Background : This study was carried out to understand the effect of seedling weight (SW) on growth and flowering in Panax ginseng. Methods and Results : The testing materials were Chunpoong (CP), Yunpoong (YP) and Jakyeongjong (JK). The increase of seedling (1yr) weight led to an increase in ratio of flowering plant and in number of flower per plant. The seed setting rate of two year-old plant (CP, YP, JK) increased with increase of SW at the planting time (PT) and number of flower per plant of three year-old plant (CP, YP) increased also. In the two year-old plant (JK), the ratio of three leaves per plant was 8.8, 19.6, 31.0, 42.0, 44.7 and 58.2%, respectively, in the SW of >0.6, 0.6~0.8, 0.8~1.0, 1.02~1.2, 1.2~1.4 and 1.4g<. The growth of ginseng plant was good with increase of SW at the PT. Conclusion : There was a highly positive correlation between seedling weight and flowering characteristics.

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.

Background : This study was conducted to determine the impact of temperature elevated and the effect of transplanting times based on climate change scenario on growth of 2-year-old korean ginseng (Panax ginseng C. A. Meyer.) in temperature gradient chambers (TGC). Methods and Results : As a plant materials, ‘Yunpoong’ was cultivated in TGC at ambient temperature(Amb), Amb+2℃, Amb+4℃ and Amb+6℃ respectively. Ginseng was also transplanted on March 29, April 12 and 26 respectively. Investigation on characteristic of aerial parts were carried out on 28, 56, 84 and 112 days after transplanting and characteristic of roots were conducted on October 19. As transplanting time was faster and temperature was higher, the growth of aerial parts were increased. Compared with those of ginseng transplanted on March 29 with Amb, the root weight which tend to decrease depending on late transplanting time and high temperature decreased about 11.1%, 35.4% and 42.4% in Amb+ 2℃, Amb+4℃ and Amb+6℃ respectively. Ginseng transplanted on April 12 and 26 decreased about 20.9%, 33.9% respectively. Conclusion : Consequently, the more transplanting time extend, the more quantity increased in all temperature treatment. So, it is possible to increase in quantity to advance transplanting time although high temperature will be caused by the climate change.

Background : When ginseng seeds were gathered, the seeds were unripe. To grow immature embryo definitely, special treatment called dehiscence must be performed. Even though dehiscence is completed, most ginseng seeds are on enforced dormancy. The breaking seed dormancy is generally achieved using cold treatment. Also it is reported that gibberellin treatment can replace the treatment. It is very time consuming process in order to develop new ginseng cultivar because ginseng flowers after 3 years of growth. To shorten the ginseng breeding period, it is necessary to establish fast generation progress. Therefore, this study examined the possibility of breaking seed dormancy of ginseng using GA3 treatment and alternating temperature. Methods and Results : Seeds were obtained from local variety fruit which is not inbred. Gibberellin of 100 ppm was treated at seeds for 24 hours. Fixed cold condition was treated on both –2℃ and 2℃. Alternating cold condition was treated on 2℃ and then –2℃, finally 2℃. Fixed and alternating temperature was continued for 15, 30, 45, 60, 90 days that 15 days of alternating temperature is first 2℃ for 5days and then -2℃ for 5days, finally 2℃ for 5days. The other treatment periods such as 30, 45, 60, 90 days mean 10, 15, 20, 30 days respectively. Each of 48 seeds were sowed on tray in greenhouse at 3 replication. Experimental plot was completely randomized. Conclusion : Seeds untreated with GA3 were germinated little and there is no difference between 2℃ and –2℃. Alternating temperature until 60days made no difference with fixed temperature but germination rate increased up to 70.8% when seeds were treated for 90days. Germination of seeds treated with GA3 is much higher than untreated seeds especially combined with alternating temperature.

Background : The effect of ridge-up bed cultivation and stem training method on the plant growth and yield of Cynanachum willfordii was investigated. Methods and Results : Domestic variety of Cynanachum willfordii was tested at Chungbuk ARES under the following conditions. Nonsupporting (custom) and I-form supporting was used for stem training method, and ridge height was set as 30 (custom) and 60 cm (high ridge). Planting spacing was adjusted to 30 (interrow spacing) × 20 cm (intrarow spacing), and other major management was followed the method of standard cultivation for medicinal crop. Investigated result from leaf characteristics, leaf length was longer in high ridge cultivation (HRC) as 11.1 cm than custom cultivation (CC) as 10.6 cm. Leaf width is proved to be 12.8 (HRC) > 11.2 cm (CC). Leaf number is proved to be 294 (HRC) > 254/plant (CC), with higher number of 44/plant at HRC and weight/10 leaves were more heavy at HRC (4.9 g) than that of CC (2.6 g). It was more fruit setting at HRC over 15/plant. According to the stem training method and ridge height from nonsupporting cultivation, main root was 4.0 (CC), higher than that of HRC over 0.5/plant. However, root length was more longer in HRC (28.6) than that of CC (25.0 cm). Main root diameter was also showed more thicker pattern in HRC. From staking cultivation, root number of HRC was 7/plant, it was recorded more 3/plant than that of CC, and it was also same pattern in main root length and root diameter. By the standard of commercial root, yield of living roots in nonsupporting cultivation were 59.0g/plant (HRC), it was more heavy over 10.4g/plant than that of CC. In staking cultivation, HRC were recorded as 74.2g/plant, more heavy 6.9g/plant than that of CC. Yield from I-form support stem training and ridge-up bed cultivation of HRC was higher approxmiately 52.7% than that of CC. Conclusion : Overall growth by high ridge cultivation of I-form support was good and yield of HRC was also increased over 52.7% than that of CC.

Seed germination is a key developmental transition that initiates the plant life cycle. The timing of germination is determined by coordinated action of two phytohormones, gibberellin (GA) and abscisic acid (ABA). In particular, ABA plays a key role in integrating environmental information and inhibiting the germination process. Utilization of embryonic lipid reserves contributes to seed germination by acting as an energy source, and ABA suppresses lipid degradation to modulate the germination process. Here, we report that the ABA-responsive R2R3-type MYB transcription factor MYB96, which is highly expressed in embryo, regulates seed germination by controlling the expression of ABA-INSENSITIVE 4 (ABI4). In the presence of ABA, germination was accelerated in MYB96-deficient myb96-1 seeds, whereas the process was significantly delayed in MYB96-overexpressing activation-tagging myb96-ox seeds. Consistently, myb96-1 seeds degraded a larger extent of lipid reserves even in the presence of ABA, while reduced lipid mobilization was observed in myb96-ox seeds. MYB96 directly regulates ABI4, which acts as a repressor of lipid breakdown, to define its spatial and temporal expression. Genetic analysis further demonstrated that ABI4 is epistatic to MYB96 in the control of seed germination. Taken together, the MYB96-ABI4 module regulates lipid mobilization specifically in the embryo to ensure proper seed germination under suboptimal conditions.

Acyl-acyl carrier protein (ACP) thioesterase (TE) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. A full-length cDNA of an acyl-ACP thioesterase, named CvFatB, was isolated from oil plant Cuphea viscosissima accumulating up to 90% caprylate (8:0) and caprate (10:0) in its seed oil. This cDNA contains a 1,245-bp open reading frame that encodes a protein of 415 amino acids. The deduced sequence also contains two essential residues (H317 and C352) for TE catalytic activity and a putative chloroplast transit peptide at the N-terminal. Overexpression of the CvFatB cDNA in Arabidopsis resulted in increased levels of saturated fatty acid, especially palmitate, and reduced levels of unsaturated fatty acids. The findings suggest that CvFatB from oil plant C. viscosissima can function as a saturated acyl-ACP TE and can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

The influences of ethylene inhibitors (AgNO3 and silver thiosulfate) and cytokinins (BAP and TDZ) on shoot regeneration from cotyledon and hypocotyl explants of B. napus cv. Youngsan were investigated. The presence of 50 μM Silver thiosulfate (STS) in shoot regeneration medium formed shoots at 60-68% after 3-4 weeks of culture, which was enhanced by 2-fold compared to that of Silver nitrate (AgNO3). Moreover, cotyledon explants were more regenerative than hypocotyls; shoots from cotyledon explants began to occur 4-5 days earlier than that of hypocotyl explants. TDZ at a concentration of 8-10 μM was effective for shoot regeneration, compared with BAP. Consequently, the optimal shoot regeneration response was observed in medium supplemented with 50 μM STS + 8 μM TDZ. In transmission electron microscopy (TEM) analysis, higher density of silver nanoparticles was shown to be accumulated widely inside the cell wall and plasmodesmata of regenerating leaf cultured in medium supplemented with AgNO3. By contrast, in the cell cultured in medium with STS, fine-grained deposits were partly observed in the surroundings of the cell wall.

To understand molecular mechanisms underlying adaptation of plant cells to saline stress and stress memory, we developed Arabidopsis callus suspension-cultured cells adapted to high salt. Adapted cells to high salt exhibited enhanced tolerance compared to control cells. Moreover, the salt tolerance of adapted cells was stably maintained even after the stress is relieved, indicating that the salt tolerance of adapted cells was memorized. Salt-adapted and stress memorized cells were densely aggregated and formed multi-layered cell lump. Cell morphology analysis using transmission electron microscopy indicated that cell wall thickness of salt-adapted cells was significantly induced compared to control cells. In order to characterize metabolic responses of plant cells during adaptation to high salt stress as well as stress memory, we compared metabolic profiles of salt-adapted and stress-memorized cells with control cells by using NMR spectroscopy. A principle component analysis showed clear metabolic discrimination among control, salt-adapted and stress-memorized cells. Compared with control cells, metabolites related to shikimate metabolism such as tyrosine, and flavonol glycosides, which are related to protective mechanism of plant against stresses were largely up-regulated in adapted cell lines. Moreover, coniferin, a precursor of lignin, was more abundant in salt-adapted cells than control cells. The results provide new insight into metabolic level mechanisms of plant adaptation to saline stress as well as stress memory.

To identify novel signaling components involved in regulation of plant responses to phosphate (Pi) starvation, we screened an Arabidopsis T-DNA activation tagging library for mutants with altered Pi-starvation responses. Here, we report the identification and characterization of novel activation-tagged mutant involved in Pi starvation signaling in Arabidopsis. The hpd (hypersensitive to Pi deficiency) mutant exhibits enhanced phosphate uptake and altered root architectural change under Pi starvation compared to wild type. Expression analysis of auxin-responsive DR5::GUS reporter gene in hpd mutant indicated that both auxin biosynthesis and auxin translocation under Pi starvation are suppressed in hpd mutant plants. Impaired auxin translocation in roots of hpd mutant was attributable to abnormal root architecture changes in Pi starvation conditions. Mis-regulation of auxin translocation in hpd mutant was further confirmed by analysis of expression patterns of auxin efflux carrier proteins, PIN-FORMED (PIN) 1, 2, and 3 fused with GFP. Not only expression levels but also expression domains of PIN proteins were altered in hpd mutant in response to Pi starvation. Molecular genetic analysis of hpd mutant revealed that the mutant phenotype is caused by the lesion in ENHANCED SILENCING PHENOTYPE4 (ESP4) gene whose function is proposed in mRNA 3’-end processing. The results propose that mRNA processing plays crucial roles in Pi homeostasis as well as developmental reprograming in response to Pi deprivation in Arabidopsis.

The aim of this study was to analyze the effect of antifreeze proteins (AFPs) on vitrification of mouse mature (MII) oocytes. We studied about 3 types of AFPs from different origins (FfIBP, LeIBPand Type III AFP). The MII oocytes were obtained from 4-week-old BD-F1 mice. Vitrification of oocyte was performed by 2 steps using the Cryotop (equilibration: 7.5% EG + 7.5% PROH for 5 min, vitrification: 15% EG + 15% PROH + 0.5M sucrose for 1 min). The concentrations of AFPs added to these solutions were 0.05 mg/ml for FfIBP and 0.1 mg/ml for LeIBP and Type III AFP. After fertilization, embryo development was assessed up to 5 days. Through immunostaining of vitrified-warmed oocytes, we assessed the normal meiotic spindle. Also, intracellular ROS and mitochondrial activity was analyzed. In the developmental stages, FfIBP and LeIBP groups showed significantly higher survival rates. In the blastocyst and apoptotic blastomere rates were significant differences in AFPs treated groups. AFPs treated groups were significantly higher in blastocyst cell numbers than control group. Among the AFPs treated groups, FfIBP, LeIBP groups were significantly higher rates. And, in cleavage rates, FfIBP group was significantly higher rates than the other groups. In vitrified-warmed MII oocytes, the normal meiotic spindle organization and chromosome alignment rate was significantly higher in FfIBP and LeIBP groups. And in intracellular ROS levels, control group was significantly increased than AFPs treated groups. However, in the mitochondrial activity, LeIBP group was significantly higher than control, FfIBP and LeIBP groups. AFPs treated groups were significant differences in development, meiotic spindle organization and intracellular ROS levels. And in the AFPs treated groups, FfIBP and LeIBP groups were significantly higher rates in normal meiotic spindle and mitochondrial activity than Type III AFP group respectively. In conclusion, FfIBP and LeIBP can be thought to improve oocyte cryopreservation efficiency.

The follicle loss of transplanted ovarian tissue (OT) is caused by ischemia and slow revascularization. To shorten the ischemic period and promote angiogenesis, some angiogenic factors have been treated for transplanted tissues. Angiopoietin-2 (ANG-2) is one of the major angiogenic factors and has been reported to promote blood vessels and increase vascular permeability in the ischemic and/or hypoxic environment. So, this study was designed to assess the impact of ANG-2 on follicle integrity and revascularization of mouse OT grafts. The 5-week-old B6D2F1 female mice were divided into 3 groups (a control and 2 ANG-2 groups) followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without ANG-2 injection (50 or 500 ng/kg), and then killed at day(D)2, 7, 21 and 42 after transplantation. Total 2,437 follicles in OT grafts were assessed for the follicular density, integrity and classification by H&E staining. Apoptosis, revascularization, and serum FSH levels were evaluated by TUNEL assay, CD31 immunohistochemistry, and ELISA respectively. All the ANG-2 groups showed remarkable increase of morphologically intact follicle ratio across all the grafting duration except D21 (no statistical difference). The numbers of CD31(+) vessels (the sum of 3 fields at ×400 magnification) were significantly increased in both ANG-2 groups compared with the control group at all the grafting duration. Especially at D42, the 500ng ANG-2 group showed significantly more vessels than the 50 ng ANG-2 group as well as the control group. However the mean follicle numbers of grafts, apoptosis ratio and serum FSH levels showed no significant difference among the groups. In this study, remarkably well preserved follicles and larger amount of vessels were appeared in ANG-2 treated groups. So we thought that ANG-2 treatment is effective for OT transplantation and improve transplantation outcomes.

Polyethyleneglycol-adsorbed–superoxide dismutase (PEG-SOD), has been proposed as an effective agent for reducing free radical-mediated injury. The objective of this study was to investigate a protective effect of PEG-SOD supplementation on ovarian tissue during transplantation. Ovaries from F-1 mice were collected and vitrified. After warming, ovaries were autotransplanted under kidney capsule. Mice were randomly divided into four groups according to dose of PEG-SOD, (0 U/ml, 100 U/ml, 1,000 U/ml and 10,000 U/ml respectively). Grafted ovaries were retrieved 2, 7 and 21 days later. PEG-SOD was treated by intraperitoneal injection once every 48 hours and especially for 21 days group, after first week treatment, PEG-SOD was treated once every 4 days. Morphology of ovaries was assessed histological analysis and ELISA for FSH was performed to evaluate restoration of ovarian function. In 2 days groups, morphologically intact follicle ratio of 10,000 U/ml group was significantly higher than other groups. In 7 days groups, morphologically intact follicle ratio was significantly higher in all treatment groups. In 21 days groups, there was no significant difference of intact follicle ratio in total follicles in all groups but intact primordial, primary and secondary follicles ratio was higher in 10,000 U/ml group. FSH levels in blood serum were decrease as time goes on, but there is no statistical difference in each groups. In conclusion, the data of the present study show that PEG-SOD has a beneficial effect on preservation of the morphologically intact follicle.

Ovarian tissue cryopreservation and transplantation causes follicle depletion. To overcome this problem, we investigate the effect of Anti-Müllerian hormone (AMH), a follicle recruitment control hormone, supplementation before and/or after mouse ovarian transplantation. A total of 120 5-week-aged BD F-1 female mice were used. The mice were randomly divided into four groups according to AMH doses (0, 5, 25, 125 μg/mL, respectively). AMH was injected intraperitoneally on every other day for a week before, after, or before and after transplantation of ovaries under kidney capsules was performed. One week after transplantation, follicular normality was evaluated by histological analysis and TUNEL assay. In Group A and C, morphologically intact follicle (G1) ratios of AMH treated groups showed no statistically significant difference. In Group B, G1 ratios of 25 and 125 μg/mL of AMH treated groups were higher than those of 5 μg/mL treated group, but there was no improvement in G1 ratio after AMH treatment. In every group, apoptotic follicle ratios did not show any trend according to AMH treatment. Proportions of primordial follicle were not significantly different according to AMH treatment in all groups. The result of the present study demonstrated that AMH treatment during on transplantation of cryopreserved ovaries has no significant effect on follicle survival and prevention of follicle depletion.

Objective : To investigate the effects of Simvastatin and Methylprednisolone on ovarian tissue cryopreservation and transplantation using mouse models. Methods : The mice were randomly distributed into 1 control and 3 experimental groups. The B6D2F1 mice were given oral Simvastatin (5 mg/kg), intravenous Methylprednisolone (15 mg/kg), or a combination of both at 2 hours before ovariectomy. Same volume of normal saline was given perorally in the control group at 2 hours before ovariectomy. The ovarian tissues were vitrified accrording to our protocols. The vitrified ovaries were warmed 1 week later and auto-transplanted under bilateral kidney capsules. The ovaries and blood sera were collected at 2, 7 or 21 days after transplantation. Histological analysis, TUNEL assay, immuno-histochemistry for CD31, serum AMH level and embryonic development after in vitro fertilization were assessed for evaluation. Results : With regard to the total grade 1 follicle rate, both Simvastatin or Methylprednisolone treated groups were significantly increased at 2, 7 or 21 days after transplantation (except Simvastatin treated group at 7 days). A combination of Simvastatin and Methylprednisolone group was significantly improved in terms of the total G1 follicle rate, apoptotic follicle rate, CD31 positive area and serum AMH after ovarian tissue transplantation. However, there were no statistically difference with respect to the oocyte maturation rate, blastulation rate, and the other embryonic development parameters after in vitro fertilization procedure among the four groups. Conclusion : Our results suggest that combined donor Simvastatin and Methylprednisolone have beneficial effects on the quality and function of transplanted ovarian tissues.

Study question: What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? Summary answer: The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. What is known already: Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified–.warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. Study design, size, duration: A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. Participants/materials, setting, methods: The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of a-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified–.warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. Main results and the role of chance: The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4–.58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6–.92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. Limitations, reasons for caution: The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. Wider implications of the findings: We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

It has been known that adenomyoma is a rare non-neoplastic lesion of the biliary tract. In literature, adenomyoma was managed by extensive surgical procedures for its diagnosis and treatment because adenomyoma showed symptoms like obstructive jaundice and epigastric pain, which made the decision for the lesion difficult to distinguish from malignant neoplasm.We successfully treated a patient who had 12 mm sized adenomyoma of the major papilla with incomplete pancreas divisum by using an endoscopic papillectomy in the asymptomatic stage. The final diagnosis with an immunohistochemical stain revealed a lesion for adenomyoma of the major papilla.