Protocol optimization for efficient in vitro micro-propagation of Stevia rebaudiana Bertoni

Muhammad Aslam Javed, Imran Habib, Muhammad Waqas Jamil, Muhammad Jamsheid Anwer, Shahid Nazir, Sajid-ur- Rahman, Muhammad Zaffar Iqbal


Background: Stevia rebaudiana Bertoni is one of the most amazing herb that is an abundant source of diterpene glycoside a non-caloric sweetener. In recent years alongside its utilization in herbal and folk medicine, commercial exploitation of this herb for weight control and anti-diabetic treatment has started.

Methods: In present study healthy nodal segments of Stevia rebaudiana Bertoni were used as explants for optimization of in vitro micro-propagation and regeneration protocol for rapid, fast and efficient multiplication under in-vitro conditions. For shoot proliferation and rooting various concentrations cytokinins NAA (Naphthalene acetic acid), BAP (Benzyl amino purine), IAA (Indole acetic acid) and kinetin (Kn) were employed.

Results: Shoots without roots were regenerated from nodal segments via auxiliary shoot induction. The production of multiple shoots from nodal portion was maximum on MS medium complemented with 0.5 mg/L of BAP and 1.5 mg/L of Kn. Maximum rooting was recorded on MS medium supplemented with 0.25 mg/L NAA and 1.0 mg/L of IAA. Successful hardening was achieved under controlled growth room conditions followed by transfer to glass house and open field.

Conclusion: Standardization of explant source and culturing media for multiplication of shoot resulted in mass proliferation of homogenous and healthy plantlets of Stevia clones under local conditions.


Keywords: Stevia rebaudiana Bertoni; BAP; Kinetin, NAA; IAA; Micro-propagation; Regeneration

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Ahmed A, Huda MN, Mandal C, Alam KA, Reza MSH, Wadud A. In vitro morphogenic response of different explant of Stevia (Stevia rebaudiana). International Journal Agricultural Research, (2007); 2: 1006-1013.

Andolfi L, Ceccarini L, Macchia M. Bio‐agronomic characteristics of Stevia rebaudiana.Informatore. Agrario, (2002); 58:c48-51.

Banerjee M. Sarkar P. In vitro callusing in Stevia rebaudiana Bertoni using cyanobacterial media-a novel approach to tissue culture. International Journal Integrative Biology, (2008); 3: 163-168.

Barathi, N. Stevia-the calorie free natural sweetener. Natural Product Radiance, (2003); 2: 120-122.

Bespalhok-Filho JC, Hattori K. Embryogenic callus formation and histological studies fromStevia rebaudiana (Bert.) Bertoni floret explants. Revista Brasileira de Fisiologia Vegetal (1997); 9: 185-188.

Bespalhok-Filho JC, Hashimoto JM, Vieira LGE. Induction of somatic embryogenesis from leaf explants of Stevia rebaudiana. Revista Brasileira de Fisiologia Vegetal (1993); 5:51-53.

Borie KB. Sweet Stevia: Nature’s own non‐caloric sweetener: One leaf. . . Or two. National Gardening Association (2000).

Carneiro JWP, Muniz AS, Guedes TA. Greenhouse bedding plant production of Stevia rebaudiana (Bert) bertoni. Canadian Journal of Plant Sciences, (1997); 77:473-474.

Das A, Gntait S, Mandal N. Micropropagation of an Elite Medicinal Plant:Stevia rebaudianaBertoni. International Journal of Agricultural Research, (2011); 6:40-48.

Chalapathi MV, Thimmegowda S, Rama K, Prama VR, Prasad TG. Natural non‐calorie sweetener stevia (Stevia rebaudiana Bertoni): A future crop of India. Crop Research, (1997); 14: 347-350.

Abu-Romman SM, Al-Hadid KA and Arabiyyat AR. Kinetin Is the Most Effective Cytokinin on Shoot Multiplication from Cucumber. Journal of Agricultural Science, (2005); 7(10): 159-165.

Abdul-Razak UN, Ong CB, Yu TS and Lau LK. In vitro Micropropagation of Stevia rebaudianaBertoni in Malaysia. Braz. Arch. Biol. Technol., (2014); 57 (1): 23-28.

Shekhawat MS, Kannan N, Manokari M, Ravindran CP. In vitro regeneration of shoots and ex vitro rooting of an important medicinal plant Passiflora foetida L. through nodal segment cultures. Journal of Genetic Engineering and Biotechnology, (2015); 13: 209-214.

Sevik H. and Kerim G. Effects of IAA, IBA, NAA, and GA3 on Rooting and Morphological Features of Melissa officinalis L. Stem Cuttings. The Scientific World Journal, (2013), Article ID 909507, 5 pages. 10.1155/2013/909507

Kanungo S, Pradhan C, Sahoo SL, and Sahu RK. Role of auxins in the in vitro rooting and micropropagation of Holarrhena antidysenterica Wall., a woody aromatic medicinal plant, through nodal explants from mature trees. Journal of Medicinal Plants Research, (2012); 6(31): 4660-4666.

Victorio CP, Lage CLS, and Sato A. Tissue culture techniques in the proliferation of shoots and roots of Calendula officinalis. Revista Ciência Agronômica, (2012); 43 (3): 539-545

Tolera B. Effects of Naphthalene Acetic Acid (NAA) and Indole -3- Butyric Acid (IBA) on In Vitro Rooting of Sugarcane (Saccharum officinarum L.) Micro-Shoots. Journal of Biotechnology and Biomaterials, (2016); 6: 215. doi:10.4172/2155-952X.1000215.


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