Journal of Applied Biosciences, 2011; 49: 3383– 3387Tissue culture derived plants are often vulnerable to abiotic stresses but mechanisms underlying such responses are hardly known. This study was conducted to determine mechanisms underlying drought stress vulnerability of in vitro derived banana cv. ‘Uganda’. Methodology and results: Suckers of in vitro derived off-type, in vitro micropropagation (MP) derived normal plants and conventionally propagated (CP) plants with no tissue culture history in their ancestry were collected in the field at Sokoine University of Agriculture and planted in 20-litre containers for drought stress evaluation. The mechanisms underlying the drought stress vulnerability were determined when banana plants reached 1.5 m tall based on leaf global cytosine DNA methylation, stomatal density and leaf senescence. Global cytosine DNA methylation was determined from cigar leaves by a reversed phase HPLC analysis. Leaf stomatal density was determined as the number of stomata per unit area of both upper and lower leaf surfaces. Leaf senescence was estimated as a number of leaves with dying margins when soil moisture level decreased to minus 630 millibars. The off-type and MP derived plants had lower (P < 0.05) global cytosine DNA methylation of 11.3 and 17.5 % compared with 22.5 % of the CP derived plants. On the contrary, the off-type and MP derived plants had higher stomatal density of 78.2 and 78.8 stomata per mm2 on the lower leaf surface compared to 72.0 stomata per mm2 of the CP derived banana plants. The leaf senescence of the off-type and MP derived plants was significantly (P < 0.05) higher with 87.7 and 79.5 % compared to 66.7 % of the CP derived plants at soil moisture of minus 630 millibars. Conclusion and application: These findings provide evidence that tissue culture process increases the vulnerability to water stress of in vitro banana regenerants as a consequence of increased leaf stomatal density which is possibly under the control of cytosine DNA hypomethylation. The vulnerability of the in vitro derived banana cv. ‘Uganda’ limits the use of tissue culture derived planting materials among small-scale farmers with limited water resource and irrigation facilities but provides an opportunity for further studies to minimise water stress susceptibility of in vitro derived banana suckers.