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 minutes in running tap water, surface sterilized for 5 minutes in 1.5 % (v/v) sodium hypochlorite, and rinsed three times in sterile distilled water for 5 minutes. As described by Fatemi et al. (2019 and 2020), the nodal segments were cultured in solid MS medium containing 2 mg/L 6-benzylaminopurine (BAP), 3 % (w/v) sucrose, and 0.2 % (w/v) polyvinylpyrrolidone (PVP). The plants were grown under in vitro conditions in a growth chamber (24 ± 2 °C, 16 h light/8 h dark) at Bu-Ali Sina University, Hamedan, Iran. The cultured nodes were incubated in the growth chamber and 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 four-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, 3 mg/L BAP, 0.25 mg/L IBA, and 3% sucrose. Using distilled water, several CaCl2.2H2O 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.
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 CaCl2.2H2O was prepared in distilled water and used as a complexing agent.
The effect of different concentrations of 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 CaCl2.2H2O combination in the 1/2 MS culture medium containing different concentrations of thidiazuron (0, 0.1, 0.25, 0.45, and 0.5 mg/L) as the gel matrix. Then, some capsules were transferred to Petri dishes containing solid MS culture medium without any hormones. Other ones were transferred to Erlenmeyer flasks containing 50 ml of liquid MS culture medium (without hormone). 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 two weeks. Shoot length, number of shoots, and number of nodes were all measured after another four weeks.
The effect of different concentrations of IBA in gel matrix on root induction
In this experiment, nodal segment explants were first pre-cultured 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 CaCl2.2H2O was also utilized in this experiment. Two-week-old germinated capsules were put on a planting substrate of coco peat and peat moss (1:1) and generated roots, after two months of encapsulation. The root induction efficiency was assessed by counting the number of roots, root percentage, and root length.
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 ºC for various time periods (0, 2, 4, 8, and 12 weeks). After each storage period, encapsulated nodal segments were transferred to a hormone-free MS culture medium for regrowth. After 2 weeks, the regrowth percentage and regrowth rate of capsules were calculated for each treatment.
Effect of different planting substrates on plant conversion
Various planting substrates, including MS culture medium without growth regulators, coco peat, perlite, sand, and 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 free hormone solid MS medium for two weeks, and then the germinated capsules were transferred to the sterilized planting substrates. 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.
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 ngμl−1 with TE (Tris–EDTA) buffer before use and stored at 4 ºC for the ISSR experiment. In ISSR analysis, six primers were used for the genetic stability assessment of nine synthetic seed-derived plants compared to their mother plant. The PCR reactions were performed in a 20 μl volume containing 6 µL H2O, 10 µL master solution (Taq 2X, 2 mM MgCL2), 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 ºC 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 ºC for 30 s. A final extension at 72 ºC 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 1X TBE buffer and stained with ethidium bromide). The gel documentation system (DigiDoc H110) was used for photography.
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. Regenerated plants from synthetic seeds made with 3 % SA and 100 mM CaCl2.2H2O 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 2020). 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 (H2O) 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.
All experiments were conducted as factorials based on completely randomized designs 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).