Hyperhydricity is a physiological disorder that leads to seriously affects the quality and multiplication rate of tissue cultured crops. To elucidate what regulates the occurrence of hyperhydricity, we investigated the effects of media components, in particular cytokinin and gelling agents which among the causal factors in in vitro propagation and in hyperhydricity development. We found that high concentrations of cytokinin showed more hyperhydricity symptoms as compared to the cytokinin-free (control) on Arabidopsis thaliana seedlings. Gelrite as gelling agent raised more severe hyperhydric symptoms as demonstrated by an increase in accumulated water in the apoplast compared to Micro-agar-grown seedlings. The relative volume of apoplastic water in seedlings from cytokinin-containing media, both Micro-agar and gelrite-solidified, was also increased, along with the development of hyperhydric symptoms. Stomata closure was found in hyperhydric leaves which contributed to a higher water retention capacity and declining transpiration compared to their corresponding controls. Besides, histological observations in hyperhydric leaf cross sections showed a disorganized cellular organization and large intercellular spaces. In addition, hyperhydric seedlings exhibited defective cuticular formation compare to normal seedlings.

Keywords: Apoplast, Cytokinin, Gelling agents, Hyperhydricity, In vitro culture, Micropropagation