Background : Due to immature development of embryo in ripened berries, dehiscence process is required for the proper germination of ginseng seeds. Such process involves the preparation of the container with alternating layers of seed and moist sand. In order to make sand fully moist, water sprayer has been usually used by farmers, which is labor intensive, time consuming and causing uneven sand moisture. Methods and Results : In this study, we investigated the effects of different stratification methods on dehiscence ratio of ginseng seeds. Ginseng seeds were stratified for 90 days in a total of 12 different treatments and the dehiscence ratios were compared; drainage methods, drainage time, the ratio between ginseng seeds and sand, and etc. Seed stratification process was performed according to the guideline of ginseng GAP. One thousand ginseng seeds were used for each treatment. It was found that the average of dehiscence of the 12 treatments was 84.6 %. The highest dehiscence ratio (90.3 %) was observed in the seeds that were treated with water soaking, immediately followed by drainage. Higher ratio was also observed in the seeds that were soaked for 60 min, followed by drainage. Therefore, our findings indicate that ginseng seeds soaked in water less than 60 min could dehisce more efficiently than traditional method. Conclusion : Our study demonstrated that ginseng seeds that are subjected to water soaking and then drainage showed better ration of dehiscence. This method will eventually decrease the time and labor used for seed stratification.

Reynoutria japonica and R. sachalinensis have been used as medicinal resources in Korea. However, it is difficult to identify and determine these medicinal herbs correctly because they are usually customized and purchased as the fragmented rhizomes types. To develop molecular markers for distinguishing two species, we analyzed and compared the chloroplast DNA sequences of seven loci (atpB, matK, ccD-psaI, atpF-H, trnL-trnF, psbK-I and rpl32-trnL). Among them, we found two effective SNPs in psbK-I region for R. japonica and atpF-H region for R. sachalinensis. Based on these SNP sites, we designed the new R. japonica- specific primer which is able to amplify 300 bp fragment in psbK-I region. A similar strategy was applied for the atpF-H region of R. sachalinensis. These molecular markers would be successfully applied to recognize R. japonica and R. sachalinensis.

Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the treatment of liver disease and the development of effective in vitro toxicity screening tool. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized differentiation protocol, which produces more than 90% albumin-positive HLCs with no purification process. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating three-dimensional (3D) spheroidal aggregate of HLCs. The 3D differentiation method increased expressions of nuclear receptors that regulate the proper expression of key hepatic enzymes. Furthermore, a significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids and HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated the functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic maturation of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells have received extensive attention in the development of drug screening and toxicity testing. However, it has been reported that stem cell-derived HLCs showed hepatic functions that were too limited to be of use in drug screening and toxicity testing, possibly due to the lack of sufficient intercellular communication under conventional two-dimensional (2D) culture conditions. Therefore, a 3D differentiation system may overcome the in vitro limitation of 2D culture to produce stem cell-derived hepatocytes with mature metabolic functions. In this study, the feasibility of using a silicone-based spherofilm, specifically designed to produce spherical cell clusters, to generate uniformly sized 3D hepatic spheroids from hESCs was investigated. Hepatic spheroids generated on the spherofilm showed more homogenous size and shape than those generated in conventional low-attachment suspension culture dishes. Results of immunohistochemical analysis showed that expression of the mature hepatic marker albumin (ALB) increased over time during the hepatic maturation process. Furthermore, the 3D culture system mimicked the in vivo 3D microenvironment. Laminin, which is an important component of hepatic ECM, was expressed in hepatic spheroids. The results of immunohistochemical analysis indicated that the 3D culture environment is capable of generating an in vivo-like microenvironment. In addition, quantitative PCR analysis showed that the mature hepatic marker ALB and cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A7 were expressed at higher levels in 3D culture than in 2D culture. This indicates that the 3D culture system is suitable for hepatic maturation and that our size-controlled 3D culture conditions might accelerate hepatic function. These results suggest that 3D hepatic spheroids significantly enhance metabolic maturation of hepatocytes derived from hESCs

Previously we have shown that human abdominal adipose derived-stem cells (ADSCs) could aggregate during the high-density culture in the presence of human serum (HS). In the present study, we observed that human cord blood serum (CBS) and follicular fluid (HFF) also induced aggregation. Similarly, porcine serum could induce aggregation whereas bovine and sheep sera induced little aggregation. qRT-PCR analyses demonstrated that, compared to FBS-cultured ADSCs, HScultured cells exhibited higher level of mRNA expression of CLDN3, -6, -7, -15, and -16 genes among the tight junction proteins. ADSCs examined at the time of aggregation by culture with HS, BSA, HFF, CBS, or porcine serum showed significantly higher level of mRNA expression of JAM2 among JAM family members. In contrast, cells cultured in FBS, bovine serum or sheep serum, showed lower level of JAM2 expression. Immunocytochemical analyses demonstrated that the aggregates of HS-cultured cells (HS-Agg) showed intense staining against the anti-JAM2 antibody whereas neither nonaggregated cells (HS-Ex) nor FBS-cultured cells exhibited weak staining. Western blot results showed that HS-Agg expressed JAM2 protein more prominently than HS-Ex and FBS-cultured cells, both of latter reveled weaker intensity. These results suggest that the aggregation property of ADSCs during high-density culture would be dependent on the specific components of serum, and that JAM2 molecule could play a role in the animal sera-induced aggregation in vitro.

Treatment of spontaneous retroperitoneal hemorrhage (SRH) is mainly conservative. However, if intra-abdominal hypertension (IAH) or abdominal compartment syndrome (ACS) develops, the treatment strategy should be more aggressive. Surgical decompression has been the gold standard; however, it is very invasive and highly morbid. Thus, percutaneous catheter drainage (PCD) has been introduced as an alternative therapeutic option. We report on a case of successful PCD for prevention of progression of IAH to ACS in a patient with SRH after coronary stent implantation. This case showed that PCD can be an efficacious and safe method for treatment of IAH with impending ACS.

We determined intravitreal levels of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in patients with early phase rhegmatogenous retinal detachment (RRD). Twenty five eyes of 25 RRD patients with symptom onset from one day to 21 days prior to vitrectomy were selected. Concentrations of VEGF and PEDF in vitreous were determined by enzyme-linked immunosorbent assay and relationships between these concentrations and durations and involved quadrants were analyzed. Duration of RRD was found to show significant correlation with PEDF concentration. However, number of involved quadrants did not show correlation with PEDF concentration in vitreous.

Chronic gamma irradiation can be used an alternative mutation breeding methods for induction of many useful mutants. Seedlings of purple-colored wheat plants were irradiated with wide range doses of chronic gamma-rays (20, 25, 30, 40, 50, 70, 100, 125, 150, 200, 250, 300 Gy) during 6 weeks at gamma-phytotron in the Korea Atomic Energy Research Institute, respectively. To identify the biological responses purple-colored wheat, we examined the plant height, chlorophyll, carotenoid and total anthocyanin contents in leaf. Plant growth, chlorophyll and carotenoid contents in leaf were decreased when the dose rate increased. Anthocyanin contents were increased with the increase of the radiation dose until 50 Gy treatment. To confirm the real contents of anthocyanin, we also investigated cyanidin-3-glucoside in purple-colored wheat leaf by using UPLC analysis. These results indicate that anthocyanin accumuation was observed under chronic gamma irradiation.

Genome sequencing researches for considerable numbers of crops and wild plants are being developed. Cytogenetic researches according to chromosome number and size are essential to confirm and comprehend ploidy level and genome size before genome sequencing project is actually conducted. Cytogenetic researches on six food crop plants were carried out by DAPI staining and fluorescence in situ hybridization (FISH) method. Fagopyrum esculentum Moench showed 2n=2x=16, each chromosome length of 1.42㎛ to 1.77㎛, total chromosome length of 13.31㎛, and karyotypic formula of 2n=8m; Phaseolus angularis W.F. Wight, 2n=2x=22, 2.01㎛ to 3.84㎛, total 28.03㎛, 2n=9m+2sm, Perilla frutescens var. japonica Hara, 2n=2x=40, 1.73㎛ to 2.76㎛, total 44.36㎛, 2n=5m+13sm+2st. Chromosome sizes of the other three species such as, Panicum miliaceum L., 2n=2x=36, total chromosome length of 30.83㎛, Sesamum indicum L., 2n=2x=26, 27.39㎛, lpomoea batatas L., 2n=2x=30, total 33.51㎛ were too small for each chromosome type to be identified and analyzed. The result of FISH analysis using 5S and 45S rDNA probe showed species-specific chromosome locations in the genome. These preliminary analyses were carried out to decide which food crop to prioritize for genome sequencing. This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (No.PJ009837), Rural Development Administration, Republic of Korea.

Human serum (HS) has been reported to induce aggregation of human eyelid adipose-derived stem cells (HEACs) during high-density culture in vitro. The present study focused on the role of cell adhesion molecules and gelatinases during HS-induced aggregation of HEACs. HS-induced aggregation occurred between 9-15 days of culture. Cells aggregated by HS medium (HS-agg) showed stronger expression of α2, α2B, αX, and CEACAM1 genes compared to non-aggregated cells in HS medium (HS-ex) or in control FBS-cultured cells. HS-agg were distinctly labeled with antibodies against α2, α2B, and αX proteins. Western blot results demonstrated that the two integrin proteins were greatly expressed in HS-agg compared to HS-ex and control FBS-cultured cells. Treatment of HEACs with anti-integrin α2 antibody during culture in HS medium delayed aggregation formation. HS-agg exhibited strong expression of MMP1 and MMP9 compared to HS-ex or FBS-cultured cells. Conditioned media from HS-culture showed remarkable increase of MMP9 gelatinolytic activity in comparison to those from FBS-culture. However, there was no change of TIMP mRNA expression in relation to the HS-induced aggregation. Based on these results, it is suggested that integrin α2, α2B, and αX, and MMP9 might play an important role in the HS-induced aggregation of HEACs.

The study was carried out to determine the gel pasting properties of barley (Hordeum vulgare L. cv. Geoncheonheugbori) as affected by different proton beam irradiation. The λmax, blue value, and amylose content were significantly associated with increasing proton beam irradiation. The pasting time in barley flour irradiated with proton beam ranged 0.09 to 0.16 min shorter than nonirradiated barley flour. Gel pasting temperature ranged 57.4 to 60.5℃. Gel pasting temperature in barley flour decreased with increasing proton beam irradiation. Proton beam irradiation caused a significant decrease in the onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and enthalpy change (ΔH). Gelatinization range (R) in barley starch was more broaden than that of non-irradiated barley starch. Barley starches gave the strong diffraction peak at around 2θ values15°, 18°, 20°, and 23° 2θ. Peak intensity tended to increase with increased proton beam irradiation. The granule crystallinity is closely associated with decreased amylose and increased amylopectin component. The crystallinity degree of barley starch irradiated with proton beam was significantly increased and it ranged from 24.9 to 32.9% compared to the non-irradiated barley starches. It might be deduced that proton beam irradiation causes significant changes of properties of starch viscosity in rice, especially at high irradiation of proton beam.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Alfalfa (Medicago sativa L.) is one of the most important forage crops in the world and it’s has been known as the best feed materials for dairy cows and other high valued animals. The new uses of alfalfa are being explored as bio-energy, food, medical and biochemical uses. R2R3-type MYB transcription factors play important roles in transcriptional regulation of anthocyanin biosynthesis. The R2R3-type IbMYB1 is known to be a key regulator of anthocyanin biosynthesis in the storage roots of sweetpotato. We previously showed that the expression of IbMYB1a led to anthocyanin pigmentation in tobacco and Arabidopsis. In this study, we generated transgenic alfalfa plants expressing the IbMYB1a gene under the control of CaMV 35S promoter. Overexpression of IbMYBa in transgenic alfalfa produced strong anthocyanin pigmentation in seedlings and generated a deep purple color in leaves, stems, roots, and even in seeds. High performance liquid chromatography (HPLC) analysis revealed that IbMYB1a expression led to the production of cyanidin as a major core molecule of anthocyanidins in alfalfa, as occurs in the purple leaves of sweetpotato (cv.Sinzami). We also examined expression of several structural genes in the anthocyanin biosynthetic pathway in alfalfa by RT-PCR analysis. In this presentation, we will further present molecular and biochemical characterization in IbMYB1a-overexpression lines. This result shows that the IbMYB1a transcription factor is sufficient to induce anthocyanin accumulation in the forage legume alfalfa plants.

Compared to wide ranges of genetic variation of natural populations, very limited Miscanthus cultivar has been released. This study was the first report on the development of Miscanthus cultivar by means of radiation breeding. Seeds of M. sinensis were initially exposed to gamma rays of 250 Gy for 24 h, generated from a 60Co gamma-irradiator. The irradiated seeds were sown and then the highly tiller-producing mutants were selected for this study. Biomass-related parameters including tiller number, plant height, stem diameter, and leaf number were measured. Ploidy level and internal transcribed spacer (ITS) were investigated to characterize the mutants compared to wild type (WT) Miscanthus. Plant height and tiller number were negatively related, where multi-tillering mutants were relatively short after 4 month growth. However stem diameter and leaf number were greater in mutants. All the materials used in this study were diploid, implying that the mutants with greater tiller numbers and stem diameter were not likely related to polyploidization. Based on the sequence of ITS regions, the mutants demonstrated base changes from the gamma irradiation where G+C content (%) was decreased in the ITS1, but increased in ITS2 when compared to WT sequence. ITS2 region was more variable than in ITS1 in the mutants, which collectively allows identification of the mutants from WT. Those mutants having enhanced tillers and allelic variations might be used as breeding materials for enhanced biomass-producing Miscanthus cultivars.

Soybean (Glycine max, 2n = 2x = 40) is broadly distributed throughout East and South East Asia, and important crop as a source of protein, oil, food and animal feed. In order to better understand the morphological differentiation of soybean germplasm collected from China, Japan, Korea, Philippines, America, we analyzed the morphological variabilities among 629 soybeans with 11 morphological traits, such as growth type, leaflet, flower color, trichome, seed coat color, color inside seed etc. and measured the fatty acid composition. The result of the principal coordinates analysis (PCoA) based on the 11 morphological traits revealed diversity among all accessions. The PCoA separated the accessions into two main groups, each group with distinctive features. Among tested germplasm, the contents of five fatty acids were as follows: linolenic acid (2.8%－16.23%), linoleic acid (27.4%－56.6%), oleic acid (9.2%－35.0%), stearic acid (2.9%－8.8%), and palmitic acid (8.7%－17.1%). The fatty acid composition has not shown significant variation among all accessions. IT 22268 was the highest linolenic acid composition (16.2%), while IT 154687 was the lowest (2.8%). Forty three of 629 accessions showed the arachidic acid (0.5%-3.6%), which is the saturated fatty acid with a 20 carbon chain and is as a minor constituent of peanut oil (1.1%-1.7%). This result of this characterization served as reliable resources for detailed description and new functional plant breeding of soybean varieties.

During the last decade, considerable progress has been made to understand the molecular mechanisms of M. grisea infection in rice plants and 10 rice blast R genes have been identified and characterized via map-based cloning methods. In case of rice germplasm, the genetic backgrounds of each germplasm accessions are not uniform and the evaluation for pathogenicity is difficult. To solve these problems, we applied the single resistance gene markers to rice germplasm accessions. A molecular survey was conducted to identify the presence of major blast resistance (R) gene in 363 accessions of Korea landrace rice germplasm. The results revealed that the resistance gene Pik-p (100%), Pib (98%), Pi-d(t)2 (98%) and Piz (76%) were widely observed in tested rice germplasm, but Pita-2, Pik and Pi39 gene were identified in less than 10 accessions. Most of landrace contain the four or five different resistant genes, but these results was not consist of field nursery screening. 13 accessions were shown the blast resistance in field nursery screening and Pik-p, Pib, Pi-d(t)2 and Piz genes were observed in these accessions. The evaluation results of blast resistance genes in rice germplasm will help in breeding of multi disease resistant varieties.

Cryopreservation has been known as an efficient method for long-term preservation of clonally propagated plants, and several cryopreservation methods have been developed. Among them, a droplet-vitrification method for potato using axillary shoot tips in vitro has been established previously. In this study, we have optimized the procedure in which explants were submitted to a step-wise pre-culture in liquid sucrose-enriched medium (0.3 and 0.7 M for 7 and 17 h, respectively). The pre-cultured explants were dehydrated with PVS3 (w/v, 50% glycerol + 50% sucrose) for 90 min or modified PVS2 vitrification solution (w/v, 37.5% glycerol + 15% DMSO + 15.0% ethylene glycol + 22.5% sucrose) for 30 min. This two dehydration solutions produced post-cryopreservation regeneration percentages of 57.2% and 80.9%, respectively. We also compared a new post-culture medium (0.1 mg L ･ -1 GA3, 0.1 mg L ･ -1 kinetin) with the conventional one (0.15 mg L ･ -1 IAA, 0.2 mg L ･ -1 zeatin, 0.05 mg L ･ -1 GA3); the shooting initiation rates were 80.9% and 43.5%, respectively. The results suggest that the modified droplet-vitrification protocol described in this study is more effective, easier to implement, and more economical than the droplet-vitrification protocols currently used for potato.