Micropropagation and GC–MS analysis of bioactive compounds in bulbs and callus of white squill

Medicinal herbs are the main source of bioactive compounds used in the medical industry. White squill (Urginea maritima) is an important medicinal and ornamental plant cultivated in the Mediterranean region. This study reports an efficient protocol for in vitro propagation of Urginea and investigates important bioactive compounds present in the bulbs and in vitro–produced callus. The least number of days for callus induction and shoot regeneration was achieved with Murashige and Skoog (MS) basal media supplemented with 1.0 mg L−1 1-naphthaleneacetic acid (NAA) plus 0.1 mg L−1 6-benzylaminopurine (BAP) and 1.0 mg L−1 NAA plus 0.4 mg L−1 BAP, respectively, while the highest number of shoots and fresh weight were obtained at medium supplemented with 1.0 mg L−1 NAA plus 0.5 mg L−1 BAP. Gas chromatography-mass spectrometry (GC–MS) analysis of Urginea bulb methanol extract showed the existence of important secondary metabolites, such as palmitic acid (C16H32O2), 9-hexadecenoic acid (C16H30O2), phthalic acid 2-ethylhexyl propyl ester (C19H28O4), tetradecanoic acid (C14H28O2), undecanoic acid (C11H22O2), and oleic acid (C18H34O2), in addition to other important compounds, such as 13-heptadecyn-1-ol, 9,12-octadecadienoic acid, 1-monolinoleoylglycerol trimethylsilyl ether, 2-methyl-1-hexadecanol, and octadecanoic acid. Callus methanol extracts showed a reduction in the percentages of most phyto-components compared to bulb extract except for oleic acid, 3-(octadecyloxy) propyl ester and 3-hydroxydodecanoic acid; on the other hand, some important compounds were detected only in callus extract possessing anti-cancer, antiviral, and anti-inflammatory effects, such as farnesol (C15H26O), 7-methyl-Z-tetradecen-1-ol acetate (C17H32O2), ethyl iso-allocholate (C26H44O5), 4-trifluoroacetoxypentadecane (C17H31F3O2), and 2-hydroxyhexadecanoic acid (C16H32O3).


Introduction
Medicinal plants are considered the main reservoir for useful and new chemical compounds that can be used in modern drug formulation. Urginea maritima (L.) Baker or Drimia maritima, also known as white squill, which belongs to the family Asparagaceae, is a medicinal and ornamental plant abundant in the Mediterranean area and North Africa in sandy soils and hills. It is a winter perennial and onionlike bulbous plant with large dark green leaves, grows from autumn to spring, and produces flowers after the growth of leaves; the flowers are found in dense clusters. After spring, the bulbs enter a dormancy stage during the summer (Aasim et al. 2008). The plant is used as a folk medicine in different countries, and it is a source of many bioactive compounds present in its bulbs (Reddy et al. 2013).
Genus Urginea includes approximately 110 bulbous geophytic species geographically distributed across Africa, Madagascar, the Mediterranean basin, and Asia. The majority of the species (93 in total) are native to Africa. In India, eight species of the genus Drimia have been identified (Yadava et al. 2021). Drimia species share striking morphological similarities, leading to taxonomic misunderstandings. The principal bufadienolides (scillaren A and proscillaridin A) were isolated from four Indian squill species, D. indica, D. coromandeliana, D. polyantha, and D. razii, where D. coromandeliana possessed the highest bufadienolide content while 41 bufadienolides were isolated from the African squill, D. maritima (Partha and Sumita 2019). Jha and Sen (1981) reported that scillaren A and proscillaridin A have been isolated from the bulbs of all cytotypes of Indian squill, but scilliphaeoside and anhydroscilliphaeosidin have only been found in the bulbs of tetraploid cytotypes. These compounds, however, were not present in the leaves. Urginea closely resembles Digitalis as it diminishes the force and frequency of the heart's action (Kopp et al. 1996). From the phytochemical point of view, U. maritima contains 1.0 to 3.0% cardiac glycosides, anthocyanins, fatty acids, flavonoids, polysaccharides, and calcium thus justifying their tonic action and diuretic actions (Iizuca et al. 2001). Prabakaran et al. (2016) reported that Urginea bulbs are used for arthritis and the removal of free carcinogenic radicals through the existence of antioxidants.
Plant tissue culture becomes a powerful technique for propagation, production of secondary metabolites, and protection of threatened and wild-growing species (Reddy et al. 2013). Callogenesis proffered a new and continuous method for secondary metabolite production (Isahm et al. 2018).
Using bulb scales as a traditional method for Urginea propagation is very slow and time-consuming. After 6 yr of growth, the Urginea bulbs are harvested, yielding approximately 25,000 bulbs per ha. If the bulb is cut into wedge sections, only a limited number of propagules can be obtained; therefore, using tissue culture as an alternative way for propagation becomes essential, and research on the micropropagation of U. maritima species is so limited (Aasim et al. 2008).
Tissue culture of plant material has several advantages over traditional plant propagation, including a constant supply of fresh material regardless of the season or the plant's reproductive cycle, a continuous source of plant pharmaceuticals that is not limited by time or space, conserving plant species subjected to extinction, larger-scale extracted metabolites free of pathogens and environmental pollutants, and plant propagation is possible in areas where agriculture is not normally feasible (Mohamed et al. 2022).
GC-MS is a powerful technique used for the identification and quantification of bioactive compounds in plant extract. GC-MS had been applied to the analysis of different medicinal herbs and proved to be a valuable method for detecting different hydrocarbons, volatile oil, acids, esters, alkaloids, fatty acids, and amino and nitro-compounds (Srivastava et al. 2015;Saravanakumar et al. 2016). Analysis of U. maritima bulbs using GC-MS showed the existence of various secondary metabolites such as organic acids, phenolic compounds, phytosterols, esters, and ketones (Prabakaran et al. 2016). The present study was undertaken to establish an efficient micropropagation and callus induction protocol for Urginea maritima and to detect the most important bioactive compounds in the bulb and in in vitro-produced callus extract through GC-MS analysis.

Plant Material
The present study was conducted at the tissue culture and biotechnology lab, Department of Floriculture, Faculty of Agriculture, Alexandria University, Egypt, during the period from 2016 to 2021. Urginea maritima bulbs were collected at a depth of 50 to 70 cm under the soil surface from coordinates 30° 55′ 25.4″ N, 29° 15′ 6.84″ E North coast, Matrouh governorate, Egypt, in December and January 2016/2017 and repeated the next year at the same time. All collected bulbs were homogenous in size with an average weight (100 g) and average dimensions (11.4 cm height and 9.3 cm diameter) and maintained in the nursery of the Floriculture Department, Faculty of Agriculture, Alexandria University, Egypt, where they were used later as the source of the explants.

Source and Sterilization of Explants
Disc plates were excised from U. maritima bulbs and used as explants. The average number of bulb leaves per bulb ranged from 25 to 35 scaly leaves. The bulbs were first washed thoroughly under running tap water for 15 min to remove any foreign materials (dirt, soil, and dry scales). The excised explants were washed using a home detergent for 30 min, followed by washing with fungicide 5% Benlate (Benomyl 50%WP 17,804-35-2, Awiner Biotech Co. Ltd., Yuhua District, Shijiazhuang City, China) with 2 drops of the wetting agent Tween 20 (Sigma-Aldrich, Saint Louis, MO) for 30 min, then surface sterilized by immersing in 70% ethyl alcohol for 5 min, followed by 0.1% mercuric chloride (Chemajet Chemical Co. Alexandria, Egypt) with 2 drops Tween 20 for 15 min, and finally rinsed three times with sterilized distilled water to remove any residues of ethanol or mercuric chloride.

Micropropagation-Stages Callus Initiation Stage
Disc plates of about 1.0 cm 2 of U. maritima were inoculated individually into solidified Murashige and Skoog (MS) medium (MSP09-50L, Caisson Labs, Smithfield, UT) (Murashige and Skoog 1962) supplemented with 30.0 g·L −1 sucrose, 2.0 g·L −1 phytagel, and a combination of two plant growth regulators with a total of 16 treatments (6-benzylaminopurine (BAP) at 0.0, 0.1, 0.2, and 0.3 mg L −1 and naphthaleneacetic acid (NAA) at 0.0, 1.0, 2.0, and 3.0 mg L −1 ). The pH of the media was adjusted to 5.8 ± 0.1 using a pH meter (211 Hanna Instruments, Cluj-Napoca, Romania), then the media were autoclaved at 121 ± 1 °C for 20 min under the pressure of 1.5 bar cm -2 (Daihan Labtech Co., LTD., model LAC-5082SE, Namyangju City, Kyonggi-Do, Korea). Explants were inoculated on MS medium, a single explant per jar of dimensions 9 cm height and 5 cm diameter. All jars were capped and sealed with parafilm (Laboratory zfilm Pechiney Plastic Packaging, Chicago, IL) and incubated in the growth room. The ability of the explants to develop callus under various plant growth regulator concentrations was measured after 12 wk of inoculation while the callus fresh weight was measured after 2 wk from the beginning of the callus induction in each treatment (Fig. 1).

Multiplication Stage and Culture Conditions
The calluses from the initiation stage were sectioned into pieces about 0.5 cm and re-cultured on MS basal medium containing 30 g L −1 sucrose and 2 g·L −1 phytagel, and supplemented with 0.0, 0.3, 0.4, or 0.5 mg L −1 BAP and 0.0, 1.0, 2.0, or 3.0 mg L −1 NAA with a total of 16 treatments. The pH was adjusted to 5.8 ± 0.1 and autoclaved at 121 ± 1 °C for 20 min (Fig. 1). Culture jars were incubated in the growth room at 25 ± 1 °C and illuminated with fluorescent lamps located 40 cm above the culture jars, giving an average irradiance of 55 to 56 µmole m −2 S −1 depending on the age of the lamp, for 16/8 h light/dark photoperiod respectively and relative humidity was 80%. Bozorgi et al.'s (2015) method was used for extraction with a modification, and fresh squill bulbs were cleaned thoroughly to remove any soil or debris. Bulbs were dried in an oven at 70 °C for approximately 72 h. The dried plant material was ground to a fine powder, and 30.0 g of the powder was suspended in 100 mL of 95% methanol. The obtained solution was incubated at 50 to 60 °C for 1 h and concentrated by evaporating methanol. Obtained calluses were used for the extraction and quantification of bioactive compounds by the same method. The modification was done using centrifugation at 3000 rpm for 5 min to purify the extract.

GC-MS Analysis
A diluted volume of 1.00 μL was injected into the system using an AI-3000 auto-injector with split injection mode (1:30); the injector temperature was 250 °C. The test was run using an ultra-trace G.C ISQMS, Thermo Fisher, Dreieich, Germany, with a TG5sil/ms capillary column of 30 m length. Helium gas was used as the carrier gas with a total flow of 1.5 mL (column flow of 1.5 mL·min −1 ). The oven temperature was programmed from 40 °C (1 min hold time) to 180 °C (1 min hold time) and then to 200 °C (2 min hold time). The total running time was 50 min for the ISQ mass spectroscopic test; through the transfer line (250 °C), the ion-source temperature was 230 °C. Solvent cut time was 2.00 min; the detector gain was 0.70 kV. Compounds were identified by their mass spectra and retention indices using the NIST Mass Spectral Library and the Retention Index Database. GC-MS Insight xcalibur software package was used to process the data (Shwaish and Al-Imarah 2017).

Total Phenolic Content
The total phenolic content of methanolic extract of U. maritima bulbs was determined according to the Folin-Ciocalteu method. The methanol extract of the samples (0.2 mL, 100.0 µg·mL −1 ) was mixed with Folin-Ciocalteu reagent (2.0 mL, 1:10 diluted with distilled water). After 5 min, a saturated solution of NaHCO 3 (1.5 mL, 60 g·L −1 distilled water) was added. At room temperature, all mixtures were allowed to stand for 90 min, then the absorption was measured at 725 nm using a spectrophotometer (Unico W49376 Spectrophotometer 1200, Shanghai, China). The total phenolic content was calculated as milligrams of gallic acid equivalents (GAE) per gram of dried extract; the same procedures were repeated for the callus samples (Tofighi et al. 2016).

Statistical Analysis
All the experiments carried out during this study were designed as factorial arrangement of treatments with NAA as the main factor and BAP as the sub-factor (4 NAA levels × 4 BAP levels = 16 treatments, 8 replicates were used per treatment with a total of 128 experimental units for each of the initiation and multiplication stages). The experiment layout was a completely randomized design (CRD) according to Snedecor and William (1967). Recorded data were analyzed statistically by using analysis of variance (ANOVA) with SAS software (copyright 2002 by SAS Institute Inc., Cary, NC), the averages were compared by the least significant difference (L.S.D.) (Steel and Torrie 1981), and the significance level was determined at p ≤ 0.05.

Results
The Effect of BAP and NAA Levels in Medium on Callus Induction and Shoot Regeneration. Table 1 shows the effect of BAP and NAA levels on the day for callus induction, callus fresh weight, and total phenolic content. Days for callus induction were inversely proportional to NAA concentrations, and the least number of days for callus induction (87 d) was obtained at 1.0 mg L −1 NAA plus 0.1 mg L −1 BAP (Fig. 2a). The callus fresh weight increased significantly by increasing NAA concentration reaching the highest value (2.24 g) after culture on medium containing 2.0 mg L −1 NAA plus 0.1 mg L −1 BAP. NAA at 3.0 mg L −1 caused a reduction in fresh weight and delayed callus induction. The greatest total phenolic content was obtained at the lowest concentrations of NAA and BAP (1.0 and 0.1 mg L −1 ), respectively. Increasing the NAA or BAP caused a significant reduction in phenolic content in callus. The fastest callus with the highest phenolic content produced at 1.0 mg L −1 NAA plus 0.1 mg L −1 BAP from the callogenesis stage was used as explant for the multiplication stage. Table 2 shows the effect of BAP and NAA levels on the number of days for shoot proliferation, the number of shoots, shoot height, shoot fresh weight, and total phenolic content; 1.0 mg L −1 NAA plus 0.4 mg L −1 BAP gave the least number of days for shoot formation (42.5 d) (Fig. 2b, c) while the highest number of shoots was obtained at 1.0 mg L −1 NAA plus 0.5 mg L −1 BAP, reaching 5.5 shoots with the highest fresh weight (Fig. 2d). The shoot height increased significantly by increasing NAA concentration and then declined at the highest BAP concentration. The total phenolics increased by increasing NAA and BAP concentrations, reaching their peak at 2.0 mg L −1 NAA plus 0.5 mg L −1 BAP. Tables 3 and 4 show the GC-MS analysis of U. maritima bulbs and callus methanolic extracts indicating a list of 28 and 25 different bioactive compounds, respectively, which are used pharmaceutically as antibacterial, antimicrobial, anti-inflammatory, antiasthma, diuretic, and antioxidant agents; for the prevention of certain cancers and for heart protection; as antimalaria agent and dermatologic agent against acne; for the prevention of hypocholesterolemia; and for the prevention and treatment of diabetic retinopathy (Saravanakumar et al. 2016;Prabakaran et al. 2016;Ambrin et al. 2019    2.70%; 9,12-octadecadienoic acid, 2.51%; 1-monolinoleoylglycerol trimethylsilyl ether, 2.46%; 2-methyl-1-hexadecanol, 1.99%; deacetylvindoline, 1.86%; octadecanoic acid, 1.53%; 3-(octadecyloxy) propyl ester, 1.00%; and ajmalicine, 0.4%, along with different compounds identified by GC-MS (Table 3).

The Identification of Bioactive Compounds in Callus and Bulbs of White Squill
On the other hand, the phyto-components identified in callus methanol extracts showed a reduction in the percentages of most active ingredients compared to the bulb extracts except for oleic acid, 3-(octadecyloxy) propyl ester, 3-(octadecyloxy) propyl ester, and 3-hydroxydodecanoic acid as their concentrations were higher in the callus extract. Also, some compounds were detected only in callus extract, such as farnesol, 5.87%; 7-methyl-Z-tetradecen-1-ol acetate, 1.70%; ethyl iso-allocholate, 1.15%; 4-trifluoroacetoxypentadecane, 0.46%; and 2-hydroxyhexadecanoic acid, 0.31% (Figs. 3 and 4). Shah et al. (2022) reported that auxins have a very important role in callus formation while suppression in the percentage of callus induction occurred at high auxin concentrations. It was observed in this study that medium containing 1.0 mg L −1 NAA plus 0.1 mg L −1 BAP accelerated callus induction while 3.0 mg L −1 NAA caused a delay in callus induction, and the maximum callus fresh weight was obtained with 2.0 mg L −1 NAA plus 0.1 mg L −1 BAP (Table 1).

Discussion
In this study, it was found that increasing the concentration of BAP with or without NAA promoted shoot number and height except for the highest BAP concentration (0.5 mg L −1 ) as it suppressed shoot height. The highest number of shoots was obtained at 1.0 mg L −1 NAA plus 0.5 mg L −1 BAP (Table 2, Fig. 2d). Similar results were obtained by Han et al. (2004) and Nakano et al. (2000). They mentioned that generally cytokinins at high concentration reduce and inhibit shoot formation from bulb scales.
Increasing NAA concentration caused a delay in days for shoot proliferation while 1.0 mg L −1 NAA plus 0.4 mg L −1 BAP accelerated shoot formation (Table 2, Fig. 2c). Similarly, days for shoot formation in Peperomia obtusifolia produced in vitro were delayed at 5 mg L −1 NAA while declining NAA concentration led to a significant reduction in the number of days for shoot formation (El-Naggar and Osman 2014).
The greatest total phenolic content accumulation was detected in callus and regenerated shoots cultured on medium containing 1.0 mg L −1 NAA plus 0.1 mg L −1 BAP and 2.0 mg L −1 NAA plus 0.5 mg L −1 BAP, respectively (Tables 1 and 2). Taiz and Zeiger (2002) mentioned that phenylalanine and phenylalanine ammonia-lyase (PAL) are important in the biosynthesis of phenolic compounds as well RT retention time   as natural auxins, like indoleacetic acid (IAA), through the shikimic pathway; meanwhile, synthetic auxins, such as NAA, have a similar effect to IAA, and adding NAA to the media may affect the PAL enzyme, which is a precursor of phenolic compounds, resulting in an increase in total phenol production. Reddy et al. (2013) indicated that combining NAA and BAP is effective in callus induction as well as increasing bioactive compound production. GC-MS analysis for U. maritima bulbs and callus methanol extract showed the existence of 28 and 25 important bioactive products, respectively, including alkaloids, terpenes, fatty acids, and antioxidants, possessing many pharmaceutical, medicinal, and antimicrobial properties.
Among the bioactive compounds found in both bulbs and callus methanol extract, n-hexadecanoic (C 16 H 32 O 2 ), octadecanoic acid (C 18 H 36 O 2 ), 9-hexadecenoic acid or palmitoleic acid (C 16 H 30 O 2 ), and 9,12-octadecadienoic acid linoleic acid ester (C 18 H 32 O 2 ) were found to have anticancer, antioxidant, anti-inflammatory, nematicide, antihistaminic, and antiarthritic activities (Prabakaran et al. 2016;Saravanakumar et al. 2016). Similar reported compounds were made in Croton tiglium seeds and found to have potential insectifuge, hypocholesterolemic, hepatoprotective, antiacne, and antieczemic effects (Prabakaran et al. 2016). The methyl esters and phenolic acids in the U. maritima extract have been reported to possess antioxidant and anti-inflammatory properties due to their redox activities inhibiting and scavenging lipid peroxidation (Dhivya and Kalaichelv 2017). Ajmalicine possesses anti-hypertensive and antimicrobial activities; also, it is used in treating circulatory diseases and high blood pressure (Ambrin et al. 2019) while Ajmaline is an antiarrhythmic compound used in the treatment of acute atrial or ventricular tachycardia (Boga et al. 2019).
The phyto-components identified in callus methanol extracts showed a reduction in the percentages of active ingredients compared to bulb extract except for oleic acid, 3-(octadecyloxy) propyl ester, and 3-hydroxydodecanoic acid, which possess an antifungal effect for plant and human fungal pathogens (Abubacker and Devi 2014). The same results were reported by Rech et al. (1998) who mentioned that the alkaloids in Rauwolfia serpentina were accumulated in the suspension cultures, but the total alkaloid level was only 62% of that of the intact plants, and they related that to the concentration and nature of inorganic nitrogen and phosphate, growth regulators, carbon supply in media, and the stage of growth of the culture at which these changes could reduce secondary metabolites. Also, they related the reduction of total alkaloids in media to the limited differentiation of the cells in the callus culture and the better cell-to-cell contact in intact plants. Secondary compounds in plants accumulate in vitro at the end of the growth cycle when growth slows, and excess carbohydrates and nitrogen are redirected into secondary pathways. The most important findings were that the secondary product accumulation was inhibited by high auxin levels, increased by high carbohydrate concentrations, and occurred at the end of the RT retention time Anti-cancer and anti-inflammatory effects, alleviate allergic asthma, gliosis, and edema, and decrease tumors (Jung et al. 2018) exponential phase of growth (Collin 2001). Enzymes of some metabolic pathways may not be active within a plant cell culture environment; cultures can convert such substrates into derivatized important products with the addition of exogenously supplied precursors (Mohamed et al. 2022). Important compounds were detected in U. maritima callus extraction only, such as farnesol 5.87% (which is sesquiterpene alcohol possessing anti-cancer and anti-inflammatory effects and that can modulate various tumorigenic proteins by downregulating the expression of interleukin-6 in humans (Jung et al. 2018)), oxirane hexadecyl 5.22% and 7-methyl-Z-tetradecen-1-ol acetate 1.70% (which acts as anticancer and anti-inflammatory), and hepatoprotective and 2-hydroxyhexadecanoic acid 0.31% (an important compound against varicella-zoster virus (VZV, human herpesvirus 3)), and the human immunodeficiency virus type 1 (HIV-1) (Harper et al. 1996). Similar results were obtained by Reddy et al. (2013) who mention that in vitro callus culture of Urginea congesta enhanced the formation of many important secondary metabolites. Anjum et al. (2017) reported that the exogenous application of plant growth regulators not only controls plant growth and development but also regulates the synthesis of secondary metabolite in plant species in in vitro cultures, and the secondary metabolism in plants is strongly influenced by both the plants' growth environment and tissue type. Ferdausi et al. (2021) discovered a differential expression pattern of genes or transcripts, biosynthetic pathways leading to various secondary metabolites, and their molecular regulation in  Narcissus pseudonarcissus field-grown bulb (basal plate) and tissue culture-derived callus; the initial pathways of secondary metabolite biosynthesis and stress response factors were mostly upregulated in the callus only.

Conclusion
For in vitro production of Urginea maritima, using MS medium amended with 1.0 mg L −1 NAA plus 0.1 mg L −1 BAP gave the least number of days for callus induction (87 d) while 1.0 mg L −1 NAA plus 0.4 mg L −1 BAP gave the least number of days for shoot formation (42.5 d), and the highest number of shoots was obtained at 1.0 mg L −1 NAA plus 0.5 mg L −1 BAP reaching 5.5 shoots with the highest fresh weight. Phyto-components identified using GC-MS analysis showed great potential for U. maritima as an important medicinal plant due to the presence of bioactive medicinal compounds. In the present study, twentynine and twenty-five bioactive compounds were determined and identified in methanol extract of U. maritima bulbs and callus culture, respectively. Important compounds were detected in callus extraction only, possessing anticancer, antiviral, anti-inflammatory, and hepatoprotective properties. U. maritima-derived bioactive compounds are used as a source of antibiotic, antioxidant, anti-inflammatory, and anti-cancer and possess pharmaceutical activities used for drug formulation; these compounds on the pharmacological activity should be evaluated. Further phytochemical and pharmacological investigation for bulbs and callus culture of U. maritima is needed for the development of new drugs.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research was self-funded.

Declarations
All the authors declare that they have no conflicts of interest.
Ethical statement All the authors declare and certify that all the work done in this research is the authors' original work and has not been submitted to any other journal for publication, and the paper is not considered for publication elsewhere. All the data in this paper are original and reflect the active contribution of the author and coauthors leading to the manuscript.
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