Stevia rebaudiana Bertoni produces sweet steviol glycosides; these are regarded as the most promising substitute for sucrose and have a wide range of applications with high economic value. To improve steviol glycosides yields, we established an efficient method for inducing polyploidy in S. rebaudiana. Treatment with 0.05% colchicine for 48 h or with 0.1% colchicine for 24 h efficiently induced polyploidy in germinating seeds and polyploidy could be accurately identified using flow cytometry. Most of the tetraploid S. rebaudiana plants exhibited apparent variations in cytological and morphological characteristics, and had higher contents of the two main steviol glycosides, stevioside and rebaudioside A, than the diploid controls. These tetraploid plants may be further selected for breeding purposes or micropropagated for commercial production. Stevia rebaudiana Bertoni is an herbaceous perennial plant of the Asteraceae family. It is native to the Amambay region of northeastern Paraguay and neighboring countries Brazil and Argentina (Madan et al. 2010;Yadav et al. 2011). Its leaf and stem tissues accumulate a mixture of more than 20 different glycosides derived from the tetracyclic diterpene steviol (Ohta et al. 2010). The main steviol glycosides are stevioside and rebaudioside A, which are the sweetest compounds that are 250-300 and 350-450 times sweeter than sucrose, respectively (Crammer and Ikan 1986). Because the steviol glycosides are naturally occurring compounds with high sweetness and little to no calorific value, they have been regarded as the most promising substitutes for sucrose and have a wide range of applications and high economic value (Soejarto et al. 1983;Yadav et al. 2011). Applications include dietary, diabetic, and dental consumer markets, and health-promoting uses as antitumor, antihypertensive, antihyperglycemic and antioxidant agents (Singh et al. 2015). S. rebaudiana has been introduced and is grown as a crop in a number of countries, including Japan, China, Korea, Indonesia, Tanzania, Mexico, the United States and Canada (Madan et al. 2010).To improve steviol glycosides yields in S. rebaudiana, polyploidy breeding may be a promising approach.Polyploidy is widely acknowledged as a major mechanism of adaptation and speciation in the evolution of higher plants (Ramsey and Schemske 1998). Polyploid plants can arise spontaneously in nature by several mechanisms, including meiotic or mitotic failures and fusion of unreduced (2n) gametes (Comai 2005). In general, polyploid plants are large, healthy and vigorous. Some chemicals induce polyploidy in plants and cell cultures; the best known of these chemicals is colchicine, which frequently causes chromosome doubling (Blakeslee and Avery 1937). Polyploidy has been used to develop new crop cultivars; including wheat, cotton, potato and sugarcane.The primary objectives of this study were to establish a protocol for induction of polyploidy in S. rebaudiana. Seeds of S. rebaudiana cultivar Zhongshan No. 2 were germinated by placing them on p...