This study has been conducted to establish the bioreactor culture of adventitious roots in Eleutherococcus koreanum for the production of biomass and bioactive compounds. To maximize root biomass and bioactive compounds production, various chemical and physical factors were optimized in flask and bi...
This study has been conducted to establish the bioreactor culture of adventitious roots in Eleutherococcus koreanum for the production of biomass and bioactive compounds. To maximize root biomass and bioactive compounds production, various chemical and physical factors were optimized in flask and bioreactor cultures. Based on the results, profiles of the adventitious roots growth and the accumulation of bioactive compounds were determined, followed by elicitations to increase the target compounds (eleutheroside B and E, chlorogenic acid, total phenolics and flavonoids). Finally, ploidy levels and bioactive compounds of the adventitious roots and field-grown plants were compared. 1. Establishment of adventitious root culture conditions Adventitious roots were cultured in 1/2 MS medium supplemented with various concentrations of auxins (IBA, NAA, IAA) and cytokinins (TDZ, BA, Kinetin) in 250 mL flasks for 5 weeks. The maximum accumulation of root biomass and bioactive compounds was obtained with 1/2 MS medium containing 7.0 mg · L-1 IBA and 0.1 mg · L-1 TDZ. When the adventitious roots were cultured in 3 L balloon type bioreactors, inoculum density of 5.0 g · L-1 and aeration volume of 0.1 vvm were optimal for root biomass and bioactive compounds production. In addition, the highest growth rate and the lowest root death were also observed under those conditions. Fresh and dry weight of the root was maximized at half-strength MS medium, but the content of total bioactive compounds gradually declined with increasing MS medium salt strength. The highest root biomass was obtained at ammonium/nitrate ratio of 5:25 and 10:20. But the content of total bioactive compounds was negatively affected by ammonium supply. Hydrogen peroxide content, antioxidant enzyme (CAT, SOD, G-POD and APX) and DPPH activities in the adventitious root were significantly enhanced with increasing ammonium concentration. The highest root biomass was obtained at 3% sucrose, while the highest content of total bioactive compounds was obtained at 5% sucrose. Sucrose concentration more than 5% increased both proline and H2O2 contents, as well as DPPH activity in the adventitious root due to low water potential in the medium. These results suggested that half-strength MS medium supplement with ammonium/nitrate ratio of 5:25 and 3% sucrose with an inoculum density of 5.0 g · L-1 and aeration volume of 0.1 vvm was found most suitable for root biomass and bioactive compounds production in E. koreanum adventitious roots. 2. Enhancement of bioactive compound production Profiles of root biomass and the accumulation of bioactive compounds were investigated in 250 mL flasks and 3 L balloon type bioreactors for 7 weeks under optimal conditions determined in the previous experiment. In bioreactors, the most intensive root growth was observed from 2 to 5 weeks of culture, and the maximum growth rate was achieved after 5 weeks of culture. In flasks, the growth rate was stable during the entire culture periods. Bioreactor culture of the adventitious root resulted in much higher production of bioactive compounds compared to flask culture, which suggested that bioreactor culture was efficient for bioactive compound production in large-scale. Sugar and mineral contents in the medium decreased in parallel with increasing of root biomass, inducing decrease of electrical conductivity of the medium. CO2 concentration inside a flask was much higher than that inside a bioreactor after 5 weeks of culture, but no relationship was found between CO2 concentration and bioactive compound accumulation. The growth pattern of the adventitious root in bioreactors was investigated for 7 weeks, determined as a lag phase (0~2 weeks), exponential phase (2~5 weeks), stationary and death phases (after 5 weeks). Five-week-old adventitious roots cultured in 3 L balloon type bioreactors were treated with various concentrations of methyl jasmonate (MJ) and salicylic acid (SA) for a week to increase bioactive compounds in the adventitious root. The maximum total production of bioactive compounds (per 1 L medium) was obtained at 50 μmol MJ without decreasing root biomass. PAL and DPPH activities of the adventitious root were also highest at 50 μmol MJ treatment. 50 μmol SA resulted in the highest production of bioactive compounds but the effect was less significant than that of MJ. SA concentration up to 400 μmol significantly enhanced accumulation of eleutheroside B in the adventitious root, confirming that SA was an effective elicitor for eleutheroside B production in E. koreanum adventitious roots. 3. Comparison of characteristics between adventitious roots and mother plants Content of eleutheroside B was higher in all field-grown plant samples (leaves, stems, thin roots and thick roots) compared with the adventitious roots. However, eleutheroside E, chlorogenic acid, total phenolics and flavonoids were higher in the adventitious roots compared with field-grown plant samples. The ploidy levels of the adventitious roots and field-grown plants were tested, resulted in two typical DNA histograms at the same retention time. The result indicated genetic stability of the adventitious root. 4. Conclusion Bioreactor culture conditions and elicitation method were optimized for production of biomass and bioactive compounds in E. koreanum adventitious roots. The adventitious root with elicitation was found to contain higher bioactive compounds. In addition, typical DNA histogram confirmed genetic stability of E. koreanum adventitious roots. These results suggested that the adventitious root culture of E. koreanum has advantages of large-scale production of bioactive compounds over field-cultivation.
This study has been conducted to establish the bioreactor culture of adventitious roots in Eleutherococcus koreanum for the production of biomass and bioactive compounds. To maximize root biomass and bioactive compounds production, various chemical and physical factors were optimized in flask and bioreactor cultures. Based on the results, profiles of the adventitious roots growth and the accumulation of bioactive compounds were determined, followed by elicitations to increase the target compounds (eleutheroside B and E, chlorogenic acid, total phenolics and flavonoids). Finally, ploidy levels and bioactive compounds of the adventitious roots and field-grown plants were compared. 1. Establishment of adventitious root culture conditions Adventitious roots were cultured in 1/2 MS medium supplemented with various concentrations of auxins (IBA, NAA, IAA) and cytokinins (TDZ, BA, Kinetin) in 250 mL flasks for 5 weeks. The maximum accumulation of root biomass and bioactive compounds was obtained with 1/2 MS medium containing 7.0 mg · L-1 IBA and 0.1 mg · L-1 TDZ. When the adventitious roots were cultured in 3 L balloon type bioreactors, inoculum density of 5.0 g · L-1 and aeration volume of 0.1 vvm were optimal for root biomass and bioactive compounds production. In addition, the highest growth rate and the lowest root death were also observed under those conditions. Fresh and dry weight of the root was maximized at half-strength MS medium, but the content of total bioactive compounds gradually declined with increasing MS medium salt strength. The highest root biomass was obtained at ammonium/nitrate ratio of 5:25 and 10:20. But the content of total bioactive compounds was negatively affected by ammonium supply. Hydrogen peroxide content, antioxidant enzyme (CAT, SOD, G-POD and APX) and DPPH activities in the adventitious root were significantly enhanced with increasing ammonium concentration. The highest root biomass was obtained at 3% sucrose, while the highest content of total bioactive compounds was obtained at 5% sucrose. Sucrose concentration more than 5% increased both proline and H2O2 contents, as well as DPPH activity in the adventitious root due to low water potential in the medium. These results suggested that half-strength MS medium supplement with ammonium/nitrate ratio of 5:25 and 3% sucrose with an inoculum density of 5.0 g · L-1 and aeration volume of 0.1 vvm was found most suitable for root biomass and bioactive compounds production in E. koreanum adventitious roots. 2. Enhancement of bioactive compound production Profiles of root biomass and the accumulation of bioactive compounds were investigated in 250 mL flasks and 3 L balloon type bioreactors for 7 weeks under optimal conditions determined in the previous experiment. In bioreactors, the most intensive root growth was observed from 2 to 5 weeks of culture, and the maximum growth rate was achieved after 5 weeks of culture. In flasks, the growth rate was stable during the entire culture periods. Bioreactor culture of the adventitious root resulted in much higher production of bioactive compounds compared to flask culture, which suggested that bioreactor culture was efficient for bioactive compound production in large-scale. Sugar and mineral contents in the medium decreased in parallel with increasing of root biomass, inducing decrease of electrical conductivity of the medium. CO2 concentration inside a flask was much higher than that inside a bioreactor after 5 weeks of culture, but no relationship was found between CO2 concentration and bioactive compound accumulation. The growth pattern of the adventitious root in bioreactors was investigated for 7 weeks, determined as a lag phase (0~2 weeks), exponential phase (2~5 weeks), stationary and death phases (after 5 weeks). Five-week-old adventitious roots cultured in 3 L balloon type bioreactors were treated with various concentrations of methyl jasmonate (MJ) and salicylic acid (SA) for a week to increase bioactive compounds in the adventitious root. The maximum total production of bioactive compounds (per 1 L medium) was obtained at 50 μmol MJ without decreasing root biomass. PAL and DPPH activities of the adventitious root were also highest at 50 μmol MJ treatment. 50 μmol SA resulted in the highest production of bioactive compounds but the effect was less significant than that of MJ. SA concentration up to 400 μmol significantly enhanced accumulation of eleutheroside B in the adventitious root, confirming that SA was an effective elicitor for eleutheroside B production in E. koreanum adventitious roots. 3. Comparison of characteristics between adventitious roots and mother plants Content of eleutheroside B was higher in all field-grown plant samples (leaves, stems, thin roots and thick roots) compared with the adventitious roots. However, eleutheroside E, chlorogenic acid, total phenolics and flavonoids were higher in the adventitious roots compared with field-grown plant samples. The ploidy levels of the adventitious roots and field-grown plants were tested, resulted in two typical DNA histograms at the same retention time. The result indicated genetic stability of the adventitious root. 4. Conclusion Bioreactor culture conditions and elicitation method were optimized for production of biomass and bioactive compounds in E. koreanum adventitious roots. The adventitious root with elicitation was found to contain higher bioactive compounds. In addition, typical DNA histogram confirmed genetic stability of E. koreanum adventitious roots. These results suggested that the adventitious root culture of E. koreanum has advantages of large-scale production of bioactive compounds over field-cultivation.
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