Abstract Photoautotrophic micropropagation (PA) in a sugar-free medium can facilitate acclimatization and increase success rates when transplanting tissue cultured plants to open fields. To identify savings in production costs (time and energy) during acclimatization in wasabi plants, we used in vitro PA with four kinds of supporting material (agar, perlite, rockwool, and vermiculite) and compared their effects on subsequent ex vitro growth (in vermiculite). The study revealed that, in all aspects, the plantlets in agar and vermiculite exhibited the highest growth, whereas plantlets in rockwool showed the worst growth performance. Growth performance in vitro seemed to be correlated with the volumetric water content of the supporting material. Agar and vermiculite promoted root growth and development with increases in number of roots, root length, and root fresh weight, and the root systems in these treatments also exhibited the best growth during the ex vitro stage. In culture vessels with plantlets, dissolved oxygen concentration (DO) in agar abruptly decreased from day 0 to day 7 but then recovered by day 28. DO decreased during the culture period very slightly, slightly, and steadily, with perlite, vermiculite, and rockwool, respectively. All the wasabi plantlets from the in vitro treatments survived after transplanting to the ex vitro condition, and subsequent growth was strongly influenced by the in vitro growth performance. Growth parameters of plantlets in the agar and vermiculite treatments were highest, whereas those in rockwool were lowest.