Factors affecting organogenesis of Stevia rebaudiana and in vitro accumulation of steviol glycosides

Abstract

The aim of this research was to investigate the effect of explant type and cytokinin type and concentration on stevia direct organogenesis in vitro, and to determine the effect of cytokinin type on stevioside and rebaudioside A content in regenerated shoots. Stem and leaf segments of stevia (Stevia rebaudiana Bertoni), taken from 6-week-old in vitro grown plants, were inoculated on Murashige and Skoog (MS) medium supplemented with different (1.0-6.0 µM) concentrations of 6-benzylaminopurine (BAP), thidiazuron (TDZ) and kinetin (KIN) for shoot regeneration. The content of steviol glycosides (stevioside and rebaudioside A) in S. rebaudiana donor plants and regenerated shoots was evaluated using high-performance liquid chromatography (HPLC). Results revealed a significant effect (p < 0.01) of explant type, cytokinin type and cytokinin concentration on shoot regeneration frequency. The mean value of regeneration frequency from stem segments was significantly higher in comparison with leaf segments. Thidiazuron significantly promoted the mean regeneration frequency in comparison to 6-benzylaminopurine and kinetin. The highest mean shoot formation frequency was obtained on the medium supplemented with 3.0 µM of TZD. Incorporation of 4.0 µM BAP and 4.0 µM TZD into culture media resulted in significant increase in rebaudioside A and stevioside ratio, while a decrease in rebaudioside A and stevioside ratio was recorded when the medium was supplemented with 4.0 µM KIN. Rebaudioside A and stevioside ratio in the medium supplemented with 4.0 µM TZD was significantly higher in comparison with 4.0 µM BAP and 4.0 µM KIN.