Enhancement of ex vitro acclimation of walnut plantlets through modification of stomatal characteristics in vitro

Propagation of plants in vitro provides a fast technology for producing a large number of plants in a limited time and space. However, low survival rate of micropropagated plantlets after transfer to natural ex vitro condition limits the application of tissue culture for many plant species. In the current study, saturated KCl solution was applied to reduce the relative humidity (RH) in the culture vessels during last 2 weeks of growth (low RH-2W)and last four weeks of growth (low RH-4W) Low RH-2W mainly decreased stomatal aperture and density, while almost all stomatal characteristics were influenced by low RH-4W treatment. Low RH-4W caused more closed stomata with lower densities in comparison with its control and also with stomata in low RH-2W treatments. The heterogeneity in stomatal area was decreased by increasing exposure time to reduced RH condition. Decreasing RH caused lower transpiration rate and higher RWC during ex vitro desiccation. This improvement was due to decreased stomatal aperture in the first phase of water loss and increased stomatal functionality in the second phase of water loss during ex vitro acclimation of in vitro-plantlets. In conclusion, decreasing RH in the culture vessels can be an efficient method for increasing ex vitro acclimation of in vitro plants without negative effects on growth.