SUBCULTURE repeats was essential prerequisite for mass micropropagation in stevia but it accompanied by vitrification of the cultured shoots. Increase the number of shoots/explant without vitrification was detected when nodal segments of in vitro obtained shoots were subcultured twice, three weeks each, on Murashige and Skoog (MS) medium with 0.25mg/L BAP. From the third subculture, shoot multiplication decreased and vitrification increased as the number of subcultures increased; where 40% of the formed shoots of 10th subculture were vitrified. Decrease of water potential by polyethylene glycol (PEG) increased the incidence of shoot vitrification but shortened the period of subculture from three to two weeks delayed its appearance up to sixth subcultures. Also, application of anti-ethylene agents [AgNO₃, CoCl₂ or salicylic acid (SA)] decreased vitrification problems up to six subcultures. The best results were obtained by 1.7mg/L AgNO₃ where verification was reduced without significant reduction in the number of shoots/explant and shoot growth. The equivalent concentration of AgNO₃ in nano-particles form (AgNPs) showed a drastic increase in shoot vitrification (20%). When shoots of the 1^st, 4^th, 7^th and 10th subcultures were subjected to genome amplification using RAPD or ISSR primers, the registered polymorphism under the influence of 1.7mg/L AgNO3 was lower than that of 1.7mg/L AgNPs; both of them were lower than that of shoots cultured on anti-ethylene free medium (control).