Clonal propagation and cryogenic storage of the medicinal plant Stevia rebaudiana

Abstract

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.