Selection and optimization of a high-producing tissue culture of <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Biomass growth, ginsenoside and polysaccharide production in different ginseng tissue cultures, including callus culture, adventitious root culture and hairy root culture, were studied, and the active component contents were compared with that of native ginseng roots. The adventitious root culture was confirmed to be a very nice system, which grew fast and contained a rather high content of ginsenosides. Then, the culture conditions of adventitious root culture were optimized. The results showed that salt strength, various sucrose concentrations, ammonia/nitrate ratios and phosphate concentrations had significant influences on adventitious roots growth, secondary metabolite and polysaccharide synthesis in ginseng. The best culture conditions for ginsenoside production seemed to be 0.75 salt strength Murashige and Skoog medium, 4% sucrose, 9 mM ammonia to 36 mM nitrate, and 1.25 mM phosphate, while the optimization for polysaccharide accumulation seemed to be 0.75 salt strength, 6% sucrose, 9 mM ammonia to 36 mM nitrate and 3.75 mM phosphate source. Appropriate conditions allowed for a maximum ginsenoside yield of up to 132.90 mg/L and polysaccharide yield of 407.63 mg/L to be obtained after 4 weeks of culture.     

Production of saponins from <Emphasis Type="Italic">Panax ginseng</Emphasis> suspension and adventitious root cultures

Biomass growth and ginsenoside production in cell suspension and adventitious roots of Panax ginseng C.A. Meyer cultures cultivated both in Erlenmayer flasks and a 3 dm³ bioreactor were studied. The maximum content of ginsenosides was found in the suspension culture cultivated in the bioreactor (4.34 % dry mass), however the saponin content was limited to two major ginsenosides, Rb₁ and Rg₁. The production of ginsenosides in adventitious roots was lower (1.45 or 1.72 % dry mass), nevertheless, the full range of ginsenosides was detected.This work was supported by 521/02/P064, COST 843.10, ME671 and Z4 055 905 projects.     

Cryopreservation of <Emphasis Type="Italic">Panax ginseng</Emphasis> Adventitious Roots

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L⁻¹ was recorded for post-freeze-regenerated cultures versus 9.1 g dw L⁻¹ for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

<Emphasis Type="Italic">Pedobacter panacis</Emphasis> sp. nov., isolated from <Emphasis Type="Italic">Panax ginseng</Emphasis> soil

A novel strain, DCY108^T was isolated from soil of a Panax ginseng field, Yeoncheon province (38°04′N 126°57′E), Republic of Korea. Strain DCY108^T is Gram-negative, non-motile, non-flagellate, rod-shaped, and aerobic. The bacterium grows optimally at 25–30 °C, pH 6.5–7.0 and 1 % NaCl. Phylogenetically, strain DCY108^T is closely related to Pedobacter jejuensis JCM 18824^T, Pedobacter aquatilis JCM 13454^T, Pedobacter kyungheensis LMG 26577^T and the type strain of the genus Pedobacter heparinus DSM 2366^T. The DNA–DNA relatedness values between strain DCY108^T and its close phylogenetic neighbors were below 30.0 %. The DNA G+C content of strain DCY108^T was determined to be 45.1 mol%. The predominant quinone was menaquinone 7 (MK-7). The major polar lipids were identified as phosphatidylethanolamine and three unidentified aminolipids AL1, AL13 and AL17. Iso-C_15:00, iso-C_17:03OH and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) were identified as the major fatty acids present in strain DCY108^T. The results of physiological and biochemical tests allowed strain DCY108^T to be differentiated phenotypically from other recognized species belonging to the genus Pedobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Pedobacter panacis sp. nov is proposed with the type strain designated as DCY108^T (=CCTCCAB 2015196^T = KCTC 42748^T).     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Advances in study of ginsenoside biosynthesis pathway in <Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

Panax ginseng C. A. Meyer is one of the important nutraceutical and medicinal plants, which is used worldwide. Until now, ginseng has been reported to contain saponins, antioxidants, peptides, polysaccharides, fatty acids, vitamins, alkaloids, lignans, and flavonoids. The saponins, known as ginsenosides, are widely believed to be the major bioactive compounds of ginseng. In this article, ginsenoside biosynthesis pathway and key enzymes regulation are also described. This review provides a reference for improving ginsenoside contents through regulation of ginsenoside biosynthesis pathway.     

<Emphasis Type="Italic">Achromobacter panacis</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Panax ginseng</Emphasis>

A novel strain DCY105^T was isolated from soil collected from the rhizosphere of ginseng (Panax ginseng), in Gochang, Republic of Korea. Strain DCY105^T is Gram-reaction-negative, white, non-motile, non-flagellate, rod-shaped and aerobic. The bacteria grow optimally at 30°C, pH 6.5–7.0 and in the absence of NaCl. Phylogenetically, strain DCY105^T is most closely related to Achromobacter marplatensis LMG 26219^T (96.81%). The DNA G+C content of strain DCY105^T was 64.4 mol%. Ubiquinone 8 was the major respiratory quinone, and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were amongst the major polar lipids. C_16:00, C_8:03OH and iso-C_17:03OH were identified as the major fatty acids present in DCY105^T. The results of physiological and biochemical tests allowed strain DCY105^T to be differentiated phenotypically from other recognized species belonging to the genus Achromobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Achromobacter panacis sp. nov. is proposed with the type strain designated as DCY105^T (=CCTCCAB 2015193^T =KCTC 42751^T).     

Modulation of copper toxicity-induced oxidative damage by nitric oxide supply in the adventitious roots of <Emphasis Type="Italic">Panax ginseng</Emphasis>

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important signalling molecule and antioxidant. Here we investigated the protective effect of NO against the toxicity caused by excess CuSO₄ (50 μM) in the adventitious roots of mountain ginseng. It was found that NO donor, sodium nitroprusside (SNP), was effective in reducing Cu-induced toxicity in the mountain ginseng adventitious roots. Protective effect of SNP, as indicated by extent of lipid peroxidation, was reversed by incorporation of 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (CPTIO), a NO scavenger, in the medium suggesting that the protective effect of SNP is attributable to NO released, which was revealed from in situ confocal laser scanning microscopic localization of NO in the adventitious roots of mountain ginseng. Results obtained in the present study suggest that reduction of excess Cu-induced toxicity by SNP is most likely mediated through the modulation in the activities of antioxidant enzymes involved in H₂O₂ detoxification (catalase, peroxidase, ascorbate peroxidase) and in the maintenance of cellular redox couples (glutathione reductase), and contents of molecular antioxidants (particularly non-protein thiol, ascorbate and its redox status). Exogenous NO supply also improved the activity of superoxide dismutase, an enzyme responsible for O₂ ^·− dismutation, and NADPH oxidase, an enzyme responsible for O₂ ^·− generation, in excess Cu supplied adventitious roots of mountain ginseng.     

Emulsifying Properties of Natural Extracts from <Emphasis Type="Italic">Panax ginseng</Emphasis> L.

One of the major trends within the food industry is the replacement of synthetically-derived food additives (e.g. emulsifiers) by natural alternatives. A promising approach is the utilization of saponins that have attracted attention due to their effective emulsifying properties and their natural origin from plants. Panax ginseng is well known in Asian countries for its health benefits that are mainly attributed to amphiphilic triterpene saponins, namely ginsenosides. In this study, we characterized two food-grade ginseng extracts (Finzelberg: FB; CheilJedang: CJ) regarding their chemical composition, surface activity, and effectiveness as emulsifier. Both ginseng extracts reduced the interfacial tension appreciably by up to 80%, and formed negatively charged oil-in-water emulsions at a low emulsifier-to-oil ratio. Ginseng FB formed small submicron-sized emulsions, whereas the mean particle sizes with ginseng CJ were much larger (up to 25 μm). Both ginseng extract-stabilized emulsions were stable towards a range of stresses (pH 4–9, ≤100 mM NaCl) or when stored at ≤25 °C for four weeks. However, the emulsions showed instability at highly acidic conditions (pH 2–3), during the 4-week storage at an elevated temperature (55 °C), and at high ionic strengths (≥250 mM NaCl, >10 mM CaCl₂), which was mainly attributed to the reduction or screening of electrostatic repulsion. Emulsion formation and stabilization was proposed to occur via formation of a saponin or biogenic saponin-protein complex layer leading to a stronger interfacial network. In conclusion, both ginseng extracts were able to form emulsions, although ginseng FB extract showed especially remarkable emulsifying properties, similar to the highly effective Quillaja saponaria extract. The results may therefore be helpful in replacing other emulsifiers and formulating emulsion products with varying particle size ranges.

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Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.     

Nucleotide substitutions in <Emphasis Type="Italic">rolC</Emphasis> and <Emphasis Type="Italic">nptII</Emphasis> gene sequences during long-term cultivation of <Emphasis Type="Italic">Panax ginseng</Emphasis> cell cultures

It has been shown previously that the rolC gene from Agrobacterium tumefaciens gene was stably and highly expressed in 15-year-old Panax ginseng transgenic cell cultures. In the present report, we analyze in detail the nucleotide composition of the rolC and nptII (neomycin phosphotransferase) genes, which is the selective marker used for transgenic cell cultures of P. ginseng. It has been established that the nucleotide sequences of the rolC and nptII genes underwent mutagenesis during cultivation. Particularly, 1–4 nucleotide substitutions were found per sequence in the 540 and 798 bp segments of the complete rolC and nptII genes, respectively. Approximately half of these nucleotide substitutions caused changes in the structure of the predicted gene product. In addition, we attempted to determine the rate of accumulation of these changes by comparison of DNA extracted from P. ginseng cell cultures from 1995 to 2007. It was observed that the frequency of nucleotide substitutions for the rolC and nptII genes in 1995 was 1.21 ± 0.02 per 1,000 nucleotides analyzed, while in 2007, the nucleotide substitutions significantly increased (1.37 ± 0.07 per 1,000 nucleotides analyzed). Analyzing the nucleotide substitutions, we found that substitution to G or to C nucleotides significantly increased (in 1.9 times) in the rolC and nptII genes compared with P. ginseng actin gene. Finally, the level of nucleotide substitutions in the rolC gene was 1.1-fold higher when compared with the nptII gene. Thus, for the first time, we have experimentally demonstrated the level of nucleotide substitutions in transferred genes in transgenic plant cell cultures.     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

A Comparative Analysis of Genetic Variability and Differentiation in <Emphasis Type="Italic">Panax vietnamensis</Emphasis> Ha et Grushv. and <Emphasis Type="Italic">P. ginseng</Emphasis> C.A. Meyer Using ISSR Markers

A comparative analysis of the genetic variability and differentiation of rare medicinal ginseng species, Panax vietnamensis Ha et Grushv. and P. ginseng C.A. Meyer, was carried out using inter-simple sequence repeat markers. It was demonstrated that all the genetic diversity parameters of Vietnamese ginseng were high and considerably exceeded those of P. ginseng. On the contrary, the level of genetic differentiation was higher in true ginseng. It is suggested that the differences in the levels of genetic variability and differentiation of the two ginseng species were influenced by the demographic history, peculiarities of the reproductive system, and human activity.