الفهرس 
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Abstract
This study was carried out in the Agricultural Botany Department, Faculty of Agriculture, Ain Shams University Shoubra El-Kheima, Egypt and the Tissue Culture Lab of the Central Laboratory for Date Palm Research and Development, Agricultural Research Center (ARC), Egypt during 2019 – 2023. To determine the impacts of certain components that might stimulate the development of high-quality date palm somatic embryos during the indirect somatic embryogenesis. Moreover, instead of using the shoot tip explants, young leaves (0.5–1 cm long) of aseptic formed somatic embryos were used for direct organogenesis.
The study included two categories divided as follows
1. Callus enhancement
The standard procedure for date palm callogenesis involves using healthy offshoots Barhee cv. as explant material. The current investigation aims to achieve high-quality date palm somatic embryos by adding nitrogenous compounds (proline and spermine 100-200-300 mg/l), antioxidants (glutathione GSH and ascorbic acid ASC 50- 100- 150 mg/l) and microelements (copper sulphate CuSO4 at 0.08- 0.32- 0.64 mg/l and cobalt chloride CoCl2 at 0.065- 0.26- 0.52 mg/l and combination of CoCl2 + CuSO4 (0.26 + 0.32 mg/l) to the maturation medium. The medium was supplemented with ABA at 0.5 mg/1, polyethylene glycol (PEG-4000), glutamine, and phytagel, allowing the friable embryogenic callus to develop into normal somatic embryos with high quality. All data were collected from formed SE after 8 weeks.
The obtained results are summarized in the following:
1.1. Nitrogenous compounds
1.1.1. Morphological Parameters
1. Nitrogenous compounds enhanced all recorded morphological parameters, including relative growth/jar percentage, differentiation percentage, and number of embryos/jars compared to the control.
2. The maximum increase in these parameters was observed with spermine at 300 mg/l, followed by 200 and 100 mg/l, respectively.
3. The addition of proline showed less effect than spermine, but it was found to be beneficial as it significantly enhanced all recorded morphological parameters compared to the control.
1.1.2. Biochemical analyses
1. All nitrogenous compound treatments increased the determined biochemical; total sugars (TS), total amino acids (TAA), total phenols (TP), and indole acetic acid (IAA).
2. The maximum increase in TS was recorded with spermine 300, 200, and 100 mg/l, respectively.
3. Amino acids were highly increased with all spermine concentrations, with ascending increases observed in media supplemented with 100, 200, and 300 mg/l, respectively.
4. Spermine addition significantly increased IAA. The highest increment was recorded with spermine at 300 mg/l followed by 200 and 100 mg/l with insignificant difference.
1.1.3. Enzyme Activity
1. Ascorpate peroxidase was highly represented in spermine treatment at 300 mg/l with significant difference compared with other treatments. An insignificant increase was recorded between all other treatments and control.
2. The highest increase in PPO activity was detected when media supplemented with nitrogenous compounds either with spermine or proline, with the highest increase recorded with the highest concentration of spermine (300 mg/l).
3. The highest concentration of spermine was the most efficient to increase PAL activity with significant difference in compare with all other treatments and control.
1.2. Antioxidant compounds
1.2.1. Morphological parameters
1. The relative growth percentage (RG%) of somatic embryos significantly increased when ASC was added to the culture media at 150 mg/l, followed by GSH at 150 mg/l, ASC at loo mg/l, and ASC at 50 mg/l.
2. Adding 150 mg/l of ASC and GSH to the media increased the differentiation percentage of somatic embryos by 78 and 66%, respectively.
3. When ASC and GSH were introduced to the medium separately, the quantity of somatic embryos per Jar was considerably altered, resulting in 17 and 16 embryos per Jar. There were no discernible differences between GSH at 50 mg/l and ASC at 100 mg/l.
1.2.2. Biochemical analyses
1. The identified biochemicals in the generated somatic embryos increased in response to all treated antioxidant substances.
2. The highest level of total sugars was detected significantly at both high concentrations of GSH and ASC at 2.98 mg/l and 2.89 mg/l, respectively.
3. As for total amino acids, significant increase was recorded with GSH at 150 mg/l, whereas insignificant increase was noticed with the same concentration of ASC.
4. The highest concentration of total phenols was noticed with 100 and 150 mg/l in both treatments.
5. When ASC at 100 mg/l was added to the media, the IAA concentration reduced significantly compared with the control; otherwise, increasing the concentration to 150 mg/l enhanced the insignificant increase in IAA concentration compared with the control.
1.2.3. Enzyme Activity
1. The highest activity level of APX was detected with glutathione at 150 mg/l (7.81 units mg -1 protein) compared with ascorbic acid treatment at 150 mg/l (7.78 units mg -1 protein) with insignificant difference between them.
2. Ameliorated increases in PPO activity were recorded with all antioxidant treatments compared to control. The highest alleviation in PPO activity was recorded with the highest concentration of ASC (1480 unit).
3. Insignificant alleviation in phenylalanine ammonia lyase (PAL) activity was recorded between both GSH (50, 100 mg/l) and ASC (100, 150 mg/l).
1.3. Microelements
1.3.1. Morphological Parameters
1. The results show that date palm somatic embryos produced in media supplemented with either CuSO4 or CoCl2 showed high growth efficiency in all determined morphological parameters compared to the control medium.
2. The highest increment in relative growth was recorded when CoCl₂ with different concentrations added individually in the medium (500, 517, and 600%).
3. The highest number of SE was noticed in the medium supplemented with a combination between CuSO4 and CoCl2.
1.3.2. Biochemical analyses
1. The data indicate that adding CoCl2 as an individual or combined with CuSO4 increased the concentration of total sugars (TS) in the produced SE. The highest significant increment in TS was detected with individual CoCl₂ at 0.52 mg/l (0.68 mg/g F.Wt.), with the same value in the combination treatment of CoCl₂ and CuSO4.
2. The maximum significant increase of free amino acids was obtained with CoCl2 at 0.26 mg/l.
3. Cobalt chloride at 0.52 mg/l significantly enhanced the total soluble phenol in the regenerated SEs.
4. The combination treatment of CuSO4 + CoCl₂ enhanced the highest significant increase in IAA concentration over all other treatments.
1.3.3. Enzyme Activity
1. When CuSO4 and CoCl₂ combine, the highest significant increase in the activity of APX was achieved, followed by CoCl2 treatments at 0.52, 0.26, and 0.065 mg/l.
2. PPO activity increased especially when CoCl2 was added individually at 0.52 mg/l.
3. The addition of CoCl2, either as an individual or combined with CuSO4, significantly achieved the highest antioxidant activity in PAL compared to all other treatments.
1.3.4. Anatomical Studies
It was possible to generate both single embryos and groups of differentiated embryos in synchronized stages. Anatomical research done on developing embryos for all treatments. The date palm cultivar Barhee’s embryogenic callus, which develops from the shoot tip, has a nodule-like morphology. After 8 weeks of culturing on maturation medium, several developmental stages of somatic embryos emerge from the embryogenic callus. Take note of the clusters of differentiated embryos at synchronized stages and the clear increase in growth and differentiation rates in comparison to the control media. Subsequently, a longitudinal segment of an individual embryo that had reached full development was obtained; this section revealed a separate embryonic axis encircled by the root tip (rt), the cotyledon, and the procambium strand (ps).
2. Alternative explant
The study involved the regeneration of shoots from young leaves of date palm embryos sewi cv. in MS germination medium. Treatments like benzyl adenine (BA) as recommended 2 mg/l, thidiazuron (TDZ), and paclobutrazol (PBZ) were added to the MS culture medium at concentrations 1-2-3 mg/l.
2.1. Morphological parameters
1. The study found that both PBZ and BA treatments significantly enhanced morphological parameters in date palm Sewi cv.
2. The highest differentiation percentage was obtained with PBZ treatments at 0.5, 1 mg/l, followed by 3mg/l PBZ and 2mg/l BA, with significant differences.
3. The addition of TDZ showed less effect on differentiation, with 1 mg/l TDZ being the best in TDZ treatments.
2.2. Biochemical analyses
The data revealed that the highest value of total sugars (TS), Free amino acids (AA), total soluble phenols (TP), and indole acetic acid (IAA) was found in PBZ treatment at 1 mg/l, followed by 0.5 mg/l PBZ, 2 mg/l BA.
2.2.3. Anatomical studies
1. The in vitro leaf was greenish, 0.5-1cm long, and 1mm thick, with a whitish basal part.
2. After two weeks of culturing on treated MS medium the leaves slightly increased in size, swelled, and acquired brown color.
3. After four weeks, the morphogenesis process developed, and the divided cells differentiated into new xylem and phloem elements, forming a nest-like shape.
4. After six weeks, intensive mitotic divisions were observed in the meristemoids centers, establishing new masses embedded in the ground tissue.
5. Adventitious shoots were visible on the adaxial side of the leaf, and the main stem continued to elongate after two further weeks on medium.
CONCLUSION
It is evident that all treated substances boosted every biochemical, morphological, and enzyme activity that was measured comparing with control.
from callus enhancement treatment, microelements treatment recommended for its significant results with enhancing somatic embryos differentiation and development. Also for alternative explant experiment, PBZ is recommended with this explant along with BA for their impressive results for direct organogenesis in such short period.