In our study, we evaluated the antioxidant capacity of the N. officinale parent plant material as well as the samples from in vitro microshoot cultures using the CUPRAC, FRAP and DPPH methods. Additionally, total phenolics (F-C) were estimated. The results were expressed as mmol TE/100 g DW. Our results for N. officinale microshoot cultures showed that the PGRs, duration of the growth period and the type of in vitro culture slightly influenced antioxidant potential. The maximal antioxidant potential using CUPRAC and DPPH method for extracts of agar microshoot cultures was respectively 2.0 and 9.7 times higher in comparison to maximal potential of control sample. In FRAP method the results were similar (Table 4 and Table S13). In extracts of agitated microshoot cultures the maximal antioxidant potential for CUPRAC method was 1.3 higher and for DPPH method 1.2 lower than in extracts of agar microshoot cultures (Table 4). The maximal potential of extracts from both types of cultures measured by CUPRAC and DPPH methods was obtained for MS medium containing 1 mg/L KIN and 1 mg/L IAA after 20 days in agitated cultures and 30 days in agar cultures. In comparison to agar control culture in agitated cultures was obtained 2.5 and 8.2 higher antioxidant potential respectively for CUPRAC and DPPH method. In FRAP method the maximal results were similar (Table 4 and Table S13). In extracts of parent plant antioxidant potential compared to maximal amounts obtained in agar and agitated microshoot cultures was 1.2 times lower in CUPRAC and DPPH method and 1.9 times lower in FRAP method (Table 4 and Table 5). In the F-C method, the maximal amount of phenolics in extracts of agar cultures was respectively 1.4 and 1.7 times higher than in maximal amount of extracts for control and agitated microshoot cultures and 3.2 times higher than in extracts form parent plant (Table 4 and Table 5 and Table S13). In extracts from agitated microshoot cultures the maximal amount of phenolic obtained in F-C method was 1.2 times lower and 1.9 times higher than in extracts of respectively agar control and parent plant material. (Table 4 and Table 5 and Table S13).

Question

In our study, we evaluated the antioxidant capacity of the N. officinale parent plant material as well as the samples from in vitro microshoot cultures using the CUPRAC, FRAP and DPPH methods. Additionally, total phenolics (F-C) were estimated. The results were expressed as mmol TE/100 g DW. Our results for N. officinale microshoot cultures showed that the PGRs, duration of the growth period and the type of in vitro culture slightly influenced antioxidant potential. The maximal antioxidant potential using CUPRAC and DPPH method for extracts of agar microshoot cultures was respectively 2.0 and 9.7 times higher in comparison to maximal potential of control sample. In FRAP method the results were similar (Table 4 and Table S13). In extracts of agitated microshoot cultures the maximal antioxidant potential for CUPRAC method was 1.3 higher and for DPPH method 1.2 lower than in extracts of agar microshoot cultures (Table 4). The maximal potential of extracts from both types of cultures measured by CUPRAC and DPPH methods was obtained for MS medium containing 1 mg/L KIN and 1 mg/L IAA after 20 days in agitated cultures and 30 days in agar cultures. In comparison to agar control culture in agitated cultures was obtained 2.5 and 8.2 higher antioxidant potential respectively for CUPRAC and DPPH method. In FRAP method the maximal results were similar (Table 4 and Table S13). In extracts of parent plant antioxidant potential compared to maximal amounts obtained in agar and agitated microshoot cultures was 1.2 times lower in CUPRAC and DPPH method and 1.9 times lower in FRAP method (Table 4 and Table 5). In the F-C method, the maximal amount of phenolics in extracts of agar cultures was respectively 1.4 and 1.7 times higher than in maximal amount of extracts for control and agitated microshoot cultures and 3.2 times higher than in extracts form parent plant (Table 4 and Table 5 and Table S13). In extracts from agitated microshoot cultures the maximal amount of phenolic obtained in F-C method was 1.2 times lower and 1.9 times higher than in extracts of respectively agar control and parent plant material. (Table 4 and Table 5 and Table S13).

5. Conclusions
In this study, the initiation and optimization of the conditions for N. officinale in vitro cultures were performed for the first time. We assessed the influence of the qualitative and quantitative composition of different auxins and cytokinins in the MS culture medium, the type of culture, and the duration of the growth cycle on biomass growth; we also estimated the production of glucosinolates and phenolic acids, and the antioxidant activity of N. officinale microshoot cultures. We optimized the studied processes and therefore we can propose the best culture type and conditions for maintaining N. officinale cultures in vitro. Considering the finest biomass growth, optimal glucosinolates and phenolic acids production, were obtained in agitated cultures grown over 10-day periods on the MS medium containing 1 mg/L BA and 1 mg/L NAA. Due to high biomass increases, the best conditions for the production of phenolic acids were the agar cultures grown over 20-day periods on the same MS medium variant. For both types of cultures high antioxidant potential was proved for microshoots maintained on MS medium containing 1 mg/L KIN and 1 mg/L IAA. The studies have also shown that in agitated in vitro cultures, liquid medium and aeration of microshoots are the promoting conditions for microshoot growth. That could be because these conditions are similar to natural environment of this species.
The in vitro microshoots exhibited very interesting differences in comparison with the parent plant and provided alternative sources for obtaining health-promoting compounds.
write the main points

Answer 1

1. The antioxidant capacity of N. officinale parent plant material and in vitro microshoot cultures was evaluated using the CUPRAC, FRAP, and DPPH methods, with total phenolics (F-C) estimated as well.

2. The results showed that the PGRs, duration of the growth period, and type of in vitro culture slightly influenced the antioxidant potential of the microshoot cultures.

3. Agar microshoot cultures had significantly higher antioxidant potential compared to control samples, with maximal potential observed in MS medium containing 1 mg/L KIN and 1 mg/L IAA after 20 days in agitated cultures and 30 days in agar cultures.

4. The F-C method showed that extracts from agar cultures had higher phenolic content compared to control and agitated microshoot cultures.

5. Optimal conditions for biomass growth, glucosinolate production, and phenolic acid production were found in agitated cultures grown over 10-day periods on MS medium containing 1 mg/L BA and 1 mg/L NAA.

6. In agitated cultures, liquid medium and aeration promoted microshoot growth, suggesting similarities to the natural environment of N. officinale.

7. Overall, in vitro microshoots showed interesting differences in antioxidant potential and provided alternative sources for health-promoting compounds compared to the parent plant.