<p>Micropropagation of sugarcane is important for obtaining elite plantlets with high genetic and phytosanitary quality. This study aimed to establish an alternative method for the in vitro multiplication and rooting of sugarcane using a temporary immersion bioreactor (TIB) based on polyethylene terephthalate (PET). Different culture systems were employed using PET containers in semisolid culture medium and TIB in horizontal and vertical positions. At six weeks in the multiplication phase, the number and length of shoots, number of leaves per shoot, chlorophyll and carotenoid content, stomatal density, and percentage of closed stomata were evaluated, while at four weeks in the rooting phase, the number and length of shoots, number of leaves per shoot, number and length of roots per explant, chlorophyll and carotenoid content, stomatal density, percentage of closed stomata and plantlet survival rate during acclimatization were evaluated. The results showed the effects of the different culture systems on all variables evaluated for both the multiplication and rooting stages. The TIB-horizontal was the best culture system in multiplication and rooting stages, with a higher number of shoots per explant (29.30 and 13.50 shoots, respectively) and a higher number of leaves per shoot (3.92 and 4.57 leaves, respectively). The highest number of roots per explant (6.89 and 7.51 roots, respectively) and the longest roots were observed in the TIB-horizontal and TIB-vertical systems (4.18 and 4.28&#xa0;cm in length, respectively), while the highest plantlet survival rates during acclimatization were obtained in the TIB-horizontal and vertical systems (99.5 and 99.0%, respectively). The TIB-horizontal position was the most efficient system for the multiplication and rooting of sugarcane. Culture systems in PET containers are an innovative and sustainable alternative for large-scale sugarcane micropropagation.</p>