Ick Hyun Jo

Optimal Harvesting Time of Ginseng Seeds and Effect of Gibberellic Acid (GA3) Treatment for improving Stratification Rate of Ginseng (Panax ginseng C. A. Meyer) Seeds

This study was performed to identify optimal harvesting time of ginseng seeds and to examine the effect of GA3 treatment for improvement of seed stratification rate. Ginseng seeds harvested from Land race, Chunpoong and Yun- poong cultivar in July 20 were tested for stratification rate. It was shown that stratification rates of land race, Yunpoong and Chunpoong cultivar were 94.1%, 93.1%, and 82.6%, respectively. Seeds of Chunpoong cultivar harvested 10-15 days later showed a comparable stratification rate to that of Land race, indicating that late harvest of Chunpoong seeds is beneficial for the increase of stratification rate. The higher stratification rate was found in mature seeds (92.3%) than immature seeds (37.8%), both of which were harvested in July 20. Stratification rate of mature seeds harvested in July 15 was 87.5%, dem- onstrating optimal harvesting time of ginseng seeds with higher stratification rate is after mid-July. An exponential growth of endosperms of ginseng seeds was observed from early June to mid-June and then slow growth was observed. There was no obvious growth of embryos from fertilization to mid-August. After the this time, embryos quickly grew until late October. Thus, appropriate stratification control is essential during the period (from early September to late October) in order to optimize embryo growth and development. While no increase of stratification rate was observed in seeds treated with 50 ppm of GA3, significant increases were observed in seeds treated with 100 ppm of GA3. At this concentration of GA3, the stratification rate of Land race, Chunpoong and Yunpoong cultivar was 95.0%, 95.3%, and 96.5%, respectively.

An Integrated Biochemical, Proteomics, and Metabolomics Approach for Supporting Medicinal Value of Panax ginseng Fruits

Panax ginseng roots are well known for their medicinal properties and have been used in Korean and Chinese traditional medicines for 1000s of years. However, the medicinal value of P. ginseng fruits remain poorly characterized. In this study, we used an integrated biochemical, proteomics, and metabolomics approach to look into the medicinal properties of ginseng fruits. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)] assays showed higher antioxidant activities in ginseng fruits than leaves or roots. Two-dimensional gel electrophoresis (2-DE) profiling of ginseng fruit proteins (cv. Cheongsun) showed more than 400 spots wherein a total of 81 protein spots were identified by mass spectrometry using NCBInr, UniRef, and an in-house developed RNAseq (59,251 protein sequences)-based databases. Gene ontology analysis showed that most of the identified proteins were related to the hydrolase (18%), oxidoreductase (16%), and ATP binding (15%) activities. Further, a comparative proteome analysis of four cultivars of ginseng fruits (cvs. Yunpoong, Gumpoong, Chunpoong, and Cheongsun) led to the identification of 22 differentially modulated protein spots. Using gas chromatography-time of flight mass spectrometry (GC-TOF MS), 66 metabolites including amino acids, sugars, organic acids, phenolic acids, phytosterols, tocopherols, and policosanols were identified and quantified. Some of these are well known medicinal compounds and were not previously identified in ginseng. Interestingly, the concentration of almost all metabolites was higher in the Chunpoong and Gumpoong cultivars. Parallel comparison of the four cultivars also revealed higher amounts of the medicinal metabolites in Chunpoong and Gumpoong cultivars. Taken together, our results demonstrate that ginseng fruits are a rich source of medicinal compounds with potential beneficial health effects.

Analysis of Mitochondrial DNA Sequence and Molecular Marker Development for Identification of Panax Species

This study describes the identification of Panax species using mitochondrial consensus primers. Initially, a total of thirty primers were tested in ten Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, three primers (cox1, nad1/2-3 and nad2/1-2) generated co-dominant polymorphic banding patterns discriminating Korean ginseng cultivars from P. quinquefolius and P. notoginseng. However, these primers could not generated polymorphisms among the Korean ginseng cultivars, and simply represented species-specific polymorphisms for P. quinquefolius and P. notoginseng. Primers PQ91 and PN418 were designed from the consensus sequence of nad1/2-3 region. Two banding patterns (A or B) were detected in PQ91. Korean ginseng cultivars and P. notoginseng shared the same banding pattern (A type) and P. quinquefolius was identified another banding pattern (B type). In the case of PN418, two banding patterns (A or B) were detected in the Korean ginseng cultivars and two foreign Panax species. Korean ginseng cultivars and P. quinquefolius shared the same banding pattern (A type) and P. notoginseng was identified another banding pattern (B type). The combination banding patterns of three Panax species, Korean ginseng cultivars (Panax ginseng C. A. Mey.), P. quinquefolius and P. notoginseng, was identified as `AA`, `BA` and `AB`, respectively. Consequently, PQ91 and PN418 primer sets can be used to distinguish among Panax species.

Effects of Salt in Soil Condition on Chlorophyll Fluorescence and Physiological Disorder in Panax ginseng C. A. Meyer

Background : Excessively high concentration of sodium ion causednutrient deficiency and significantly decrease growth. This study was carried out to determine the limiting concentration range of sodium ion in the soil of ginseng field. Methods and Results : The growth of the ginseng cultivar Chunpoong reduced with increase in salinity, and the rate of growth reduction was higher in shoots than that of roots. Particularly, ginseng plants cultivated at high level of nitrate nitrogen or sodium may suffer delayed development and stunted growth. Chlorophyll damage occurred on the leaves of ginseng planted in relatively high levels (> ) of sodium ion, as determined by the fluorescence reaction. The incidence of physiological disorder in ginseng cultivated at 249 sites was correlated with the concentration of sodium ion in the soils. About 74% of ginseng fields in which physiological disorders occurred had concentrations of sodium ion in soil greater than . In contrast, the concentration of sodium ions at 51 of 85 sites where no damage occurred was relatively (). Conclusions : The concentration of sodium ion in soil of ginseng fields can be classified into three levels optimum (), permissible allowance (0.15 - 0.2) and excessive (> 0.2).

Proteomics Analysis of Early Salt-Responsive Proteins in Ginseng (Panax ginseng C. A. Meyer) Leaves

*Department of Plant Bioscience, Pusan National University, Miryang 627-706, Korea.**Ginseng Research Division, Department of Herbal Crop Research, NIHHS, RDA, Eumseong 369-873, Korea.ABSTRACT : Salt stress is one of the major abiotic stresses affecting the yield of ginseng (Panax ginseng C. A. Meyer). Theobjective of this study was to identify bio-marker, which is early responsive in salt stress in ginseng, using proteomicsapproach. Ginseng plants were exposed to 5 ds/m salt concentration and samples were harvested at 0, 6, 12 and 18 hoursafter exposure. Total proteins were extracted from ginseng leaves treated with salt stress using Mg/NP-40 buffer and wereseparated on high resolution 2-DE. Approximately 1003±240 (0 h), 992±166 (6 h), 1051±51 (12 h) and 990±160 (18 h)spots were detected in colloidal CBB stained 2D maps. Among these, 8 spots were differentially expressed and were identi-fied by using MALDI-TOF/TOF MS or/and LC-MS/MS. Ethylene response sensor-1 (spot GL 1), nucleotide binding protein(spot GL 2), carbonic anhydrase-1 (spot GL 3), thylakoid lumenal 17.9 kDa protein (spot GL 4) and Chlorophyll a/b bindingprotein (spot GL 5, GL 6) were up-regulated at the 12 and 18 hour, while RuBisCO activase B (spot GL 7) and DNA helicase(spot GL 8) were down-regulated. Thus, we suggest that these proteins might participate in the early response to salt stressin ginseng leaves.Key Words : Ginseng, Protein Biomarker, Proteomics, Salt Stress, 2-DE

Applications of molecular markers in the discrimination of Panax species and Korean ginseng cultivars (Panax ginseng)

The development of molecular markers is one of the most useful methods for molecular breeding and marker-based molecular associated selections. Even though there is less information on the reference genome, molecular markers are indispensable tools for determination of genetic variation and identification of species with high levels of accuracy and reproducibility. The demand for molecular approaches for marker-based breeding and genetic discriminations in Panax species has greatly increased in recent times and has been successfully applied for various purposes. However, owing to the existence of diverse molecular techniques and differences in their principles and applications, there should be careful consideration while selecting appropriate marker types. In this review, we outline the recent status of different molecular marker applications in ginseng research and industrial fields. In addition, we discuss the basic principles, requirements, and advantages and disadvantages of the most widely used molecular markers, including restriction fragment length polymorphism, random amplified polymorphic DNA, sequence tag sites, simple sequence repeats, and single nucleotide polymorphisms.

Internal Transcribed Spacer Barcoding DNA Region Coupled with High Resolution Melting Analysis for Authentication of Panax Species

Background : Correct identification of Panax species is important to ensure food quality, safety, authenticity and health for consumers. This paper describes a high resolution melting (HRM) analysis based method using internal transcribed spacer (ITS) and 5.8S ribosomal DNA barcoding regions as target (Bar-HRM) to obtain barcoding information for the major Panax species and to identify the origin of ginseng plant. Methods and Results : A PCR-based approach, Bar-HRM was developed to discriminate among Panax species. In this study, the ITS1, ITS2, and 5.8S rDNA genes were targeted for testing, since these have been identified as suitable genes for use in the identification of Panax species. The HRM analysis generated cluster patterns that were specific and sensitive enough to detect small sequence differences among the tested Panax species. Conclusion : The results of this study show that the HRM curve analysis of the ITS regions and 5.8S rDNA sequences is a simple, quick, and reproducible method. It can simultaneously identify three Panax species and screen for variants. Thus, ITS1HRM and 5.8SHRM primer sets can be used to distinguish among Panax species.

Development of a Simple and Reproducible Method for Removal of Contaminants from Ginseng Protein Samples Prior to Proteomics Analysis

This study describes the effects of activated charcoal on the removal of salts, detergents, and pigments from protein extracts of ginseng leaves and roots. Incubation of protein extracts with 5% (w/v) activated charcoal (100-400 mesh) for 30 min at 4°C almost removed the salts and detergents including NP-40 as can be observed on SDS-PAGE. In addition, analysis of chlorophyll content showed significant depletion of chlorophyll (~33%) after activated charcoal treatment, suggesting potential effect of activated charcoal on removal of pigments too along with the salts and detergents. 2-DE analysis of activated charcoal treated protein samples showed better resolution of proteins, further indicating the efficacy of activated charcoal in clearing of protein samples. In case of root proteins, although not major differences were observed on SDS-PAGE, 2-DE gels showed better resolution of spots after charcoal treatment. In addition, both Hierarchical clustering (HCL) and Principle component analysis (PCA) clearly separated acetone sample from rest of the samples. Phenol and AC-phenol samples almost overlapped each other suggesting no major differences between these samples. Overall, these results showed that activated charcoal can be used in a simple manner to remove the salts, detergents and pigments from the protein extracts of various plant tissues.

Label-free quantitative proteomic analysis of Panax ginseng leaves upon exposure to heat stress

Background Ginseng is one of the well-known medicinal plants, exhibiting diverse medicinal effects. Its roots possess anticancer and antiaging properties and are being used in the medical systems of East Asian countries. It is grown in low-light and low-temperature conditions, and its growth is strongly inhibited at temperatures above 25°C. However, the molecular responses of ginseng to heat stress are currently poorly understood, especially at the protein level. Methods We used a shotgun proteomics approach to investigate the effect of heat stress on ginseng leaves. We monitored their photosynthetic efficiency to confirm physiological responses to a high-temperature stress. Results The results showed a reduction in photosynthetic efficiency on heat treatment (35°C) starting at 48 h. Label-free quantitative proteome analysis led to the identification of 3,332 proteins, of which 847 were differentially modulated in response to heat stress. The MapMan analysis showed that the proteins with increased abundance were mainly associated with antioxidant and translation-regulating activities, whereas the proteins related to the receptor and structural-binding activities exhibited decreased abundance. Several other proteins including chaperones, G-proteins, calcium-signaling proteins, transcription factors, and transfer/carrier proteins were specifically downregulated. Conclusion These results increase our understanding of heat stress responses in the leaves of ginseng at the protein level, for the first time providing a resource for the scientific community.

Difference in Growth Characteristics of 5-Year-Old Ginseng Grown by Direct Seeding and Transplanting

Background : In order to determine the effects of planting methods on root growth of ginseng varieties, two different methods, direct seed sowing and transplanting were compared in terms of their effects on different root growth characteristics. Methods and Results : Higher fresh root weight was observed in ginseng grown by direct seed sowing. Direct seed sowing of three cultivars (Sunhyang, Chungsun and K-1) resulted in higher yield, whereas no difference was observed in the yield of one cultivar (Chungsun). Gumpoong was highly tolerant to physiological stress, as it showed fewer symptoms of rusty and rough skin root diseases in both direct seed sowing and transplanting. The average main root length per total root length of ginseng grown by direct seed sowing was 33.6%, whereas that of ginseng grown by the average of those by transplanting was 22.4%. Other root growth characteristics, including root length, main root diameter, and number of side roots, improved when the direct seed sowing method was used. Conclusions : To our knowledge, this is the first study reporting the differences in root growth parameters of ginseng varieties grown by direct seed sowing or transplanting at the same planting density. Because of the advantages of direct sowing during ginseng planting, developing new varieties and improving cultivation methods are imperative.