Alginate encapsulation of micro-cuttings in endangered Satureja khuzistanica species: a promising method for obtaining genetically stable plants with high rosmarinic acid content

Synthetic seed technology is a suitable approach for the rapid, and uniform mass production of Satureja khuzistanica Jamzad, a valuable medicinal plant of the Lamiaceae family. In this study, the encapsulation of micro-cuttings of S. khuzistanica using sodium alginate (SA) was investigated. In order to determine the best conditions for encapsulation, different concentrations of SA and CaCl2·2H2O, different types of explants and matrix culture media, different types and concentrations of plant growth regulators, and different cold storage conditions of synthetic seeds were tested. Also, the genetic stability of regenerated plants from synthetic seeds using the inter simple sequence repeat (ISSR) molecular marker as well as the content of rosmarinic acid in synthetic seed-derived plants were evaluated. The use of 3% SA and 100 mM CaCl2·2H2O was found to be optimal for gel complexation. Encapsulation of nodal segments with 1/2 Murashige and Skoog (MS) medium containing 2.5 µM 6-Benzylaminopurine resulted in the highest regrowth percentage (72.22%) and regrowth rate (0.173). Pre-culture of nodal segments in 1/2 MS medium containing 5 µM indole-3-butyric acid (IBA) and 0.2% activated charcoal for 10 days and the use of 2.5 µM IBA in the rooting medium resulted in the highest rooting percentage (88.88%) in synthetic seed-derived plants. The highest regrowth percentage (61.11%) and regrowth rate (0.173) of synthetic seeds were obtained using 2.0 µM thidiazuron (TDZ) in MS liquid medium. The placement of germinated seeds on the coco peat substrate resulted in the highest conversion rate (61.11%) of synthetic seeds. Also, storage of encapsulated nodal segments at 4 °C in MS culture medium, compared to cold storage without using MS medium, resulted in better regrowth of synthetic seeds. The highest regrowth percentage (44.44%) and regrowth rate (0.092) for cold-stored synthetic seeds occurred after 2 weeks. The genetic stability testing by ISSR molecular marker showed that synthetic seed-derived plantlets were genetically similar to their mother plants. Also, encapsulated nodal segments and shoot tip-derived plants significantly enhanced the rosmarinic acid content up to 7.77 times that of the natural seed-derived plants. Genetic restoration programs, short-term storage, and germplasm distribution in S. khuzistanica plants could all benefit from the encapsulation regeneration strategy reported here. By encapsulating Satureja khuzistanica micro-cuttings in alginate, we were able to produce genetically stable plants with high rosmarinic acid content for the first time.


Introduction
Satureja khuzistanica Jamzad is an aromatic and valuable medicinal plant of the Lamiaceae family and an endemic species in southern Iran (Jamzad 1994). There are various chemicals such as tannins, sugars, and essential oils in this plant, making it a valuable plant species for human consumption (Hadian et al. 2011). The most important chemical compounds in the essential oil of S. khuzistanica are: carvacrol (over 90%), paracymene, limonin, 1,8-cineole, eugenol, myrcene and alphatogen (Farsam et al. 2004). The carvacrol in the essential oil of this plant species has anti-inflammatory, anti-agonal, anti-nociceptive, antibacterial, anti-fungal, and anti-oxidant properties (Liolios et al. 2009;Abid et al. 2014;Dai et al. 2016). Also, rosmarinic acid (RA) in the extract of this plant shows antimicrobial activity and prevents Alzheimer's disease (Hamaguchi et al. 2009). In general, the medicinal and biological properties of S. khuzistanica make it a valuable plant for use in the pharmaceutical and food industries (Farsam et al. 2004;Hosainzadegan and Delfan 2009). Due to the overuse of this wild plant species for commercial purposes and its low propagation rate in nature, S. khuzistanica has been reported as an endangered plant species (Hadian et al. 2017;Khojasteh et al. 2019) and also a highly threatened species in the Red Data Book of Iran (Jalili and Jamzad 1999). Over the past years, herbal drugs of this endemic plant have been collected from wild habitats to supply the demands of industries, leading to problems such as the threat of species extinction and the production of heterogeneous plant material. To resolve these concerns, the domestication and cultivation of this plant are emphasized, which aids in germplasm conservation as well as the quality and homogeneity of plant raw materials in this species (Hadian et al. 2011). Plant tissue culture techniques are very useful methods for the production of uniform plants in a short period. These systems are also efficient and faster methods for the biotechnological production of some plant secondary metabolites (Khojasteh et al. 2020). Due to the presence of dormancy in S. khuzistanica seeds and its low potential for seed production and seed germination, plant tissue culture methods can be used effectively for plant propagation and secondary metabolite production in this plant species (Ramak et al. 2011). "Artificial seed technology" is one of the important micropropagation methods in plant tissue culture. This technology is one of the effective alternatives for preserving plant species that generate non-viable seeds and are difficult to reproduce through conventional methods (Daud et al. 2008). This method has the ability to provide genetically identical, virus-free germplasm, as well as ease of transportation, long-term storage, and inexpensive production costs (Ghosh and Sen 1994). Synthetic seed technology can be a viable alternative conservation approach for endangered rare plants like S. khuzistanica that cannot be propagated effectively through traditional methods. In this method, various explants, such as somatic embryos, shoot tips and axillary buds, as well as other vegetative parts of the plant, can be encapsulated in an artificial hydrogel (Redenbaugh et al. 1986). However, the encapsulation of somatic embryos in medicinal plants is limited to species in which the induction of somatic embryogenesis has been established (Verma et al. 2010). Therefore, encapsulation of non-embryonic vegetative propagules such as shoot tips and nodal segments has been used as a suitable alternative for micropropagation and short-term storage of valuable medicinal plants (Lisek and Orlikowska 2004;Singh et al. 2010;Katouzi et al. 2011;Gantait et al. 2015;Siddique and Bukhari 2018). In addition to the choice of initial explants, several other factors, such as encapsulating agent and matrix, addition of growth regulators and nutrients to the capsules, and also experimental conditions, substantially influence the success of synthetic seed production, their storage and regeneration (Saiprasad 2001;Gantait et al. 2015). On the other hand, as synthetic seeds become more popular for germplasm conservation and multiplication, the genetic stability of stored plant material must be evaluated. Inter-simple sequence repeat (ISSR) is a simple and polymerase chain reaction (PCR) based marker system that is increasingly being used in crop plants and medicinal plants to analyze the genetic stability and instability of in vitro culture derived plants (Williams et al. 1990).
In recent years, some research groups (Sahraro et al. 2014(Sahraro et al. , 2016Fatemi et al. 2020) have developed different plant tissue culture systems, such as callus, cell suspension, and nodal segment cultures in S. khuzistanica, for further scaling up and commercial production of RA as a valuable natural antioxidant. However, so far, the micropropagation system through synthetic seed production has not been studied to increase the amount of secondary metabolites, especially RA, in S. khuzistanica. Therefore, the main objective of this research was to develop an efficient system for synthetic seed production of S. khuzistanica and their germination and conversion into plants for further propagation. In the following, the genetic stability of synthetic seed-derived plants and also the RA content of these plants were investigated.

Plant material preparation
Multiple shoot cultures of S. khuzistanica were established by culturing the nodal shoot explants obtained from greenhouse-grown plants. Suitable nodal segments (each segment containing one node) were cut from plants at the reproductive stage and transferred to the laboratory. The nodal segments were washed for 10 min in running tap water, surface sterilized for 5 min in 1.5% (v/v) sodium hypochlorite, and rinsed three times in sterile distilled water for 5 min. As described by Fatemi et al. (2019 and, the nodal segments were cultured in solid MS medium (Murashige and Skoog 1962) containing 9 µM 6-benzylaminopurine (BAP), 3% (w/v) sucrose, and 0.2% (w/v) polyvinylpyrrolidone (PVP). The cultured nodes were placed at 24 ± 2 °C, in a growth chamber with a photosynthetic photon flux density of 40 µmol m −2 s −1 and a 16/8 h light/dark photoperiod, at Bu-Ali Sina University, Hamedan, Iran. The cultures were sub-cultured every 3 weeks. The micro-cuttings, viz., apical tips and nodal segments (3 mm) of in vitro-maintained shoots, were excised aseptically from 4-week cultures, and these micro-cuttings were used for encapsulation.

Effect of encapsulation matrix
Different concentrations of SA (2, 3, 4, and 5%, w/v) were prepared for encapsulation using liquid MS media containing 0.2% PVP, 13.3 µM BAP, 1.2 µM IBA, and 3% sucrose. Using distilled water, several CaCl 2 .2H 2 O solutions (25, 50, 75, and 100 mM) were produced for complexation. Both the gel matrix and the complexing agent were sterilized by autoclaving at 121 °C for 20 min after adjusting the pH to 5.8. Encapsulation was accomplished by mixing the nodal segments or shoot tips into the SA solution and dropping them into the calcium chloride solution. The droplets of gel matrix, each containing a single nodal segment or shoot tip, were dropped in the complexing agent and allowed for polymerization and the formation of capsules for 20 min. The alginate beads were then collected, rinsed with sterile water, and transferred to sterile filter paper for 5 min under laminar airflow to remove excess water, before being placed in Petri plates with MS culture media without plant growth regulator for regrowth. As previously stated, the MS culture medium was solidified with 0.8% agar and autoclaved to sterilize it. All the developed cultures were maintained in a growth chamber at 24 ± 2 °C with a 16 h light/8 h dark photoperiod for regrowth. This experiment was performed as a factorial based on a completely randomized design with 3 replications. Different concentrations of SA and various levels of CaCl 2 ·2H 2 O were considered as the first and second factors, respectively.

Effect of MS culture medium strength, explant type, and concentration of BAP in gel matrix on regrowth
In this experiment, for the encapsulation of two different explant types (nodal segment and shoot tip), a 3% SA solution prepared in MS culture medium (half and full strength) supplemented with 3% sucrose, and different concentrations of BAP (0, 1, 2.5, 5 and 10 µM), were used as matrix agents. Also, 100 mM CaCl 2 ·2H 2 O was prepared in distilled water and used as a complexing agent. This experiment was also performed as a factorial based on a completely randomized design with 3 replications. Two types of explants, two levels of MS culture medium strength and different concentrations of BAP in the gel matrix were considered as the first, second and third factors, respectively.

The effect of different concentrations of thidiazuron (TDZ) in gel matrix and type of culture medium on regrowth
In this experiment, nodal segment explants were encapsulated with a 3% SA and 100 mM CaCl 2 ·2H 2 O combination in the 1/2 MS culture medium containing different concentrations of TDZ (0, 0.5, 1.1, 2.0, and 2.3 µM) as the gel matrix. Then, half of the capsules were transferred to petri dishes containing MS solid culture medium without any growth regulators and the other half were transferred to Erlenmeyer flasks containing 50 mL of MS liquid culture medium (Growth regulator-free). The Erlenmeyer flasks were continuously shaken at 120 rpm and kept at a temperature of 24 °C in the growth chamber. The regrowth percentage and rate were calculated after 2 weeks (Regrowth percentage was defined as the percentage of encapsulated nodal segments that regenerated and emerged out of the capsule wall to produce in vitro plantlets. Regrowth rate was calculated according to Maguire's equation (Maguire 1962): M = n 1 / t 1 + n 2 /t 2 + …+ n n /t n , where n 1 , n 2 , … n n are the number of germinated capsules at times t 1 , t 2 , …,t n measured in days.). Shoot length, number of shoots, and number of nodes were all measured after another 4 weeks. This experiment was conducted as a factorial based on a completely randomized design with 3 replications. Five different concentrations of TDZ used in the gel matrix, were considered as the first factor and two types of MS culture medium (solid and liquid) were used for regrowth of synthetic seeds as second factor.

The effect of different concentrations of IBA in gel matrix on root induction
In this experiment, nodal segment explants were first precultured on 1/2 MS culture medium supplemented with 0.2% activated charcoal and 5 µM IBA for 10 days. After 10 days, these explants were encapsulated with a 3% SA solution prepared in a half-strength MS culture medium containing 2.5 µM BAP and different concentrations of IBA (0, 2.5, 5, and 10 µM) in the gel matrix. A complexing agent of 100 mM CaCl 2 ·2H 2 O was also utilized in this experiment. Two-weekold germinated capsules were put on a planting substrate of coco peat and peat moss (1:1) and generated roots, after 2 months of encapsulation. The root induction efficiency was assessed by counting the number of roots, rooting percentage, and root length. This experiment was performed in a completely randomized design with 3 replications.

The effect of cold storage periods and conditions on regrowth traits of encapsulated nodal segments
To assess the effect of cold storage on regrowth traits, encapsulated nodal segments were transferred to two glass jars containing solid MS culture medium and without MS culture medium. The samples were then stored in a refrigerator at 4 °C for various time periods (0, 2, 4, 8, and 12 weeks). After each storage period, encapsulated nodal segments were transferred to a growth regulator-free MS culture medium for regrowth. After 2 weeks, the regrowth percentage and regrowth rate of capsules were calculated for each treatment. This experiment was performed as factorial based on a completely randomized design with 3 replications. Two storage conditions of synthetic seeds (glass jars with solid MS culture medium and without MS culture medium) were considered as the first factor and different storage times of artificial seeds in cold condition were considered as the second factor.

Effect of different planting substrates on plant conversion
Various planting substrates, including MS culture medium without growth regulators, coco peat, perlite, sand, and a loamy soil, were assessed for the conversion of encapsulated nodal segments into whole plantlets. Some of the capsules were directly transferred to the sterilized planting substrates immediately after encapsulation. Other capsules were first placed on the growth regulator-free solid MS medium for 2 weeks, and then the germinated capsules were transferred to the sterilized planting substrates (indirect method). The planting substrates were irrigated with quarter strength MS culture medium (1/4 MS). After 4 weeks, different traits such as the percentage of plant conversion, shoot length, stem diameter, and the number of leaves per plant were measured. This experiment was conducted as factorial based on a completely randomized design with 3 replications. Different planting substrates were considered as the first factor while two methods of transferring synthetic seeds to the planting substrates (direct and indirect) were second factor.

Evaluation of genetic uniformity of synthetic seeds derived plants using ISSR molecular marker
Total genomic DNA was extracted using the CTAB extraction method (Doyle and Doyle 1990) from the synthetic seed-derived plants and the mother plant. The quantification of DNA was done using a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA). The DNA samples were diluted to 50 ng µL −1 with TE (Tris-EDTA) buffer before use and stored at 4 °C for the ISSR experiment. In ISSR analysis, six primers were used for the genetic stability assessment of nine synthetic seed-derived plants (Plants obtained from experiment comparing planting substrates) compared to their mother plant. The PCR reactions were performed in a 20 µL volume containing 6 µL H 2 O, 10 µL master solution (Taq 2×, 2 mM MgCl 2 ), 2 µL primers, and 2 µL template DNA. The PCR reactions were done with a thermal cycler (MJMini Bio-Rad, USA) using a single primer in each reaction. The PCR amplification was adjusted as an initial denaturation at 94 °C for 5 min, followed by 35 cycles of PCR reaction consisting of denaturation at 94 °C for 1 min, primer annealing at a specified temperature for each primer for 30 s, and an elongation stage at 72 °C for 30 s. A final extension at 72 °C for 7 min was also done. The resolution of the PCR products by ISSR primers was studied by electrophoresis (1% agarose gel for 100 min in 1× TBE buffer and stained with ethidium bromide). The gel documentation system (DigiDoc H110) was used for photography.

HPLC analysis
In this experiment, the rosmarinic acid content of two types of plants derived from the encapsulation of nodal segments and shoot tips was compared to that of natural seed-derived plants in a completely randomized design with three replications. Regenerated plants from synthetic seeds made with 3% SA and 100 mM CaCl 2 ·2H 2 O in a half-strength MS culture medium supplemented with 3% sucrose and 2.5 µM BAP were used for HPLC analysis. HPLC analysis was done according to the protocol described by Fatemi et al. (2019 and. The leaves of S. khuzistanica plants derived from natural seeds, encapsulated nodal segments, and encapsulated shoot tips were harvested and dried in a dark condition at room temperature. The samples (1000 mg) were grinded and suspended in 250 mL Erlenmeyer flasks containing 40 mL methanol/water (80/20 v/v), and then transferred to darkness for 2 days. The mixtures were continuously shaken at 80 rpm for 5 h, followed by sonication for 30 min. Finally, the homogenate was filtered through Whatman paper (No. 1) and the filtered solution was evaporated at 50 °C using a rotary evaporator (Heidolph, Germany). The residues were dried, and the hydroalcoholic extract was stored in darkness. Different concentrations of rosmarinic acid standard were prepared in 1 mL of methanol/water (50/50 v/v), ranging from 1 to 200 mg/L. Injections derived peak areas were used to calculate the calibration curve. The Spherisorb ODS-2 (5 mg/L) reversed phase 4.6 mm × 250 mm was used as the HPLC column. Elution was done at a flow rate of 1.0 mL/ min at 25 °C and detection at 333 nm. The injection volume was 20 µL. Two mobile phases, A (H 2 O) and B (methanol), were used. The solvent composition of the gradient was at low pressure, 75% A and 25% B for the first 5 min, followed by 50% A and 50% B for the next 10 min, and finally 100% B for an additional 15 min. Each extract (2 mg) was dissolved in 1 mL of methanol/water (50/50 v/v) and filtered through a 0.45 mm filter. The chromatography peak of rosmarinic acid was confirmed according to the retention time of the reference standard (Fig. S1A). Using Agilent ChemStation software, the quantitative analysis was carried out using external standardization by measuring the peak areas.

Data analysis
All experiments were performed in a completely randomized design with 3 replications. Each Petri dish containing six capsules (synthetic seeds) was considered one replication. All data were subjected to analysis of variance (ANOVA) using SPSS software (version 16) and significant differences between the means were assessed by Duncan's multiple range test (Duncan 1955) at P ≤ 0.05. Before being analyzed, percentage data was square root transformed (√x + 0.5).

Results
In the present study, after establishment of in vitro shoot cultures of S. khuzistanica plants (Fig. 1A), the 3 mm micro-cuttings, viz. nodal segments (Fig. 1B) and apical tips (Fig. 1C) were used to produce synthetic seeds. Uniform beads in size and shape were produced when the 3% (w/v) SA solution was exposed to 100 mM CaCl 2 .2H 2 O solution for 20 min (Fig. 1D). The regrowth of synthetic seeds occurred after 2 weeks from initial encapsulation (Fig. 1E, F). Germinated synthetic seeds produced roots after 2 months of transfer to a mixture of coco peat and peat moss (1:1) as a planting substrate (Fig. 1G). The 2-week old, germinated synthetic seeds converted into whole plants after 4 weeks from transferring to the planting substrates ( Fig. 1 H, I).

Effect of encapsulation matrix on synthetic seed regrowth properties
In this experiment, nodal segments were encapsulated using different concentrations of SA and calcium chloride. The combination of 2% SA with 100 mM CaCl 2 .2H 2 O resulted in capsules with a higher regrowth percentage (88.88%) and regrowth rate (0.22) ( Fig. 2A, B) compared to other treatments. However, using 2% SA produced soft and fragile beads that were too difficult to handle. Application of 3% SA with 100 mM CaCl 2 ·2H 2 O was the optimal combination for regrowth percentage (72.22%) and regrowth rate (0.18) of synthetic seeds ( Fig. 2A, B), creating uniform, easy-to-handle, firm, and clear beads. Due to the bead hardiness and delay in shoot emergence, the regrowth percentage and regrowth rate significantly decreased at higher concentrations (4 and 5%) of SA ( Fig. 2A, B).

Effect of MS culture medium strength, explant type, and concentration of BAP on regrowth properties of synthetic seeds
In this experiment, we evaluated the influence of MS culture medium salt strength, explant type, and BAP concentration in the gel matrix on the regrowth properties of synthetic seeds. In comparison with other treatments, the higher regrowth percentage (72.22%) and regrowth rate (0.173) were obtained when nodal segments were encapsulated with a half-strength MS culture medium containing 2.5 µM BAP ( Table 1). Use of full-strength MS culture medium containing 5 µM BAP and also half-strength MS culture medium containing 2.5 µM BAP in the gel matrix of the encapsulated shoot tips resulted in the highest shoot length (1.93 cm) ( Table 1). Encapsulation of shoot tips with a half-strength MS culture medium containing 10 and 2.5 µM BAP produced the maximum number of shoots compared to other treatments (1.83 and 1.78 shoots, respectively). The maximum number of nodes (3.19 nodes) was obtained by using the half-strength MS culture medium and 2.5 µM BAP in the gel matrix of the encapsulated shoot tips (Table 1).

Effect of TDZ and type of culture medium on regrowth properties of encapsulated nodal segments
The results of this experiment showed that the use of 2.0 µM TDZ in the gel matrix of the encapsulated nodal segments and transferring them to the liquid half-strength MS culture medium resulted in the highest regrowth percentage (61.11%) and regrowth rate (0.173) compared to other treatments ( Table 2). The use of a solid culture medium for regrowth of the synthetic seeds supplemented with 2.3 µM TDZ and also a liquid culture medium for regrowth of synthetic seeds containing 2.0 µM TDZ produced the longest shoots (2.75 and 2.68 cm, respectively) in comparison with other treatments ( Table 2). The maximum number of shoots (2 shoots per explant) was obtained from synthetic seeds containing 0.5 µM TDZ in the liquid culture medium (Table 2). Also, regrowth of synthetic seeds in both solid and liquid culture media resulted in the highest numbers of nodes (4.5 nodes), when higher concentrations of TDZ (2.3 µM and 2.0 µM, respectively) were used in their gel matrix ( Table 2).

The effect of IBA on the rooting of encapsulated nodal segments
In this experiment, the use of different concentrations of IBA in the gel matrix of the encapsulated nodal segments was evaluated for root induction. The optimal IBA concentrations for root induction were 2.5 µM and 5 µM, which provided the highest percentages of rooting (88.88% and 77.77%, respectively, Fig. 3A). Various concentrations of IBA used in this experiment (2.5, 5, and 10 µM) did not show significant differences (P ≤ 0.05) in term of mean root length and mean root number traits, resulted in longer roots (1.58, 1.33, and 1.50 cm, respectively) and higher root numbers (1, 1, and 0.66, respectively) compared with their control (Fig. 3B, C).

Effect of low temperature storage on regrowth of encapsulated nodal segments
To evaluate the effect of cold storage on the germination properties of encapsulated nodal segments, the prepared synthetic seeds were stored within glass jars containing MS medium and also without MS medium at 4 °C for  Fig. 4B) were obtained for control synthetic seeds (without cold storage, 0 week) in both conditions, with and without MS culture medium. Among the cold storage times, storage of the encapsulated nodal segments on the MS culture medium for 2 weeks showed the higher regrowth percentage (44.44%, Fig. 4 A) and regrowth rate (0.092, Fig. 4B) compared to other cold storage times. The regrowth percentage as well as the regrowth rate of the synthetic seeds stored at 4 °C gradually decreased with increasing storage period in both conditions (with and without MS culture medium), and in the 12th week, regrowth of the cold   (Fig. 4A, B).

Effect of different planting substrates on conversion of encapsulated nodal segments to plantlets
As shown in Table 3, the highest percentage of plant conversion (61.11%), was obtained when synthetic seeds were pre-cultured on the MS culture medium for 2 weeks before being transferred to coco peat as a planting substrate (indirect transfer). Transfer of germinated synthetic seeds to coco peat after 2 weeks of pre-culture on MS culture medium and also, direct transfer of them to coco peat (without pre-culture on MS culture medium) resulted in longer shoots (2.40 and 1.75 cm, respectively) than perlite, sand or soil mixture (Table 3). Indirect transfer of synthetic seeds to coco peat and perlite substrates and also direct transfer of them to coco peat substrate resulted in the highest stem diameter (1.33, 1.33 and 1.17 mm, respectively) and number of leaves per plant (11.17, 6.42 and 7.33, respectively) ( Table 3). Transferred synthetic seeds (indirect and direct) to the soil mixture had the lowest percentage of plant conversion as well as other growth features (Table 3).

Evaluation of genetic stability of synthetic seed derived plants by ISSR molecular markers
The number of bands formed by each primer used in this study, as well as the percentage of polymorphic bands, are listed in Table 4. Each ISSR primer amplified a different number of bands, ranging from 3 (IS1) to 11 (IS19), with an average of 7 bands per ISSR primer. A monomorphic banding pattern (Fig. 5) was observed in all nine synthetic seed-derived plants and their mother plant. According to the findings, each primer's produced bands were monomorphic and similar to the mother plant.

Plants grown from artificial seeds have higher levels of RA than plants grown from natural seeds
HPLC analysis with a standard compound was used to assess the amount of RA in hydro-methanolic extracts of encapsulated nodal segments-derived plants, encapsulated shoot tips-derived plants, and natural seed derived plants of the same age (Figs. S1 and 6). Figure 6 shows that the content of RA in leaves of encapsulated nodal segments-derived plants was 78.99 mg/g dry weight (DW), which was 7.77 times higher than the content of RA in natural seed-derived plants (10.17 mg/g DW). Also, plants grown from encapsulated shoot tips had a RA content of 77.12 mg/g DW, which was 7.58 times higher than plants grown from natural seed (Fig. 6).

Discussion
The use of SA to encapsulate nodal segments of S. khuzistanica was investigated in this report. The effects of SA and CaCl 2 .2H 2 O concentrations, explant type, culture medium salt-strength, various levels of plant growth regulators BAP, IBA, and TDZ, methods and times of synthetic seed storage in cold conditions, and type of substrate for seed conversion were evaluated in order to optimize the conditions for synthetic seed production in S. khuzistanica plants. The genetic stability of synthetic seed-derived plantlets was also studied, as well as the rosmarinic acid content of synthetic seedderived plants. One of the most important factors in the production and germination of S. khuzistanica synthetic seeds is choosing the right concentration of SA and CaCl 2 ·2H 2 O. In this study, gel complexation with 3% SA and 100 mM CaCl 2 ·2H 2 O resulted in firm, clear, isodiametric, and easyto-handle capsules. According to previous findings, 3% SA has been used to produce firm and well-shaped capsules with the shoot tips of Solanum nigrum (Verma et al. 2010), Glochidion velutinum (Mallikarjuna et al. 2016), Rhinacanthus nasutus (Elangomathavan et al. 2017), and Withania coagulans (Rathore and Kheni 2017). Furthermore, we discovered that lower SA (2%) produced fragile beads that were difficult to handle, but greater SA (4-5%) produced hard beads that caused a significant delay in shoot emergence, which is consistent with earlier research findings (Adhikari et al. 2014;Parveen and Shahzad 2014). Also, the use of an appropriate culture medium with desired hormone content in a synthetic seed matrix is one of the most essential and effective elements in germination efficiency and growth of encapsulated explants. In both explants of S. khuzistanica, we found that using half-strength MS culture media led to the highest regrowth percentage, regrowth rate, and other growth features. The best regrowth frequency of synthetic seeds was also recorded using a half-strength MS culture media in Dendrobium white fairy (Siew et al. 2014) and Curcuma amada Roxb (Raju et al. 2016). In this study, we used two explants, nodal segment and shoot tip, for the production of synthetic seeds in S. khuzistanica, and showed that encapsulating the nodal segments in a 1/2 MS culture medium resulted in the highest regrowth percentage and rate. A similar finding was obtained in Phyllanthus fraternus by previous researchers (Upadhyay et al. 2014).
We also conducted an experiment to evaluate rooting in S. khuzistanica synthetic seeds using different concentrations of IBA in the gel matrix. Pre-culturing nodal segments in root culture medium (1/2 MS media supplemented with 2% activated charcoal and 5 µM IBA for 10 days) before encapsulation increased root induction of the resultant synthetic seeds on a mixture of coco peat and perlite as planting substrate. In consistent with these results, Chand and Singh (2004) reported that pre-culture of explants on 1/2 MS culture medium supplemented with 5 µM IBA for 10 days before encapsulation resulted in simultaneous development of shoot and root. The highest rooting percentage in halfstrength (MS) culture medium has also been reported for artificial seed production in Arnebia euchroma (Manjkhola et al. 2005). In another study by Saha et al. (2015), microshoots of Ocimum kilimandscharicum Guerke, recovered from encapsulated shoot tips, were best rooted on a halfstrength MS culture medium containing 1.5 mg/L IBA. Also, the highest rooting frequency was achieved using 15 µM IBA in the gel matrix of encapsulated protocorm-like bodies (PLBs), resulting in the successful acclimatization of the plantlets in Ansellia africana (Leopard orchid) (Bhattacharyya et al. 2018). Recently, in the encapsulation of nodal segments of Allamanda cathartica, the regenerated shoots rooted on half-strength MS medium containing 0.5 µM IBA (Khanam et al. 2021). As described by previous researchers, IBA is an ideal auxin for in vitro rooting in Salvia fruticosa (Arikat et al. 2004), Salvia nemorosa (Skala and Wysokinska 2004), and Salvia hispanica (Bueno et al. 2010).
In another part of this research, the effect of solid and liquid MS culture media on the regrowth and development of encapsulated nodal segments of S. khuzistanica containing different concentrations of TDZ in their gel matrix was evaluated. The majority of features, such as regrowth percentage, regrowth rate, node and shoot numbers, were all higher when liquid culture medium was utilized instead of Fig. 6 Contents of rosmarinic acid in different Satureja khuzistanica plant types derived from natural seeds, encapsulated nodal segments and shoot tip nodal segment. The levels of rosmarinic acid in each sample were analysed by HPLC. Different letters indicate significant differences according to Duncan's multiple range test (P ≤ 0.05). Bars represent means ± SD solid culture medium, especially when larger concentrations of TDZ were used in the gel matrix. Similarly, the maximum conversion of encapsulated nodal segments into plantlets was obtained in guava (Psidium guajava L.) in a liquid MS culture medium (Rai et al. 2008). Also, in synthetic seed production of Stevia rebaudiana, when full-strength liquid MS medium was compared with the full-strength solid MS medium with or without any growth regulator for the conversion of encapsulated shoot tips and nodal segments, it was found that in liquid medium the frequency of conversion was higher (Ali et al. 2012). Better ventilation and oxygenation of explants, as well as better nutrient absorption from this type of culture medium, may explain the better regrowth results of encapsulated nodal segments in liquid medium. According to the findings of this study, an effective shortterm storage protocol for Ansellia africana was established using encapsulated protocorm-like bodies (PLBs) induced from seedling nodal segments, with the best response reported on MS medium supplemented with 10 µM TDZ (Bhattacharyya et al. 2018). Since its first experimental demonstration of cytokinin-like activity by Mok et al. (1982), TDZ has been used to induce PLBs in a variety of plant species and has proven to be superior to traditional purine-based cytokinins like BAP (Asghar et al. 2011).
The cold storage duration and condition significantly influenced the regeneration potential of encapsulated nodal segments of S. khuzistanica plants. The regeneration potential of synthetic seeds stored in containers without MS culture medium was significantly decreased after 2 weeks when compared to control and completely lost after 12 weeks of storage in these conditions. Artificial seeds stored in containers with MS culture medium had a much higher potential for regeneration than the previous synthetic seeds, and their ability to regenerate slightly decreased after 2 weeks. However, after 4 weeks in MS culture medium, regeneration ability of recent synthetic seeds gradually decreased, but these seeds were still able to regenerate after 12 weeks. According to previous research (Danso and Ford-Lloyd 2003), the decrease in regrowth parameters of cold-stored synthetic seeds in this study could be attributed to the alginate matrix inhibiting tissue respiration or a loss of moisture due to partial desiccation during storage. On the other hand, the higher regrowth of synthetic seeds stored in jars containing MS medium could be due to the availability of water and nutrients in MS culture media, which prevents moisture loss in cold-stored synthetic seeds (Redenbaugh et al. 1993). Similar to the findings of this study, short-term storage of synthetic seeds under cold conditions (4 °C) has previously been reported in various plant species such as Withania somnifera (Singh et al. 2006), Decalepis hamiltonii (Sharma and Shahzad 2012), Cassia angustifolia (Parveen and Shahzad 2014), Withania coagulans (Rathore and Kheni 2017), Solanum tuberosum (Ghanbarali et al. 2016), Plectranthus amboinicus (Arumugam et al. 2019), and Decalepis salicifolia (Rodrigues et al. 2020).
The method of synthetic seed conversion and the kind of planting substrate were both crucial in the establishment and growth of S. khuzistanica encapsulated nodal segments. Synthetic seeds that were planted directly on planting substrates after encapsulation had a lower conversion rate and developed slower than those that were sown indirectly. The increased percentages of conversion and growth features were seen after the pre-culture of synthetic seeds on MS culture medium for 2 weeks (indirect conversion method). The findings of this experiment also demonstrated that coco peat is the optimum planting substrate for converting encapsulated nodal segments. Similar to these findings, Ghanbarali et al. (2016) reported that pre-culture of synthetic seeds in basic MS culture medium for 2 weeks before planting in coco peat resulted in the best regrowth and conversion among several commercial substrates used to convert potato artificial seeds. Faisal et al. (2012) also demonstrated that synthetic Rauvolfia serpentine seeds in a garden soil mixture had the lowest rate of plant conversion, confirming our findings. When mass propagation of plants is done through synthetic seed production, it is critical to maintain the genetic fidelity of the mother plant. To test the genetic fidelity of clonal plants, molecular markers, especially ISSR markers, are commonly used. The ISSR-based bands generated in this study were all monomorphic, and no genetic variation was observed between the mother plant and the nine randomly selected synthetic seed-derived plants. Several publications have proposed the use of molecular marker techniques, particularly ISSR, for clonal fidelity of in vitro grown plants in various plant systems (Mehrotra et al. 2012;Faisal et al. 2012;Saha et al. 2015;Viehmannova et al. 2016;Kundu et al. 2018;Hatzilazarou et al. 2021). Our findings are consistent with previous researches on the genetic stability of synthetic seed-derived plantlets of Albizia lebbeck (Perveen and Anis 2014), Malus domestica (Li et al. 2014), Limonium hybrid (Bose et al. 2017), Erythrina variegata (Javed et al. 2017), Sphagneticola calendulacea (L.) (Kundu et al. 2018), Sphagneticola cale (Hatzilazarou et al. 2021).
As indicated in the title, one of the most remarkable achievements of this work is the regeneration of plants with high rosmarinic acid content from encapsulated S. khuzistanica explants. Plants derived from encapsulated nodal segments and shoot tips produced significantly more rosmarinic acid in their leaves than plants derived from natural seeds. In vitro culture techniques seem to be a promising method to explore sustainable rosmarinic acid production in S. khuzistanica under controlled conditions (Sangwan et al. 2001). Establishment of callus and cell suspension cultures, and also nodal segment cultures for rosmarinic production in this plant species has been reported by previous researchers (Sahraroo et al. 2014(Sahraroo et al. , 2016Fatemi et al. 2020). However, 1 3 increased production of rosmarinic acid using synthetic seeds in this medicinal plant or any other medicinal species has not been reported. In a similar experiment, Rodrigues et al. (2020) investigated the level of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the roots of Decalepis salicifolia artificial seed-derived plants. In contrast to the findings of our work, synthetic seed-derived plants produced 2H4MB in amounts comparable to seed-derived field-grown plants of the same age. Also, consistent with the results of this experiment, many researchers have reported an increase in secondary metabolites, particularly rosmarinic acid, in shoot cultures of some plant species, such as Thymus lotocephalus (Gonçalves et al. 2019), Melissa officinalis L. (Vanda et al. 2019) and Eryngium alpinum L. (Kikowska et al. 2020). As described by other researchers (Khan et al. 2020), the production of bioactive secondary metabolites can be enhanced in medicinal plants with micropropagation techniques. During micropropagation, tiny parts of plants, commonly called explants, excised from different plant species can be micropropagated under optimized growth conditions of culture media, temperature, and photoperiod (Abbasi et al. 2016). The process of plant cell growth in vitro, as well as the rapid but controlled secondary metabolism in these cells, causes ontogenetic changes in regenerated plants. These ontogenetic changes can be an important reason for the enhanced biosynthesis of secondary metabolites in micropropagated plants (Khan et al. 2020).

Conclusions
This is the first report of synthetic seed production in S. khuzistanica using shoot tip and nodal segment explants. The current methodology offers a promising way for producing large-scale synthetic seeds of this endangered medicinal plant, as well as a simple and efficient approach of producing rosmarinic acid in this plant species. By optimizing the encapsulation conditions, determining the optimal growth regulators for synthetic seed regrowth and rooting, and using a suitable substrate for plant conversion, we were able to obtain entire plants from synthetic seeds in a plant species whose natural seeds are hard to germinate. Short-term storage of synthetic seeds at 4 °C also preserved the regeneration potential of synthetic seeds for up to 30 days. However, if additional research is done on the production and storage of synthetic seeds in this plant, as well as optimizing storage conditions and employing different storage temperatures, long-term storage of these seeds might be possible.
Author contributions MRA conceived and designed the experiments. RA performed the experiments. MRA and RA analyzed the data and wrote the manuscript. MRA, SSM and AM contributed in preparation of reagents, materials and analysis tools. All authors read and approved the final manuscript.
Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.