Rhodophiala C. Presl (Amaryllidaceae) is a genus of attractive flowering geophytes native to South America. They have ornamental value, but most species are little known and have conservation problems. The objective of this study was to optimize a micropropagation process to support the use and preservation of three Chilean native species, R. montana (Phil.) Traub, R. splendens (Rengifo) Traub., and R. ananuca (Phil.) Traub. The research evaluated the feasibility of implementing liquid medium culture and assessed the influence of different tissue culture systems on the shoot production and biomass increment of small bulbs. Three experiments were carried out. The first one determined the influences of flask size and volume of media; the second compared liquid and solid media, and in the third experiment, a temporary immersion system (TIS), and conventional culture in static liquid, shaken liquid and gelled Murashige and Skoog (MS) media were compared. By using larger (350 mL) flasks with higher (50 mL) media volume, 100% more fresh weight of microbulb was obtained that treatment with smaller flasks (45 mL) and media volume (10 mL). In gelled medium, hyperhydricity affected only 5% of explants, while in liquid medium was 16-40%. Survival to acclimatization reached 87-94% for plants from gelled medium; from liquid medium only 38-69%. TIS yielded higher propagation rate (1.9 shoots in 30 d) compared with shaken liquid medium (1.0) (P < 0.05) in R. ananuca only. Current procedures are appropriate for the support of ex situ conservation and germplasm bank establishment.