An Enhanced Method for Studying Wheat Stomata Physiology

High sensitivity and rapid closure of wheat stomata to environmental stimuli make it difficult and inconvenient when investigating stomatal physiology and morphology using epidermal peels. This is due to inevitable mechanical stress to stomatal guard cells when separating epidermis from mesophyll cells, which induced a vast majority of stomatal closure in wheat. Stomata are more open and active in detached leaves than in epidermal peels. Based on these observations, we proposed a simple method, which promotes stomatal opening using detached leaves rather than epidermis for physiological observations. Stomatal response to stimuli was significantly increased when using intact leaf segment. The method was used to investigate stomatal behaviours of two wheat genotypes with contrasting salt tolerance to salinity stress. The effects of salt stress and exogenous abscisic acid (ABA) treatment on stomatal behaviours were also assessed. The salt-tolerant genotype, H-135, demonstrated a greater stomatal closure rate than the salt-sensitive genotype, H-093, in response to exogenous ABA under salt stress, highlighting the potential of stomatal responsiveness as an indicator for breeding salt-resistant crops. This method not only facilitates the effective initiation of stomatal opening but also ensures the continued responsiveness of stomata to subsequent treatments in wheat.