1. Introduction
Ruta chalepensis L. is a perennial aromatic herb belongs to Rutaceae family, known by the common name “fringed rue” and the Arabic name “Al-Shathap”. It is spread in the Mediterranean Sea area [
1] and widely distributed in the Kingdom of Saudi Arabia. Nowadays, it is cultivated in many regions of the world, especially in temperate and equatorial countries [
2], and usually growing on rocky slopes [
3,
4].
R. Chalepensis has various pharmacological properties, attributed to its high contents of phytochemicals such as alkaloids, flavonoids, cardiac glycosides, coumarins, tannins, saponins, anthraquinones, volatile oil, cynagenic glycosides, glucosinolates, triterpenes, sterols, amino acids, phenols, and furocoumarins [
2,
4,
5,
6,
7]. In many countries, including Saudi Arabia, Yemen, Algeria, China, and India,
R. Chalepensis is used in traditional medicine because of its biological activities, which include antioxidant, anti-bacterial [
8], anti-fungal [
9], anti-inflammatory [
1,
10], and anthelmintic [
11] properties. It has been used extensively in folk medicine as an antipyretic and analgesic, as well as for the treatment of mental disorders, convulsions, rheumatism, dropsy, neuralgia, and bleeding problems [
4]. Moreover, the leaves are used for treatment of epilepsy, vertigo, colic, intestinal worms, toxicity, headache, and eye problems [
5]. It is used internally as an antispasmodic and antihypertensive [
1,
12,
13].
Tissue culture has emerged as a key component of biotechnology because it allows for the propagation and mass multiplication of plants from small explants of the plant, such as segments of roots, leaves, or stems, on artificial media under controlled conditions [
14,
15]. This technology has played a significant role in the production of competitive and sustainable agriculture, reforestation, and biomedical applications, and has been extensively applied in breeding programs for the generation of better plants with desired traits [
16,
17,
18,
19]. Currently, it is a well-established technology for cultivating and investigating the physiological behavior of isolated plant organs, tissues, cells, protoplasts, and even cell organelles under precisely controlled chemical and physical conditions [
20]. Several factors, including the selection of explant, type of medium and composition [
21], plant growth regulators (PGRs), and the presence of a carbon energy source [
22] that maintains osmotic potential [
23], may significantly affect shoot multiplication. In tissue cultures, several different types of basal media have been formulated, and the response of different plant species to these varied media is dependent on their nutritional needs [
24]. MS medium has been widely used for micropropagation purposes in a number of medicinal plants such as
Vitex negundo [
25],
Albizia lebbeck [
26] and
Centella asiatica [
27],
Artemisia sieberi [
28],
Artemisia pallens [
29],
Vitex trifolia [
30],
Ruta graveolens [
31],
Thymus persicus [
32],
Cassia alata [
33],
Bacopa monnieri [
34], and
Tecoma stans [
35,
36]. WPM medium induced the highest response in
Rauvolfia tetraphylla [
37], and B5 medium had the best morphogenic response in
Saraca asoca [
24].
Plant regeneration in vitro is also influenced by the pH of the medium, which has a substantial effect on the morphogenic activity of plant tissues [
25,
38]. The pH of a culture medium must be within the acceptable limits, such that it does not disrupt the plant tissue [
38]. The pH influences gelling efficiency of agar, where pH above 6 makes the medium very hard, and the medium does not solidify properly at less than 5 pH [
39]. Appropriate pH governs several processes such as concentration of salts, uptake of PGRs, and chemical reactions, especially those catalyzed by enzymes [
38]. Several studies were conducted by many researchers where a wide range of pH levels (5.5–6) was successfully tested for in vitro propagation of different plant species [
25,
26,
40,
41,
42,
43].
The type and concentration of carbon sources supplied to the medium as the energy source which maintain the osmotic potential are essential for efficient micropropagation [
44]. Carbon sources are unavoidable as they have a partial effect on growth and morphogenesis because of their nutritional value, which impacts the rate of cell division or the level of morphogenesis of the cells [
45]. Furthermore, carbon sources have an important role in the synthetic pathway of many compounds, acting as building blocks of macromolecules, and may control many developmental processes in the cell [
46,
47]. Carbon sources and the amount of carbohydrates act together to determine the amount of sucrose hydrolysis and the medium pH after autoclaving [
48]. Hence, sugars are of prime importance for in vitro morphogenesis, a process requiring high energy [
49]. Different sugars were used as a carbon source in tissue culture, such as monosaccharides (fructose, glucose, and galactose), disaccharides (sucrose, lactose, and maltose) and trisaccharide (raffinose). Meanwhile, sucrose is the most effective and promising carbon source since it is the most frequent carbohydrate found in the phloem sap of many plants [
50,
51], as well as because it is inexpensive, readily available, and highly efficient [
52]. It has been used in the tissue culture of a number of plant species, such as
Vitex negundo [
25],
Rauvolfia serpentina [
53],
Psidium guajava [
54],
Artemisia abrotanum [
55],
Ruta graveolens [
18],
Bacopa monnieri [
34], and
Tecoma stans [
35]. Sucrose at 3% was the most effective compared to glucose, fructose, and maltose for micropropagation of
Harpagophytum procumbens [
56].
To ensure clonal stability, it is essential to evaluate the genetic integrity study between in vitro propagating plants and mother plants. In vitro culture of plants may lead to the development of somaclonal difference as a result of exposure to certain stresses during culture conditions, such as the type of PGRs used, the regeneration pathway, long-term cultures, and a large number of sub-cultures, all of which have the potential to damage DNA via cytosine methylation, nucleotide substitutions (SNS), or changes in chromosome number or structure [
33,
57,
58,
59,
60]. Clonal stability in plantlets can be assessed using various techniques based on morphophysiological, biochemical, and molecular attributes. Polymerase chain reaction (PCR)-based approaches such as amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), directed amplification of minisatellite DNA (DAMD), and simple sequence repeats (SSR) molecular markers have been considered to be quite suitable because they are reliable, easily detectable, cost-effective, do not require any prior nucleotide sequence information, and are not affected by environmental factors [
60]. RAPD and DAMD marker techniques have been successfully used to assess the genetic fidelity in several medicinal plants such as
Aconitum violaceum [
61],
Gloriosa superba [
57],
Withania somnifera [
62],
Artemisia nilagirica [
63],
Cassia alata [
33],
Zanthoxylum armatum [
64],
Ruta graveolens [
18],
Bacopa monnieri [
34],
Rauvolfia tetraphylla [
59,
65],
Hildegardia populifolia [
66], and
Thalictrum foliolosum [
67]. Homogeneity of in vitro propagated plants can also be ascertained using flow cytometry, which offers a quick, accurate, and simple method for assessing the ploidy level, genome size, cell cycle, and DNA content within plant nucleus homogenates [
60,
68]. This technique has been successfully employed to determine the nuclear DNA content, genome size, and ploidy level in
Mentha arvensis [
69],
Pongamia pinnata [
70],
Nardostachys jatamansi [
71],
Bacopa monnieri [
34],
Curcuma zedoaria [
72], and
Lippia lacunosa [
73].
Micropropagation studies have been reported for another species of
Ruta genus, i.e.,
R. graveolens [
18,
31,
74,
75,
76,
77]. Nevertheless, no in vitro propagation studies of
R. chalepensis have been reported. The purpose of this work was to design a successful strategy for producing
R. chalepensis on a large scale from nodal explants by optimizing different attributes of micropropagation processes. The aptitude of the in vitro plants to survive in the ex vitro environment, as well as their genetic fidelity, were also assessed using DNA-based molecular markers (RAPD and DAMD) and flow cytometry to ensure the supply of authentic planting materials.
3. Discussion
PGRs play a crucial role in in vitro morphogenesis such as callus induction, shoot regeneration, and somatic embryogenesis, and the levels of PGRs needed in plant tissue culture may differ across species. According to the findings of this investigation, nodal explants of
R. chalepensis that were cultivated in a nutrient-rich medium supplemented with a variety of BA or Kin concentrations (1–10 M) demonstrated a variable response when exposed to different concentrations of BA or Kin. The frequency of shoot regeneration from node explants was the highest in MS medium containing (5.0 µM) BA. The type of exogenously administered cytokinins, as well as their concentration, absorption, and transport, have an effect on the success of the in vitro propagation method [
78,
79]. The effect of BA on shoot induction and multiplication from node explants has been reported by many authors [
67,
74,
80,
81,
82,
83,
84]. The effect of optimized level (5.0 µM) of BA with lower concentration (1.0 µM) of auxins (NAA) produced a better response in terms of greatest shoot induction and proliferation from nodal explant of
R. chalepensis. The findings of this study demonstrate that a proper balance of cytokinin and auxin (BA/NAA) is required for optimum shoot regeneration. When a relatively high ratio of cytokinins is used in addition to the low ratio of auxins, it affects cell division, promoting a higher frequency of shoot bud induction and inducing a greater number of shoots/explants over cytokinin alone [
85,
86]. The positive effects of auxin additions on regeneration rates could be attributable to the potential synergistic effects between auxins and cytokinins that enhance the physiological response of regenerated plants and promote their development and proliferation [
87]. Furthermore, the combined action of cytokinins and auxins has an important role in cell division and multiplication to form new cells, each of which appears to influence different phases of the cell cycle. In addition to having an influence on DNA replication, cytokinin seems to have some control over the processes leading up to mitosis. Therefore, using cytokinin and auxin in combination increases the efficacy of culture media for maximal shoot multiplication [
88]. Based on our findings, nodal explants
R. chalepensis cultivated on MS basal medium with NAA (1.0 µM) and BA (5.0 µM) exhibited the highest response percentage (96.3%) and the maximum number of shoots (40.3 per explant) when compared to other treatments. These findings are consistent with previous studies in which the highest levels of shoot proliferation were observed in a wide range of plant species, including
Ruta graveolens [
18,
74,
76],
Syzygium cumini [
80],
Hildegardia populifolia [
66], and
Mondia whitei [
82].
Plant regeneration success is contingent upon the kind of nutritional medium employed, as well as its composition, pH, and carbon supply. To maximize differentiation and growth of explants, the culture medium’s mineral salts, organic additions, and pH of the media must be carefully balanced. However, plant species usually differ in their requirements, and therefore, they respond differently to different basal media. The pH of medium may be ascribed to medium components, autoclaving, ion exchange, and ambient conditions, among other factors. The pH of the medium may be altered both before and after autoclaving depending on the medium components [
89,
90]. For in vitro shoot regeneration and tissue culture, MS media with a pH of 5.8 has been shown in a number of studies to be the most appropriate medium [
34,
44,
53]. Shoot regeneration from explants cultivated in MS medium with a pH of 5.8 was determined to be optimal in this study, according to the findings. However, shoot differentiation and growth were found less effective in B5, WPM, NN, and White media with a pH 5.8. MS medium with a pH 5.8 has also been found significantly more effective than other media in various plants species, e.g.,
Albizia lebbeck [
26],
Vitex negundo [
25],
Centella asiatica [
27],
Plumbago zeylanica [
91], and
Physocarpus opulifolius [
92].
The pH of the medium may be influenced by a variety of events, including hydrolysis, enzyme breakdown, photooxidation, and photolysis on light-sensitive medium components. Water splitting and glycosidic bond breaking are frequent requirements for sucrose hydrolysis. Because tissue culture media is frequently adjusted to slightly acidic conditions (5.8), autoclaving offers a temperature that is adequate for catalyzing sucrose hydrolysis. It has been shown that acid-facilitated autocatalyzed sucrose hydrolysis is both pH- and temperature-dependent, with lower pH at a given temperature promoting greater sucrose hydrolysis [
93,
94]. Carbon sources and the amount of carbohydrates act together to determine the amount of sucrose hydrolysis and the medium pH after autoclaving [
48]. Carbohydrates such as sucrose, glucose, fructose, and maltose are often used in the establishment of plant tissue culture. Sucrose is broadly applied in in vitro culture because of its positive effects on plant growth and development and low cost. Successful in vitro organogenesis, on the other hand, is highly dependent on the concentration of sucrose used and its interaction with other medium compositions. In the current study, the effects of different types of carbohydrates, i.e., sucrose, glucose, fructose, and maltose, at various concentrations were tested. Sucrose was shown to be the most effective sugar for promoting shoot regeneration from nodal explants in this study, followed by fructose, maltose, and glucose. Similar results were obtained in micropropagation of
Harpagophytum procumbens [
56],
Aquilaria malaccensis [
95],
Plumbago zeylanica [
91],
Pterocarpus marsupium [
96], and
Lupinus albus [
97]. Sucrose, the most prevalent carbohydrate in plant phloem sap [
50,
51], is often used in tissue culture due to its inexpensive cost, easy availability, and ability to quickly absorb through the plasma membrane [
74], which aids in sugar hydrolysis as well as maintain the pH of the medium [
48].
One of the most essential aspects in the micropropagation process is the development of a robust root system. In this study, microshoot rooting (5.4 roots per microshoot) was best obtained in ½ MS basal medium with 0.5 M of IBA. Similar results were reported in
Ruta graveolens [
18,
74],
Rauvolfia serpentina [
53],
Hemidesmus indicus [
98],
Maerua oblongifolia [
99],
Rauvolfia tetraphylla [
59],
Artemisia vulgaris [
100],
Thalictrum foliolosum [
67], and
Asystasia gangetica [
101]. In a wide range of plant species, IBA was shown to be the most suited for in vitro root induction over IAA or NAA because of its greater resilience to photodegradation, adsorption to microshoots, and deactivation with biological action [
102]. IBA is also quickly absorbed, retained, and transported throughout plant tissues, and it has the potential to activate the gene responsible for rhizogenesis [
103]. IAA and NAA were shown to be less efficient in inducing roots than IBA, owing to the fact that IAA is readily oxidized by light and denatures rapidly in culture media, but NAA may survive longer in plant tissues [
88].
The development of somaclonal anomalies between the regenerated plants may limit the effectiveness of the micropropagation protocol [
33,
104]. Explant preparation and subsequent sub-culturing of sub-clones for extended durations in tissue culture systems might lead to these changes [
105,
106,
107]. Because of this, evaluating the genetic integrity of micropropagated plants is critical. PCR-based markers are one of the most significant approaches being used test genetic stability in several plant species, since it is not affected by culture conditions and may be acquired at any stage of plant development. ISSR, RAPD, AFLP, SSR, and DAMA are the most frequently employed techniques since they do not need any previous DNA sequence information [
33,
106]. In the present investigation, RAPD and DAMD markers were applied to assess the genetic homogeneity of
R.
chalepensis plantlets. All RAPD and DAMD-generated bands were monomorphic and identical, confirming the complete absence of somaclonal variations in regenerated plantlets. It has been shown that the use of RAPD and DAMD molecular markers is efficient in determining the genetic stability of regenerated plants in a variety of medicinal plant species, including
Withania somnifera [
108],
Henckelia incana [
109],
Avicennia marina [
110],
Ruta graveolens [
18],
Curcuma zedoaria [
72],
Artemisia vulgaris [
100],
Anarrhinum pubescens [
111], and
Anthurium andraeanum [
106]. In recent years, it has been shown that a flow cytometry-based approach for evaluating in vitro raised plantlets is an excellent technique for assessing the clonal integrity and ploidy status in micropropagated plants [
34,
71,
72,
112,
113]. In the present investigation, no major differences between fluorescence peak derived from nuclei of
Ruta chalepensis plantlets and ex vitro plants were found. The findings of flow cytometric analysis are consistent with those obtained from earlier research on
Mentha arvensis [
69],
Puya berteroniana [
112],
Solanum lycopersicum [
113],
Cucumis melo [
114],
Bacopa monnieri [
34],
Curcuma zedoaria [
72], and
Juglans regia [
115].
4. Materials and Methods
4.1. Plant Material and Surface Sterilization
The young, healthy stem segments of R. chalepensis were harvested from a plant growing in the Botany and Microbiology Department of the King Saud University in Riyadh, Saudi Arabia. The explants were washed for 25 min in laboratory tap water, then treated for 5 min in a 5% (v/v) liquid detergent, followed by 4–5 rinses with sterile ultrapure (Milli-Q) water to remove any detergent residue. Inside a laminar air flow hood (ESCO Labculture® Class II Type A2 Biological Safety Cabinet, Esco Micro Pte. Ltd., Singapore), the plant materials were surface sterilized with 0.1 percent (w/v) mercuric chloride (HgCl2) for 3 min at room temperature. The HgCl2-treated explants were finally rinsed 4–5 times with sterile ultrapure (Milli-Q) water and cut into pieces that were about 0.5–0.7 cm in diameter, which were then utilized for further in vitro assays.
4.2. Preparation of Media and Culture Conditions
The sterile nodal segment explants of
R. chalepensis were aseptically cultured onto Murashige and Skoog (MS) [
116] agar medium with 3% (
w/v) sucrose and various combinations and concentrations of growth regulators (auxins and cytokinins), as specified below. pH of the nutrient media was adjusted to 5.8 by the addition of 1 M NaOH or HCl before adding 0.8% (
w/v) agar. The media was then autoclaved at 121 °C and 15 psi for 20 min. The media was dispensed into petri dishes, each of which contained 25 mL of medium. Following the inoculation of the explants, the dishes were sealed with one layer of parafilm. All cultured plates/vials were maintained at a temperature of 24 ± 2 °C, a photoperiod of 16/8 h (day/night), a photon flux density of 50 mol m
−2 s
−1 provided by cool LED tubes, and a relative humidity of 50–60%.
4.3. In vitro Shoot Initiation and Proliferation
For shoot initiation and multiplication, the nodal explants of R. chalepensis were cultured on MS medium supplied with varying concentrations of cytokinins, i.e., 6-benzyladenine (BA) or Kinetin (Kin) (1.0, 2.5, 5.0, 7.5, or 10 μM), individually or in combination with auxins, i.e., indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and indole 3-butyric acid (IBA), at various concentrations (0.5, 1.0, 1.5, or 2.0 μM). All cultures were subcultured in the same fresh medium every three weeks.
4.4. Effect of Various Media and pH
The use of the optimal growth regulators and a suitable media in in vitro culture is essential for overall growth response of the explants. In order to find the best basal media for shoot initiation and development from nodal explants of
R. chalepensis, we examined MS medium, Gamborg’s Medium (B5) [
117], Woody Plant Medium (WPM) [
118], White’s medium [
119], and Nitsch and Nitsch (NN) Medium [
120]. Each basal medium comprised 3% sucrose, 0.8% agar, and the optimized auxin and cytokinin (5.0 μM BA and 1.0 μM NAA) combination. Effect of different pH levels (4, 4.8, 5.8, and 6.8) of the optimized nutrient medium on shoot development were also evaluated. After three weeks, all the responding explants with shoot clumps were sub-cultured on the fresh culture medium, and the data on the number of shoots and the length of the shoots were collected after eight weeks of cultivation.
4.5. Effects Carbon Sources
Response of explants to several carbon sources, such as sucrose, glucose, maltose, and fructose, at concentrations of 2, 3, and 4 percent (w/v), were assessed in MS media augmented with 5.0 μM BA, 1.0 μM NAA. Following 8 weeks of culture, the frequency with which R. chalepensis explants produced shoots, number of shoots per explant, and the shoot length were determined.
4.6. Rooting of Shootlets
Rooting of the regenerated microshoots of R. chalepensis was accomplished by the use of an in vitro rooting approach. Isolated healthy microshoots (4–5 cm) were transplanted to culture tubes with half-strength MS without PGRs (as a control) or supplemented with 0.1, 0.5, 1.0, or 2.0 μM of an auxin such as IAA, IBA, or NAA. To find the optimal medium for root induction, different strengths of MS nutrient agar media (¼, ¾, and full strength) coupled with the better dose of an auxin (0.5 μM IBA) were examined.
4.7. Acclimatization
Plantlets with a well-developed shoot and root systems were carefully removed from the culture tube and rinsed with normal water to eliminate any remaining agar. A high level of humidity was maintained around the plant by implanting plantlets in pots containing potting soil (Planta Guard, Germany) and covering the pots with transparent plastic covers. The plantlets were kept in a growth environment under a 16/8 h (day/night) photoperiod with a photon flux density of 50 mol m−2 s−1, high humidity (50–60 percent), and watered with ¼ MS devoid of organic nutrients every three days for three weeks before being irrigated with regular water. The coverings were perforated and progressively removed over a period of 12–20 days in order to allow the plantlets to become more acclimated to field conditions. After 6 weeks, these well-acclimated plantlets were transplanted into pots filled with standard garden soil and kept in a greenhouse under natural day light conditions.
4.8. Flow Cytometric Analysis
Flow cytometric analysis portable Muse Cell Analyzer (Muse
® Cell Analyzer, Merck Millipore, USA) was used to compare the ploidy level of
R.
chalepensis donor and in vitro regenerated plants. Using a sharp scalpel blade (No. 21), approximately 100 mg of fresh leaf samples was chopped in 1 mL of Galbraith buffer (pH 7.0) containing 0.1% (
v/v) Triton X-100, 20 mM MOPS, 30 mM sodium citrate, and 45 mM MgCl
2 [
68] to isolate the nuclei. The isolated nuclei in buffer were filtered through double-layered 28 µm nylon meshes, and then, 50 µg/mL of propidium iodide (PI, Sigma, USA) solution was added and mixed for 15 min. Finally, 50 µg/mL RNase (Sigma, USA) were added to avoid staining of double-stranded RNA molecules and the nuclei samples were passed through the Muse Cell Analyzer. Each run had at least 5000 nuclei, and each experiment was repeated three times.
4.9. DNA Extraction and PCR Amplification
Young leaf samples were collected from the donor plant and randomly selected micropropagated plants. DNA was extracted from leaf samples (approximately 250 mg) by the cetyltrimethylammonium bromide (CTAB) method [
121]. DNA purity and concentration were determined using a Nanodrop spectrophotometer (Nanodrop 2000, Thermo Scientific, USA). The purity and quality of the DNA were also checked using 1% agarose gel (1X TBE buffer) stained with ethidium bromide using gel electrophoresis. DNA samples were diluted to a final concentration of 25 ng/μL in DNAse, RNAse free ultrapure water (Milli QR, Millipore, USA) for PCR reactions. Twelve RAPD (GeneLink, Inc., Orlando, FL, USA) and five DAMD (GeneLink, Inc., Orlando, FL, USA) primers were used for screening of DNA amplification. Then, 20 µL of PCR reaction comprising 10 µL of PCR master mix (GoTaq
® Green Master Mix, 2X, Promega, USA), 1 µL genomic DNA (50 ng/μL), 1 µL primer, and 8.0 µL ultrapure (Milli-Q) water. Thermal Cycler, Bio-Rad, USA) was used to perform the PCR reactions, which were configured to complete 40 cycles after an initial denaturation cycle of 5 min at 94 °C. Each cycle included a denaturation step at 94 °C for one minute, an annealing step at 29.5–57 °C for one and a half minutes, an extension step at 72 °C for two minutes, and a final extension cycle at 72 °C for seven minutes. All of the experiments were repeated three times in order to avoid false outcomes and to validate the reproducibility of the RAPD and DAMD markers. The PCR products were separated by electrophoresis on 1.5 percent agarose gels with 5 µL ethidium bromide in 1X Tris-borate-EDTA (TBE) buffer at 75 V for 2 h and photographed using the gel documentation system (G:BOX F3, Syngene, Cambridge, UK).
4.10. Statistical Analysis
IBM-SPSS software for Macintosh version 26.0 (IBM-SPSS Inc., IL, Chicago, USA) was used for statistical data analysis in one of the experiments, which had a totally randomized design with 20 replicates and was performed three times. The collected data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests were used to evaluate the significant differences (p ≤ 0.05) between the treatment values.