2.1.2. Influence of Elicitation on Biomass Growth
In this study, the effect of the application of all elicitors on the biomass growth of agitated microshoot cultures of F and M lines of
S. rubriflora was found. The highest biomass growth for line M was found 24 h after the addition of YeE. It caused the highest biomass gain, 2.06 times higher than the control. For line F, the highest biomass increase was found at 8 days after MeJA addition. It caused the highest biomass gain, 2.39 times higher biomass gain compared to the control (
Table 1).
At 24 h after the addition of the elicitor, the biomass gains of elicited cultures, except for MeJA elicitation for lines M, were higher than those of the control cultures. The highest biomass gain of lines M at 24 h was recorded for YeE elicitation (max. Gi = 41.76), it was 2.06 times higher compared to the control. At 24 h after addition, elicitors did not affect the biomass gains of lines F except for YeE elicitation (max. Gi = 23.35). This gain was 1.28 times higher compared to the control (
Table 1).
At 48 h after the addition of the elicitor, higher biomass gains were recorded for lines M except for those elicited with ETH and MeJA. The highest biomass gain of lines M was found for CH elicitation at 48 h (max. Gi = 37.57), it was 1.43 times higher compared to the control. The elicitors at 48 h after their addition also affected the biomass gain of the female lines except for MeJA elicitation. A high value of Gi coefficient was also found after ETH elicitation (max. Gi = 27.47). This gain was 1.94 times higher compared to the control (
Table 1).
Supplementation with elicitors had a negative effect on the biomass gains of the experimental cultures harvested after 4 days. Only after CH elicitation for the biomass of line F, the Gi value (26.67) was 1.07 times higher compared to the control (
Table 1).
For microshoots of agitated cultures of lines M 6 days after the addition of elicitors, only CH and MeJA showed a slight increase in Gi values compared to the control. The highest increase in biomass of line M was found after CH elicitation (max. Gi = 52.63), it was 1.16 times higher compared to the control (
Table 1). For line F, only two elicitors, CH and YeE, affected biomass growth after 6 days of addition. The highest Gi value was found after YeE elicitation (max. Gi = 30.00). This increase was 1.32 times higher compared to the control (
Table 1).
After 8 days of elicitation application for line M cultures, only CH and MeJA were found to have a higher Gi value compared to the control cultures. The highest biomass gain for lines M was found for MeJA elicitation (max. Gi = 69.26), this was 1.5 times higher than for the control. On the other hand, for line F, the highest Gi value was found after MeJA elicitation (Gi = 42.99). This gain was 2.39 times higher compared to the control (
Table 1).
The notable Gi values in the same time points are diverse. That is caused by the type of elicitor and the duration time of elicitor treatment. The elicitor addition often caused a decrease in in vitro culture biomass growth while the production of secondary metabolites increase. Such a fact is well known in plant biotechnology studies and has been described before, e.g., after CH elicitation in
Hypericum perforatum root cultures [
25], MeJA elicitation in
Fagonia indica adventitious root cultures [
26], YeE elicitation in
Panax ginseng cell culture [
27], or
Aspergillus flavus fungus elicitation in
Catharanthus roseus callus cultures [
28].
Analysis of the literature allows a direct comparison of data on the effect of elicitation with two elicitors: YeE and CH on the value of the Gi index for in vitro cultures of
S. chinensis species, and the experiment carried out in the present work. The highest recorded Gi index values for YeE elicitation of
S. chinensis species were observed after YeE addition at a concentration of 1000 mg/L. The Gi value ranged from 33.37 to 42.89, where the value for the control sample was 40.74. The other, higher concentrations of YeE, 3000 mg/L and 5000 mg/L, caused a decrease in biomass growth especially when YeE was supplemented on the first day of culture [
22]. In the course of our experiment, a decrease in the biomass growth of in vitro cultures of
S. rubriflora was found 4 days after the addition of YeE at a concentration of 3000 mg/L. For line M it was also observable on day 6 and day 8 of culture. Line M of
S. rubriflora was found to be much more sensitive to YeE compared to line F. Elicitation of
S. chinensis with CH at concentrations in the range of 25–200 mg/L did not adversely affect microshoots growth. The Gi index was comparable to control cultures. The highest Gi value was found for CH supplementation at concentrations of 50 and 100 mg/L on the first day of culture. It was 57.39 and 56.87, respectively; where the Gi value for the control sample was 40.00 [
22]. In the course of our experiment for in vitro cultures of
S. rubriflora, the increase in CH elicited biomass for cultures of line F occurred only at 48 h after elicitor addition, it was the highest at day 8 after elicitor addition, while cultures of line M showed the highest increase at 24 h after elicitor addition (
Table 1).
2.1.3. The Influence of Elicitation on Lignan Production
The study proved the significant effect of the applied elicitation on the accumulation of lignans in cultures of lines F and M of S. rubriflora. In biomass extracts, 22 compounds from four groups of lignans were qualitatively and quantitatively determined: dibenzocyclooctadiene lignans (schisantherin A and B, schisandrin, schisandrin C, gomisin A, D, G, J, N, O, 6-O-benzoylgomisin O, schisandrin A, rubrisandrin A, epigomisin O, schisanhenol, interiotherin C, angeloylgomisin H and O), aryltetralin lignans (wulignan A1), dibenzylbutane lignans (pregomisin, mesodihydroguaiaretic acid), and tetrahydrofuran lignans (fragransin A2). In addition, 2 compounds from the dihydrobenzofuran group of neolignans (licarin A and B) were also found in the analyzed extracts.
The study confirmed the effect of the elicitation schemes used on the accumulation of metabolites in the microshoot culture biomass extracts analyzed, while only trace amounts were found in the culture media (<5 mg/L).
Detailed results of quantitative analyses in the control sample and depending on the elicitation scheme used and on the time of tissue harvesting are presented in
Supplementary Data (Tables S1–S5).
The highest obtained contents of the analyzed compounds in the course of the whole experiment were as follows: wulignan A1 (max. 0.36 mg/100 g DW; CH, line F, 6 days, 2.37 times higher compared to the control), rubrisandrin A (max. 0.19 mg/100 g DW; YeE; line M, 6 days, 3.82 times higher compared to the control), interiotherin C (max. 0.36 mg/100 g DW; CH; line F, 6 days, 7.16 times higher compared to the control), schisandrin (max. 71,98 mg/100 g DW; MeJA, line M, 24 h, 3.13 times higher compared to the control), gomisin D (max. 28.80 mg/100 g DW; MeJA, line M, 24 h, 4.44 times higher compared to the control), gomisin J (max. 15.71 mg/100 g DW; MeJA, line M, 24 h, 4.78 times higher compared to the control), gomisin A (max. 91.53 mg/100 g DW; MeJA, line M, 24 h, 3.31 times higher compared to the control), gomisin G (max. 6.93 mg/100 g DW; MeJA, line M, 24 h, 3.91 times higher compared to the control), licarin B (max. 0.41 mg/100 g DW, ETH, line F, 48 h, 3.33 times higher compared to the control), epigomisin O (max. 1.54 mg/100 g DW; ETH, line F, 48 h, 2.40 times higher compared to the control), gomisin O (max. 4.80 mg/100 g DW; MeJA, line F, 48 h, 1.38 times higher compared to the control), mesodihydroguaiaretic acid (max. 0.23 mg/100 g DW; MeJA, line M, 8 days, 4.62 times higher compared to the control), schisantherin A (max. 5.42 mg/100 g DW; ETH, line F, 48 h, 1.99 times higher compared to the control), schisantherin B (max. 16.66 mg/100 g DW; MeJA, line M, 24 h, 5.03 times higher compared to the control), licarin A (max. 37.54 mg/100 g DW; YeE, line F, 8 days, 3.23 times higher compared to the control), schisanhenol (max. 10.11 mg/100 g DW; ETH, line F, 48 h, 1.79 times higher compared to the control), deoxyschisandrin (max. 94.86 mg/100 g DW; YeE, line M, 4 days, 1.65 times higher compared to the control), gomisin N (max. 28.07 mg/100 g DW; MeJA, line M, 24 h, 4.14 times higher compared to the control), 6-O-benzoylgomisin O (max. 1.40 mg/100 g DW; ETH, line F, 48 h, 2.64 times higher compared to the control), and schisandrin C (max. 5.24 mg/100 g DW; YeE, line M, 24 h, 3.64 times higher compared to the control) (
Tables S1–S5 and
Table 2,
Table 3,
Table 4,
Table 5 and
Table 6).
The obtained results were compared with elicitation in in vitro cultures of well-established in phytotherapy
S. chinensis which affected the production of dibenzocyclooctadiene lignans [
22]. Cultures of
S. chinensis were maintained on MS medium containing 3 mg/L BA and 1 mg/L NAA. Cultures were treated with elicitors at 10 and 20 days of culture. The elicitors tested were cadmium chloride (CdCl
2), YeE, CH, MeJA and the permeabilizing agent dimethyl sulphoxide (DMSO). The duration of culture was 30 days. In the biomass extracts of the experimental cultures, the content of dibenzocyclooctadiene lignans was determined by DAD-HPLC. The total content of lignans obtained in cultures elicited with CdCl
2 was twice as high as in non-elicited control samples. The content of secondary metabolites after the use of this elicitor was about 730 mg/100 g DW. Elicitation with CH increased lignan production by 1.35 times (500 mg/100 g DW). The use of YeE led to a 1.8-fold increase in the content of the tested compounds. The identified lignans differed from those determined in in vitro cultures of
S. rubriflora species during the present experiment. In the
S. chinensis species, only lignans from the dibenzocyclooctadiene lignan group were determined: schisandrin, gomisin A, gomisin G, schisantherin A, schisantherin B, Schisanhenol, deoxyschizandrin, γ-schisandrin, schisandrin C, angeloylgomisin H and Q, schisandrin B, benzoylgomisin P and schisantherin D. Only the effect of elicitation on the compounds found in both species, i.e., dibenzocyclooctadiene lignans, was evaluated comparatively in this study (compounds from the other labeled lignan groups in
S. rubriflora were not considered): schisandrin, gomisin A, G, schisantherin A, B, schisanhenol, deoxychizandrin and the total content of these compounds (
Table 7). Schisandrin, gomisin A and G, and schisantherin B showed higher content multiplicity relative to the control due to elicitation in
S. rubriflora species (3.13, 3.31, 3.91, 5.03 times higher for MeJA, respectively, 50 µM, 24 h,) than in
S. chinensis species (2.28, 2.80 times more for CdCl
2, respectively, 1000 µM, 10 days, 3.03 times more for YeE, 3000 mg/L, day 10; 2.61 times more for YeE, 1000 mg/L, 20 days). The content of these compounds was lower in elicited cultures of
S. rubriflora species. Schisantherin A, schisanhenol, deoxyschizandrin showed lower multiplicity of content relative to control upon elicitation in
S. rubriflora species (1.99, 1.79 times more, respectively, ETH, 25 µM, 48 h, 1.65 times more, YeE, 3000 mg/L, 4 days) in cultures of
S. rubriflora species than in those for
S. chinensis (3.11 times more, YeE, 3000 mg/L, day 0; 4.44 times more, CdCl2, 1000 µM, 10 days; 1.95 times more, YeE, 5000 mg/L, days 10). The contents of schisantherin A and schisanhenol were lower in
S. rubriflora cultures than in S.
chinensis. The only compound that showed a higher content in
S. rubriflora cultures was deoxyschizandrin (max. 94.86 mg/100 g DW). The total content of lignans was higher in
S. chinensis cultures (max. 730.60 mg/100 g DW) than in
S. rubriflora (max. 368.50 mg/100 g DW), while the multiplicity relative to the control was higher in
S. rubriflora cultures (3.29 times higher) (
Table 7).
Studies on the effect of elicitation on the production of specific compounds from the lignan group are a rather difficult subject of research, and that is probably why they are not a frequent object of study. Studies on a popular compound with anticancer activity-podophyllotoxin are of greatest interest. Kasparova et al. conducted a study on the effect of MeJA elicitation on podophyllotoxin accumulation in suspension cultures of
Juniperus virginiana. Cultures were elicited on day 14 of culture and harvested at 6, 24, 48 and 168 h after the addition of the elicitor. The highest maximum podophyllotoxin content was determined at 168 h after the addition of 5 mmol/L MeJA (max. 0.68 mg/g DW) and was about 2.83 times higher than the control sample [
29]. Anbazhagan et al. conducted a study on the effect of MeJA elicitation on podophyllotoxin accumulation in suspension and adventitious root cultures of
Podophyllum peltatum species. Day “0” (inoculation) was the day of addition of the elicitor 20 μM MeJA. Cultures were harvested 5 weeks after elicitor addition. Podophyllotoxin content in suspension cultures was 4.1 times higher than in the control samples (max. 0.3625 mg/g DW), and in adventitious root cultures was 1.62 times higher than in the control samples (max. 0.588 mg/g DW) [
30]. Bhattacharyya et al. conducted a study on the effect of MeJA elicitor on podophyllotoxin accumulation in
Podophyllum hexandrum species. Elicitor; 100 µM MeJA, was added on day 3 of culture. Cultures were harvested after 9 days. Podophyllotoxin content was 80 µg/g DW and was 8 times higher than in the control samples [
31].
Sasheva et al. conducted a study on the effect of MeJA elicitation on podophyllotoxin accumulation in
Linum thracicum species. MeJA was used at concentrations of 50 and 100 µM and added on day 7 of culture. Cultures were harvested 24 h and 72 h after the addition of the elicitor. Cultures were grown on MS media differing in sucrose content. The highest content of podophyllotoxin was 1.6 mg/g DW, which was 1.14 times higher than in the control sample and was obtained in biomass cultured on medium with 20 g/L sucrose. The researchers found no significant effect of elicitation on the production of podophyllotoxin [
32].
Waqar et al. studied the effect of CH elicitation on the accumulation of specific lignans and neolignans: secoisolariciresinol diglucoside, lariciresinol diglucoside, dehydrodiconiferyl alcohol glucoside and guaiacylglycerol-β-coniferyl alcohol ether glucoside in suspension cultures of
Linum usitatissimum. Cultures were elicited with CH at a concentration of 10 mg/L on day 10 of culture. The duration of culture was 30 days. Material was harvested at 8, 24 and 48h. Maximum enhancements of 7.3-fold (28 mg/g DW) occurred for lariciresinol diglucoside, 3.5-fold (58.85 mg/g DW) in dehydrodiconiferyl alcohol glucoside and while the least enhancement of 2-fold (18.42 mg/g DW) for secoisolariciresinol diglucoside was observed in CH treated cell cultures than to controls [
33]. Nadeem et al. also studied the effect of elicitation, but with a different elicitor, YeE, on the accumulation of lignans and neolignans in suspension cultures of
L. usitatissinum species. YeE was added to the medium on day “0” (inoculation) at concentrations of 10, 50, 100, 200, 500 and 1000 mg/L. YeE at a concentration of 200 mg/L caused the highest increase in the accumulation of the compounds: secoisolariciresinol diglucoside (3.36-fold, max. 10.1 mg/g DW), lariciresinol diglucoside (1.3-fold, 11.0 mg/g DW) and dehydrodiconiferyl alcohol glucoside (4.26-fold, max. 21.3 mg/g DW) [
34].
Wawrosch et al. studied the effects of elicitation with AgNO
3, MeJA and YeE on the accumulation of specific furan-type lignans: leoligin and 5-methoxy-leoligin in hairy root cultures of
Leontopodium nivale. Elicitors were added to 3-week-old cultures at concentrations of: 15, 30 and 60 µM AgNO
3, 50, 100, 200, 300 µM MeJA and 1, 2, 5 g/L YeE. The duration of culture was 4 weeks. The highest content of leoligin was recorded after elicitation with 100 µM MeJA (max. 0.05 in %), it was 8.33 times higher than that of the control sample. The content of 5-methoxy-leoligin was highest after supplementation with 15 µM AgNO
3 (max. 0.026 in %), and was 6.5 times higher than that of the control sample [
35].
Schmitt and Petersen examined the effect of MeJA elicitation on the accumulation of tetrahydrofuran lignans, pinoresinol and matairesinol, in
Forsythia ×
intermedia suspension cultures. MeJA was added on day “0” at a concentration of 100 µM. Cultures were harvested every other day from the addition of the elicitor. Pinoresinol content increased 3-fold (max. 0.8 mg/g DW) and matairesinol content increased 7-fold (max. 2.7 mg/g DW) relative to the control sample [
36].
Sanchez-Sampedro et al. conducted an experiment proving the effect of elicitation with YeE, SA, CH and chitin on the synthesis of flavonolignan: silymarin in suspension cultures of
Silybum marianum. SA, CH and chitin did not stimulate silymarin production even at higher concentrations. YeE caused intense browning and significant loss of cell viability after 48 h (at concentrations of 100 and 200 μg/mL). A slight increase in silymarin content was observed following YeE supplementation. MeJA at a concentration of 10 µM/mL was ineffective, but at a concentration of 100 µM/mL caused significant accumulation of silymarin in cells. MeJA alone or in combination with YeE gave the best results. Three-day cultures were treated for 48 h with 50 g/mL YeE, 100 μM MeJA or both elicitors simultaneously. Silymarin content for control samples was max 2.01 mg/g DW. The combination of MeJA and YeE yielded nearly 600% higher accumulation of silymarin in biomass [
37].