Clonal propagation and cryogenic storage of the medicinal plant Stevia rebaudiana

  • M. A. Shatnawi Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt
  • R. A. Shibli Faculty of Agriculture. University of Jordan. Amman
  • S. M. Abu-Romman Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt
  • M. S. Al-Mazra’awi Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt
  • Z. I. Al Ajlouni Jordan University of Science and Technology. Irbid
  • W. A. Shatanawi Hashemite University. Zarqa
  • W. H. Odeh Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt
Keywords: cryopreservation, in vitro conservation, micropropagation, osmoticum, root formation


Successful clonal propagation of Stevia rebaudiana was achieved using microshoots as a primary step for in vitro conservation. Maximum proliferation was obtained on Murashige and Skoog (MS) medium supplemented with 1.5 mg L–1 benzyl amino purine and 0.2 mg L–1 indole-3-butyric-acid (IBA). Auxin increased rooting percentage of shoots at concentration of 0.4 mg L–1 IBA, indole-3-acetic-acid or naphthalene acetic acid and no rooting occurred without plant growth regulator. A survival of 90% was achieved when rooted explants were acclimatized in vivo in 1 soil: 1 perlite: 1 peat. In vitro S. rebaudiana shoots were successfully stored for up to 32 weeks on MS medium supplemented with an appropriate concentration of sucrose, sorbitol or mannitol, at 24 ± 2°C. After 32 weeks, 93.6% of the shoots were able to survive. Moreover, 89.3% of them were able to regrow when stored under light conditions. Cryopreservation by vitrification was successfully achieved (65.6% regrowth) when shoot tips were precultured on a medium supplemented with 0.4 M sorbitol for 2 d, followed by loading shoot tips with 80% concentrated plant vitrification solution 2 (PVS2) for 20 min; then dehydrated with 100% PVS2 for 60 min at 0°C prior to storage in liquid nitrogen. This procedure is easy to handle and produced a high levels of shoot formation. This protocol could be useful for longterm storage of S. rebaudiana germplasm.


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Author Biographies

M. A. Shatnawi, Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt

Biotechnology Department

R. A. Shibli, Faculty of Agriculture. University of Jordan. Amman

Department of Horticulture and Agronomy

S. M. Abu-Romman, Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt

Biotechnology Department

M. S. Al-Mazra’awi, Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt

Biotechnology Department

W. H. Odeh, Faculty of Agricultural Technology. Al-Balqa Applied University. Al-Salt

Biotechnology Department


Ahmed M.B., Salahin M., Karim R., Razvy M.A., Hannan M.M., Sultana R., Hossain M., Islam R., 2007. An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni.) in Bangladesh. Amer-Euras J Sci Res 2, 121-125.

Anabzhagan M., Kalpana M., Rajendran R., Ntaarajan V., Dhanavewl D., 2010. In vitro production of Stevia rebaudiana Bertoni. Emirates J Food Agr 222, 216-222.

Armstrong G., Johnson K., 2003. Micropropagation of Ceratopetalum gummiferum, an important Australian cut flower crop. In vitro Cell Dev-Plant 37, 173-177.

Assy-Bah B., Engelmann F., 1992. Cryopresevation of mature embryos of coconut (Cocos nucifera L.) and subsequent regeneration of plantlets. Cryo-Letters 13, 117-126.

Bachiri Y., Gazeau C., Hansz J., Morisset C., Dereuddre J., 1995. Successful cryopreservation of suspension cells by encapsulation dehydration. Plant Cell Tiss Org 43, 241-248.

Bertrand-Desbrunais A., Noirot M., Charrier A., 1992. Slow growth of in vitro conservation of coffee (Coffea spp) 2: influence of reduced concentrations and low temperature. Plant Cell Tiss Org 31, 105-110.

Brown D.C.W., Leung D.W.M., Thrope T.A., 1979. Osmoticum requirements for shoot formation in tobacco callus. Plant Physiol 46, 36-41.

Chalapathi M.V., Thimmegowda S., 1997. Natural non-calorie sweetener stevia (Stevia rebaudiana Bertoni), a future crop of India. Crop Res 14, 347-350.

Debnath M., 2008. Clonal propagation and antimicrobial activity of an endemic medicinal plant Stevia rebaudiana. J Med Plant Res 2, 45-51.

Durkovic J., Misalova A., 2008. Micropropagation of temperate noble hardwood: an overview. In: Functional plant science and biotechnology (Durkovic J., Misalova A., eds). Global Science Books, UK. pp. 1-19.

Fang J.Y., Wetten A., Hadley P., 2004. Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage. Plant Sci 166, 669-675.

González-Benito M.E., Clavero-Ramírez I., López-Aranda J.M., 2004. Review. The use of cryopreservation for germplasm conservation of vegetatively propagated crops. Spanish J Agric Res 2, 341-351.

Ibrahim A.I., Nazer M.A., Mohammed B.R., El Zefazafi M.M., 2008. Plant growth regulators affecting in vitro cultivation of Stevia rebaudiana. Tech Sugar 10(3), 254-259.

Jha A.K., Parjash S., Jain N.A., Gupta S.C., 2002. Production of adventitious shoot and plantlets from the hypocotyls explant of Sebania rostrata (Bremek and Obrem). In Vitro Cell Dev-Pl 38, 430-434.

Kornilova O.V., Kalashnikova E.A, 1997. Clonal micropropagation of stevia (Stevia rebaudiana). Cercetari Agrton Moldova 30, 80-85.

Liu J., Li S.F.Y., 1995. Separation and determination of stevia sweeteners by capillary electrophoresis and high chromatography. Planta Medica 18, 1703-1719.

Mitra A., Pal A., 2007. In vitro regeneration of Stevia rebaudiana (Bert.) from nodal explants. J Plant Biochem Biotechnol 16, 59-62.

Murashige T., Skoog F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15, 473-497.

Nakamura S., Tamura Y., 1985. Variation in the main glycosides of stevia (Stevia rebaudiana Bertoni). Jpn J Tropic Agr 29, 109-116.

Orlikawoska T., 1992. Effect of in vitro storage at 4°C on survival and proliferation of two apple rootstocks. Plant Cell Tiss Org 31, 1-7.

Panis B., Lambardi M. 2005. Status of cryopreservation technologies in plants (crops and forest tree). In: The role of biotechnology, Villa Gualino, Turin, Italy. 5-7 March, pp. 43-54.

Sakai A., Kobayshi S., Oiyama I., 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb var brasiliensis Tanaka) by vitrification. Plant Cell Rep 9, 30-33.

Savita S.M., Shella K., Sunanda S., Shankar A.G., Ramakrishna P., 2004. Stevia rebaudiana – a functional component for food industry. J Human Ecol 15, 261-264.

Shatnawi M., Shibli R., Qrunfleh I., Bataeinhe K., Obeidat M., 2007. In vitro propagation and cryopreservation of Prunus avium using verification and encapsulation dehydration methods. J Food Agr Environ Sci 5, 204-208.

Shibli R.A., Shatnawi M.A., Subaih W., Ajlouni M.M., 2006. In vitro conservation and cryopreservation of plant genetic resources: a review. World J Agr Sci 2, 372-382.

Sivaram L., Mukundan U., 2003. In vitro culture studies on Stevia rebaudiana. In Vitro Cell Dev-Pl 39, 520-523.

Statsoft Inc., 1995. Statistica for Windows. Tulsa, Ok, USA.

Takagi H., Tien-Thinh N., Islam O.M., Senboku T., Sakai A., 1997. Cryopreservation of in vitro grown shoot tips of taro [Colocasia esculenta (L) Schott] by vitrification. 1. Investigation by basic conditions of the vitrification procedure. Plant Cell Rep 16, 594-599.

Wang Y.L., Fan M.J., Loaw S.L., 2005. Cryopreservation of in vitro-grown shoot tips of papaya (Carica papaya L.) by vitrification. Botanical Bulletin of Academia Sinica 46, 29-34.

Zarghami R., Pirseydi M., Harsak S., Sardrood B.P., 2008. Evaluation of genetic stability in cryopreserved Solanum tuberosum. Afr J Biotechnol 7, 2798-2802.

How to Cite
ShatnawiM. A., ShibliR. A., Abu-RommanS. M., Al-Mazra’awiM. S., Al AjlouniZ. I., ShatanawiW. A., & OdehW. H. (1). Clonal propagation and cryogenic storage of the medicinal plant Stevia rebaudiana. Spanish Journal of Agricultural Research, 9(1), 213-220.
Plant physiology