Cell Cultures Of Panax Ginseng Research Articles

Elicitation of ginsenoside biosynthesis in cell cultures of Panax ginseng by vanadate

Signal transduction engineering is important to enhance secondary metabolite production. In this work, the effect of several heavy salts including NH4VO3, NaVO3, VOSO4, NiSO4, CuSO4 and MnSO4 on the ginsenoside biosynthesis in suspension cultures of Panax ginseng was investigated and vanadate was demonstrated most efficient. The optimal concentration and time of vanadate addition was 50μM and on day 4, respectively. The maximal content of ginsenosides (Rg1+Re+Rb1+Rc+Rd) was 564.3±12.4μg/100mg DW (day 14) in vanadate treated cultures, while that was only 136.2±13.3μg/100mg DW (day 17) in the control. The ginsenoside content enhancement coincided with increased activities of UDPG-ginsenoside Rd glucosyltransferase and protopanaxadiol 6-hydroxylase. To further understand the mechanism of vanadate elicitation, jasmonic acid (JA) content and transcriptional levels of genes encoding enzymes related to the triterpene biosynthesis were examined. Upon vanadate treatment, the induction of endogenous JA biosynthesis and up-regulation of transcription levels of sqs, se and ds genes were observed. Interestingly, addition of ibuprofen, an inhibitor of JA biosynthesis, reversed all those effects. The results indicate that vanadate may induce the ginsenoside biosynthesis by inducing the signal molecule JA, and vanadate addition was an effective strategy for increasing ginsenoside production.

Enhancement of ginsenoside biosynthesis in cell cultures of Panax ginseng by N,N′-dicyclohexylcarbodiimide elicitation

In this work, the effect of N,N′-dicyclohexylcarbodiimide (DCCD) on ginsenoside biosynthesis in suspension cultures of Panax ginseng cells was investigated. The optimal concentration and timing of DCCD addition were found to be 10μM and on day 4 of cultivation. Under this condition, the maximal content of total ginsenosides increased to 3.0-fold that of untreated control, and the contents of Rg-group (Rg1 and Re) ginsenosides and Rb1 were 2.5- and 8.9-fold higher, respectively, which coincided with elevated activities of protopanaxatriol biosynthetic enzyme protopanaxadiol 6-hydroxylase and UDPG-ginsenoside Rd glucosyltransferase that converts Rd to Rb1. In addition, DCCD treatment induced the activity of defense response enzyme, phenylalanine ammonia lyase. To gain a better understanding of the molecular processes underlying the elicitation, we examined nitric oxide (NO) content and expression levels of the triterpene biosynthetic genes encoding squalene synthase (sqs), squalene epoxidase (se), and dammarenediol-II synthase (ds). It was found that DCCD up-regulated NO generation and transcription levels of sqs, se and ds. Interestingly, these effects of DCCD were compromised by an NO biosynthetic inhibitor, while an NO donor alone recapitulated the elicitation effect of DCCD on ginsenoside biosynthesis. These results suggest that DCCD may induce the ginsenoside biosynthesis via NO signaling in the P. ginseng cells. The information obtained might also be helpful to hyperproduction of valuable secondary metabolites in other plant cell cultures.

DNA mutagenesis in 2- and 20-yr-old Panax ginseng cell cultures

Previously, Panax ginseng var. Mimaki C.A. Meyer had been shown to accumulate genetic mutations during long-term propagation of a callus culture over a period of 20 yr. In this study, we analyzed the mutation types and frequency in a 2-yr-old P. ginseng callus culture and compared it with the 20-yr-old callus culture, and leaves of cultivated plants. We analyzed the sequence variability between the Actin genes, which are a family of housekeeping genes; phenylalanine ammonia-lyase (PAL) and dammarenediol synthase (DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinases (SERK), which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-yr-old P. ginseng callus culture was markedly higher than in cultivated plants, but lower than in the 20-yr-old callus culture. Most of the mutations in the 2-yr-old P. ginseng calli were A↔G and T↔C transitions, as in the 20-yr-old calli and intact P. ginseng plants. The number of nonsynonymous mutations was higher in the 2- and 20-yr-old callus cultures than the number of nonsynonymous mutations in cultivated P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using a methylation-sensitive DNA fragmentation assay, we showed the level of methylcytosine to be higher in the DNA of the 20-yr-old P. ginseng calli that than in the DNA of the 2-yr-old cultures.

Mutation of Panax ginseng genes during long-term cultivation of ginseng cell cultures

It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during the long-term cultivation of transgenic cell cultures of Panax ginseng. In the present report, we analyzed the nucleotide sequences of selected plant gene families in the 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We sequenced the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase ( PAL) and dammarenediol synthase genes ( DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase ( SERK) genes, which control plant development. We demonstrate that the plant genes also developed mutations during long-term cultivation. The highest level of nucleotide substitution was detected in the sequences of the SERK genes (2.00 ± 0.11 nt per 1000 nt), and the level was significantly higher when compared with the cultivated P. ginseng plant. Interestingly, while the diversity of Actin genes was similar in the P. ginseng cell culture and the cultivated plants, the diversity of the DDS and SERK genes was less in the 20-year-old cell culture than in the cultivated plants. In this work, we detail the level of nucleotide substitutions in different plant genes during the long-term culture of plant cells.

CDPK gene expression in salt tolerant rolB and rolC transformed cell cultures of Panax ginseng

CDPKs (calcium-depended protein kinases) are of great importance for the activation of defense reactions in plants. In this study, we aimed to find a connection between CDPK expression and increased salt tolerance in Panax ginseng. Treatment of P. ginseng cell cultures with W7 (CDPK protein inhibitor) showed that CDPK proteins were necessary for salt tolerance. Expression of PgCDPK1c, PgCDPK2c and PgCDPK4a was significantly increased in the cells treated with 60 mM NaCl compared to control cells, whereas expression of PgCDPK1b and PgCDPK3a was decreased. In the NaCl-treated cells, new CDPK transcripts also appeared (PgCDPK3c, PgCDPK4as). We also used rolC and rolB transformed cultures and the effects of the rol genes on CDPK expression were similar to the effects of salt stress: they caused a significant increase in the expression of PgCDPK1c, PgCDPK2c, and PgCDPK4a and decreased expression of PgCDPK3a, in addition to the appearance of the "short" CDPK transcripts.

Structure and expression profiling of a novel calcium-dependent protein kinase gene PgCDPK1a in roots, leaves, and cell cultures of Panax ginseng

Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study, we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator.

Stability of the rolC gene and its expression in 15-year-old cell cultures of Panax ginseng

After 15 years of cultivation of Panax ginseng, transgenic cell cultures were shown to express rolC gene at a high level. We determined that the rolC gene underwent mutagenesis. In particular, from one to four nucleotides were changed for the whole gene sequence (540 bp). These substitutions were synonymous in half of the cases or resulted in the modification of single amino acids in the rolC-encoded protein. With the example of beta-1,3-glucanases we showed that long cultivation and the observed changes in nucleotide sequences of the transgene did not inhibit the activating effect of rolC on enzymatic activity of beta-1,3-glucanases.

Nucleotide substitutions in rolC and nptII gene sequences during long-term cultivation of Panax ginseng 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.

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8-50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mgL(-1) indolebutyric acid, 0.5 mgL(-1) kinetin and 30 gL(-1) sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g(DW)L(-1), 4.5mg(gDW)(-1) on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.

Enhancement of saponin production in Panax ginseng cell culture by osmotic stress and nutrient feeding

Osmotic stress at elevated osmotic pressure was used to stimulate saponin production in Panax ginseng cell cultures. The addition of 0.3 M sorbitol to culture at the time of inoculation, raising the medium osmolality by about 75% (from 150 to 260 mmol/kg), enhanced the saponin accumulation but depressed the cell growth, resulting in no improvement of the volumetric saponin yield of culture. The feeding of sorbitol (0.2–0.3 M, 36.4–54.6 g/L) to culture during the stationary growth phase increased both the saponin content of cell (45–50%) and the volumetric yield (36–38%). With combined feeding of sorbitol (0.2 M) and growth nutrients, sucrose (30 g/L) + casein hydrolysate (0.5 g/L), about 2 days before the stationary phase, the volumetric saponin yield was increased 3.5-fold, 1130.8 mg/L versus the control culture of 251.2 mg/L. Sorbitol in the culture also induced two characteristic stress responses of plant cells, the increase in phenylalanine ammonium-lyase (PAL) activity and the production of reactive oxygen species. This suggests that the stimulated saponin accumulation was part of the cell response to the osmotic stress.

Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng

Chitosan (CHN) specially induced the activities of 39 kD and 42 kD protein kinases in ginseng cells, which could be suppressed by an inhibitor of mitogen-activated protein kinase (MAPK) pathway, PD98059. The immunoprecipitation (IP) using MAPK antibody or kinase assay in vitro also showed that CHN-induced 42 kD and 39 kD protein kinases belonged to the MAPK family. PD98059 suppressed CHN-induced transcriptions of ginseng squalene synthase and ginseng squalene epoxidase genes (gss and gse), CHN-induced accumulation of beta-Amyrin synthase (beta-AS) and synthesis of saponin. These results showed that CHN-induced activities of MAPKs were necessary for the CHN-induced saponin synthesis. EGTA and LaCl3 suppressed CHN-induced 39 kD and 42 kD MAPK activities. Ruthenium red (RR) could suppress CHN-induced 39 kD activity. All of them suppressed CHN-induced saponin synthesis. These results indicated that CHN-induced increment of cytosolic calcium was necessary for CHN-induced saponin synthesis. PD98059 also suppressed CHN-induced oxidative burst (including the increment of activity of plasma membrane NADPH oxidase and production of H2O2), but diphenylene iodonium (DPI), dimethylthiourea (DMTU) and 2,5-dihydroxycinnamic acid methyl ester (DHC) could not suppress CHN-induced MAPK activities, which indicated that MAPK was possibly function upstream of CHN-induced oxidative burst.

Nitric oxide mediates elicitor-induced saponin synthesis in cell cultures of Panax ginseng.

The elicitor oligogalacturonic acid (OGA) stimulated nitric oxide (NO) accumulation in the growth medium of ginseng suspension cultures and induced increased nitric oxide synthase (NOS) activity in ginseng cells. OGA also stimulated accumulation of saponin, transcription of genes encoding squalene synthase (sqs) and squalene epoxidase (sqe), two early enzymes of saponin synthesis, and the accumulation of β-amyrin synthase protein (β-AS). Saponin accumulation, sqs and sqe gene expression, and increases in β-AS content were also induced by exposure to NO via the NO donor sodium nitroprusside (SNP). Inhibitors of mammalian nitric oxide synthase reduced both OGA-induced NO accumulation and NOS activity, suggesting that OGA-induced NO production occurs via a NOS-like enzyme. OGA-induced accumulation of β-AS and saponin, and transcription of sqs and sqe, were suppressed by treatments that removed NO or inhibited its production, indicating a role for NO in mediating OGA effects on these defence responses. NO accumulation and increased NOS activity were inhibited by calcium channel inhibitors and a protein kinase inhibitor, but not by a protein phosphatase inhibitor, indicating the requirement for calcium and protein phosphorylation during OGA-induced NO production. Saponin production and transcription, and accumulation of saponin biosynthetic genes and enzymes were also suppressed by these treatments, as well as by the protein phosphatase inhibitor okadaic acid.

Ultrasound-induced physiological effects and secondary metabolite (saponin) production in Panax ginseng cell cultures

This work was aimed at the effects of ultrasound (US) on the growth and secondary metabolite biosynthesis of cultured plant cells. Suspension cultures of Panax ginseng cells were exposed to US at power density below 82 mW/cm 3 for short periods of time (1–4 min) in a US bath (38.5-kHz fixed frequency and 810 W maximum peak power). Under most exposure conditions, US stimulated the biosynthesis of secondary metabolites, the ginsenoside saponins of ginseng cells, increasing the total saponin content of the cell by up to 75%. The growth and viability of ginseng cells were usually depressed immediately after the exposure to US, but recovered gradually to levels similar to those of a normal culture in a few days, with virtually no net loss of biomass yield at the end of the culture period. At some lower US doses, sonicated cultures could even reach slightly higher biomass yields than that of normal cultures. The effects of US on cell growth and secondary metabolite yield showed a significant correlation with the total US energy emitted ( i.e., the product of US power and exposure time). Mechanical stress and microstreaming induced by acoustic cavitation were considered as the most possible causes of the various physiological effects of US on ginseng cells. In particular, the stimulation of secondary metabolite production by US may be a result of US-induced plant cell defense response. (E-mail: bcjywu@pdyu.edu.hk)

Phosphate effect on production of ginseng saponin and polysaccharide by cell suspension cultures of Panax ginseng and Panax quinquefolium

The effects of inorganic phosphate on cell growth, accumulation of ginseng saponin (secondary metabolite) and ginseng polysaccharide (primary metabolite), together with nutrient utilisation, were investigated in suspension cultures of Panax ginseng (PG) and Panax quinquefolium strain Q91625 (PQ). The results indicate that the crucial concentration of phosphate for cell growth of PG and PQ was 1·04 and 0·65 m m, respectively. Bound phosphorus content for PG and PQ cells was determined to be 4 and 7 mg P/g DW, respectively, which suggested a more urgent demand of phosphate during cell cultivation of PQ. For simultaneous production of ginseng saponin and polysaccharide, the optimal concentration of phosphate in PG cell cultures was 0·42 m m, and saponin and polysaccharide accumulation was 643 mg litre −1 and 2·18 g litre −1, respectively; 1·25 m m of medium phosphate was the best for biosyntheses of saponin (960 mg litre −1) and polysaccharide (1·8 g litre −1) by PQ cell cultures.

Effects of long-term preservation on growth and productivity of Panax ginseng and Catharanthus roseus cell cultures

Cell cultures of Panax ginseng and Catharanthus roseus producing secondary metabolites were preserved in liquid nitrogen or under mineral oil for six months. The growth behaviour and the ability of the cultures to produce ginsenosides or indole alkaloids were measured after a recovery period and compared with cultures maintained by frequent subcultivation during the same period. Neither growth kinetics nor the degree of vacuolization during growth were affected by the long term preservation. Some changes in secondary metabolism were however found, indicating that preservation under mineral oil does not preserve the productivity of cell cultures whereas the cryogenic method does.

Growth and Ginsenoside Patterns of Cryopreserved Panax ginseng Cell Cultures

Summary Cryopreservation experiments were performed using ginsenoside-producing cell cultures of Panax ginseng. We obtained viable cells after storage at the temperature of liquid nitrogen. The protocol was optimised with regard to preeculture conditions and cryoprotectants. The relatively low freezing tolerance of ginseng cells was enhanced from less than 10 % to about 40 % as expressed by cell viability immediately after thawing. Though less than 50 % of the cell population survived, no selection occurred. The growth patterns of frozen-thawed and control cultures were identical. This is shown on the basis of fresh and dry weight, cell number, and packed cell volume. In addition, frozen-thawed ginseng cultures were shown to retain their capacity for producing ginsenosides. Using HPLC analysis the following components were determined: ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, and Rd. Neither ginsenoside yield nor ginsenoside pattern were influenced by cryostorage.