IN VITRO PROPAGATION AND CRYOPRESERVATION OF STEVIA REBAUDIANA USING A VITRIFICATION METHOD

Abstract

The present study aims at developing a reliable protocol for in vitro propagation and cryostorage shoot tips of Stevia rebaudiana using a vitrification method. The highest multiplication rate (8.9) was achieved on Murashige and Skoog (MS) medium supplemented with 1.5 mg/L benzyl amino purine (BAP) and 0.2 mg/L Indole-3-butryic acid (IBA). For cyropreservation of shoot tips in liquid nitrogen, several pretreatment trials prior to cryopreservation were conducted to enhance shoot tips recovery after storage in liquid nitrogen. Culturing plantlets in vitro for four weeks in MS medium containing 30 g/L sucrose, excision of shoot tips; preculture of shoot tips on media supplemented with 0.4 M sorbitol for 2 d, followed by loading shoot tips with 80% PVS2 for 20 mint, then dehydrated with a highly concentrated vitrification solution (100% PVS2) for 60 min at 0°C prior plunge into liquid nitrogen produced the highest re-growth (up to 68.8%) post thawing at 45°C for 2 min. After six months post cryopreservation none of the growth parameters had been significantly affected by the cryopreservation process. The procedure developed in this study is easy to handle and produced high levels of shoot formation. Thus, the protocol is applicable for long-term storage of S. rebaudiana germplasm in liquid nitrogen.