Production of Biomass and Ginsenosides from Adventitious Roots of Panax ginseng in Bioreactor Cultures

Organic nutrients play a central role during Panax ginseng adventitious root culture in bioreactor systems. To understand how the nutrient elements were uptaken during the adventitious root growth as well as the production of biomass and natural ginsenosides, a biotechnological approach to identifying the nutritional physiology of ginseng in a commercial‐scale bioreactor was necessary. Normal MS medium nutrient in the bioreactor culture of adventitious roots resulted in slow growth, low biomass, and Rg and Rb ginsenoside contents. When the ginsenoside production increased to higher levels, a group of regulatory nutritional elements that have the potential to interact with biomass was identified. The effects of the salt strength of the medium, of macroelements, metal elements, the ammonia/nitrate ratio, sucrose concentration, and osmotic agents on the growth, the formation of biomass and the production of ginsenosides from adventitious roots were investigated. Appropriate conditions allowed for a maximum ginsenoide production of up to 12.42 [mg/g DW] to be obtained after 5 weeks of culture. The results demonstrated that the key organic nutrients can be regulated to improve the biomass and growth, and increase the ginsenoside yield in bioreactor cultures of P. ginseng adventitious roots.     

Optimization and quality assessment of adventitious roots culture in Panax quinquefolium L.

In this study, adventitious roots of Panax quinquefolium L. have been successfully established. The highest induction rate of roots was obtained in MS medium containing 3 mg L^?1 IBA after 4 weeks of culture. The culture conditions of adventitious root were optimized and evaluated with response surface methodology. The best culture conditions for root growth seemed to be 0.75 salt strength MS medium, 4.70 g L^?1 inoculum size and 40 days of culture. The active component contents of adventitious roots were compared with those of native roots. The total saponins content was found to be 16.28 mg g^?1 in native root and 4.64 mg g^?1 in adventitious root. The polysaccharide content of the adventitious root was 1.5 times higher than that in the native P. quinquefolium (30.54 vs. 20.28 mg g^?1).     

Gradually scale-up culture in a bioreactor promotes radical scavenging activity of <Emphasis Type="Italic">Panax ginseng</Emphasis> (C. A. Meyer) adventitious roots on 1,1-diphenyl-2-picrylhydrazyl

A scale-up culture of adventitious roots of ginseng was established using a balloon-type bubble bioreactor (BTBB). Maximum growth rates of ~52-fold and ~50-fold in 3 and 5 L BTBBs were obtained, respectively after 40 days of inoculation, which was significantly higher than that in 0.5 L conical flask (~15-fold). Gradually scale-up culture of adventitious roots increased the root biomass, while the contents of ginsenoside and polysaccharides were not affected. This study also revealed that radical scavenging activity of dried adventitious roots on 1,1-diphenyl-2-picrylhydrazyl was higher than that of native roots at 20–100 mg L⁻¹ methanolic extract.     

Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of <Emphasis Type="Italic">Periploca sepium</Emphasis> Bunge

In order to study the capabilities of Periploca sepium adventitious root induction in different types of explants, we selected leaves, roots and stems with or without buds. The growth of adventitious roots and periplocin content in these roots were determined. In order to investigate the suitable media salt strength, we cultured the adventitious roots in different salt strength (0.25, 0.50, 1.0, 1.5, 2.0) of Murashige and Skoog (MS) media supplemented with 1 mg/l indole butyric acid (IBA) and 30 g/l sucrose. The results showed that both leaf and root explants were proven suitable for the adventitious root induction; however, the stems could hardly induce adventitious roots no matter whether the stems had buds or not. Further studies reported that adventitious root proliferation and periplocin production derived from root explants were higher than those derived from leaf explants. So the root explants were the optimum explants for adventitious root induction, growth and periplocin production. The salt strength experiment showed that with the increasing salt strength (1.0–2.0 MS), adventitious root growth decreased significantly, as well as periplocin content in comparison with lower (0.25–0.5 MS) salt strength media.     

Influences of polyunsaturated fatty acids (PUFAs) on growth and secondary metabolite accumulation in Panax ginseng C.A. Meyer adventitious roots cultured in air-lift bioreactors

The present study relates to different polyunsaturated fatty acids (PUFAs) which were used as elicitors to enhance biomass accumulation and ginsenoside production in Panax ginseng. Adventitious root cultures of ginseng were elicited with oleic and linolenic acid at 0, 1, 5, 10 or 50 µmol/l concentrations respectively. Elicitors were added to the medium of adventitious roots on the 40th day of culture and roots were harvested on day 47. Cultures supplemented with oleic acid decreased root biomass and ginsenoside accumulation. Cultures supplemented with 1 µmol/l linolenic acid enhanced ginsenoside accumulation, without the decrease of adventitious root biomass. Linolenic acid enhanced the biosynthesis of both protopanxatriols (2.95 ± 0.048 mg/g DW) and protopanxadiols (5.66 ± 0.043 mg/g DW) compared to that of control at (1.41 ± 0.002 mg/g DW) and (1.58 ± 0.006 mg/g DW) respectively. No changes in polysaccharides and phenolics content have been noticed upon elicitation with PUFAs. This is the first report on linolenic acid as an elicitor for ginsenoside accumulation in ginseng adventitious root cultures.     

Medium salt strength induced changes in growth, physiology and secondary metabolite content in adventitious roots of Morinda citrifolia: the role of antioxidant enzymes and phenylalanine ammonia lyase

In an attempt to improve growth and secondary metabolite production, and to understand the possible mechanism involved in relation to the changes in physiology and activities of antioxidant enzymes, we cultured Morinda citrifolia adventitious roots in different strength (0.25, 0.50, 0.75, 1.0, 1.5 and 2.0) of Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ indole butyric acid and 30 g l⁻¹ sucrose. Quarter-strength MS medium was proven suitable for the production of both root biomass and secondary metabolites [anthraquinone (AQ), phenolics and flavonoids]. With the increasing salt strength, root growth and AQ accumulation decreased significantly. Higher (1.5 and 2 MS) salt strength provoked osmotic stress resulted in more than twofold free proline accumulation than lower salt strength treated roots and induced free radical scavenging activity. Phenylalanine ammonia lyase activity showed a positive correlation in relation to salt strength that leads to an increase in phenol biosynthesis in expense of AQ formation. The elevated catalase (CAT), guaiacol peroxidase (G-POD) and superoxide dismutase activities and decreased ascorbate peroxidase (APX) activities were observed in roots treated with 2.0 MS. On the other hand, APX activity was strongly activated along with considerable increase in CAT activity at 0.25 MS treated culture. However, the joint functions of CAT, G-POD and APX at 0.25 MS treated cultures were efficient to eliminate the potential danger of hydrogen peroxide (H₂O₂) as evidenced from low H₂O₂ accumulation and low level of lipid peroxidation.     

小型生物反应器内人参不定根的人参皂苷累积

对小型生物反应器(3~10 L)培养人参不定根的生长和人参皂苷(Rg1、Re、Rb1)的累积规律,以及蔗糖浓度、初始接种量对其生长和人参皂苷累积的影响进行研究。结果表明:小型生物反应器内人参不定根的最佳收获周期为7周。初始接种量和蔗糖浓度影响生物反应器内人参不定根的生长和人参皂苷的累积,20或40 g/L蔗糖对人参不定根的生长和人参皂苷的累积优于60 g/L蔗糖;5和10 L生物反应器内最佳初始接种量分别为15和30g,其不定根的生长量分别为9.29和19.17 g,人参皂苷含量分别为5.16和4.58 mg/g。生物反应器内培养7周的人参与栽培4年的人参相比,人参皂苷Rg1和Re含量相差不大,但栽培人参中Rb1的含量远高于生物反应器中所培养的人参不定根。     

Podophyllotoxin production via cell and adventitious root cultures of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis>

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH₄NO₃. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization–tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.     

Induction,characterization, and NMR-based metabolic profiling of adventitious root cultures from leaf explants of <Emphasis Type="Italic">Gynura procumbens</Emphasis>

Gynura procumbens is a medicinal plant used in South East Asia to treat various ailments such as rash, hemorrhoids, inflammation, and diabetes. In order to develop a large-scale culture system for G. procumbens biomass containing bioactive compounds, adventitious root cultures were initiated from leaf explants. Murashige and Skoog (MS) media containing different compositions of indole-3-butyric acid (IBA), 1-naphthalene-acetic acid (NAA), and combinations of both plant growth regulators (PGRs) were evaluated for root induction. A combination of 3 mg/l NAA + 1 mg/l IBA gave the highest root induction (48%) as compared to other PGRs treatments after 9 weeks of incubation period. Subsequently, the adventitious roots were established in liquid culture containing MS medium and the combination of 3 mg/l NAA + 1 mg/l IBA. A study on the medium strength, sucrose concentration, pH, and light versus dark was conducted to optimize the in vitro culture conditions. The results showed that differences in MS medium strength from half to double strength, and light or dark condition did not significantly affect the biomass production, while the initial medium pH of 5.5 and 2% w/v sucrose concentration were most suitable for the root culture growth. Nuclear magnetic resonance (NMR) spectroscopy was performed to characterize the metabolite content in the root cultures of G. procumbens. Among the elucidated metabolites were some phenylpropanoids identified as caffeic acid, chlorogenic acid, and 3,5-di-O-caffeoylquinic acid which might be the bioactive compounds associated to the folk use of this plant.     

Establishment of adventitious root culture of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> Bertoni in a roller bottle system

Cultures of adventitious roots of Stevia rebaudiana (Bert.) Bertoni were performed in a roller bottle system for the production of both primary and secondary metabolites. Adventitious roots were induced from 1-cm-long root tip explants derived from in vitro regenerated plantlets on solid Murashige and Skoog (MS 1962) media supplemented with 10.7 μM of α-naphthaleneacetic acid. These cultures were successfully maintained in the same medium for 6 months with regular subcultures after 4 weeks. Thereafter, the roots were cut into 1.0- to 1.5-cm-long segments and transferred to the roller bottle system containing a fresh root tissue culture on liquid MS medium supplemented with 10.7 μM NAA. The apparatus consisted of a flask rolling system adjusted to 4g, and 3° of flask inclination. The roots were allowed to grow in the absence of light for adaptation and adventitious root formation. The best conditions for cultivation were investigated, considering culture volume (25 ml), culture period (4 weeks), salt concentrations in the nutrient medium (33%) and optimal initial inoculum (0.2 g) of S. rebaudiana roots. These results could give important information on how to improve the development of adventitious roots of S. rebaudiana for the production of primary and secondary metabolites.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Establishment of culturing conditions and assessment of antioxidant activity and somaclonal variation in the adventitious root suspension cultures of <Emphasis Type="Italic">Oplopanax elatus</Emphasis> Nakai

Oplopanax elatus Nakai, a plant traditionally used in folk medicine, is currently in population decline due to uncontrolled harvesting. In the present study, we investigated the factors affecting O. elatus adventitious root production, including hormones (alone or in combination), explant type, basal salt type and strength, sucrose concentration, pH, and temperature. Results revealed that adventitious root formation was optimal with root explants grown on 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^?1 Indole-3-butyric acid (IBA) (pH 5.8) at 25 °C. Chlorogenic acid concentration was highest in roots propagated in 1/2 MS medium containing 0.5 mg L^?1 IBA; vanillin, another phenolic compound, was also detected in cultures. Liquid media containing 3% sucrose exhibited the highest radical scavenging activity and total phenolic compound contents. X-ray diffraction revealed significant differences in the elemental intensity between adventitious root and field-grown plantlet extracts. Analysis of simple sequence repeats confirmed that adventitious roots regenerated in vitro were genetically similar to their mother plant. Thus, we identified the optimal conditions for proliferation of O. elatus adventitious roots in liquid culture, from which, secondary metabolites, particularly bioactive compounds associated with the medicinal use of this plant, can be mass produced without further population deterioration.     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Influence of sucrose concentration and phosphate source on biomass and metabolite accumulation in adventitious roots of Pseudostellaria heterophylla

In this study, we investigated the induction of Pseudostellaria heterophylla adventitious root and the effects of sucrose concentration and phosphate source on biomass increase and metabolites accumulation. These roots were initially cultured in Murashige and Skoog medium for 4 weeks. IBA 3.0 mg L^?1 proved to be the best auxin for inducing adventitious roots and the frequency of adventitious roots induced from roots (100 %) was higher than that from leaves (78 %) and stems (27 %). The medium with 4 % sucrose resulted in the optimum biomass i.e. 1.04 g/flask DW, and the content of saponin and polysaccharides reached the peak i.e. 0.676 and 24.4 %, respectively. With regards to phosphate source, 1.25 mM phosphate concentration was more favorable for biomass of roots (0.87 g/flask of DW), whereas the optimum saponin (0.74 %) and polysaccharides (22.09 %) were achieved with 2.5 mM phosphate. However, the saponin content at 2.5 mM phosphate did not show significant difference from the saponin content at 0.625 mM (0.69 %) or 3.75 mM phosphate (0.69 %).     

Induction and Characterization of Adventitious Roots Directly from the Explants of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

Adventitious roots from leafstalks and lateral roots were obtained directly from explants of Panax notoginseng. The lateral root explants were more sensitive to the induction of adventitious roots using indole-3-butyric acid. HPLC analysis of saponins extracted from the adventitious roots indicated that several protopanaxatriol saponins were present but ginsenoside Rd was missing, compared with the saponins extracted from the raw herbs. The dry weight of primary adventitious root culture of Panax notoginseng increased 5.25 times during multiplication in a classical shaking-flask system, suggesting that it is a culture system with great potential for scale-up. Revisions requested 13 July 2005; Revisions received 1 September 2005     

Establishment of adventitious root co-culture of Ginseng and Echinacea for the production of secondary metabolites

We have successfully established the co-culture of ginseng (Panax ginseng C.A. Meyer) and echinacea [Echiancea purpurea (L.) Moench.] adventitious roots for the production secondary metabolites. Adventitious roots of ginseng and echinacea were cultured in different proportions (5 g L⁻¹; 4:1, 3:2 and 2:1 ginseng and echinacea, respectively) in 5-L capacity airlift bioreactors containing 4 L Murashige and Skoog medium supplemented with 25 μM indole-3-butyric acid and 50 g sucrose L⁻¹ and maintained at 25°C in the dark for 40 days. Results showed the negative effect of echinacea adventitious roots on the growth of ginseng roots, however, by limiting the inoculum density of echinacea, it was possible to establish the co-cultures. To enhance the accumulation of secondary metabolites, co-cultures were treated with 200 μM methyl jasmonate after 30 days of culture initiation. Methyl jasmonate elicitation promoted the accumulation of ginsenosides in the co-cultures. It was possible to produce ginsenosides and caffeic acid derivatives in higher amounts by establishing co-cultures with higher inoculum proportion of ginseng to echinacea (4:1 and 3:2) followed by elicitation treatment. This work demonstrates the effectiveness of interspecies adventitious root co-cultures for the production of plant secondary metabolites.     

The influence of methyl jasmonate,cholesterol and l‐arginine on solasodine production in hairy root culture of Solanum mammosum

The increasing demand of diosgenin for high‐revenue synthesis of steroid hormones by the pharmaceutical industries has driven researchers to look for other alternatives. Solasodine which was reported to be present in Solanum mammosum is known to be a potential source. The present study highlighted that added methyl jasmonate, cholesterol and l ‐arginine into the modified liquid full‐strength Murashige and Skoog (MS) medium (with ammonium to nitrate ratio 10.3 mM: 39.4 mM, and 4% (w/v) sucrose) could influence the solasodine production in the hairy roots of S. mammosum. The findings showed that both hairy root line‐ATCC31798 and line‐A4 (which were separately induced by Agrobacterium rhizogenes strain ATCC31798 and A4) acquired solasodine productivity of 4.5 mg/g dry weight roots with average dry biomass of 190 mg after 32 days culture, when using 50 mg fresh weight roots as initial inoculum size, with 100 mM cholesterol, 1000 μM l ‐arginine and 300 μM methyl jasmonate added simultaneously into the culture medium on day 20 of culture. The amount of solasodine obtained was five times higher than those without both the elicitor and precursor treatment. The improved solasodine production with a high‐biomass growth could reduce the production cost of steroid synthesis in the long run.     

Pilot-scale culture of adventitious roots of ginseng in a bioreactor system

A pilot-scale culture of multiple adventitious roots of ginseng was established using a balloon-type bubble bioreactor. Adventitious roots (2 cm) induced from callus were cultured in plastic Petri dishes having 20 ml of solid Schenk and Hildebrandt (1972) medium containing 3% sucrose, 0.15% gelrite, and 24.6 μM indole-3-butric acid. An average of 29 secondary multiple adventitious roots were produced after 4 weeks of culture. These secondary roots were elongated on the same medium, reaching a length of 5 cm after 6 weeks of culture. A time course study revealed that maximum yields in 5-l and 20-l bioreactors were approximately 500 g and 2.2 kg at day 42 with 60 g and 240 g inoculations, respectively. Cutting twice during the culture increased the total amount of biomass produced. The root biomass in a 20-l balloon-type bubble bioreactor was 2.8 kg at harvest with 240 g of inoculum after 8 weeks of culture. The total saponin content obtained from small-scale and pilot-scale balloon type bubble bioreactors was around 1% based on dry weight. Inoculation of 500 g fresh weight of multiple adventitious roots into a 500 l balloon-type bubble bioreactor with cutting at 4 and 6 weeks after inoculation produced approximately 74.8 kg of multiple roots. The ginsengnoside profiles of these multiple adventitious roots were similar to profiles of field-grown ginseng roots when analyzed by HPLC. This revised version was published online in June 2006 with corrections to the Cover Date.