Vol.:(0123456789) 1 3 Trees https://doi.org/10.1007/s00468-020-01992-x ORIGINAL ARTICLE Stomatal features and desiccation responses of Persian walnut leaf as caused by in vitro stimuli aimed at stomatal closure Sasan Aliniaeifard 1  · Zeinab Maleki Asayesh 1  · John Driver 2  · Kourosh Vahdati 1 Received: 12 August 2019 / Accepted: 20 May 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract 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 fnding paves the way for discovering a solution for the uncontrollable water loss problem of in vitro-generated walnut plantlets. Abstract 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. Diferent concentrations of PEG (0, 3 and 5%) and ABA (0, 1 and 10 µM) were tested, and their efects were evaluated in two separate experiments. In the frst 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 (ψ 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 infuence 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 µM) 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 efect was observed on morpho-physiologic parameters. Our fndings are signifcant because they can ultimately be useful for preventing the uncontrollable water loss problem of in vitro-generated walnut plantlets. Keywords Abscisic acid (ABA) · Tissue culture · Polyethylene glycol (PEG) · Stomata · Acclimatization  Introduction The acclimation of micropropagated plantlets to ex vitro conditions involves exposing them to environmental con- ditions that are very diferent from those in their in vitro environment. Plantlets naturally fail to be established suc- cessfully after shifting from an in vitro environment to an ex vitro one because the evaporative demand is higher in the latter. Therefore, a period of acclimatization to the ex vitro environment is necessary for the successful establish- ment and survival of plantlets (Chandra et al. 2010). Aseptic culturing media and unusual environmental conditions such as light, temperature, nutrient levels as well as the gase- ous composition [e.g., CO 2 , ethylene and relative humid- ity (RH)] in the in vitro media and vessel head space can Communicated by S.A. Merkle. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00468-020-01992-x) contains supplementary material, which is available to authorized users. * Kourosh Vahdati kvahdati@ut.ac.ir 1 Department of Horticulture, College of Aburaihan, University of Tehran, 3391653755 Tehran, Iran 2 Driver Consulting, Inc, Waterford, CA, USA