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Few reports explain the mechanism of PEG action on stomatal behavior and anatomical structure and analyze the photosynthetic pigments of in vitro date palm plantlets for better tolerance to ex vitro exposure. The main challenge for in vitro micropropagation of date palm techniques remains restricted to high survival rates and vigorous growth after ex vitro transplantation. In vitro hardening is induced by Polyethylene glycol PEG (0.0, 10, 20, 30 g L−1) for 4 weeks. Leaf anatomy, stomatal behavior, water loss %, photosynthetic pigments, and reducing sugars were examined in date palm plantlets (Phoenix dactylifera L.) cv. (Sewi) after 4 weeks from in vitro PEG treatment and after 4 weeks from ex vitro transplanting to the greenhouse. Leaf anatomy and the surface ultrastructure of in vitro untreated leaves showed a thin cuticle layer, wide opened malfunctioning stomata, and abnormal leaf anatomy. Furthermore, addition of PEG resulted in increasing cuticle thickness, epicuticular wax depositions, and plastids density, improving the stomatal ability to close and decreasing the stomatal aperture length while reducing the substomatal chambers and intercellular spaces in the mesophyll. As a result, a significant reduction in water loss % was observed in both in vitro and ex vitro PEG treated leaves as compared to untreated ones, which exhibited rapid wilting when exposed to low humidity for 4 h. PEG application significantly increased Chlorophylls a, b and carotenoids concentrations, especially 10, 20 g L−1 treatments, which were sequentially reflected in increasing the reducing sugar concentration. However, leaves of plantlets treated with PEG at 30 g L−1 became yellow and had necrosis ends with death. In vitro hardening by 20 g L−1 PEG increased the survival rate of plantlets to 90% after ex vitro transfer compared to 63% recorded for the untreated plantlets. Therefore, this application provides normal date palm plantlets developed faster and enhances survival after ex vitro transfer.

The study of morpho-anatomical aspects, metabolic changes of proteins, antioxidant substances, as well as phenolic compounds in embryogenic callus (EC) and degenerative embryogenic callus (DEC) was the aim of the present investigation. Ability to form somatic embryos (SEs) was associated with the softness of the EC, which exhibited a white or creamy color and was composed of isodiametric cells containing dense cytoplasm, conspicuous nuclei and minimal vacuoles with observed mitotic activity. Furthermore, protein, reduced glutathione (GSH) and ascorbic acid (ASC) concentrations and the ratio between ASC and dehydroascorborbic acid (DHA) were increased significantly in the EC in comparison to the DEC. In addition, the phenolic extract of the EC was proved to have higher scavenging activity than the extract from the DEC. A loss of embryogenic competence in the DEC was correlated with the presence of more rigid clumps and such calli had a yellowish to brown color and no cell division could be observed in the cells of such aggregates as the cells had large vacuoles and they have very thick walls. Moreover, these morphological and anatomical observations of the DEC were accompanied by accumulations of the oxidized form of ascorbic acid (DHA), H2O2, total soluble phenolic compounds and overaccumulation of naringenin. Alternations in cellular metabolism can affect and regulate the morphogenesis of somatic embryos.