Morphological stages of date palm cell suspension culture and production of biomass, phenolics, and proteins under elicitors of activated charcoal and glutamine.

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Mansour A. Abohatem, Mohmmed Baaziz, Fatima Jaiti This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6574056/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study presents an efficient protocol for the large-scale production of somatic embryos from embryogenic cell suspension cultures of Phoenix dactylifera (date palm). The morphological development of somatic embryos was documented across distinct stages from globular, cotyledonary and somatic embryos forms, with synchronized growth over an eight-week period. The cells of suspension cultures divided actively and formed globular embryos within 2nd and 3rd week, the globular embryos started to conversion to cotyledonary embryos in the 3rd week and continued until the 4th week, while the somatic embryos formed within the 5th and 6th week, followed by a stationary phase in the week 7 and 8. Effects of activated charcoal and glutamine on production of biomass, phenolics and proteins in cell suspension culture were studied. The accumulation of biomass increased approximately eightyfold in the medium with charcoal and glutamine at the end of 8th week. The culture medium free of activated charcoal showed significant increased browning of tissue and spent liquid medium, and significant increased phenolics production and peroxidase activity in somatic embryos compared to the medium with charcoal. Overall, the findings contribute valuable insights into optimizing in vitro culture conditions for date palm, with implications for enhanced production of biomass and secondary metabolites such as phenolics and proteins. Biological sciences/Biochemistry Biological sciences/Biotechnology Phoenix dactylifera Cell suspension culture Phenolics production Activated charcoal Glutamine Peroxidase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Somatic embryogenesis of date palm is a critical technique for micropropagation of elite date palm cultivars 1 , 2 , 3 . Recently, considerable progress has been made in the development and optimization of this technique from embryogenic cell suspension cultures 4 , 5 . In vitro cell suspension culture offers the best method to get large-scale of somatic embryos in date palm 6 , 1 , 5 . Our protocol allows the production of a large number individual somatic embryos of uniform physiological and growth characteristics, with synchronized development 7 . In vitro cell suspension cultures show great potential to produce commercially useful bioactive compounds with great antioxidant activity 8 , 9 , 10 . The growth condition variation impacts plants primary and secondary metabolism and induces many changes in the internal and external features of the dates 11 , 12 . In vitro of cells plant synthesizes phenolic compounds, this is due to the tissue culture specificity as synthetic biological system in which the phenols function is to interfere in cell proliferation 13 . It is known that secondary metabolites like phenolic compounds are produced as a defense mechanism against stress conditions. In this regard, the culture medium is provided with stress-inducing compounds known as elicitors, such as activated charcoal (AC), which is widely used in tissue culture media, influences the environment of a plant grown in vitro by adsorption of inhibitory or toxic substances, and adsorption of plant growth regulators from the culture medium 14 . Activated charcoal can employ harmful and beneficial effects on the tissues in vitro because, according to García and Tabarez 15 , charcoal addition to the medium can interfere in the absorption of nutrients essential, such as nitrogen, and in the action of endogenous hormones, which are fundamental for plant tissue culture. However, due to the hormone’s adsorption exuded by plants and toxic metabolites, activated charcoal acts as a potential antioxidant 16 . Organic nitrogen sources seem to be a limiting factor in in vitro date palm culture and their addition as amino acids has a beneficial effect, the glutamine stimulated growth better of callus and formation of somatic embryos 17 . The medium with 0.1 mg l − 1 2,4-D and 6.7 ×10 − 4 M glutamine has improved the proliferation of date palm somatic embryos and obtained the highest protein accumulation 4 . Amino acids have been used for in vitro cultures as organic nitrogen source of several types as date palm, rice, and pineapple to induce regeneration and somatic embryogenesis 18 , 19 , 20 , 21 . Numerous studies have shown that glutamine alone or combined with casein hydrolysate has been most frequently used in the different stages of somatic embryogenesis 22 , 23 , 24 . Glutamine and asparagine at 3.42 mM have been proved to be the most favorable for the formation of somatic embryos and the induction of secondary somatic embryos of Moroccan cork oak 25 . The addition of l-glutamine, 0.1% casein acid hydrolysate, and abscisic acid (ABA) greatly increased the number of somatic embryos in Spruce ( Picea asperata ) 26 . The objective of this study is to describe the morphological growth stages of embryogenic cells in the cell suspension cultures and to compare the effect of activated charcoal and glutamine on the production of biomass, phenolics, proteins, and peroxidase activities of somatic embryos in suspension cultures. Methods Callus induction and multiplication Shoot tips of date palm cultivars, namely Bouskri (BSK), were disinfected using a 20% solution of sodium hypochlorite, containing for 20 min. They were cultured on a callogenesis induction medium as described by our previous research 5,7 , which contained MS salts 27 and Fossard vitamins 28 with 5 mg/l of 6-benzylaminopurine (BAP), 5 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose, and 150 mg/l activated charcoal. For embryogenesis induction, the friable callus formed after 6–8 months of culture was selected and transferred onto a multiplication medium containing 0.1 mg/L of BAP and 0.5 mg/L of 2,4-D. The cultures were incubated at 25±2 ◦C in the dark and subcultured to fresh medium every 5 weeks until the production of embryogenic callus. Establishment of cell suspension culture To establish the cell suspension, the method described by Fki et al. 6 , and Abohatem et al. 7 has been used in this study. For that, 0.5 g of embryogenic callus were cut with sterile scalpel into small pieces as possible and then transferred in 50 ml of liquid medium in 250 ml Erlenmeyer. The Erlenmeyer content was passed through sieves with a 500 µm mesh size and the filtrate is incubated on a rotary shaker (100 rpm) at 25 ± 2ºC under a 16/8-h (light/dark) photoperiod. The liquid medium MS/2 was supplemented with 0.3 mg L -1 BAP and 0.1 mg L -1 2,4-D. Two elicitors—activated charcoal (AC) and glutamine (G)—were tested in the culture medium. AC was evaluated at 150 mg L⁻¹ and in a control treatment without AC. Glutamine was tested at 100 mg L⁻¹ and in a control treatment without glutamine. Development and growth stages of embryogenic cell suspension culture The division and development of embryogenic cell was observed under a microscope during the 2 weeks of cell suspension culture . The characteristics morphological growth stages of somatic embryogenesis at the different developmental stages (globular, elongation and cotyledonary) in date palm suspension cultures were recorded. To measure the embryogenic cell growth, cells were collected after every week of subculture. Cell growth was followed with flasks harvested at one-week intervals from the first week of subculture until cell weight was stable (8th week). The fresh weight of the samples was measured and recorded by a digital scale. Maturation and germination of somatic embryos Somatic embryos maturation was carried out on MS liquid medium diluted a half in the absence of plant growth regulator for two weeks. Subsequently, somatic embryos are transferred to a germination medium consisting of MS solid medium supplemented with NAA (0.1 mg L -1 ). Extraction and analysis of phenolics Phenolic compounds were extracted and analyzed as described by Macheix et al. 29 . At the end of 6th week of suspension culture, 250 mg fresh tissues of somatic embryo were homogenized with 2 ml of 80% methanol at 4ºC and centrifuged three times for 3 min at 7000 × g. The supernatants were recovered each time, and 100 μL of the supernatant was added to Folin-Ciocalteu reagent (250 μL) and sodium carbonate (20%). The mixture was incubated for 30 min at 40º C and the blue color was determined at 760 nm. The intensity of browning in the spent liquid medium was visually estimated and noted ++, + and − for high, medium, and minimal intensity, respectively according to the rate of scaling by Pottino 30 . Extraction and analysis of proteins Total proteins were extracted according to the method described by Lecouteux 31 . At the end of 6th week of suspension culture, fresh somatic embryogenesis tissues (250 mg) were homogenized with 2mL Tris maleate buffer (0.1M, PH 6.5) and centrifuged for 6 min at 7000g. The supernatant was used as the extract of crude proteins. The total proteins were measured by spectrophotometer at 595 nm according to the method described by Bradford 32 . Peroxidase extraction, activity assays and electrophoresis At the end of 6th week of suspension culture, 250 mg fresh tissue of somatic embryos were homogenized in 1 mL of Tris maleate buffer pH 6.5 (0.1M). After centrifugation at 10 000 g for 10 min, the supernatant was used for determination of enzymatic activities. Peroxidase (POX) activity was tested by measuring the oxidation of guaiacol at 470 nm. Afterward, 10 μL of enzyme extract were added to 2ml of reaction mixture consisting of a solution of 25mM guaiacol and 0.1M Tris-maleate buffer (pH 6.5). Reactions were initiated with 10 μL of hydrogen peroxide - H 2 O 2 (10 %) and stopped after 3 min. To separate isoenzyme of peroxidase, polyacrylamide gel electrophoresis for soluble proteins was conducted according to Baaziz 33 using a vertical gel electrophoresis. The resolving gel was 11 % and the stacking gel 5 % (w/v). The electrode buffer was Tris (0.02 M) - glycine (0.129M) - SDS (0.1 %) pH 8.3. Each gel slot was loaded with 30 μg and 60 μg (POX) proteins samples. Electrophoresis was conducted at a constant voltage of 100 V. For POX staining, the gel was incubated for 15 min in 100 ml of 0.1M sodium acetate buffer (pH 5) containing 0.1 ml 10% H 2 O 2 and 0.1g of benzidine. Gels with the substrates were incubated for 30 min in dark until dark bands appeared. Statistical analysis The results were analyzed by variance analysis (ANOVA), followed by SNK test at P = 0.05 level to compare the means. The repetitions number are three replicates for the two independent experiments. Results and Discussion Development and growth stages of embryogenic cell suspension culture During the first 2 weeks of cell suspension culture, the microscopic observation was conducted to describe the embryogenic cell development (Fig.1 A, B). In the first week, single cells of suspension cultures divided slowly to form small clumps, those cells showed dense cytoplasm and large vacuolar cells (Fig. 1A) whereas in the second week, cells were dividing actively to form large clumps, those cells showed dense cytoplasm with small vacuoles (Fig. 1B). Similar results were obtained in oil palm by Kramut and Te-chato 34 who described two types of cell aggregates: one consisting of 5 to 10 cells that consisted of large vacuolar cells and another with more than 10 cells that showed dense cytoplasm. Roowi et al. 35 found that cell suspensions contained cellular aggregates composed of round cells with dense cytoplasm that were small in diameter at 10–20 µM. De Touchet et al. 36 reported that embryogenic cells were dividing actively and showed a round prominent nucleus, and a dense cytoplasm with small vacuole. The morphological observations were carried out to describe the embryogenic cell proliferation and the growth stages of embryogenic cell suspension cultures during period of somatic embryos formation. During the first week, embryogenic cells of suspension cultures (Fig. 2A) divided slowly and formed small spherical proembryos (Fig. 2B), whereas, within the second and third weeks, cells were dividing actively and formed large spherical embryos (globular stage) (Fig. 2C, D). The globular embryos started the process of elongating to conversion to cotyledonary embryos in the 3rd week and continued until to 4th week (Fig. 2D, E). Cotyledonary embryos (Fig. 2E) were formed in the 4th and 5th week, meanwhile the somatic embryos formed in the 5th and 6th week (Fig. 2F) and reached to full somatic embryos in the 7th and 8th week at stationary stage (Fig. 2G). To our knowledge, we are the first described of the time course of embryogenic cell stages in the date palm suspension cultures 7 . The morphological description growth stages of somatic embryos in date palm suspension cultures are going to be an efficient biotechnological importance in the secondary metabolites production from plant cell suspension culture, which helps in determining the appropriate growth stage of embryogenic cells for adding elicitors. Therefore, the obtained results of this study represent novel and efficient biotechnological advancements in the woody plant cell suspension culture. Growth kinetics of cell suspension cultures All date palm cell suspension cultures showed sigmoid growth curves during the period of embryogenic cell suspension development into the somatic embryos which lasted 6 weeks. After one week of the lag phase, the cells of cultures were dividing and grew vigorously entering their exponential phase. The growth rate of cells continued to increase exponentially until week 6, followed by a stationary phase in the week 7 and 8 where the growth of somatic embryos ceased. (Fig. 3, Table 1). Table 1 : Cell biomass accumulation per week in date palm suspension culture under elicitors of charcoal and glutamine. Elicitors Cell biomass per week (g) 1 2 3 4 5 6 7 8 With charcoal + glutamine 1.8±0.3 4.5±0.9 9.7±1.8 15.4±2.9 24.1±3.1 35±2.5 38.7±3.6 42.4±4.6 Without charcoal 1.5±0.2 3.4±0.8 7.3±1.9 11.9±2.7 18.9±1.8 28.7±3.8 33.3±2.9 36.9±5.3 Without glutamine 1.5±0.2 3.3±0.9 6.1±1.9 9.5±1.7 15.3±2.9 25.3±2.6 29.3±3.1 32.9±4.4 This kinetic behavior is in agreement with some previous date palm studies, which reported the maximum growth (fresh weight) was in the 6-week-old cell suspension culture 4,37,5 . While in contrast with other reports, which reported the highest growth biomass (fresh weight) was in the11-week-old cell suspension culture 38,9,39 . This difference is due to the type and concentration of auxin and cytokinin used in those studies. In the studies that took 6 weeks to reach the maximum level of embryo growth used low concentrations of auxin were used (0.1 mg L -1 2,4-D and 0.5 mg L -1 BAP), and the concentration of cytokinin was slightly higher than auxin. Studies that took 11 weeks to reach the maximum level of embryo growth used high concentrations of auxin (10 mg L -1 NAA and 1.5 mg L -1 2-iP), and the concentration of cytokinin was slightly lower. Cell biomass accumulation The evaluation of embryogenic cell biomass accumulation is the most important methods to measure growth. The accumulation of biomass (fresh weight of cells) in date palm cell suspension cultures showed a progressive increase during the period of cell suspension development into the somatic embryos. The maximum fresh weight of biomass accumulation was observed in the 7th and 8th week when somatic embryos reached the stationary growth stage. The highest fresh weight (FW) accumulation of somatic embryos biomass was 42.4±4.6 g in the medium with activated charcoal (AC) and glutamine (G), which increased approximately eightyfold than the first week, followed by 36.9 ± 5.3 g in the medium without activated charcoal (AC), while the least fresh weight was 32.9 ± 4.4 g in the medium without glutamine (G) (Table 1,2). Similar result was obtained by Zouine and El Hadrami 4 , which found that when glutamine concentration increased from 3.35× 10 -4 to 6.7 ×10 -4 M, the date palm somatic embryos production increased from 14 to 56 embryos. The glutamine stimulated better growth callus and formation of somatic embryos of date palm 17 . The addition of arginine at a concentration of 3 mM caused an early development of date palm somatic embryos to spherical entities with regular and smooth outlines 37 . Table 2: Effect of activated charcoal and glutamine on biomass, phenolics and browning of tissue and culture medium in date palm suspension cultures. Elicitor Cell biomass per 0.5 g callus (g) Intensity of browning Phenolic content mg / g FW With charcoal + glutamine 42.4±4.6a - 0.12 ± 0.025bc Without charcoal 36.9 ± 5.3b ++ 0.37 ± 0.035a Without glutamine 32.9 ± 4.4bc - 0.15 ± 0.03b Values are means ± standard error of three replicates. Mean values followed by the same letters (a–c) are not significantly different at p = 0.05 level according to SNK test. Production of phenolics in the tissue and the liquid culture medium of suspension cultures To limit phenolic production that causes an appreciable loss of cell culture viability, activated charcoal (AC) are commonly used in the date palm tissue and the culture medium to trap phenols and oxidized phenols. In this study, at the addition of 150 mg L -1 activated charcoal (AC) caused a significant decrease in phenolic content (browning) of tissue and spent liquid medium and improved the growth rate of somatic embryos (Fig. 4, Table 1). At the same time, in the liquid culture medium without AC, there was observed significant increase in the level of phenolic content (browning) in the tissue and the spent liquid medium (Fig.4, Table 2). Activated charcoal (AC) was found to be the best antibrowning factor particularly during the first months of tissue culture and suspension cultures. This result agrees with the known facts concerning the role of the activated charcoal in trapping phenols and oxidized polyphenols in palm tissue culture 40.41 . The biochemical analysis of phenolics content showed the medium without charcoal (AC) a significant increase in phenolic content in somatic embryos tissues from 0.12 mg/g FW to 0.37 mg/g FW. This represents an approximately threefold compared to the medium with AC (Fig. 4, Table 2). In previous studies, the primary objective of adding activated charcoal (AC) to the culture medium was to adsorb phenolic compounds and reduce the accumulation of oxidized phenols in plant tissue cultures 15,42,43 . Based on this, our hypothesis is that the absence of AC in the culture medium will lead to a significant increase in phenolic production, as well as enhanced exudation of these compounds into the medium. The positive and negative effects of activated charcoal mainly depend on its adsorptive properties 44 . Activated charcoal (AC) has a very fine pores network with large inner surface area on which many substances can be adsorbed 45 . The most crucial impact of adding AC to the culture media is a drastic dip in concentration of plant growth regulators and other organic supplements, which are used as elicitors to produce secondary metabolites such as phenolics and flavonoids 46 . This is due to the adsorption of these elicitor chemicals by AC. Date palm cell suspension cultures evidenced a considerable antioxidant activity under without AC treatment which was related to the high phenolic production. This is the first report on the important role of activated charcoal (AC) as elicitor on the production phenolics in the suspension cultures that will to contribute to establishing an efficient protocol for date palm cell suspension culture for large-scale production of phenolic compounds. Proteins content and peroxidases activities in suspension cultures Addition 100 mg/L glutamine to culture medium led to increased proteins content in somatic embryos from 75.6± 2.8 μg / g FW to 87.46 ± 3.6 μg / g FW compared for medium without G, and decreased peroxidase activity from 56.8 ± 14.9 U E/g FW to 35.9±9.6 U E/g FW (Table 2). In addition, activated charcoal (AC) decreased proteins content from 87.46 ± 3.6 μg /g FW to 77.43 ± 2.9 μg/g FW compared for medium without AC and increased peroxidase activity from 35.9±9.6 UE/ g FW to 67.2± 10.4 UE/g FW (Table 3). Zouine and El Hadrami 4 found that the use of 0.1 mg L -1 2, 4-D with 6.7 ×10 –4 M glutamine showed a significant increase in protein content in date palm somatic embryos of suspension cultures. In oil palm somatic embryos, the use of glutamine alone or combined with arginine was found to be a factor in enhancement accumulation of protein 22 . Table 3: Effect of activated charcoal and glutamine on proteins content and peroxidases activities of date palm somatic embryos Elicitor Total protein μg / g FW Peroxidase activity UE / g FW With charcoal + glutamine 87.46 ± 3.6a 35.9±9.6c Without charcoal 77.43 ± 2.9b 67.2± 10.4a Without glutamine 75.6± 2.8bc 56.8 ± 14.9b Values are means ± standard error of three replicates. Mean values followed by the same letters (a–c) are not significantly different at p = 0.05 level according to SNK test. Effect of activated charcoal and glutamine on peroxidase isoforms Extracts of peroxidase from somatic embryos of date palm, when separated by gel electrophoresis showed a major migration zone with Rf value interval 0.1- 0.16 (Fig. 4), where the stain intensity increased with the high extract volume to dauble volume (60 µL). Extracts without AC and G were characterized by a peroxidase isoform of relatively high migration speed (Rf = 0.16). This latter disappeared in extracts of peroxidase prepared from somatic embryos cultivated on medium with AC and G. This result confirms that AC and G modulate browning by their action on qualitative aspect of peroxidases. They induce the enzyme isoform formation, which migrates at Rf = 0.16 on 11% polyacrylamide gels, this isoform correlated with maturation of somatic embryo. (Fig. 5). Germination of somatic embryos The germination rate of somatic embryos increased significantly to 32% when they were first cultured in half-strength MS liquid medium without plant growth regulators and then transferred to solid MS medium. In contrast, only 8% germination was achieved when embryos were transferred directly to solid MS medium (Fig. 6). Conclusions Our study presents an efficient protocol for the large-scale production of somatic embryos in suspension culture. The detailed description of the morphological growth stages of somatic embryos in date palm suspension cultures provides valuable insights with significant biotechnological relevance, particularly for the production of secondary metabolites in woody plant cell suspension systems. Our results in this study confirmed that the culture medium free of activated charcoal led to significantly increased phenolics production and peroxidase activity of somatic embryos in suspension cultures and increased tissue browning and medium browning. This will be an efficient method for large-scale production of phenolics from cell date palm. Abbreviations AC Activated charcoal G Glutamine 2,4-D 2,4-Dichlorophenoxyacetic acid BAP 6-Benzylaminopurine NAA α-Naphthalene acetic acid MS Murashige and Skoog (1962) medium Declarations Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Author contributions M.A.A. wrote the manuscript, performed, and designed the experiments, and performed the biochemical data analysis. M.B. contributed to performed the biochemical data analysis, reviewing and editing the manuscript. Ethics approval and consent to participate All the plant experiments/protocols were performed with relevant institutional, national, and international guidelines and legislation. Consent for publication The authors declare no objection in consent to publication. Competing interests The authors declare that there are no conflicts of interest. Clinical trial number Not applicable. Funding Not applicable. References Othmani, A., Bayoudh, C., Drira, N., & Trifi, M. In vitro cloning of date palm Phoenix dactylifera L., cv. Deglet Bey by using embryogenic suspension and temporary immersion bioreactor (TIB). Biotechnology & Biotechnological Equipment., 23(2),1181-1188. https://doi.org/10.1080/13102818.2009.10817635 (2009). Aslam, J., Khan, S.A., Cheruth, A.J., Mujib, A., Sharma, M.P., & Srivastava, P.S. Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm ( Phoenix dactylifera L.) cultivars. 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Methods in plant tissue culture. Dept. of Hort., Agric., Maryland Univ., College Park. pp 8–29. (1981). Lecouteux, C.G., Lai, F.M., & McKersie, B.D. Maturation of alfalfa ( Medicago sativa L.) somatic embryos by abscisic acid, sucrose and chilling stress. Plant Science, 94(1-2), 207-213. https://doi.org/10.1016/0168-9452(93)90021-Q (1993). Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem . 72, 248–254 https://doi.org/10.1016/0003-2697(76)90527-3 (1976). Baaziz, M. the activity and preliminary characterization of peroxidase in leaves of cultivars of date palm, Phoenix dactylifera L. New Phytol , 111: 403-411 (1989). Roowi, S.H., Ho, C.L., Alwee, S.S.R.S., Abdullah, M.O., & Napis, S. Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm ( Elaeis guineensis Jacq.) in response to different concentration of auxins. Molecular Biotechnology , 46, 1-19. https://doi.org/10.1007/s12033-010-9262-9 (2010). Kramut, P., & Te-Chato, S. Effect of culture media, plant growth regulators and carbon sources on establishment of somatic embryo in suspension culture of oil palm. Journal of Agricultural Technology . Vol.6(1): 159-170 (2010). De Touchet, B., Duval, Y., & Pannetier, C. Plant regeneration from embryogenic suspension cultures of oil palm ( Elaeis guineensis Jacq.). Plant Cell Reports, 10, 529-532. https://doi.org/10.1007/BF00234588 (1991). Sghaier, B., Kriaa, W., Bahloul, M., Novo, J.V.J., & Drira, N. Effect of ABA, arginine and sucrose on protein content of date palm somatic embryos. Scientia Horticulturae , 120(3), 379-385. https://doi.org/10.1016/j.scienta.2008.11.035 (2009). Al-Khayri, J.M., & Naik, P.M. Elicitor-induced production of biomass and pharmaceutical phenolic compounds in cell suspension culture of date palm ( Phoenix dactylifera L.). Molecules , 25(20), 4669. doi:10.3390/molecules25204669 (2020). Al-Khayri, J.M. Determination of the date palm cell suspension growth curve, optimum plating efficiency, and influence of liquid medium on somatic embryogenesis. Emirates Journal of Food & Agriculture (EJFA ), 24(5). http://ejfa.info/index.php/ejfa/article/view/13502 (2012). Teixeira, J.B., Söndahl, M.R., & Kirby, E.G. Somatic embryogenesis from immature inflorescences of oil palm. Plant Cell Reports , 13, 247-250. https://doi.org/10.1007/BF00233313 (1994). El Hadrami, I., & Baaziz, M. Somatic embryogenesis and analysis of peroxidases in Phoenix dactylifera L. Biologia plantarum , 37, 197-203. https://doi.org/10.1007/BF02913210 (1995). de Oliveira Paiva, P.D., Paiva, R., & Pasqual, M. Controle de oxidação no cultivo in vitro de embriões de estrelícia ( Strelitzia reginae). Ornamental Horticulture , 13(2). https://doi.org/10.14295/rbho.v13i2.213 (2007). Azofeifa-Delgado, Á. Problemas de oxidación y oscurecimiento de explantes cultivados in vitro. Agronomía Mesoamericana , 20(1), 153–175. (2009). Kim, D.H., Kang, K.W., Enkhtaivan, G., Jan, U., & Sivanesan, I. Impact of activated charcoal, culture medium strength and thidiazuron on non-symbiotic in vitro seed germination of Pecteilis radiata (Thunb.) Raf. South African Journal of Botany , 124, 144-150. https://doi.org/10.1016/j.sajb.2019.04.015 (2019). Thomas, T.D. The role of activated charcoal in plant tissue culture. Biotechnology advances , 26(6), 618-631. https://doi.org/10.1016/j.biotechadv.2008.08.003 (2008). Bienaimé, C., Melin, A., Bensaddek, L., Attoumbré, J., Nava-Saucedo, E., & Baltora-Rosset, S. Effects of plant growth regulators on cell growth and alkaloids production by cell cultures of Lycopodiella inundata. Plant Cell, Tissue and Organ Culture (PCTOC), 123, 523-533. https://doi.org/10.1007/s11240-015-0856-6 ( 2015). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6574056","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471680127,"identity":"d9051cee-5861-4d64-99a2-c5e69afb4878","order_by":0,"name":"Mansour A. Abohatem","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYFACHgZmMM3eAGUQqcUASB8gWYtEApFadNvPHnxcUPNHTnfmG8PPBRU2DPzt3Ql4tZidyUs2nnHMwNjsdo6x9IwzaQwSZ85uwK/lQI6ZNA+bQeK22zkG0rxthxkMJHIJaDn/Bqjln0H9tptnjH8Tp+UG0BbeNoMEsxs8ZkTacuNdsjFvn7HhtjNpZdY8Z9J4CPvlfO7Bxzzf5OTNjh/efJunwkaOv70XvxYkwGEAInmIVQ4C7A9IUT0KRsEoGAUjCAAA8+VExXsHzXgAAAAASUVORK5CYII=","orcid":"","institution":"Amran University","correspondingAuthor":true,"prefix":"","firstName":"Mansour","middleName":"A.","lastName":"Abohatem","suffix":""},{"id":471680128,"identity":"75cc0736-981e-4db5-92ee-cd0fccaefc7e","order_by":1,"name":"Mohmmed Baaziz","email":"","orcid":"","institution":"Cadi Ayyad University","correspondingAuthor":false,"prefix":"","firstName":"Mohmmed","middleName":"","lastName":"Baaziz","suffix":""},{"id":471680129,"identity":"91a33c1f-cd15-4d8b-a7d7-afb92710119d","order_by":2,"name":"Fatima Jaiti","email":"","orcid":"","institution":"Université Moulay Ismail de Meknes","correspondingAuthor":false,"prefix":"","firstName":"Fatima","middleName":"","lastName":"Jaiti","suffix":""}],"badges":[],"createdAt":"2025-05-01 20:08:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6574056/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6574056/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84796882,"identity":"283805f2-ca74-4f15-a812-880cfac5606d","added_by":"auto","created_at":"2025-06-17 12:30:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":776793,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic view of date palm embryogenic cell suspension. (A) Small clumps of cells during the first week under ×100 enlargement with light microscope. (B) Large clumps of cells during the second week under ×100 enlargement with light microscope. Scale bar: 0.1mm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/9bd3db032165805a3dbd4e72.png"},{"id":84796883,"identity":"79222771-f4f3-4862-a070-a40af3d41709","added_by":"auto","created_at":"2025-06-17 12:30:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1630279,"visible":true,"origin":"","legend":"\u003cp\u003eThe morphological growth stages of date palm embryogenic cells in the cell suspension cultures. (A) Embryogenic callus. (B) Embryogenic cell suspension after 1st week of culture. (C) Embryogenic cell in globular stage after 2nd week of culture. (D) Conversion of globular stage to elongated embryos after 3rd week of culture. (E) Cotyledonary embryos after 4th week of culture. (F) Somatic embryo after 5th week of culture.\u003cstrong\u003e \u003c/strong\u003e(G) Full somatic embryos at stationary growth stage. Scale bar: 0.1mm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/d0796e92f38cfb6165dfd562.png"},{"id":84796880,"identity":"533c569a-5c11-494c-b703-0682c230f12c","added_by":"auto","created_at":"2025-06-17 12:30:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38624,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth kinetics of date palm cell suspension cultures under elicitors of charcoal and glutamine.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/733ad4ae8ff16a84271d0083.png"},{"id":84796885,"identity":"c011fef2-ef71-4565-be8b-92331513d2ba","added_by":"auto","created_at":"2025-06-17 12:30:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1357540,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AC on phenolics and browning of tissue and spent\u003cstrong\u003e \u003c/strong\u003eliquid medium in date palm suspension cultures. Scale bar: 0.1mm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/cd88217876abf8c3316221ca.png"},{"id":84797256,"identity":"10b05cf0-c8d7-4994-a8b1-bd220a2a2ff1","added_by":"auto","created_at":"2025-06-17 12:38:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":438110,"visible":true,"origin":"","legend":"\u003cp\u003eZymogram of peroxidases extracted from somatic embryos of date palm (cultivar BSK), separated by polyacrylamide gel electrophoresis (11% gels) and revealed with benzidine, as substrate. Extract samples (30 µL and 60 µL) are loaded for cultures ‘without AC (1), ‘without G’ (2) and ‘with AC and G’ (3). Arrow indicates peroxidase isoform of rf 0.16.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/bdf570f25368a72c96b0fb00.png"},{"id":84796889,"identity":"de731e33-9a7b-47d8-91a0-6b226a8f3e65","added_by":"auto","created_at":"2025-06-17 12:30:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":627371,"visible":true,"origin":"","legend":"\u003cp\u003eGermination of date palm somatic embryos\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/657d0c1808f4ad0418b41f90.png"},{"id":85459749,"identity":"b04666da-c080-4055-964e-bd223f9db467","added_by":"auto","created_at":"2025-06-26 07:17:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7337053,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6574056/v1/25e26b47-b09f-4c01-8827-a021144195ac.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Morphological stages of date palm cell suspension culture and production of biomass, phenolics, and proteins under elicitors of activated charcoal and glutamine.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSomatic embryogenesis of date palm is a critical technique for micropropagation of elite date palm cultivars\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recently, considerable progress has been made in the development and optimization of this technique from embryogenic cell suspension cultures\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In vitro cell suspension culture offers the best method to get large-scale of somatic embryos in date palm\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Our protocol allows the production of a large number individual somatic embryos of uniform physiological and growth characteristics, with synchronized development\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vitro cell suspension cultures show great potential to produce commercially useful bioactive compounds with great antioxidant activity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The growth condition variation impacts plants primary and secondary metabolism and induces many changes in the internal and external features of the dates\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In vitro of cells plant synthesizes phenolic compounds, this is due to the tissue culture specificity as synthetic biological system in which the phenols function is to interfere in cell proliferation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is known that secondary metabolites like phenolic compounds are produced as a defense mechanism against stress conditions. In this regard, the culture medium is provided with stress-inducing compounds known as elicitors, such as activated charcoal (AC), which is widely used in tissue culture media, influences the environment of a plant grown in vitro by adsorption of inhibitory or toxic substances, and adsorption of plant growth regulators from the culture medium\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Activated charcoal can employ harmful and beneficial effects on the tissues in vitro because, according to Garc\u0026iacute;a and Tabarez\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, charcoal addition to the medium can interfere in the absorption of nutrients essential, such as nitrogen, and in the action of endogenous hormones, which are fundamental for plant tissue culture. However, due to the hormone\u0026rsquo;s adsorption exuded by plants and toxic metabolites, activated charcoal acts as a potential antioxidant\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOrganic nitrogen sources seem to be a limiting factor in in vitro date palm culture and their addition as amino acids has a beneficial effect, the glutamine stimulated growth better of callus and formation of somatic embryos\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The medium with 0.1 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 2,4-D and 6.7 \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M glutamine has improved the proliferation of date palm somatic embryos and obtained the highest protein accumulation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Amino acids have been used for in vitro cultures as organic nitrogen source of several types as date palm, rice, and pineapple to induce regeneration and somatic embryogenesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Numerous studies have shown that glutamine alone or combined with casein hydrolysate has been most frequently used in the different stages of somatic embryogenesis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Glutamine and asparagine at 3.42 mM have been proved to be the most favorable for the formation of somatic embryos and the induction of secondary somatic embryos of Moroccan cork oak\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The addition of l-glutamine, 0.1% casein acid hydrolysate, and abscisic acid (ABA) greatly increased the number of somatic embryos in Spruce (\u003cem\u003ePicea asperata\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe objective of this study is to describe the morphological growth stages of embryogenic cells in the cell suspension cultures and to compare the effect of activated charcoal and glutamine on the production of biomass, phenolics, proteins, and peroxidase activities of somatic embryos in suspension cultures.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCallus induction and multiplication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShoot tips of date palm cultivars, namely Bouskri (BSK), were disinfected using a 20% solution of sodium hypochlorite, containing for 20 min. They were cultured on a callogenesis induction medium as described by our previous research\u003csup\u003e5,7\u003c/sup\u003e, which contained MS salts\u003csup\u003e27\u003c/sup\u003e and Fossard vitamins\u003csup\u003e28\u003c/sup\u003e with 5 mg/l of 6-benzylaminopurine (BAP), 5 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose, and 150 mg/l activated charcoal. For embryogenesis induction, the friable callus formed after 6\u0026ndash;8 months of culture was selected and transferred onto a multiplication medium containing 0.1 mg/L of BAP and 0.5 mg/L of 2,4-D. The cultures were incubated at 25\u0026plusmn;2 ◦C in the dark and subcultured to fresh medium every 5 weeks until the production of embryogenic callus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of cell suspension culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish the cell suspension, the method described by Fki et al.\u003csup\u003e6\u003c/sup\u003e, and Abohatem et al.\u003csup\u003e7\u003c/sup\u003e has been used in this study. For that, 0.5 g of embryogenic callus were cut with sterile scalpel into small pieces as possible and then transferred in 50 ml of liquid medium in 250 ml Erlenmeyer. The Erlenmeyer content was passed through sieves with a 500 \u0026micro;m mesh size and the filtrate is incubated on a rotary shaker (100 rpm) at 25 \u0026plusmn; 2\u0026ordm;C under a 16/8-h (light/dark) photoperiod. The liquid medium MS/2 was supplemented with 0.3 mg L\u003csup\u003e-1\u003c/sup\u003e BAP and 0.1 mg L\u003csup\u003e-1\u003c/sup\u003e 2,4-D. Two elicitors\u0026mdash;activated charcoal (AC) and glutamine (G)\u0026mdash;were tested in the culture medium. AC was evaluated at 150 mg L⁻\u0026sup1; and in a control treatment without AC. Glutamine was tested at 100 mg L⁻\u0026sup1; and in a control treatment without glutamine.\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment and growth stages of embryogenic cell suspension culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe division and development of embryogenic cell was observed under a microscope during the 2 weeks of cell suspension culture\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e The characteristics morphological growth stages of somatic embryogenesis at the different developmental stages (globular, elongation and cotyledonary) in date palm suspension cultures were recorded.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo measure the embryogenic\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecell growth, cells were collected after every week of subculture. Cell growth was followed with flasks harvested at one-week intervals from the first week of subculture until cell weight was stable (8th week). The fresh weight of the samples was measured and recorded by a digital scale.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaturation and germination of somatic embryos\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSomatic embryos maturation was carried out on MS liquid medium diluted a half in the absence of plant growth regulator for two weeks. Subsequently, somatic embryos are transferred to a germination medium consisting of MS solid medium supplemented with NAA (0.1 mg L\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction and analysis of phenolics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhenolic compounds were extracted and analyzed as described by Macheix et al.\u003csup\u003e29\u003c/sup\u003e. At the end of 6th week of suspension culture,\u0026nbsp;250 mg fresh tissues of somatic embryo were homogenized with 2 ml of 80% methanol at 4\u0026ordm;C and centrifuged three times for 3 min at 7000 \u0026times; g. \u0026nbsp;The supernatants were recovered each time, and 100 \u0026mu;L of the supernatant was added to Folin-Ciocalteu reagent (250 \u0026mu;L) and sodium carbonate (20%). The mixture was incubated for 30 min at 40\u0026ordm; C and the blue color was determined at 760 nm. The intensity of browning in the spent\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eliquid medium was visually estimated and noted ++, + and \u0026minus; for high, medium, and minimal intensity, respectively according to the rate of scaling by Pottino\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtraction and analysis of proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted according to the method described by Lecouteux\u003csup\u003e31\u003c/sup\u003e. At the end of 6th week of suspension culture,\u0026nbsp;fresh somatic embryogenesis tissues (250 mg) were homogenized with 2mL Tris maleate buffer (0.1M, PH 6.5) and centrifuged for 6 min at 7000g. The supernatant was used as the extract of crude proteins. The total proteins were measured by spectrophotometer at 595 nm according to the method described by Bradford\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeroxidase extraction, activity assays and electrophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of 6th week of suspension culture,\u0026nbsp;250 mg fresh tissue of somatic embryos were homogenized in 1 mL of Tris maleate buffer pH 6.5 (0.1M). After centrifugation at 10 000 g for 10 min, the supernatant was used for determination of enzymatic activities. Peroxidase (POX) activity was tested by measuring the oxidation of guaiacol at 470 nm. Afterward, 10 \u0026mu;L of enzyme extract were added to 2ml of reaction mixture consisting of a solution of 25mM guaiacol and 0.1M Tris-maleate buffer (pH 6.5). Reactions were initiated with 10 \u0026mu;L of hydrogen peroxide - H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (10 %) and stopped after 3 min.\u003c/p\u003e\n\u003cp\u003eTo separate isoenzyme of peroxidase, polyacrylamide gel electrophoresis for soluble proteins was conducted according to Baaziz\u003csup\u003e33\u003c/sup\u003e using a vertical gel electrophoresis. The resolving gel was 11 % and the stacking gel 5 % (w/v). The electrode buffer was Tris (0.02 M) - glycine (0.129M) - SDS (0.1 %) pH 8.3. Each gel slot was loaded with 30 \u0026mu;g and 60 \u0026mu;g (POX) proteins samples. Electrophoresis was conducted at a constant voltage of 100 V. For POX staining, the gel was incubated for 15 min in 100 ml of 0.1M sodium acetate buffer (pH 5) containing 0.1 ml 10% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eand 0.1g of benzidine. Gels with the substrates were incubated for 30 min in dark until dark bands appeared.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results were analyzed by variance analysis (ANOVA), followed by SNK test at P = 0.05 level to compare the means. The repetitions number are three replicates for the two independent experiments.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eDevelopment and growth stages of embryogenic cell suspension culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the first 2 weeks of cell suspension culture, the microscopic observation was conducted to describe the embryogenic cell development (Fig.1 A, B). In the first week, single cells of suspension cultures divided slowly to form small clumps, those cells showed dense cytoplasm and large vacuolar cells (Fig. 1A) whereas in the second week, cells were dividing actively to form large clumps, those cells showed dense cytoplasm with small vacuoles (Fig. 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar results were obtained in oil palm by Kramut and Te-chato\u003csup\u003e34\u003c/sup\u003e who described two types of cell aggregates: one consisting of 5 to 10 cells that consisted of large vacuolar cells and another with more than 10 cells that showed dense cytoplasm. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRoowi et al.\u003csup\u003e35\u003c/sup\u003e found that cell suspensions contained cellular aggregates composed of round cells with dense cytoplasm that were small in diameter at 10\u0026ndash;20 \u0026micro;M. De Touchet et al.\u003csup\u003e36\u003c/sup\u003e reported that embryogenic cells were dividing actively and showed a round prominent nucleus, and a dense cytoplasm with small vacuole.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe morphological observations were carried out to describe the embryogenic cell proliferation and the growth stages of embryogenic cell suspension cultures during period of somatic embryos formation.\u003c/p\u003e\n\u003cp\u003eDuring the first week, embryogenic cells of suspension cultures (Fig. 2A) divided slowly and formed small spherical proembryos (Fig. 2B), whereas, within the second and third weeks, cells were dividing actively and formed large spherical embryos (globular stage) (Fig. 2C, D). The globular embryos started the process of elongating to conversion to cotyledonary embryos in the 3rd week and continued until to 4th week (Fig. 2D, E). Cotyledonary embryos (Fig. 2E) were formed in the 4th and 5th week, meanwhile the somatic embryos formed in the 5th and 6th week (Fig. 2F) and reached to full somatic embryos in the 7th and 8th week at stationary stage (Fig. 2G). To\u0026nbsp;our knowledge, we are the first described of the time course of embryogenic cell stages in the date palm suspension cultures\u003csup\u003e7\u003c/sup\u003e. The morphological description growth stages of somatic embryos in date palm suspension cultures are going to be an efficient biotechnological importance in the secondary metabolites production from plant cell suspension culture, which helps in determining the appropriate growth stage of embryogenic cells for adding elicitors. Therefore, the obtained results of this study represent novel and efficient biotechnological advancements in the woody plant cell suspension culture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth kinetics of cell suspension cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll date palm cell suspension cultures showed sigmoid growth curves during the period of embryogenic cell suspension development into the somatic embryos which lasted 6 weeks. After one week of the lag phase, the cells of cultures were dividing and grew vigorously entering their exponential phase. The growth rate of cells continued to increase exponentially until week 6, followed by a stationary phase in the week 7 and 8 where the growth of somatic embryos ceased. (Fig. 3, Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: Cell biomass accumulation per week in date palm suspension culture under elicitors of charcoal and glutamine.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eElicitors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 567px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell biomass per week (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eWith charcoal + glutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.8\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4.5\u0026plusmn;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9.7\u0026plusmn;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e15.4\u0026plusmn;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e24.1\u0026plusmn;3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e35\u0026plusmn;2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e38.7\u0026plusmn;3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e42.4\u0026plusmn;4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eWithout charcoal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.5\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3.4\u0026plusmn;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e7.3\u0026plusmn;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e11.9\u0026plusmn;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e18.9\u0026plusmn;1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e28.7\u0026plusmn;3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e33.3\u0026plusmn;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e36.9\u0026plusmn;5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eWithout glutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.5\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e3.3\u0026plusmn;0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e6.1\u0026plusmn;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e9.5\u0026plusmn;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e15.3\u0026plusmn;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e25.3\u0026plusmn;2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e29.3\u0026plusmn;3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e32.9\u0026plusmn;4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThis kinetic behavior is in agreement with some previous date palm studies, which reported the maximum growth (fresh weight) was in the 6-week-old cell suspension culture\u003csup\u003e4,37,5\u003c/sup\u003e. While in contrast with other reports, which reported the highest growth biomass (fresh weight) was in the11-week-old cell suspension culture\u003csup\u003e38,9,39\u003c/sup\u003e. \u0026nbsp;This difference is due to the type and concentration of auxin and cytokinin used in those studies. In the studies that took 6 weeks to reach the maximum level of embryo growth used low concentrations of auxin were used (0.1 mg L\u003csup\u003e-1\u003c/sup\u003e 2,4-D and 0.5 mg L\u003csup\u003e-1\u003c/sup\u003e BAP), and the concentration of cytokinin was slightly higher than auxin. Studies that took 11 weeks to reach the maximum level of embryo growth used high concentrations of auxin (10 mg L\u003csup\u003e-1\u003c/sup\u003e NAA and 1.5 mg L\u003csup\u003e-1\u003c/sup\u003e 2-iP), and the concentration of cytokinin was slightly lower.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell biomass accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of embryogenic\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecell biomass accumulation is the most important methods to measure growth. The accumulation of biomass (fresh weight of cells) in date palm cell suspension cultures showed a progressive increase during the period of cell suspension development into the somatic embryos. The maximum fresh weight of biomass accumulation was observed in the 7th and 8th week when somatic embryos reached the stationary growth stage. The highest fresh weight (FW) accumulation of somatic embryos biomass was 42.4\u0026plusmn;4.6 g in the medium with activated charcoal (AC) and glutamine (G), which increased approximately eightyfold than the first week, followed by 36.9 \u0026plusmn; 5.3 g in the medium without activated charcoal (AC), while the least fresh weight was 32.9\u0026nbsp;\u0026plusmn; 4.4 g in the medium without glutamine (G) (Table 1,2). Similar result was obtained by Zouine and El Hadrami\u003csup\u003e4\u003c/sup\u003e, which found that when glutamine concentration increased from 3.35\u0026times; 10\u003csup\u003e-4\u003c/sup\u003e to 6.7 \u0026times;10\u003csup\u003e-4\u003c/sup\u003e M, the date palm somatic embryos production increased from 14 to 56 embryos. The glutamine stimulated better growth callus and formation of somatic embryos of date palm\u003csup\u003e17\u003c/sup\u003e. The addition of arginine at a concentration of 3 mM caused an early development of date palm somatic embryos to spherical entities with regular and smooth outlines\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Effect of activated charcoal and glutamine on biomass, phenolics and browning of tissue and culture medium in date palm suspension cultures.\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"552\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eElicitor\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eCell biomass per 0.5 g callus (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003eIntensity of browning\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePhenolic content\u003c/p\u003e\n \u003cp\u003emg / g FW\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eWith charcoal + glutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e42.4\u0026plusmn;4.6a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.025bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eWithout charcoal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e36.9 \u0026plusmn; 5.3b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.37 \u0026plusmn; 0.035a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eWithout glutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e32.9 \u0026plusmn; 4.4bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e0.15 \u0026plusmn; 0.03b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eValues are means \u0026plusmn; standard error of three replicates. Mean values followed by the same letters (a\u0026ndash;c) are not significantly different at p = 0.05 level according to SNK test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of phenolics in the tissue and the liquid culture medium of suspension cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo limit phenolic production that causes an appreciable loss of cell culture viability, activated charcoal (AC) are commonly used in the date palm tissue and the culture medium to trap phenols and oxidized phenols. In this study, at the addition of 150 mg L\u003csup\u003e-1\u003c/sup\u003e activated charcoal (AC) caused a significant decrease in phenolic content (browning) of tissue and spent liquid medium and improved the growth rate of somatic embryos (Fig. 4, Table 1). At the same time, in the liquid culture medium without AC, there was observed significant increase in the level of phenolic content (browning) in the tissue and the spent liquid medium (Fig.4, Table 2). Activated charcoal (AC) was found to be the best antibrowning factor particularly during the first months of tissue culture and suspension cultures. This result agrees with the known facts concerning the role of the activated charcoal in trapping phenols and oxidized polyphenols in palm tissue culture\u003csup\u003e40.41\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe biochemical analysis of phenolics content showed the medium without charcoal (AC) a significant increase in phenolic content in somatic embryos tissues from 0.12 mg/g FW to 0.37 mg/g FW. This represents an approximately threefold compared to the medium with AC (Fig. 4, Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn previous studies, the primary objective of adding activated charcoal (AC) to the culture medium was to adsorb phenolic compounds and reduce the accumulation of oxidized phenols in plant tissue cultures\u003csup\u003e15,42,43\u003c/sup\u003e. Based on this, our hypothesis is that the absence of AC in the culture medium will lead to a significant increase in phenolic production, as well as enhanced exudation of these compounds into the medium. The positive and negative effects of activated charcoal mainly depend on its adsorptive properties\u003csup\u003e44\u003c/sup\u003e. Activated charcoal (AC) has a very fine pores network with large inner surface area on which many substances can be adsorbed\u003csup\u003e45\u003c/sup\u003e. The most crucial impact of adding AC to the culture media is a drastic dip in concentration of plant growth regulators and other organic supplements, which are used as elicitors to produce secondary metabolites such as phenolics and flavonoids\u003csup\u003e46\u003c/sup\u003e. \u0026nbsp;This is due to the adsorption of these elicitor chemicals by AC. \u0026nbsp;Date palm cell suspension cultures evidenced a considerable antioxidant activity under without AC treatment which was related to the high phenolic production.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis is the first report on the important role of\u0026nbsp;activated charcoal (AC) as elicitor on the production phenolics in the suspension cultures that will to contribute to establishing an efficient protocol for date palm cell suspension culture for large-scale production of phenolic compounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteins content and peroxidases activities in suspension cultures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAddition 100 mg/L glutamine to culture medium led to increased proteins content in somatic embryos from 75.6\u0026plusmn; 2.8 \u0026mu;g / g FW to 87.46 \u0026plusmn; 3.6 \u0026mu;g / g FW compared for medium without G, and decreased peroxidase activity from 56.8 \u0026plusmn; 14.9 U E/g FW to 35.9\u0026plusmn;9.6 U E/g FW (Table 2). In addition, activated charcoal (AC) decreased proteins content from 87.46 \u0026plusmn; 3.6 \u0026mu;g /g FW to 77.43 \u0026plusmn; 2.9 \u0026mu;g/g FW compared for medium without AC and increased peroxidase activity from 35.9\u0026plusmn;9.6 UE/ g FW to 67.2\u0026plusmn; 10.4 UE/g FW (Table 3).\u003c/p\u003e\n\u003cp\u003eZouine and El Hadrami\u003csup\u003e4\u003c/sup\u003e found that the use of 0.1 mg L\u003csup\u003e-1\u003c/sup\u003e 2, 4-D with 6.7 \u0026times;10 \u003csup\u003e\u0026ndash;4\u003c/sup\u003e M glutamine showed a significant increase in protein content in date palm somatic embryos of suspension cultures. In oil palm somatic embryos, the use of glutamine alone or combined with arginine was found to be a factor in enhancement accumulation of protein\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u003c/strong\u003e\u0026nbsp; Effect of activated charcoal and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eglutamine on proteins content and peroxidases activities of date palm somatic embryos\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eElicitor\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eTotal protein \u0026mu;g\u0026nbsp;/ g FW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003ePeroxidase activity UE / g FW\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eWith charcoal + glutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e87.46 \u0026plusmn; 3.6a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e35.9\u0026plusmn;9.6c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eWithout charcoal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e77.43 \u0026plusmn; 2.9b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e67.2\u0026plusmn; 10.4a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eWithout glutamine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e75.6\u0026plusmn; 2.8bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 227px;\"\u003e\n \u003cp\u003e56.8 \u0026plusmn; 14.9b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eValues are means \u0026plusmn; standard error of three replicates. Mean values followed by the same letters (a\u0026ndash;c) are not significantly different at p = 0.05 level according to SNK test. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Effect of activated charcoal and glutamine on peroxidase isoforms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracts of peroxidase from somatic embryos of date palm, when separated by gel electrophoresis showed a major migration zone with Rf value interval 0.1- 0.16 (Fig. 4), where the stain intensity increased with the high extract volume to dauble volume (60 \u0026micro;L).\u003c/p\u003e\n\u003cp\u003eExtracts without AC and G were characterized by a peroxidase isoform of relatively high migration speed (Rf = 0.16). This latter disappeared in extracts of peroxidase prepared from somatic embryos cultivated on medium with AC and G. This result confirms that AC and G modulate browning by their action on qualitative aspect of peroxidases. They induce the enzyme isoform formation, which migrates at Rf = 0.16 on 11% polyacrylamide gels, this isoform correlated with maturation of somatic embryo.\u0026nbsp;(Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGermination of somatic embryos\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe germination rate of somatic embryos increased significantly to 32% when they were first cultured in half-strength MS liquid medium without plant growth regulators and then transferred to solid MS medium. In contrast, only 8% germination was achieved when embryos were transferred directly to solid MS medium (Fig. 6).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study presents an efficient protocol for the large-scale production of somatic embryos in suspension culture. The detailed description of the morphological growth stages of somatic embryos in date palm suspension cultures provides valuable insights with significant biotechnological relevance, particularly for the production of secondary metabolites in woody plant cell suspension systems.\u003c/p\u003e \u003cp\u003eOur results in this study confirmed that the culture medium free of activated charcoal led to significantly increased phenolics production and peroxidase activity of somatic embryos in suspension cultures and increased tissue browning and medium browning. This will be an efficient method for large-scale production of phenolics from cell date palm.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eAC Activated charcoal \u003c/p\u003e\n\u003cp\u003eG Glutamine\u003c/p\u003e\n\u003cp\u003e2,4-D 2,4-Dichlorophenoxyacetic acid \u003c/p\u003e\n\u003cp\u003eBAP 6-Benzylaminopurine\u003c/p\u003e\n\u003cp\u003eNAA \u0026alpha;-Naphthalene acetic acid\u003c/p\u003e\n\u003cp\u003eMS Murashige and Skoog (1962) medium \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.A.A. wrote the manuscript, performed, and designed the experiments, and performed the biochemical data analysis. M.B. contributed to performed the biochemical data analysis, reviewing and editing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the plant experiments/protocols were performed with relevant institutional, national, and international guidelines and legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no objection in consent to publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eOthmani, A., Bayoudh, C., Drira, N., \u0026amp; Trifi, M. 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Plant Cell, Tissue and Organ Culture (PCTOC),\u003c/em\u003e123, 523-533. https://doi.org/10.1007/s11240-015-0856-6 (\u003cem\u003e \u003c/em\u003e2015). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Phoenix dactylifera, Cell suspension culture, Phenolics production, Activated charcoal, Glutamine, Peroxidase","lastPublishedDoi":"10.21203/rs.3.rs-6574056/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6574056/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study presents an efficient protocol for the large-scale production of somatic embryos from embryogenic cell suspension cultures of \u003cem\u003ePhoenix dactylifera\u003c/em\u003e (date palm). The morphological development of somatic embryos was documented across distinct stages from globular, cotyledonary and somatic embryos forms, with synchronized growth over an eight-week period. The cells of suspension cultures divided actively and formed globular embryos within 2nd and 3rd week, the globular embryos started to conversion to cotyledonary embryos in the 3rd week and continued until the 4th week, while the somatic embryos formed within the 5th and 6th week, followed by a stationary phase in the week 7 and 8. Effects of activated charcoal and glutamine on production of biomass, phenolics and proteins in cell suspension culture were studied. The accumulation of biomass increased approximately eightyfold in the medium with charcoal and glutamine at the end of 8th week. The culture medium free of activated charcoal showed significant increased browning of tissue and spent liquid medium, and significant increased phenolics production and peroxidase activity in somatic embryos compared to the medium with charcoal. Overall, the findings contribute valuable insights into optimizing in vitro culture conditions for date palm, with implications for enhanced production of biomass and secondary metabolites such as phenolics and proteins.\u003c/p\u003e","manuscriptTitle":"Morphological stages of date palm cell suspension culture and production of biomass, phenolics, and proteins under elicitors of activated charcoal and glutamine.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 12:30:01","doi":"10.21203/rs.3.rs-6574056/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65cb229d-addd-4b2a-8419-77a5d7e9c4b0","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50084925,"name":"Biological sciences/Biochemistry"},{"id":50084926,"name":"Biological sciences/Biotechnology"}],"tags":[],"updatedAt":"2025-06-26T07:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 12:30:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6574056","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6574056","identity":"rs-6574056","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}