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• Elicitation is an option for overproduction of steviol glycosides in Stevia. • Salicylic acid significantly increased steviol glycosides with no changes in plant performance. • Chitosan showed significant biostimulant activity on Stevia increasing leaf number. • Gene expression-associated to steviol glycosides biosynthesis different response depending on the elicitor. • Controlled elicitation is an interesting approach to increase steviol glycosides in Stevia industrial cultivation. Stevia rebaudiana (Bertoni) is a perennial shrub native from Paraguay whose leaves accumulate steviol glycosides. Plants increase the production of phytochemicals as a response to stress factors to cope with possible damage. Elicitors are biological stress factors that can induce plant response to stress. Steviol glycosides are the phytochemicals responsible of the sweetener power of stevia (Stevia rebaudiana Bertoni cv. Morita II), which is up to 300-fold stronger than sucrose. Elicitors might affect Steviol glycosides content and gene expression-associated to their biosynthesis. The aim of this research was to evaluate the effect of controlled elicitation using salicylic acid, chitosan and hydrogen peroxide on plant performance, steviol glycosides contents and gene expression-associated to steviol glycosides biosynthesis (SrKA13H, SrUGT85C2, SrUGT74G1 and SrUGT76G1) in stevia cultivated under greenhouse conditions. Weekly applications of elicitors were foliarly sprayed during four weeks, morphological measurements were made and samples were lyophilized and processed to evaluate SGs content. Gene expression-associated was analyzed by Ultra-Performance Liquid Chromatography coupled to a Quadrupole-Time of Flight. Results showed that all the evaluated elicitors (especially salicylic acid 0.1 mM) enhanced steviol glycosides content, which correlated with inducement of gene expression-associated to the biosynthesis of these compounds and increase in leaf number in stevia. Thus, controlled elicitation of stevia cultivation might be used to improve the yield of steviol glycosides for industrial purposes. [ABSTRACT FROM AUTHOR]