An ornamental hybrid of Gentiana scabra Bunge was successfully propagated in a Temporary Immersion System (TIS). In order to increase biomass and shoot production with simultaneous improvement of the plant quality, culture parameters such as flooding frequency, volume of medium, sucrose concentration and ventilation were compared. The fresh weight and multiplication rate of shoots increased using lowered flooding frequency. The uptake of sucrose was more efficient when the shoots were flooded less often. The plant quality represented by chlorophyll pigment content and the ratio of dry to fresh mass also decreased with higher flooding frequencies. Higher flooding frequencies caused pronounced hyperhydricity of the shoots which led to problems during the hardening process. Regarding the culture medium volume, shoots cultured in 150 ml medium produced a lowered biomass and fewer shoots which revealed a higher dry biomass and chlorophyll content compared to those cultivated in 300 ml medium. The additional ventilation of cultures grown with 150 ml medium increased the number of high quality shoots. While an increase of sucrose in the medium led to a reduced multiplication rate there were more shoots of better quality which also had higher levels of dry biomass. The optimisation of different cultivation parameters in TIS was shown to be an effective way to increase the multiplication rate and quality of Gentiana shoots transplantable to the greenhouse.