchids: propagation and production. North Am Native Terrestrial Orchid Conf, Germantown, Maryland, pp 63–71. Google Scholar
  35. Maloupa E, Grigoriadou K, Papanastassi K, Krigas N (2008) Conservation, propagation, development and utilization of xerophytic species of the native Greek flora towards commercial floriculture. Acta Hortic 766:205–214CrossRefGoogle Scholar
  36. Martínez MT, San-José MC, Arrillaga I, Cano V, Morcillo M, Cernadas MJ, Corredoira E (2019) Holm oak somatic embryogenesis: current status and future perspectives. Front Plant Sci 10:239CrossRefGoogle Scholar
  37. Martini AN, Papafotiou M (2013) Effects of plant growth regulators and environmental factors on in vitro propagation of X Malosorbus florentina. Propag Ornam Plants 13:112–122Google Scholar
  38. Martini AN, Papafotiou M, Vemmos SN (2013) Season and explant origin affect phenolic content, browning of explants, and micropropagation of X Malosorbus florentina (Zucc.) Browicz. HortScience 48:102–107CrossRefGoogle Scholar
  39. Minas GJ (2001) Cretan dittany apical meristem micro-propagation in-vitro. Miscellaneous Rep 80, ISSN 0253-6749 Accessed 13 Aug 2019
  40. Molassiotis AN, Dimassi K, Therios I, Diamantidis G (2003) Fe-EDDHA promotes rooting of rootstock GF-677 (Prunus amygdalus X P. persica) explants in vitro. Biol Plant 47:141–144CrossRefGoogle Scholar
  41. Monteiro ACBA, Higashi EN, Goncalves AN, Rodriguez PM (2000) A novel approach for the definition of the inorganic medium components for micropropagation of yellow passionfruit (Passiflora edulis Sims. f. flavicarpa Deg.). In Vitro Cell Dev Biol-Plant 36:527–531CrossRefGoogle Scholar
  42. Mulas M, Mulas G (2005) Cultivar selection from rosemary (Rosmarinus officinalis L.) spontaneous populations in the Mediterranean area. Acta Hortic 676:127–133CrossRefGoogle Scholar
  43. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  44. Murch SJ, Peiris SE, Liu C-Z, Saxena PK (2004) In vitro conservation and propagation of medicinal plants. Biodivers 5:19–24CrossRefGoogle Scholar
  45. Ostrolucká MG, Gajdošová A, Libiaková G (2007) Protocol for micropropagation of Quercus spp. In: Jain SM, Häggman H (eds) Protocols for micropropagation of woody trees and fruits. The Netherlands, Springer, Dordrecht, pp 85–91CrossRefGoogle Scholar
  46. Öz GC, Yüzbaşioğlu E, Erol O, Üzen E (2008) In vitro propagation of Amsonia orientalis Decne (Apocynaceae). Afr J Biotechnol 7:3635–3638Google Scholar
  47. Ozel C, Mahmood K, Arslan O, Unal F (2009) In vitro propagation of the golden grape hyacinth (Muscari macrocarpum Sweet) from twin scale explants. Propag Ornam Plant 9:169–175Google Scholar
  48. Özhatay N (1997) The wild medicinal plant trade in Turkey: information on medicinal and aromatic vascular plants collected from natural habitats for internal and international trade, with conservation recommendations. Doğal Hayatı Koruma Derneği, Istanbul, TurkeyGoogle Scholar
  49. Panagouleas C, Skaltsa H, Lazari D, Skaltsounis AL, Sokovic M (2003) Antifungal activity of secondary metabolites of Centaurea raphanina ssp. mixta, growing wild in Greece. Pharm Biol 41:266–270CrossRefGoogle Scholar
  50. Papafotiou M, Kalantzis A (2009a) Seed germination and in vitro propagation of Sideritis athoa. Acta Hortic 813:471–476CrossRefGoogle Scholar
  51. Papafotiou M, Kalantzis A (2009b) Studies on in vitro propagation of Lithodora zahnii. Acta Hortic 813:465–470CrossRefGoogle Scholar
  52. Papafotiou M, Martini AN, Vlachou G (2017) In vitro propagation as a tool to enhance the use of native ornamentals in archaeological sites of Greece. Acta Hortic 1155:301–308CrossRefGoogle Scholar
  53. Phitos D, Constantinidis T, Kamari G (2009) The red data book of rare and threatened plants of Greece, vol 1 (A–D) and vol. 2 (E–Z). Hellenic Botanical Society, Patras, Greece (in Greek)Google Scholar
  54. Phitos D, Strid A, Snogerup S, Greuter W (1995) The red data book of rare and threatened plants of Greece. WWF, Athens, Greece, pp 122–123Google Scholar
  55. Ponert J, Vosolsobě S, Kmecová K, Lipavská H (2011) European orchid cultivation – from seed to mature plant. Eur J Environ Sci 1:95–107Google Scholar
  56. Samaras G, Tantsi A, Papanastasi K, Grigoriadou K, Maloupa E (2012) In vitro propagation of the native aromatic medicinal plants Sideritis scardica Griseb. and Sideritis sipylea Boiss. Proc of the 25th conference of the Hellenic Society for Horticultural Science, 1-4 November 2011, Limassol, Cyprus, vol 2:246–248 (In Greek)Google Scholar
  57. Sarropoulou V, Maloupa E (2016a) In vitro shoot proliferation of the endemic Sideritis scardica Griseb. plant species (Tea of Olympus mountain). Proc of the 16th Congress of the Hellenic Scientific Soc of Plant Breeding, 28-30 September, Florina, Greece Accessed 18 Aug 2019
  58. Sarropoulou V, Maloupa E (2016b) Αsexual propagation with tissue culture of Sideritis syriaca L. subsp. syriaca species (Crete mountain tea). Proc of the 16th Congress of the Hellenic Scientific Soc of Plant Breeding, 28–30 September, Florina, Greece ; Accessed 13 Aug 2019
  59. Sarropoulou V, Maloupa E (2017a) In vitro propagation of the endemic Sideritis syriaca L. subsp. syriaca (Tea of Crete-Malotira-Kalokoimithia). Proc of 15th Scientific Congress of the Hellenic Botanical Society, 14-17 September, Chania, Crete, Greece Accessed 13 Aug 2019
  60. Sarropoulou V, Maloupa E (2017b) Effect of the NO donor “sodium nitroprusside” (SNP), the ethylene inhibitor “cobalt chloride” (CoCl2) and the antioxidant vitamin E “α-tocopherol” on in vitro shoot proliferation of Sideritis raeseri Boiss. & Heldr. subsp. raeseri. Plant Cell Tissue Organ Cult 128:619–629CrossRefGoogle Scholar
  61. Šatović Z (2004) Legal protection, conservation and cultivation of medicinal and aromatic plants in Croatia. In: Bernáth J, Maggioni L, Lipman E (eds) Report on a working group on medicinal and aromatic plants, second meeting. International Plant Genetic Resources Institute, Rome, ItalyGoogle Scholar
  62. Schippmann U, Leaman D, Cunningham AB (2006) Cultivation and wild collection of medicinal and aromatic plants under sustainability aspects. In: Bogers RJ, Craker LE, Lange D (eds) Medicinal and aromatic plants. Springer, Dordrecht, The Netherlands, Wageningen UR Frontis Series no, p 17 Accessed 13 Aug 2019
  63. Steele WK (1996) Large-scale seedling production of North American native terrestrial orchids. In: Allen C (ed) North American native terrestrial orchids. Propagation and production, North American Native Terrestrial Orchid Conf Proc, Germantown, Maryland, pp 11–26Google Scholar
  64. Tsiftsis S, Tsiripidis I (2016) Threat categories of the Greek orchids (Orchidaceae). Bot Chron 21:43–74Google Scholar
  65. Valletta A, Attorre F, Bruno F, Pasqua G (2008) In vitro asymbiotic germination of Orchis mascula L. Plant Biosyst 142:653–655CrossRefGoogle Scholar
  66. Zuzarte MR, Dinis AM, Cavaleiro C, Salgueiro LR, Canhoto JM (2010) Trichomes essential oils and in vitro propagation of Lavandula pedunculata (Lamiaceae). Ind Crop Prod 32:580–558CrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  1. 1.Laboratory for Conservation and Evaluation of Native and Floricultural Species-Balkan Botanic Garden of KroussiaInstitute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization – DEMETERThessalonikiGreece

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