Stomatal features and desiccation responses of Persian walnut leaf as caused by in vitro stimuli aimed at stomatal closure

Key message Functional stomata can be generated by in vitro ABA applications. This mitigates the desiccation responses of Persian walnut leaves in their early stage of ex vitro exposure. This finding paves the way for discovering a solution for the uncontrollable water loss problem of in vitro-generated walnut plantlets. Most in vitro-generated plants are susceptible to wilting when they are transferred to an ex vitro environment, which is characterized by a higher evaporative nature. In this study, two stomatal closing stimuli [abscisic acid (ABA) and polyethylene glycol (PEG)] were added to in vitro culture medium of Persian walnut (Juglans regia L.) to evaluate their leaf ability to control water loss during exposure to an ex vitro environment. Different concentrations of PEG (0, 3 and 5%) and ABA (0, 1 and 10 mu M) were tested, and their effects were evaluated in two separate experiments. In the first experiment, the proline concentration decreased in the leaves grown on media containing PEG. In contrast, glycine betaine (GB) increased only in leaves grown on a culture medium with 5% PEG. Leaf osmotic potential (psi(s)) increased parallel to the increase in PEG concentration. Adding PEG to the culture medium caused a decrease in stomatal aperture, and, as a result, the water conservation capacity of the in vitro-grown leaves was increased during detached leaf ex vitro desiccation. However, PEG-induced osmotic stress caused a negative influence on the morpho-physiologic characteristics and chlorophyll content of in vitro-grown nodal segments. In the second experiment, ABA was tested in the culture medium with the eventual aim of making in vitro leaves lose less water when they were transferred to an ex vitro environment. ABA (especially 10 mu M) improved the leaf water conservation capacity during ex vitro desiccation of detached leaves. Small stomata having narrow apertures were increased in number by supplementing the cultures with ABA, although the length of shoots decreased with its use; no serious side effect was observed on morpho-physiologic parameters. Our findings are significant because they can ultimately be useful for preventing the uncontrollable water loss problem of in vitro-generated walnut plantlets.