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), H₂O_2, total soluble phenolic compounds and overaccumulation of naringenin. Alternations in cellular metabolism can affect and regulate the morphogenesis of somatic embryos.

Many embryogenic systems have been designed to generate somatic embryos (SEs) with the morphology, biochemistry, and vigor uniformity of zygotic embryos (ZEs). During the current investigation, several antioxidants were added to the maturation media of the developing somatic embryos of date palm. Explant material was a friable embryogenic callus that was placed in maturation media containing ABA at 0.5 mg L⁻¹, 5 g L⁻¹ polyethylene glycol, and 10 g L⁻¹ phytagel. Furthermore, α-tocopherol or reduced glutathione (GSH) were used separately at (25 and 50 mg L⁻¹). These treatments were compared to a widely used date palm combination of reduced ascorbic acid (ASC) and citric acid at 150 and 100 mg L⁻¹, respectively, and to the medium free from any antioxidants. The relative growth percentage of embryogenic callus (EC), globularization degree, differentiation%, and SEs number were significantly increased with GSH (50 mg L⁻¹). Additionally, the latter treatment significantly enhanced the conversion% of SEs and the number of secondary somatic embryos (SSEs). ASC and citric acid treatment increased leaf length, while α-tochopherol (50 mg L⁻¹) elevated the number of leaves plantlet⁻¹. GSH at 50 mg L⁻¹ catalyzed the activities of polyphenol oxidase (PPO) and peroxidase (POD) in EC and enhanced the accumulation of proteins in SEs.

The survival and growth of in vitro plantlets are still problematic for the date palm; thus, the application of nanotechnology may provide date palm plantlets that develop faster with enhanced growth and biochemical parameters. Moreover, the usage of Fe-modified activated carbon (Fe-AC) prepared from date palm pits instead of activated charcoal (AC) in tissue culture media during the current investigation can be considered an innovative approach. Furthermore, the effect of zinc oxide nanoparticles (ZnO-NPs) and bulk particles (ZnO-BPs) on the rooting and growth of date palm plantlets and on some physiological indices was also investigated. X-ray diffraction, dynamic light scattering, scanning electron microscopy, and transmission electron microscopy were used to characterize Fe-AC and ZnO-NPs. As detected from the characterization, the particle size was 720 nm for Fe-AC and 60 nm for ZnO-NPs. The obtained results indicated that AC significantly enhanced plantlet growth, plantlet height, number of leaves/plantlet, root length, number of roots/plantlet, and the concentrations of IAA, chlorophyll a and chlorophyll b compared with Fe-AC. On the other hand, sugars and phenols concentrations, and POD and PAL activities were highly increased with Fe-AC. There was no significant difference in terms of plant fresh weight (FW), leaf length, leaf width, protein, amino acids, and carotenoids. ZnO nanoparticles significantly improved plant height, leaf length, leaf width, root length, and the concentrations of IAA, chlorophylls a and b, and carotenoids; in contrast, the bulk form increased roots/plantlet, amino acids, and total sugars. Supplying the MS medium with combinations of ZnO-NPs or ZnO-BPs and AC or Fe-AC significantly increased all growth and physiological parameters. Plant height, leaf length, leaf width, root length, and the concentrations of IAA, protein, and chlorophylls a and b significantly increased when ZnO-NPs were combined with AC. Amino acids, total sugars, carotenoids, and phenols concentrations and the activities of POD and PAL increased when ZnO-BPs and Fe-AC were added together.

Somatic embryogenesis is a means by which plants can regenerate bipolar structures from somatic cells. Furthermore, a high frequency and synchronous embryogenic system is necessary to obtain the complete advantages of somatic embryogenesis. Additionally, examining the regenerability of embryogenic callus (EC) and its ability to develop further into advanced stages of somatic embryos (SEs) could therefore help in the mass production of date palm SEs. For that reason; the embryogenic callus (EC), non-embryogenic callus (NEC) and the other developing SEs phases [pro-embryos (PE) and fully developed SEs (FDSEs)] were undergone to determine the endogenous plant growth regulators (PGRs) as well as different amino acids levels. Indole-3-acetic acid (IAA), gibberellic acid (GA3), zeatin (Z) and 2-isopentenyladenine (2iP) concentrations reached their lowest levels in EC in comparing with the other developing SEs phases (PE and FDSEs). However, NEC had the greatest level of ABA. Furthermore, FDSEs had high levels of GA3 and total cytokinins (CKs). In contrast to FDSEs and NEC, which had high 2iP/zeatin ratios, EC and PE had elevated IAA/CKs ratio. IAA/ABA ratio increased significantly in PE. On either hand, EC had the highest significant quantities of aspartic, alanine, histidine and lysine than PE and FDSEs. Whereas, NEC had the highest significant levels of glycine, tyrosine, and phenylalanine, but no proline was detected in NEC or PE. Serine, glutamic, and arginine levels were high in FDSEs. In PE, the level of threonine increased considerably. These determined biochemical components are highly of importance since the discovery that the induction and expression of somatic embryogenesis are totally dependent on their endogenous levels from PGRs and free amino acids.