Nova Biotechnol Chim (2021) 20(2): e652 DOI: 10.36547/nbc.652 1 Nova Biotechnologica et Chimica Optimization of callus induction with enhancing production of phenolic compounds production and antioxidants activity in callus cultures of carob (Ceratonia siliqua L.) Assia Lozzi1, , Rachid Mentag2, Driss Alami-Halimi1, Rabha Abdelwahd2, Abdelhadi Abousalim1 1Department of Plant Production, Protection and Biotechnologies, Institute of Agronomy and Veterinary Medicine Hassan II, Rabat-Instituts 6654, Morocco 2Biotechnology Unit, Regional Center of Agricultural Research, National Institute of Agricultural Research, Rabat 415, Morocco  Corresponding author: assia.lozzi@gmail.com Article info Article history: Received: 12th August 2020 Accepted: 16th August 2021 Keywords: Antioxidant activity Callus cultures Ceratonia siliqua Condensed tannin Flavonoids Polyphenols Abstract Carob (Ceratonia siliqua L.) is an important Mediterranean plant species with worldwide commercial and medicinal uses. The establishment of a callus culture protocol as an alternative system to produce polyphenols of chemical and pharmaceutical interest was made in the present study for the first time in carob. Explant type and the light regime are two important factors that influence morphogenic responses and biochemical production. Maximal callus induction (100 %) and biomass accumulation were obtained in cotyledon explants under both tested light regimes (16-h photoperiod and darkness). However, leaf-derived callus produced higher amounts of polyphenols (TPC) and flavonoids (TFC) but a lower amount of total condensed tannins (TCT) as compared to cotyledon-derived callus. The light treatment has significantly increased TCT content but decreased the antioxidant activity in carob callus cultures. Strong and positive correlations were obtained between TPC, TFC, and the antioxidant activities with correlation coefficients in range 0.68 – 0.98. The obtained results indicated that calli of C. siliqua have the potential for enhanced production of phenolic compounds with antioxidant activity that is favored by cultivation under dark condition.  University of SS. Cyril and Methodius in Trnava Introduction Ceratonia siliqua L., commonly known as ‘carob’, is an important xerophytic plant belonging to the Fabaceae family. Due to its important economic and ecologic values, it has drawn much attention in recent years. The interest in carob as an inexpensive source of several products has been increasing over the last few years. Carob seeds are being used for carob bean gum extraction. Carob pods can also be exploited for the production of sugar syrups (Fidan et al. 2020), cocoa substitutes (Akdeniz et al. 2021), bioethanols (Germec et al. 2020), and secondary metabolites (Kyratzis et al. 2021). Carob pods and leaves are being in use since olden time to cure digestive disorders, asthma, pharyngitis, bronchitis, and anemia (Rtibi et al. 2017; Sargin and Büyükcengiz 2019). Furthermore, mailto:assia.lozzi@gmail.com Nova Biotechnol Chim (2021) 20(2): e652 2 many studies have been conducted on several carob-tree parts for their bioactive components, including total phenolics which exhibit potent cytotoxic, anticancer, anti-inflammatory, hepatoprotective, nephroprotective, antidiabetic, sedative, and antioxidative properties (Benchikh et al. 2014; Custódio et al. 2015; Aboura et al. 2017; Ghanemi et al. 2017; Othmen et al. 2020; Gurel et al. 2021). The major phenolic compounds in this plant are phenolic acids, flavonoids, and tannins (Stavrou et al. 2018). However, phenolic contents in carob field-grown plants are highly variable due to genetics and environmental factors (Custódio et al. 2007; El Hajaji et al. 2011; Korkmaz et al. 2020). In vitro culture techniques emerge as efficient biotechnological tools to produce commercially important medicinal compounds such as polyphenols, under aseptic and controlled conditions. These techniques would allow continuous, economical, viable, and sustainable production of pure bioactive compounds, regardless of the climatic and geographic conditions, during a shorter and more flexible production cycle (Dias et al. 2016; Gai et al. 2020). Different factors affect phenolic production in in vitro plant cultures, such as explants type and light conditions, although responses vary with the class of secondary metabolites and among species (Estell et al. 2016; Farhadi et al. 2017; Shehzad et al. 2021). It must be noted that while studies on callus culture of carob have been reported (Ksia et al. 2008; Lozzi et al. 2015; 2018; 2019; Zouari and El Mtili 2020), the antioxidant properties of their phenolic compounds have not been investigated. To our knowledge, this study represents the first attempt to demonstrate that the callus culture of carob is a reliable tool for polyphenols production. The objectives of the present work were to: (i) optimize methods for producing vigorous carob callus by monitoring the effects of explant type and light conditions, (ii) determine the content of the total phenolics, flavonoids, and condensed tannins and (iii) evaluate the antioxidant activity in in vitro calli to elucidate the medicinal potency of in vitro cultures of this plant. Experimental Plant materials and callus culture Mature seeds were collected from selected C. siliqua trees growing in Beni Mellal, Morocco, treated with concentrated sulfuric acid (98 %) for 60 min, washed and then immersed for 48 h in sterile distilled water. Cotyledons were aseptically excised from seeds and segmented into portions of 4 – 6 mm in length. Leaf, hypocotyl, epicotyls, and root segments were cut from two-month-old seedlings, germinated on the carob culture medium (LAC) that we have developed and successfully applied in our previous research on carob micropropagation (Lozzi et al. 2019). All explants (cotyledons, leaves, hypocotyls, epicotyls, and roots) were inoculated on the LAC medium supplemented with 5 µM 2,4- dichlorophenoxyacetic acid (2,4-D), 3 % (w/v) sucrose and 0.7 % (w/v) agar. The cultures were maintained at 26 ± 2 °C, either in darkness or under a 16-h photoperiod (60 μmol.m–2.s–1) for five weeks. Extraction Callus cultures of known fresh weight were dried in a hot air oven at 60 °C to constant weight. Each finely ground dried sample (0.1 g) was mixed with 70 % (v/v) acetone (40 mL). The mixtures were then centrifuged at 4,000 rpm for 20 min. The supernatant was collected and maintained at 4 °C until used. Total phenolic content (TPC) Total phenolic content of the calli extracts was determined spectrometrically according to the Folin–Ciocalteu assay (Singleton and Rossi 1965) using the gallic acid as standard. The calli extract (0.1 mL) was mixed with 1.4 mL of deionized water and 0.1 mL of Folin-Ciocalteu reagent. After 6 min of incubation, 0.5 mL of Na2CO3 (20 %) was added and the reaction mixture was allowed to incubate in the dark for 30 min. Absorbance was measured at 750 nm and results were expressed as mg gallic acid equivalents (GAE) per gram of dry weight of the sample. Nova Biotechnol Chim (2021) 20(2): e652 3 Total flavonoid content (TFC) Total flavonoid content was evaluated in the extracts using the aluminum chloride (AlCl3) assay (Lamaison et al. 1990). 1 mL of AlCl3·6H2O (2 %) was added to 1 mL of acetonic extract and the absorbance was read 10 min later at 430 nm. Quercetine was used to make the calibration curve, and the results were expressed as mg of Quercetine equivalents (QE) per g dry matter in extracts. Total condensed tannin (TCT) Condensed tannins were analyzed using the vanillin–HCl method (Broadhurst and Jones 1978). 0.1 mL of calli extract was added to 3 mL of vanillin (4 %) and 1.5 mL of highly concentrated HCl. The absorbance was measured at 500 nm after standing for 20 min in a dark room. The results were expressed as mg of catechin equivalent (CE) per g of dry weight. DPPH radical-scavenging assay The free radicals scavenging activity of the in vitro callus of carob was evaluated spectrophotometrically using the 2,2-diphenyl-1- picryl hydrazyl (DPPH) method (Brand-Williams et al. 1995). Briefly, 1 mL of 0.1 mM DPPH solution (in ethanol) was added to 0.5 mL of calli extract diluted in 1.4 mL of ethanol. The mixtures were vortexed and incubated for 30 min in darkness. The absorbance was measured at 515 nm and the ascorbic acid was used as a standard. The radical scavenging activity was calculated according to the following formula (Eq. 1): (1) where A0 is the absorbance of the negative control (ethanol) and A1 is the absorbance of the reaction mixture containing the sample extract. Ferric-reducing power (FRAP) The reducing power of the extract was evaluated spectrophotometrically using the assay of Oyaizu (1986). 0.22 mL of the extract was added to 0.25 mL of phosphate buffer (0.2 M, pH 6.6) and 0.25 mL of potassium ferricyanide (1 %). The mixture was then incubated at 50 °C for 20 min. Afterward, 0.25 mL of 10 % trichloroacetic acid was added, and the mixture was mixed with 1 mL of distilled water and 0.25 mL of iron trichloride (0.1 %). The absorbance was read at 700 nm. The increased absorbance of the reaction mixture meant increased reducing power of the calli extract. Statistical analysis All extractions and assays were carried out in triplicate. The results were expressed as mean ± standard deviation. The experimental data were processed with the analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) at 5 % to determine significant differences among the mean values. Pearson’s correlation test was used to identify the correlation between total phenolics and antioxidant activity. Results and discussion Effect of explant type and light conditions on induction The percentage of callus induction and the dry weights (DW) of calli were significantly (P<0.001) affected by explant type, confirming the results of previous works (Gourguillon et al. 2018; Rameshkumar et al. 2018; Wu et al. 2020). A successful callus formation was achieved in both cotyledon and leaf explants (Fig. 1A, B). However, the cotyledon explants showed 100 % callus induction and a much better callus dry weight than leaf explants (Table 1). Attempts to induce callus from hypocotyls and epicotyls were completely ineffective (Fig. 1C, D). The root explants exhibited poor callus formation, turned brown and died within 5 weeks (Fig. 1E). The difference of response between the explants could be related to the difference in endogenous hormone level and the competence of cells to respond to the external signals (Jiménez and Bangerth 2001a, 2001b; Wu et al. 2020; Saeedpour et al. 2021). Besides of the explant type, light is another factor that may influence competence by modifying the concentration of endogenous hormone in explant tissues (Jiménez and Bangerth 2001a; Su et al. Nova Biotechnol Chim (2021) 20(2): e652 2 2014; Vinterhalter et al. 2020). Callus development of C. siliqua in response to light conditions is missing in the cited literature. In the present investigation, callus growth (DW) on cotyledon explants was similar under both tested light regimes (16-h photoperiod and darkness). The maximum dry weight (53.9 ± 4.9 mg.L-1) being achieved in darkness. Note that the present result is considerably higher than our previous studies (Lozzi et al. 2015, 2018) on callus induction from callus cotyledons using other basal culture media: MS (Murashige and Skoog 1962), B5 (Gamborg et al. 1968), WPM (Lloyd and McCown 1980) and DKW (Driver and Kuniyuki 1984), indicating that LAC medium is more suitable for callus induction from carob cotyledons. In the case of leaf explants, the biomass accumulation (DW) in callus formed in the dark was significantly higher than that in the light. Previous studies revealed a close relationship between light regimes and callus biomass accumulation (Ju et al. 2014; Shehzad et al. 2021) which indicate the need for optimal light conditions for optimum biomass accumulation. Table 1. Comparative response of callus induction in Ceratonia siliqua from mature cotyledons and leaves in photoperiod and darkness. Type of explant Light conditions Callus induction [%] Callus DW [mg.L-1] Mature cotyledons 16-h Photoperiod 100.0a 53.9 ± 4.9a Darkness 100.0a 52.5 ± 7.0a Leaves 16-h Photoperiod 96.3a 30.8 ± 5.0b Darkness 73.6b 24.4 ± 1.1c Significance of two-way ANOVA Type of explant (A) *** *** Light conditions (B) ** * A×B ** NS Values are mean ± SE. Mean values within a column followed by the same letter are not significantly different by Duncan’s multiple range test (5 %). DW – dry weight; ∗, ∗∗, ∗∗∗ – significance at P≤0.05, P≤0.01, and P≤0.001, respectively (two-way ANOVA). Fig. 1. Callus induction from different explants of Ceratonia siliqua L. a – mature cultyledons; b – leaf explants; c – hypocotyl explants; d – epicotyls explants; e – root explants. Arrows indicate callus tissues. 4 Nova Biotechnol Chim (2021) 20(2): e652 5 Effects of explant type and light conditions on antioxidant compounds The total phenolic contents of the extracts are shown in Fig. 2. In contrast to the finding on callus induction and biomass accumulation, the TPC, TFC, and TCT contents in leaf derived calli extracts were significantly higher when compared to cotyledon-derived calli extracts. Leaf-derived callus produced the highest amounts of phenolic compounds (21.4 ± 1.4 mg GAE.g−1 in darkness and 20.6 ± 0.9 in light) which were about three-fold higher than those of cotyledon callus. Similarly, the leaf-derived calli produced a higher amount of flavonoids than those of cotyledon explant (7.5 ± 0.9 mg.QE.g−1 in darkness and 7.0 ± 0.7 in light). Among calli derived from cotyledon explants, regardless of the light conditions, the level of TPC was not significantly different (P<0.05). Similar behavior was observed among calli derived from leaf explants, indicating that the light regime tested did not influence the accumulation of these compounds. Similar results were documented by Fazal et al. (2016) in callus culture of Prunella vulgaris where TPC production was not influenced by light conditions. Opposite results were however, obtained with calli from Tecoma stans and Cnidium officinale when illumination induced a strong accumulation of polyphenols (Rameshkumar et al. 2018; Adil et al. 2019). In contrast, the TFC level was significantly (P<0.01) influenced by the light regime. The total flavonoid content was higher in darkness (3.8 ± 0.2 mg.QE.g−1 in cotyledon-derived callus and 5.3 ± 0.3 in leaf-derived callus) than in periodic light. In contrast to the finding on TPC and TFC accumulation in carob calli extracts, the cotyledon-derived callus accumulated higher amounts of TCT, which were about two-fold higher than those of leaf-derived callus; the difference being significant (P<0.001). Furthermore, callus grown under periodic light enhanced the accumulation of TCT (1.7 ± 0.2 mg.CE.g−1 in cotyledon-derived callus and 0.84 ± 0.07 in leaf- derived callus) as compared to dark incubation. Similarly, (Castro et al. 2016) found a positive response of light on the TCT in Byrsonima verbascifolia callus cultures, whereas Favre et al. (1991) found no effect in Quercus callus. Fig. 2. Effects of explant type and light conditions on antioxidant compounds in callus cultures of carob (Ceratonia siliqua L.). A – total phenolic content; B – total flavonoid content; C – total condensed tannin. Mean values within a column followed by the same letter are not significantly different by Duncan’s multiple range test (5 %). The levels detected in the present study were found to be higher than those reported in different aerial parts of Moroccan carob, which ranged from 0.45 to 2.64 and from 15.8 to 18.08 mg.GAE.g−1 of phenolic content in intact carob leaves and pulp (Hajaji et al. 2010) and from 0.25 to 1.02 mg.CE.g−1 of condensed tannins in carob pulp (El Bouzdoudi et al. 2016). While in the aceton extract A B C Nova Biotechnol Chim (2021) 20(2): e652 2 of carob seed peel, total phenolics, flavonoids, and condensed tannins were found to be 0.16 µg.GAE.g−1, 0.5 µg.QE.g−1 and 0.5 µg.CE.g−1, respectively (Lakkab et al. 2019). Other studies on Algerian, Tunisian, Portuguese, and Cypriot carob showed different levels of total phenolic content that ranged from 1.8 to 307 and 6 to 336 mg.GAE.g−1 of leaves and pods (Papagiannopoulos et al. 2004; Custódio et al. 2009; Ortega et al. 2009; Meziani et al. 2015; Correia et al. 2018; Ayache et al. 2020; Kyriacou et al. 2021).These authors attributed the differences not only on genetic, geographical, environmental, and cultural conditions, but also on technological factors such as the extraction methodologies and the used solvent system. Effects of explant type and light conditions on antioxidant activity Total antioxidant capacity may vary depending on the assay used (Tabart et al. 2009). Therefore, using more than one method is recommended to evaluate this property (Tabart et al. 2014). In the present study, the antioxidant activities of carob calli extracts were determinate using two widely known assays, based on electron transfer reaction, the DPPH free radical-scavenging activity (DPPH) and Ferric-reducing power (FRAP). However, the two methods clearly showed that carob calli extracts contain considerable antiradical and antioxidant activities (Table 2). DPPH assay showed that all extracts were able to reduce the purple-coloured radical DPPH into pale, yellow- coloured DPPH-H. Free radical scavenging activity in different treatments ranged from 60.92 ± 4.98 to 88.82 ± 0.10 %, which levels were the highest in leaf-derived callus followed by cotyledon-derived callus. Cultivation in the light or darkness had no apparent effect on scavenging activity in leaf- derived callus. In contrast, light treatment induced a significant decrease of the scavenging activity in cotyledon-derived callus. Similar behavior was observed for the FRAP test that measures the ability of the extract to reduce Fe3+ to Fe2+ and may serve as a significant indicator of its antioxidant potential (Govindan and Muthukrishnan 2013). These findings are in line with the results reported by Ali and Abbasi (2014) in the cell culture of Artemisia absinthium, where dark-grown cultures showed maximum TPC, TFC, and antioxidant activity compared to light treatments. Weremczuk- Jeżyna et al. (2013) also reported that the hairy roots of Dracocephalum moldavica cultivated in the darkness showed the highest antioxidant activity compared to those cultivated under photoperiod. Very few studies about the effect of light and dark on secondary metabolites accumulation and the antioxidant activity in callus tissue culture are reported. The increased antioxidant activity under darkness in the present study could be attributed to the stressed callus culture conditions. Indeed, there is increasing evidence that some light conditions such as UV, high-intensity light, and continuous dark may act as a stressful factor requiring a change in the oxidative balance in plants which produce a large amount of scavenging enzymes and antioxidant molecules as an answer to the increased generation of reactive oxygen species (ROS), and thereby minimise potential oxidative damage (Gogorcena et al. 1997; Close and McArthur 2002; Ali et al. 2005; Pashkovskiy et al. 2018). Dark stress was reported to induce structural and oxidative damage in other Fabaceae species such as Soybean and Phaseolus vulgaris (Gogorcena et al. 1997; Wang et al. 2021). Our results are in good agreement with a previous report considering the antioxidant activity in intact carob leaves, pulp, and seeds (Hajaji et al. 2010; Lakkab et al. 2019; Ayache et al. 2020). The DPPH scavenging activity reached 61.17 %, 80.9 % and 79.4 % respectively. This antioxidant activity may be attributed to the levels of phenolic content, flavonoids, and condensed tannin. Based on the correlation coefficient values calculated for the total phenolic components and the antioxidant assays, it could be suggested that both the scavenging activity and the reducing power of the analyzed extracts are strongly and positively correlated with the phenolic (r2 = 0.84 and 0.98, respectively) and flavonoid accumulation (r2 = 0.71 and 0.68, respectively) (Table 3). These results agree with the earlier reports on carob pulp extract (Benchikh et al. 2014; Benchikh and Louailèche 2014). In contrast, significant (P<0.01) negative relationships were observed of TCT tannins with TPC, TFC, DPPH, and FRAP, with correlation coefficients ranging between -0.86 and -0.73. Furthermore, the FRAP was significantly 6 Nova Biotechnol Chim (2021) 20(2): e652 5 P<0.01) correlated with DPPH (r2 = 0.88) which rises the hypothesis that compounds which express reducing activity meet the criteria for exhibiting antiradical effect. Table 2. Effects of explant type and light conditions on antioxidant compounds in callus cultures of carob (Ceratonia siliqua L.). Values are mean ± SE. Mean values within a column followed by the same letter are not significantly different by Duncan’s multiple range test (5m%). FW – fresh weight; DW – dry weight; OD – optical density; GAE – gallic acid equivalents; QE – Quercetine equivalents; CE – catechin equivalent; NS – nonsignificant; ∗, ∗∗, ∗∗∗ – significant at P≤0.05, P≤0.01, and P≤0.001, respectively (two-way ANOVA). Table 3. Pearson’s correlation coefficients of different biochemical parameters of carob calli extracts. TPC TFC TCT DPPH TPC - - - - TFC 0.78** - - - TCT -0.73** -0.80** - - DPPH 0.84** 0.71** -0.86** - FRAP 0.98** 0.68* -0.78** 0.88** * Correlation is significant at the 0.05 level, ** Correlation is significant at the 0.01 level. Conclusion The present study demonstrates, for the first time, the potential of callus cultures of Ceratonia siliqua as a new source of phenolic compounds which possesses an important antioxidant activity. Leaves-derived calli were found to produce significantly higher levels of phenolics and showed higher antioxidant activity than the cotyledon- derived callus. However, cotyledon-derived callus accumulated more TCT. The light treatment has significantly increased total condensed tannins content, but significantly decreased the antioxidant activity in callus cultures. Moreover, a positive correlation between antioxidant activity and total phenolics and flavonoids contents were found in carob callus cultures. Further works on improving protocols for in vitro cultivation of carob and characterization of individual groups of bioactive components responsible for the biological activity are needed. Conflict of Interest The authors declare that they have no conflict of interest. References Aboura I, Nani A, Belarbi M, Murtaza B, Fluckiger A, Dumont A, Benammar C, Tounsi MS, Ghiringhelli F, Rialland M, Khan NA, Hichami A (2017) Protective effects of polyphenol-rich infusions from carob (Ceratonia siliqua) leaves and cladodes of Opuntia ficus-indica against inflammation associated with diet-induced obesity and DSS-induced colitis in Swiss mice. Biomed. Pharmacother. 96: 1022-1035. Adil M, Ren X, Jeong BR (2019) Light elicited growth, antioxidant enzymes activities and production of medicinal compounds in callus culture of Cnidium officinale Makino. J. Photochem. Photobiol. B: Biology 196: 111509. Akdeniz E, Yakişik E, Pirouzian HR, Akkin S, Turan B, Tipigil E, Toker O, Ozcan O (2021) Carob powder as cocoa substitute in milk and dark compound chocolate formulation. J. Food Sci. Technol. 233: 1-9. Ali M, Abbasi BH (2014) Light-induced fluctuations in biomass accumulation, secondary metabolites production and antioxidant activity in cell suspension cultures of Artemisia absinthium L. J. Photochem. Photobiol. B: Biol. 140: 223-227. Ali MB, Hahn E-J, Paek K-Y (2005) Effects of light intensities on antioxidant enzymes and malondialdehyde content during short-term acclimatization on micropropagated Phalaenopsis plantlet. Environ. Exp. Bot. 54: 109-120. Type of explant Light conditions DPPH [%] FRAP [OD] Mature cotyledons 16-h Photoperiod 60.92 ± 4.98c 0.29 ± 0.04c Darkness 76.60 ± 7.14b 0.38 ± 0.01b Leaves 16-h Photoperiod 86.93 ± 1.91a 0.65 ± 0.02a Darkness 88.82 ± 0.10a 0.61 ± 0.01a Significance of two-way ANOVA Type of explants (A) *** *** Light conditions (B) ** NS A×B * ** 7 Nova Biotechnol Chim (2021) 20(2): e652 8 Ayache S ben, Saafi EB, Emhemmed F, Flamini G, Achour L, Muller CM (2020) Biological activities of aqueous extracts from carob plant (Ceratonia siliqua L.) by antioxidant, analgesic and proapoptotic properties evaluation. Molecules. 25: 3120. Benchikh Y, Louailèche H (2014) Effects of extraction conditions on the recovery of phenolic compounds and in vitro antioxidant activity of carob (Ceratonia siliqua L.) pulp. Acta Bot. Gall. 161: 175-181. Benchikh Y, Louaileche H, George B, Merlin A (2014) Changes in bioactive phytochemical content and in vitro antioxidant activity of carob (Ceratonia siliqua L.) as influenced by fruit ripening. Ind. Crops Prod. 60: 298-303. Brand-Williams W, Cuvelier M-E, Berset C (1995) Use of a free radical method to evaluate antioxidant activity. LWT- Food Sci. Technol. 28: 25-30. Broadhurst RB, Jones WT (1978) Analysis of condensed tannins using acidified vanillin. J. Sci. Food. Agric. 29: 788-794. Castro AHF, Braga K de Q, de Sousa FM, Coimbra MĆ, Changas RCR (2016) Callus induction and bioactive phenolic compounds production from Byrsonima verbascifolia (L.) DC. (Malpighiaceae). Revista Ciência Agronômica. 47: 143-151. Close DC, McArthur C (2002) Rethinking the role of many plant phenolics-protection from photodamage not herbivores? Oikos. 99: 166-172. Correia PJ, Saavedra T, Gama F, da Graça Miguel M, de Varennes A, Pestana M (2018) Biologically active compounds available in Ceratonia siliqua L. grown in contrasting soils under Mediterranean climate. Sci. Hortic. 235: 228-234. Custódio L, Fernandes E, Romano A (2007) Quantification of polyphenols in carob tree (Ceratonia siliqua L.) fruits and leaves in Portuguese cultivars. Acta Hortic. 841: 503-506. Custódio L, Fernandes E, Romano A (2009) Quantification of polyphenols in carob tree (Ceratonia siliqua L.) fruits and leaves in portuguese cultivars. Acta Hortic. 841:503–506. Custódio L, Patarra J, Alberício F, Rosa N (2015) In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, α- amylase and α-glucosidase. Nat. Prod. Res. 29: 2155– 2159. Dias MI, Sousa MJ, Alves RC, Ferreira ICFR (2016) Exploring plant tissue culture to improve the production of phenolic compounds: A review. Ind. Crops Prod. 82: 9-22. Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut rootstock. HortScience 19: 507-509. El Bouzdoudi B, El Ansari ZN, Mangalagiu I, Mantu D, Badoc A, Lamarti A (2016) Determination of Polyphenols Content in Carob Pulp from Wild and Domesticated Moroccan Trees. Am. J. Plant Sci. 7: 71135. El Hajaji H, Lachkar N, Alaoui K, Cherrah Y, Farah A, Ennabili A, El Bali B, Lachkar M (2011) Antioxidant activity, phytochemical screening, and total phenolic content of extracts from three genders of carob tree barks growing in Morocco. Arab. J. Chem. 4: 321-324. Estell RE, Fredrickson EL, James DK (2016) Effect of light intensity and wavelength on concentration of plant secondary metabolites in the leaves of Flourensia cernua. Biochem. Syst. Ecol. 65: 108-114. Farhadi N, Panahandeh J, Azar AM, Salte SA (2017) Effects of explant type, growth regulators and light intensity on callus induction and plant regeneration in four ecotypes of Persian shallot (Allium hirtifolium). Sci. Hortic. 218: 80- 86. Favre J-M, Scalbert A, Hervé du Penhoat CLM (1991) Quercus spp. (Oak): In Vitro culture and production of tannins. In Bajaj YPS. (Eds.), Medicinal and aromatic plants V. Biotechnology in agriculture and forestry, Springer, Berlin, Heidelberg, pp. 300-312. Fazal H, Abbasi BH, Ahmad N, Ali M, Ali S (2016) Sucrose induced osmotic stress and photoperiod regimes enhanced the biomass and production of antioxidant secondary metabolites in shake-flask suspension cultures of Prunella vulgaris L. Plant Cell Tissue Organ Cult. 124: 573-581. Fidan H, Petkova N, Sapundzhieva T, Baeva M, Goranova Z, Slavov A, Hrastev L (2020) Carob syrup and carob flour (Ceratonia siliqua L.) as functional ingredients in sponge cakes. Carpathian J. Food Sci. Technol. 12: 58-68. Gai Q-Y, Jiao J, Wang X, Fu Y-J, Lu Y, Liu J, Wang Z-Y, Xu X-J (2020) Establishment of Cajanus cajan (Linn.) Millsp. cell suspension cultures as an effective in vitro platform for the production of pharmacologically active phenolic compounds. Ind. Crops Prod. 158: 112977. Gamborg OL, Miller R, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151–158. Germec M, Karhan M, Demirci A, Turhan I (2020) Implementation of flexible models to bioethanol production from carob extract-based media in a biofilm reactor. Biomass Convers. Biorefin. 1-17. Ghanemi FZ, Belarbi M, Fluckiger A, Nani A, Dumont A, De Rosny C, Aboura I, Khan AS, Murtaza B, Benammar C, Lahfa BF, Patoli D, Delmas D, Rébé C, Apétoh L, Akhtar Khan N, Ghringhelli F, Rialland M, Hichami A (2017) Carob leaf polyphenols trigger intrinsic apoptotic pathway and induce cell cycle arrest in colon cancer cells. J. Funct. Foods 33: 112-121. Gogorcena Y, Gordon AJ, Escuredo PR, Minchin FR, Witty JF, Moran JF, Becana M (1997) N2 fixation, carbon metabolism, and oxidative damage in nodules of dark- stressed common bean plants. Plant Physiol. 113: 1193- 1201. Gourguillon L, Rustenholz C, Lobstein A, Gondet L (2018) Callus induction and establishment of cell suspension cultures of the halophyte Armeria maritima (Mill.) Willd. Sci. Hortic. 233: 407-411. Govindan P, Muthukrishnan S (2013) Evaluation of total phenolic content and free radical scavenging activity of Boerhavia erecta. J. Acute Med. 3: 103-109. Gurel F, Ekici H, Dikmen BY (2021) Cytotoxic and antitumoral effect of Ceratonia siliquracta L.(carob) extract. J. Appl. Biol. Sci. 15: 225-234. Hajaji H el, Lachkar N, Alaoui K, Cherrah Y, Farah A, Ennabili A, El Bali B, Lachkar M (2010) Antioxidant properties and total phenolic content of three varieties of carob tree leaves from Morocco. Rec. Nat. Prod. 4: 193- Nova Biotechnol Chim (2021) 20(2): e652 9 204. Jiménez VM, Bangerth F (2001a) Endogenous hormone concentrations and embryogenic callus development in wheat. Plant Cell Tissue Organ Cult. 67: 37-46. Jiménez VM, Bangerth F (2001b) Endogenous hormone levels in explants and in embryogenic and non- embryogenic cultures of carrot. Physiol. Plant. 111: 389- 395. Ju HJ, Jeyakumar J, Kamaraj M, Praveen N, Chung I-M, Kim S-H, Thiruvengadam M (2014) High frequency somatic embryogenesis and plant regeneration from hypocotyl and leaf explants of gherkin (Cucumis anguria L.). Sci. Hortic. 169: 161-168. Korkmaz N, Akin M, Koç A, Eyduran SP, Ilhan g, Sagbas HI, Ercisli S (2020) Morphological and biochemical diversity among wild-grown carob trees (Ceratonia siliqua L.). Folia Hortic. 22: 69-78.. Ksia E, Harzallah-Skhiri F, Verdeil JL, Gouta H, Alemanno L, Bouzid S (2008) Somatic embryo production from immature seeds of carob (Ceratonia siliqua L.): Histological evidence. J. Hortic. Sci. Biotechnol. 83: 401- 406. Kyratzis AC, Antoniou C, Papayiannis LC, Graziani G, Rouphael Y, Kyriacou MC (2021) Pod morphology, primary and secondary metabolite profiles in non-grafted and grafted carob germplasm rre configured by agro- environmental zone, genotype, and growing season. Front. Plant Sci.11: 2199. Kyriacou MC, Antoniou C, Rouphael Y, Grazani G, Kyratzis A (2021) Mapping the primary and secondary metabolomes of carob (Ceratonia siliqua L.) fruit and its postharvest antioxidant potential at critical stages of ripening. Antioxidants.10: 1-21. Lakkab I, El Hajaji H, Lachkar N, Lefter R, Ciobica A, El Bali B, Lachkar M (2019) Ceratonia siliqua L. seed peels: Phytochemical profile, antioxidant activity, and effect on mood disorders. J. Funct. Foods. 54: 457-465. Lamaison JL, Petitjen-Freytet C, Carnat A (1990) Teneurs en acide rosmarinique, en dérivés hydroxycinnamiques totaux et activité antioxydante chez les apiacées, les borraginacées et les laminacées médicinales. Ann. Pharm. Fr. 48: 103-108. Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. In: Combined Proceedings, International Plant Propagators’ Society. pp 421–427. Lozzi A, Abdelwahd R, Alami-Halimi D, Mentag R, Abousalim A (2018) Optimization of a mature cotyledons- based in vitro culture system for embryogenic-callus induction in carob (Ceratonia siliqua L.). Rev. Chapingo Ser. Cienc. For. Ambiente. 25: 71-84. Lozzi A, Abdelwahd R, Mentag R, Abousalim A, (2019) Development of a new culture medium and efficient protocol for in vitro micropropagation of Ceratonia siliqua L. In Vitro Cell. Dev. Biol. Plant. 55: 615-624. Lozzi A, Abousalim A, Abdelwahd R (2015) Effet de 2, 4-D sur l’induction de l’embryogenèse somatique à partir de cotylédons matures de caroubier (Ceratonia siliqua L.). Rev. Mar. Sci. Agron. Vét. 3: 24-29. Meziani S, Oomah BD, Zaidi F, Simon-Levertd A, Bertrande C, Zaidi-Yahiaoui R (2015) Antibacterial activity of carob (Ceratonia siliqua L.) extracts against phytopathogenic bacteria Pectobacterium atrosepticum. Microb. Pathog. 78: 95-102. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15:473–497. Ortega N, Macià A, Romero M, Trullols E, Morello JR, Anglès N, Motilva M-J (2009) Rapid determination of phenolic compounds and alkaloids of carob flour by improved liquid chromatography tandem mass spectrometry. J. Agric. Food Chem. 57: 7239-7244. Othmen KB, Elfalleh W, Garcia Beltrán JM, Esteban MÁ, Haddad M (2020) An in vitro study of the effect of carob (Ceratonia siliqua L.) leaf extracts on gilthead seabream (Sparus aurata L.) leucocyte activities. Antioxidant, cytotoxic and bactericidal properties. Fish Shellfish Immunol. 99: 35-43. Oyaizu M (1986) Studies on products of browning reaction: antioxidative activity of products of browning reaction. Jpn. J. Nutr. 44: 307-315. Papagiannopoulos M, Wollseifen HR, Mellenthin A, Haber B, Galensa R (2004) Identification and quantification of polyphenols in carob fruits ( Ceratonia siliqua L .) and derived products by HPLC-UV-ESI / MSn. J. Agric. Food Chem. 52:3784–3791. Pashkovskiy PP, Soshinkova TN, Korolkova D V, Kartashov AV, Zlobin IE, Lyubimov VY, Kreslavski VD, Kuznetsov VV (2018) The effect of light quality on the pro- /antioxidant balance, activity of photosystem II, and expression of light-dependent genes in Eutrema salsugineum callus cells. Photosynth. Res. 136: 199-214. Rameshkumar R, Satish L, Pandian S, Kartashov AV, Zlobin IE., Lyubimov VY, Kreslavski VD, Kuznetsov VV (2018) Production of squalene with promising antioxidant properties in callus cultures of Nilgirianthus ciliatus. Ind. Crops. Prod. 126: 357-367. Rtibi K, Selmi S, Grami D, Amri M, Eto B, El-Benna J, Sebai H, Marzouki L (2017) Chemical constituents and pharmacological actions of carob pods and leaves (Ceratonia siliqua L.) on the gastrointestinal tract: A review. Biomed. Pharmacother. 93: 522-528. Saeedpour A, Godehkahriz SJ, Lohrasebi T, Esfahani K, Salmanian AH, Razavi K (2021) The Effect of endogenous hormones, total antioxidant and total phenol changes on regeneration of barley cultivars. Iran. J. Biotechnol. 19:e2838. Sargin SA, Büyükcengiz M (2019) Plants used in ethnomedicinal practices in Gulnar district of Mersin, Turkey. J. Herb. Med. 15: 100224. Shehzad M aamir, Khan MA, Ali A, Mohammad S, Noureldeen A, Darwish H, Ali A, Ahmad A, Khan T, Khan RS (2021) Interactive effects of zinc oxide nano particles and different light regimes on growth and silymarin biosynthesis in callus cultures of Silybum marianum L. Artif. Cells Nanomed. Biotechnol. 49: 523- 535. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics Nova Biotechnol Chim (2021) 20(2): e652 8 with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16: 144-158. Stavrou IJ, Christou A, Kapnissi-christodoulou CP (2018) Polyphenols in carobs: A review on their composition, antioxidant capacity and cytotoxic effects, and health impact. Food Chem. 269: 355-374. Su N, Wu Q, Cui J (2014) Effects of monochromatic LED light quality on endogenous hormone content in leaves and hypocotyl of Cucumis sativus L. Chin. J. Appl. Environ. Biol. 20: 45–49. Tabart J, Kevers C, Dardenne N, Chini-Kerth V, Albert A, Dommes J, Defraigne J-O, Pincemail J (2014) Deriving a global antioxidant score for commercial juices by multivariate graphical and scoring techniques: applications to blackcurrant juice. In Preedy V. (Eds.), Processing and impact on antioxidants in beverages, Elsevier, London, UK, pp. 301-307. Tabart J, Kevers C, Pincemail J, Defraigne J-O, Dommes J (2009) Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chem. 113: 1226-1233. Vinterhalter D, Savić J, Stanišić M, Vinterhalter B, Dobrev PI, Motyka V (2020) Diurnal rhythmicity of endogenous phytohormones and phototropic bending capacity in potato (Solanum tuberosum L.) shoot cultures. Plant Growth Regul. 90: 151-161. Wang P, Hou S, Wen H, Hou S, Wen H, Hou Q, Li G (2021) The stay-green mutation contributes to enhanced antioxidative competence and delays leaf senescence in soybean hybrid Z1. Internat. J. Agric. Biol. 25: 361-372. Weremczuk-Jeżyna I, Grzegorczyk-Karolak I, Frydrych B, Królicka A, Wysokińska H (2013) Hairy roots of Dracocephalum moldavica: rosmarinic acid content and antioxidant potential. Acta Physiol. Plant. 35: 2095-2103. Wu G-Y, Wei X-L, Wang X, Wei Y (2020) Induction of somatic embryogenesis in different explants from Ormosia henryi Prain. Plant Cell Tiss. Org. Cul. 142: 229-240. Zouari N, El Mtili N (2020) In Vitro propagation of mature carob trees (Ceratonia siliqua L.) from the axillary buds. Am. J. Plant Sci. 11: 1369. 10