Autotetraploid plants of Stevia rebaudiana Bertoni were induced by immersing buds with different concentrations of colchicines solution,then autotetraploid plants were confirmed based on morphological characters,stomatal analysis,chromosome determination and flow cytometry analysis.The results of this study showed that buds immersed in 0.20% colchicine solution for 12 h prior to culture induced as high as 32.14% autotetraploid plants.The stomatal size of autotetraploid plants was larger than that in diploid plants;Autotetraploid plants possessed larger,thicker and deeper green leaves,and distorted appearances in comparison with diploid plants;The chromosome numbers of diploid cells was 2n=2x=22,antotetraploid cells was 2n=4x=44;The relative DNA content of diploid was 100,antotetraploid was 200 by flow cytometry analysis to determine their ploidy.A total of 48 lines of autotetraploid plantlets were successfully induced by colchicine.This technique for in vitro induction of autotetraploid Stevia could provid technical basis for the induction of ploidy plants,and provid experimental material for the further research of new varieties of autotetraploid breeding programs.