Nicotinan tabacum L. shoot cultures were used as a model to evaluate the effect of bottom cooling and double layer system on plant quality and metabolism. The percentage of hyperhydric shoots produced, decreased 35% when bottom cooling was applied, while non hyperhydric, high quality plants were obtained with a combination of bottom cooling and the double layer system. However, in the latter case, the multiplication rate was completely inhibited due to the presence of charcoal in the liquid medium. Sucrose metabolism (mainly invertase activity) was significantly enhanced by the sucrose supplement present in the liquid medium. Activities of superoxide dismutase and catalase were highest in hyperhydric cultures.