Influence of Polyethylene Glycol on Leaf Anatomy, Stomatal Behavior, Water Loss, and Some Physiological Traits of Date Palm Plantlets Grown In Vitro and Ex Vitro

Abstract

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.

Keywords: Phoenix dactylifera; ex vitro transfer; in vitro hardening; leaf anatomy; polyethylene glycol (PEG); stomata; survival; water loss %.

Figure 1

Cross-section of in vitro leaves of a date palm: (A,C) control untreated leaves, (B,D) treated with 20 g L⁻¹ PEG. (A) The thin cuticle layer, larger mesophyll cells with fewer chloroplasts, greater substomatal chamber and intercellular spaces. Note the opened stomata and the same thickness of both ventral and dorsal walls of the guard cells, thin-walled of subsidiary cells. (B) The thick cuticle, compacted mesophyll cells with smaller substomatal chambers. Note the closed stomata, increasing in the thickness of ventral walls of the guard cell, and also the outer tangential walls of the two subsidiary cells appear lignified (the two stars). (C) Weak differentiation of the vascular bundle sheath compared to the well-developed one appears as lignified cells around phloem and xylem in (D). Abbreviations: Cut. cuticle; Ep. epidermis; GC. guard cells; ss.c. subsidiary cells; s. ep. sub epidermal layer; s.ch. substomatal chamber’ mes. mesophyll; b.sh. bundle sheath; ph. phloem; M.X. meta xylem; P.X. proto xylem.

Figure 2

SEM micrographs of the adaxial surface of in vitro date palm (cv. Sewi) grown in media supplemented with different levels of PEG. (A) shows that most stomata are wide opened, the leaf surface apparently smooth. (B) shows fewer opened with narrow apertures, the development of little epicuticular wax on the leaf surface. (C) shows increasing closed stomata, as well as the density of the epicuticular wax density compared to (A,D). Most stomata are closed covered with a high density of epicuticular wax crystalloids. (Magnification: 2000×, Scale Bar = 50 µm). (E–H) are the magnified micrographs from (A–D) respectively. (E) Guard cells are full, slightly raised, stomatal aperture is wide compared to narrow ones observed in (F,G). (H) shows a completely closed stomata with an increase in epicuticular wax density on the leaf surface. (I–L) are the adaxial surfaces of leaves after four weeks from ex vitro transplantation. (I) shows stomata still opened, slightly raised up from the leaf surface. (J,K) shows a decrease in stomatal apertures, guard cells are slightly depressed. (L.) showed a completely closed stomata with increase in epicuticular wax density. (Magnification: 7000×, Scale Bar = 10 µm).

Figure 2

SEM micrographs of the adaxial surface of in vitro date palm (cv. Sewi) grown in media supplemented with different levels of PEG. (A) shows that most stomata are wide opened, the leaf surface apparently smooth. (B) shows fewer opened with narrow apertures, the development of little epicuticular wax on the leaf surface. (C) shows increasing closed stomata, as well as the density of the epicuticular wax density compared to (A,D). Most stomata are closed covered with a high density of epicuticular wax crystalloids. (Magnification: 2000×, Scale Bar = 50 µm). (E–H) are the magnified micrographs from (A–D) respectively. (E) Guard cells are full, slightly raised, stomatal aperture is wide compared to narrow ones observed in (F,G). (H) shows a completely closed stomata with an increase in epicuticular wax density on the leaf surface. (I–L) are the adaxial surfaces of leaves after four weeks from ex vitro transplantation. (I) shows stomata still opened, slightly raised up from the leaf surface. (J,K) shows a decrease in stomatal apertures, guard cells are slightly depressed. (L.) showed a completely closed stomata with increase in epicuticular wax density. (Magnification: 7000×, Scale Bar = 10 µm).

Figure 3

Cross sections in in vitro date palm leaves (cv. Sewi) grown in media supplemented with different levels of PEG (A–D) and after 4 weeks from ex vitro transplantation (E–H). (A) Abnormal structures appeared as thin cuticle, large mesophyll cells and fewer chloroplasts, large intercellular spaces, and substomatal chambers. (B,C) Organized mesophyll with reduction in intercellular spaces and well developed cuticle layer. (D) Compacted mesophyll, thick cuticle. (E) Leaves still have thin cuticle, loose mesophyll, but the plastids appear in higher density than observed in (A,F). More organized mesophyll, closed stomata. (G) Well developed compacted mesophyll and slightly sunken stomata. (H) Thick cuticle, closed stomata. Abbreviations: u.ep. upper epidermis; s.ep. subepdermal layer; l.ep. lower epidermis; mes. Mesophyll; v.b. vascular bundle. Arrowheads point to stomata, stars point to substomatal chambers and intercellular spaces. Bar = 200 µm.

Figure 4

In vitro PEG hardening of date palm plantlets Sewi cv. (A) 4 weeks after in vitro treatments. Arrows point to browning regions in the external leaves treated with 30 g L⁻¹ PEG. (B) Plantlets transfer to ex vitro conditions for another 4 weeks. Note: The 20 g L⁻¹ PEG hardened plantlets appeared stronger and grew faster, and this enabled them to tolerate the low relative humidity in the greenhouse.