In this study, methyl jasmonate (MJ)-induced changes of triterpene saponins in ginseng (Panax ginseng C.A. Meyer) hairy roots and expression profiling of relevant responsive genes were analyzed. The transcription of PgSS (squalene synthase), PgSE (squalene epoxidase), and PNA (dammarenediol synthase-II) genes in hairy root cultures elicited by MJ treatment increased as compared with the controls, whereas that of PNX (cycloartenol synthase) decreased slightly. In order to select candidate genes encoding for cytochrome P450-dependent hydroxylase or glucosyltransferase associated with triterpene biosynthesis, RT-PCR analysis was conducted following MJ elicitation. No differences were observed in any expression among the five genes associated with the cytochrome P450 family, when compared to that of control. For candidates of the glucosyltransferase gene,expression of EST IDs PG07020C06, PG07025D04, and PG07029G02 was upregulated. In an effort to assess the effects of MJ elicitation on the biosynthesis of triterpene saponin, protopanaxadiol saponin (Rb group) and protopanaxatriol saponin (Rg group) contents in hairy roots were evaluated by HPLC analysis. With 7 days of MJ elicitation, levels of all ginseonsides of the two-groups increased much higher than that observed in the control. In particular, protopanaxadiol-type saponin contents increased by 5.5-9.7 times that of the control, whereas protopanaxatriol-type saponin contents were increased by 1.85-3.82-fold. In the case of Rg1 ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures.
An ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-Tof MS)-based metabolomic technique was applied for metabolite profiling of 60 Panax ginseng samples aged from 1 to 6 years. Multivariate statistical methods such as principal component analysis and hierarchical clustering analysis were used to compare the derived patterns among the samples. The data set was subsequently applied to various metabolite selection methods for sophisticated classification with the optimal number of metabolites. The results showed variations in accuracy among the classification methods for the samples of different ages, especially for those aged 4, 5, and 6 years. This proposed analytical method coupled with multivariate analysis is fast, accurate, and reliable for discriminating the cultivation ages of P. ginseng samples and is a potential tool to standardize quality control in the P. ginseng industry.
Transformed root ("hairy root") cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative beta-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.
Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius) are widely used medicinal plants with similar morphology but different medicinal efficacy. Roots, flowers, and processed products of Korean and American ginseng can be difficult to differentiate from each other, leading to illegal trade in which one species is sold as the other. This study was carried out to develop convenient and reliable chloroplast genome-derived DNA markers for authentication of Korean and American ginseng in commercial processed products. One codominant marker could reproducibly identify both species and intentional mixtures of the two species. We further developed a set of species-unique dominant DNA markers. Each species-specific dominant marker could detect 1% cross contamination with other species by low resolution agarose gel electrophoresis or quantitative polymerase chain reaction. Both markers were successfully applied to evaluate the original species from various processed ginseng products purchased from markets in Korea and China. We believe that high-throughput application of this marker system will eradicate illegal trade and promote confident marketing for both species to increase the value of Korean as well as American ginseng in Korea and worldwide.
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