Economic micropropagation of Stevia rebaudiana Bertoni and evaluation of in vitro cultures in order to improve steviol glycosides

Stevia rebaudiana Bertoni leaves produce a considerable amount of zero-calorie sweetener, which is 300 times sweeter than sucrose. S. rebaudiana is a self-incompatible plant that generates a few seeds with weak germina-tion. The present study was aimed to introduce an economic protocol for micropropagation of S. rebaudiana. By eliminating the rooting culture medium in this study, the production cost has decreased by approximately 34%. The sensory characteristics were determined by the ratio of rebaudioside A to stevioside as the main steviol glycosides. In this study S. rebaudiana tissue with a rebaudioside A to stevioside ratio of 3.76 was produced in a bioreactor by applying 0.02 mg/l NAA and 0.05 mg/l BAP. The flavor of this plant tissue was better than the shoots grown in field conditions. In cell suspension, the amount of stevioside was higher than that of rebau-dioside A, and no improvement in steviol glycosides was observed. In addition, the hairy root formation con-ditions were also optimized in this study, by using A4, ATCC15834, and LBA9402 strains of Agrobacterium rhizogenes for leaf and stem explants using two transformation methods including co-culture and injection. The highest transformation rate (60%) was observed using the ATCC15834 strain and injection method in leaf tissue. This transformation rate has not been observed in previous studies. In some hairy roots, stevioside and rebau-dioside A were detected, and in two samples of the hairy roots the amount of rebaudioside A was higher than stevioside. The use of genetic engineering and biotechnology could considerably increase the quality and quantity of steviol glycosides and improve the taste of S. rebaudiana.

Automated summary

This study aimed to develop an economic protocol for micropropagation of Stevia rebaudiana and optimize conditions to increase the quality and quantity of steviol glycosides.