Μicropropagation and cultivation of Salvia sclarea for essential oil and sclareol production in northern Greece

  • K. Grigoriadou
  • F. A. Trikka
  • G. Tsoktouridis
  • N. KrigasEmail author
  • V. Sarropoulou
  • K. Papanastasi
  • E. Maloupa
  • A. M. Makris


Salvia sclarea L. is valuable for its essential oils and other perfumery products used in the food and pharmaceutical industries. The aim of this study was to develop a rapid micropropagation protocol and cultivate S. sclarea in northern Greece for essential oil and sclareol production. The initial plant material used was S. sclarea seeds (biotype SC1) collected from a local population in Bulgaria. Apical meristems from seedlings germinated in the greenhouse were disinfected and established in vitro. For shoot proliferation, six different modified basal culture media supplemented with plant growth regulators (benzyladenine, auxins, and gibberellic acid) at different concentrations and combinations were tested. After 30 d, the highest proliferation rate (1.78) was achieved with an MS medium supplemented with 1.5 μM benzyladenine (BA) and 0.15 μM IBA. The MS medium with 5 μM IBA was the most effective treatment for rooting (40% rooting percentage, one root/microshoot, and 1.3-cm root length after 12 d of culture). Within 2 mo, rooted shoots (93–95%) were successfully acclimatized and survived ex vitro, whereas the remaining shoots rooted and survived in vivo after treatment with 5 μM IBA or α-naphthaleneacetic acid (NAA) (51–53%). During the 2-yr pilot cultivation, the yield of inflorescences in dry weight within the first year was 7 g/plant (129.50 kg/ha) and 33.3 g/plant (616.05 kg/ha) in the second year. The essential oil was found to be rich in secondary metabolites (linalyl acetate, trans-caryophyllene and its oxide, a-copaene and germacrene D). The extraction yield of sclareol from the blossoms was 1.5–2 g/100 g of dry mass (10–15 kg/ha). This micropropagation protocol results in plant propagation in 330 d and field cultivation with the first harvest in 450 d.


Clary sage Pilot cultivation Aromatic plants Medicinal plants IBA NAA 



This work has been performed in the framework of the research program titled “A systems approach into the production of plant and algal diterpenes with high industrial and pharmaceutical value” (09SYN879), funded through the Operational Programmes “Competitiveness & Entrepreneurship” (NSRF 2007–2013), National Action COOPERATION, SUB-ACTION ΙΙ: Large Scale Cooperative Projects, financed by the Ministry of Development of the Hellenic Republic.


  1. Angelova VR, Ivanova RV, Todorov GM, Ivanov KI (2016) Potential of Salvia sclarea L. for phytoremediation of soils contaminated with heavy metals. Int J Agric Biosyst Engineer 10(12):780–790Google Scholar
  2. Arikat A, Jawad FM, Karam NS, Shibli RA (2004) Micropropagation and accumulation of essential oil in wild sage (Salvia fruticosa Mill.). Sci Hortic 100:193–202CrossRefGoogle Scholar
  3. Caissard JC, Olivier T, Delbecque C, Palle S, Garry PP, Audran A, Valot N, Moja S, Nicolé F, Magnard JL, Legrand S, Baudino S, Jullien F (2012) Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae). PLoS One 7:e48253PubMedPubMedCentralCrossRefGoogle Scholar
  4. Clebsch B, Barner CD (2003) The New Book of Salvias. Timber Press, Portland, pp 115–117Google Scholar
  5. Cuenca S, Amo-Μarco JB (2000) In vitro propagation of two Spanish endemic species of Salvia through bud proliferation. In Vitro Cell Dev Biol-Plant 36:225–229CrossRefGoogle Scholar
  6. Dimas K, Papadaki M, Tsimplouli C, Hatziantoniou S, Alevizopoulos K, Fráter G, Bajgrowicz JA, Kraft P (1998) Fragrance chemistry. Tetrahedron 54:7633–7703CrossRefGoogle Scholar
  7. Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut rootstock. Hort Sci 19:507–509Google Scholar
  8. Dweck AC (2000) The folklore and cosmetics use of various Salvia species. In: Kintzios SE (ed) Sage: the genus Salvia. Harwood Academic Publishers, Amsterdam, pp 1–25Google Scholar
  9. Džamić A, Soković M, Ristić M, Grujić-Jovanović S, Vukojević J, Marin PD (2008) Chemical composition and antifungal activity of Salvia sclarea (Lamiaceae) essential oil. Arch Biol Sci (Belgrade) 60:233–237CrossRefGoogle Scholar
  10. Echeverrigaray S, Carrer RP, Andrade LB (2010) Micropropagation of Salvia guaranitica Benth. Through axillary shoot proliferation. Braz Arch Biol Technol 53:883–888CrossRefGoogle Scholar
  11. George EF (1993) Plant Propagation by Tissue Culture. Part. 1. The technology. Exegetics ltd., EdingtonGoogle Scholar
  12. George EF, Hall MA, De Klerk GJ (2008) Stock plant physiological factors affecting growth and morphogenesis. In: George EF, Hall MA, De Klerk GJ (eds) Plant propagation by tissue culture. The Background, 3rd ed, vol. 1, Springer, Dordrecht, The Netherlands, pp 403–422Google Scholar
  13. Georgiev E, Stoyanova A (2006) A guide for the specialist in the aromatic industry. UFT Academic Publishing House, PlovdivGoogle Scholar
  14. Ghanbar T, Hosseini B, Jabbarzadeh Z, Farokhzad A, Sharafi A (2016) High-frequency in vitro direct shoots regeneration from axillary nodal and shoot tip explants of clary sage (Salvia sclarea L.). Bulg J Agric Sci 22:73–78Google Scholar
  15. Ghanbari T, Hosseini B, Jabbarzadeh Z (2012) Ιmproving Salvia sclarea L. seed germination under in vitro condition. Int J Agric Res Rev 2:1051–1058Google Scholar
  16. Gochev V, Hristova Y, Girova T, Stoyanova A (2017) Composition of aroma compounds in Salvia spp. from Bulgaria and their potential practical applications. In: Georgiev V, Pavlov A (eds) Salvia biotechnology. Springer, Cham, pp 133–149CrossRefGoogle Scholar
  17. Green CE, Phillips RL (1975) Plant regeneration from tissue cultures of maize. Crop Sci 15:417–421CrossRefGoogle Scholar
  18. Gülçin Ü, Uģuz MT, Oktay M, Beydemür Ş, Frevüoúlu ÜK (2004) Evaluation of the antioxidant and antimicrobial activities of clary sage (Salvia sclarea L.). Turk J Agric For 28:25–33Google Scholar
  19. Hayet E, Fatma B, Souhir I, Waheb FA, Abderaouf K, Mahjoub A, Maha M (2007) Antibacterial and cytotoxic activity of the acetone extract of the flowers of Salvia sclarea and some natural products. Pak J Pharm Sci 20:146–148PubMedPubMedCentralGoogle Scholar
  20. Hosoki T, Tahara Y (1993) In vitro propagation of Salvia leucantha Cav. HortScience 28:226CrossRefGoogle Scholar
  21. Jonoubi P, Majd A, Hosseini RH, Mehrjardi HA (2017) Evaluation of Mwcnts effects on shoot regeneration and leaf callus cultures of Salvia sclarea (clary sage). World J Environ Biosci 6:5–10Google Scholar
  22. Khawar KM, Unver T, Özcan S (2003) In vitro induction of crown galls by Agrobacterium tumefaciens super-virulent strain A281 (pTiBo 542) in Salvia sclarea and S. pretense. Biotechnol Biotechnol Equip 17:63–69CrossRefGoogle Scholar
  23. Kulkarni M, Chaudhari R, Chaudhari A (2007) Novel tension-active microbial compounds for biocontrol applications. In: Ciancio A, Mukerji KG (eds) General concepts in integrated pest and disease management. Springer, Dordrecht, pp 295–304CrossRefGoogle Scholar
  24. Kuźma L, Kalemba D, Różalski M, Różalska B, Więckowska-Szakiel M, Krajewska MU, Wysokińska H (2009) Chemical composition and biological activities of essential oil from Salvia sclarea plants regenerated in vitro. Molecules 14:1438–1447PubMedPubMedCentralCrossRefGoogle Scholar
  25. Leifert C, Cassells AC (2001) Microbial hazards in plant tissue and cell cultures. In Vitro Cell Dev Biol-Plant 37:133–138CrossRefGoogle Scholar
  26. Leifert C, Waites B, Keetley JW, Wright S, Nicholas JR, Waites WM (1994) Effect of medium acidification on filamentous fungi, yeasts and bacterial contaminants in Delphinium tissue cultures. Plant Cell Tiss Organ Cult 36:149–155CrossRefGoogle Scholar
  27. Liu W, Chilcott CE, Reich RC, Hellmann GM (2000) Regeneration of Salvia sclarea via organogenesis. In Vitro Cell Dev Biol-Plant 36:201–206CrossRefGoogle Scholar
  28. 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
  29. Meshcheriakova NI (1984) Use of isolated meristem culture for the purpose of clonal micropropagation of Salvia sclarea. Vsesoiuznyi Nauchno-issledovatel’skii Institut Efirnomaslichnyky Kul’tur 16:84–86Google Scholar
  30. Mihalik E, Lehoczki E, Bodor Z, Németh EZ (2005) Photosynthetic and morphological characters of leaves of the annual and biennial Salvia sclarea biotypes. Acta Biol Szeged 49:161–163Google Scholar
  31. Moreno MM, Lacasta C, Meco R, Moreno C (2011) Rain-fed crop energy balance of different farming systems and crop rotations in a semi-arid environment: results of a long-term trial. Soil Till Res 114:18–27CrossRefGoogle Scholar
  32. Moretti MDL, Peana AT, Satta MA (1997) A study of anti-inflammatory and peripheral analgesic actions of Salvia sclarea oil and its main constituents. Essential Oil Res J 9:199–204CrossRefGoogle Scholar
  33. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  34. Mišić D, Grubišić D, Konjević R (2006) Micropropagation of Salvia brachyodon through nodal explants. Biol Plant 50:473–476CrossRefGoogle Scholar
  35. Olszowska O, Furmanowa M (1990) Micropropagation of Salvia officinalis by shoot buds. Planta Med 56:637CrossRefGoogle Scholar
  36. Peana AT, Moretti MD, Juliano C (1999) Chemical composition and antimicrobial action of the essentials of Salvia desoleana and S. sclarea. Planta Med 65:752–754PubMedCrossRefPubMedCentralGoogle Scholar
  37. Pitarokili D, Couladis M, Petsikos-Panayotarou N, Tzakou O (2002) Composition and antifungal activity on soil-borne pathogens of the essential oil of Salvia sclarea from Greece. J Agric Food Chem 50:6688–6691PubMedCrossRefPubMedCentralGoogle Scholar
  38. Quoirin M, Lepoivre P, Boxus P (1977) Un premier bilan de 10 années de recherches sur les cultures de méristèmes et la multiplication in vitro de fruitiers ligneux. In: Rech CR (ed) 1976–1977 et Rapports de Synthèse Stat. des Cult. Fruit. et Maraîch., Gembloux, pp 93-117Google Scholar
  39. Rota C, Carramiñana JJ, Burillo J, Herrera A (2004) In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens. J Food Prot 6:1092–1308Google Scholar
  40. Shimomura K, Kitazawa T (1991) Tanshinone production in adventitious roots and regenerates of Salvia miltiorrhiza. J Nat Prod 54:1583–1587CrossRefGoogle Scholar
  41. Souleles C, Argyriadou N (1997) Constituents of the essential oil of Salvia sclarea growing wild in Greece. Int J Pharmacogn 35:218–220CrossRefGoogle Scholar
  42. Tibaldi G, Fontana E, Nicola S (2010) Cultivation practices do not change the Salvia sclarea L. essential oil but drying process does. J Food Agric Environ 8:790–794Google Scholar
  43. Ulubelen A, Topcu G, Eriş C, Sönmez U, Kartal M, Kuruku S, Bozok-Johansson C (1994) Terpenoids from Salvia sclarea. Phytochem 36:971–974CrossRefGoogle Scholar
  44. Verma RS (2010) Chemical investigation of decanted and hydrophilic fractions of Salvia sclarea essential oil. Asian J Trad Med 5:102–108Google Scholar
  45. Wagner S, Pfleger A, Mandl M, Böchzelt H (2012) Changes in the qualitative and quantitative composition of essential oils of clary sage and Roman chamomile during steam distillation in pilot plant scale. In: Zereshki S (ed) Distillation - advances from modeling to applications, InTech Europe, University Campus STeP Ri, Croatia, pp 142–158Google Scholar

Copyright information

© The Society for In Vitro Biology 2020

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
  2. 2.Vitro Hellas SAAlexandriaGreece
  3. 3.Institute of Applied Biosciences—Centre for Research and Technology Hellas (INAB-CERTH)ThessalonikiGreece

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