Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide) assays showed that 100 nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.01). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl, two membrane surface molecule genes (P<0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.

OPU 유래 수정란 생산 및 이식은 기존의 체내 및 체외 수정란 생산과 이식의 단점을 해결하고 한우 개량을 촉진 시킬 수 있는 기술이다. 특히 살아있는 공란우를 활용하므로 모계와 부계에 대한 혈통관리가 정확하고 또한 선발된 공란우와 계획 교배를 통하여 가장 적합한 동결정액을 사용하여 개량의 효과를 극대화가 가능하고 또한 현재까지 생산효율이 가장 높은 생산기술이다. 따라서 기존의 수정란 생산방법보다 공란우의 선발강도를 더 높일 수 있어 개량의 효과를 극대화 할 수 있는 장점이 있다. 본 연구실에서는 2009 년도부터 OPU 유래 수정란을 생산하여 이식하여 수정란이식이 산업화로 전개될 수 있도록 진행하고 있다. 특히 한우에 대한 우수성 확보, 개량 및 그 다음 세대 후대의 근친적인 문제가 발생되지 않도록 수정란이식에 의해 탄생된 송아지 혈통관리를 위한 친자검정으로 수정란에 대한 신뢰성을 높여 수정란 이식이 산업화가 될 수 있도록 진행하였다. 본 연구에 대한 조사는 2013 년도 7 개의 시·군을 시작으로 ‘14 년도 11 개, ‘15 년도 15 개, ‘16년도 17 개의 시·군으로 점진적으로 확대됨에 따라 공란우의 두수도 ‘13 년도 35 두를 시작으로 ‘16 년도는 71 두로 증가되었다. 조사된 4 년동안 각 지역에서 선발된 유전능력이 우수한 공란우는 총 211 두에서 수정란을 총 18,839 개, 두당 89.3 개를 생산하였다. 두당 생산 효율도 ‘13 도에는 회당 3.0±4.5 개에서 ‘16 년도에는 4.8±2.2 로 회당 이식 가능한 수정란의 생산량이 년도에 따라 꾸준히 향상되었다. 생산된 수정란은 자연발정 또는 발정동기화를 통하여 매주 2 회 이상 이식으로 ‘13 년 49.7%, ‘14 년 51.9%, ‘15 년 52.9%와 ‘16 년 47.9%의 수태율을 확인하였다. 이는 년도가 증가되면서 수정란의 생산성의 향상과 적정 수준의 수태율의 성과는 시술자와 농가의 적극적인 참여에 의한 결과로 판단되고 또한 유전능력이 우수한 수정란의 대량생산 및 공급으로 개량의 효과가 증가되고 우수한 한우 집단 구축으로 한우산업이 지속적으로 발전 할 것으로 판단된다.

Flammulina velutipes belonging to white rot fungi is one of the commercially important edible mushrooms and is produced in large quantities due to the introduction of a automated and mechanized cultivation system in Korea. Despite the chief item of export among edible mushrooms, Flammulina velutipes has the lowest distribution rate of domestic cultivar, estimated that about 20 percent. As the result that most white cultivars of Flammulina velutipes produced and exported in Korea were introduced from Japan, farmers pay a large amount of royalties. Therefore, we try to develop a new pure domestic cultivars as a substitute for Japanese cultivars. To breed both white and gold superior strains, we selected the crossing mother groups including 10 white strains of ASI 4198 etc. and 7 brown strains of ASI 4049 etc. and mated each of the 17 strains by mon-mon hybridization. 19 white and 14 brown strains were chosen through two selection experiment over 2014 2016. In the third selection experiment this year, we finally selected one white(Fv 16 c 37) and the other gold(Fv 15 a 31) strain. Two selected strains were cultivated in the same environmental conditions. Spawn running period on the sawdust substrate required 30days at 20°C. The cultivation period and optimum temperature were 12±1 days at 14°C for primordia formation, 5 days at 4°C for inhibition phase, and 14±1 days at 7°C for fruiting body development. The length of pilei and stipes in two selected strains and Megumi as a control Japanese cultivar harvested in optimal stage was as follows: 10.5±0.81mm and 139.7±4.23mm in Fv 16 c 37, 10.8±0.43mm and 128..2±7.31mm in Fv 15 a 31, and 10.9±0.41mm and 141.8±4.64mm in Megumi respectively. The Yield of Fv 16 c 37, Fv 15 a 31 and Megumi was 271.2±11.84g, 237.7±9.05g and 270.7±16.87g per 1100ml in bottle cultivation.

Volvariella volvacea is mainly cultivated in subtropics area like South-East Asia. Because it is cultivated in rice straws, it is called a straw mushroom. That mushroom grows well in high temperature about 30~38°C and high humidity. Straw mushroom is a homothallic mushroom, so it is difficult to identify whether the offspring is different from the parents. This study was carried out to investigate RAPD primers that can be used for identification the DNA polymorphism of Volvariella volvacea genetic resources. 9 strains were collected from various countries like China, Vietnam etc. When ITS regions of their DNA were analyzed, they proved to be Volvariella volvacea. A cultivation test was conducted to measure the morphological characteristics of them 2 strains, KMCC04380 and KMCC04382 were selected for breeding resources because the mycelium of them grew well on medium and fruiting bodies were formed quickly. The Universal PCR Fingerprinting kits(UPF primers) were used to confirm the genetic polymorphisms of the 2 strains. As a result of confirming the DNA bands, 2 of 12 primers could be used to genetically distinguish 2 strains. About 50 spores were isolated from their fruiting bodies respectively and they also will be confirmed DNA polymorphisms by using UPF primers.

Tremella fuciformis has about 40 species worldwide, and is widely distributed in the subtropical, tropical, and temperate regions. Tremella fuciformis is known to have been eaten by poppy in Chinese classics and has been a symbol of beauty since ancient times. In fact, it has been known that the extract of the Tremella fuciformis induces inhibition of melanin formation of the cells to have a skin whitening effect and to prevent wrinkles caused by UV stimulation damage, thereby improving wrinkles. Tremella fuciformis was first cultivated in Sichuan province, China, and is now cultivated in China and Southeast Asia. China has succeeded in large-scale artificial cultivation of Tremella fuciformis, but there is no research on mass artificial cultivation in Korea. Therefore, this study was carried out to develop standard cultivation technique for mass production of Tremella fuciformis. In order to select the optimal medium for Tremella fuciformis, five kinds of medium such as PDA, MCM, MEA, YM and CDA were used. Tremella fuciformis showed the best growth on PDA medium, and Annulohypoxylon stygium showed the best growth on MCM. The growth of Tremella fuciformis was good at pH 5 , and Annulohypoxylon stygium showed the best growth at pH 6. The optimum temperature for Tremella fuciformis and Annulohypoxylon stygium was 25 °C. Carbon source and nitrogen source were selected to establish optimal culture conditions.

Cladobotryum mycophilum is the causal agent of cobweb disease of commercial mushrooms. Symptoms were noticed as round, fleshy, yellowish brown lesions on mushroom caps and progressed when the parasitic fungus formed white cobweb circular colonies on dead or damaged pinheads, spread on the surface of the casing. Two gram-positive bacterium were isolated from mushroom media that markedly showed the antagonistic activity against C. mycophilum.. These isolates were identified as Bacillus altitudinis and Bacillus subtilis by analysis of the cultural, morphological and physiological characteristics, and analysis of the 16S rDNA. The isolated bacteria is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacteria cell was sufficient for inhibition in vitro for C. mycophilum. Control efficacy of browning disease of Bacillus altitudinis treatment was 78% on Agaricus bisporus. The optimal culture medium was determined as follows: 3% Soluble startch, 10% Soytone, 1% (NH4)2HPO4, 1 mmol KCl, and 0.5% L-asparagin at pH 6.0 at 30°C. Control efficacy of browning disease of Bacillus subtilis treatment was 71% on Agaricus bisporus. The optimal culture medium was determined as follows: 1.5% Xylose, 2% Soytone, 1% NH4H2PO4, 7 mmol CaCl2, and 0.5% Histidine at pH 6.0 at 25°C. Accordingly, the suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by C. mycophilum.

Microsatellite SSR markers were developed and utilized to reveal the genetic diversity of 32 strains of Flammulina velutipes collected in Korea, China, and Japan. From SSR-enriched library, 490 white colonies were randomly selected and sequenced. In the 490 sequenced clones, 85 clones (17.35%) were redundant. Among the remaining 405 unique clones, 201 clones (49.6%) contained microsatellite sequences. As a result, 12 primer pairs produced reproducible polymorphic bands within diverse 4 strains and these selected markers were further characterized in 32 Flammulina velutipes strains. A total of 34 alleles were detected using the 12 markers, with an average of 3.42 alleles and the number of alleles ranged from two to seven per locus. The major allele frequency ranged from 0.42 (GB-FV-127) to 0.98 (GB-FV-166), and values for observed (HO) and expected (HE) heterozygosity ranged from 0.00 to 0.94 (mean = 0.18) and from 0.03 to 0.67 (mean = 0.32), respectively. SSR loci amplified with GB-FV-127 markers gave the highest polymorphism information content (PIC) of 0.61 and mean allele number of five, while for loci amplified with GB-FV-166 markers these values were the lowest, namely 0.03 and two. The mean PIC value (0.29) observed in the present study with average number of alleles (3.42). The genetic relationships among 32 Flammulina velutipes strains based on SSR data were generated by UPGMA cluster analysis. In conclusion, we succeeded in developing 12 polymorphic SSRs markers from SSR-enriched library of Flammulina velutipes. These SSRs are presently being used for phylogenic analysis and evaluation of genetic variations. In future, these SSR markers will be used in clarifying taxonomic relationships among the Flammulina velutipes.

The button mushroom, Agaricus bisporus, is one of the major economical crops cultivated in Korea. This mushroom showed the 5th production to 11,493 M/T in 2014. Several fungus are known as the causal agents of diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). Trichoderma harzianum is the causal agent of green mould disease of commercial mushrooms. Early symptoms were noticed as round, fleshy, brown lesions on mushroom caps. Late symptoms spread on the surface of the casing, and covered entirely fruiting bodies. A Gram-positive bacterium was isolated from mushroom media that markedly showed the antagonistic activity against Trichoderma harzianum, the destructive pathogen of cultivated mushrooms. The CH518 strain was selected as antagonistic bacterium by inhibition zone method and it was identified as Bacillus methylotrophicus by the cultural, morphological and physiological characteristics, and analysis of the 16S rDNA. The isolated bacterium is saprophytic but not parasitic nor pathogenic to cultivation mushroom. The isolated bacterium for Trichoderma harzianum cell, was sufficient for inhibition in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease in Trichoderma harzianum. Control efficacy of browning disease of strain CH518 treatment was 77.7% on Agaricus bisporus. The optimal culture medium for the antagonistic bacteria growth was determined as follows: 3.0% saccharose, 1.5% Soytone, 1% NH4H2PO4, 10 mM MgSO4, and 2.0% glutamic acid at pH 6.0 at 25°C. The suppressive bacterium may be useful in future for the development of biocontrol system and the construction of genetically modified edible fungi resistant to the disease caused by Trichoderma harzianum.

Bovine somatic cell nuclear transfer (bSCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization (IVF). However, the efficiency of somatic cell cloning has remained low, and applications have been limited, irrespective of the nuclear donor species or cell types. One possible explanation is that the reprogramming factors of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we would like to introduce the aggregation method (agSCNT), a new experimental system that enables and increase oocyte volume and examined its subsequent development. Judgement by the blastocyst formation rate or total cell number was significantly higher in the agSCNT group than that in the SCNT group, and was very similar to that in the control IVF group. Moreover, the cleavage formation rate in the agSCNT group (61.5 ± 1.3) was higher than that in the SCNT group (39.7 ± 2.1), while still less than that in the IVF group (75.4 ± 1.3). We also analyzed the epigenetic modifications in bovine IVF, agSCNT, and untreated SCNT embryos. In conclusion, the present study demonstrated that agSCNT improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell numbers (TC).

This study investigated the effect of Charcoal:Dextran Stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P<0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% vs. 36.85 ± 0.89%, respectively). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P<0.05) higher in the CDS FBS than in the HI FBS group (85.33 ± 4.84% vs. 68.67 ± 1.20%). Quantitative real-time PCR showed that the mRNA levels of acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, hydroxymethylglutaryl-CoA reductase, and insulin-like growth factor 2 receptor were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of sirtuin 1, superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen-thawed blastocysts were significantly (P<0.05) higher in the CDS FBS group than in the HI FBS group, however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. Taken together, these data suggest that supplementation of medium with CDS FBS improves in vitro bovine embryo developmental competence and cryo-tolerance.

The production of feline induced pluripotent stem cells (iPSCs) can solve the problems that are related with existing unstable supply and demand of eggs as well as ethical aspects about embryonic stem cell at the same time. On the basis of excellent proliferation, it is to facilitate the researches about human disease like FIV and Allergen at the level of cells, not experimental animals. But, a lot of advanced researches are lean too much towards on the transduction using DNA type virus that have the risk of tumorigenesis during reprogramming and on the mLIF-dependent culture condition for the production of feline iPSCs. This being so, this study shows the reprogramming results using Sendai virus vector that is RNA type virus and have no the footprint after transduction. In addition, the feline iPSCs were stably cultured in bFGF-dependent culture condition during the reprogramming step and culture step. In conclusion, we found the bFGF-dependent culture condition in feline iPSCs and suggested the approach using Sendai virus vector as an alternative for reprogramming without concern about tumorigenesis. These methods can be universally applicable to not only the researches about reconstruction and conservation of feline species, but also to a lot of deep studies related with iPSCs or LIF, bFGF to find new approaches.

The production scale of mushrooms in Korea is approximately 600 billion won, which is 1.6% of Korea’s gross agricultural output. In Korea, ca. 190,000 tons of mushrooms are harvested annually. Although the numbers of mushroom farms and cultivators are constantly decreasing, total mushroom yields are increasing owing to large-scale cultivation facilities and automation. The recent expansion of the well-being trend has caused an increase in mushroom consumption in Korea: the annual per capita mushroom was 3.9 kg (’13), whichis a little higher than that in Europe. Thus, mushroom export, mainly Flammulina velutipes and Pleurotus ostreatus, has increased since the mid-2000s. Recently, however, it is slightly reduced. Nevertheless, Vietnam, Hong Kong, the United States, and the Netherlands continue to export mushrooms, and Korea has increased its export to Australia, Canada, Southeast Asia, etc. Canned Agaricus bisporus, the first export of the Korean mushroom industry, reached it speak sales in 1977-1978. When Korea initiated trade with China in 1980, the international prices of mushrooms fell sharply, leading to shrinkage of the domestic markets. Spurred by the high demand to develop substitute goods for A. bisporus, the oyster mushroom (P. ostreatus) gained attention since it seemed to suit the taste of Korean consumers. Although the log cultivation technique for oyster mushroom was developed in the early 1970s, it required a great deal of labor. Thus, we developed the shelf cultivation technique, which is easier to manage and allows for mass production. In this technique, the growing shelf is made mafrom fermented rice straw, whichis the only P. ostreatus medium in the world and isused only in South Korea. After then, the use of cotton wastes as an additional material of medium, the productivity. Currently, we are developing a standard cultivation technique and environmental control system that can stably produce mushrooms throughout the year. The increase of oyster mushroom production may boostthe domestic market and contribute to industrial development. In addition, oyster mushroom production technology played a role in forming the basis for the development of bottle cultivation, which made mass production . In particular, bottle cultivation using liquid spawn could allow for the export of F. velutipes and Pleurotus eryngii. In addition, the white varieties of F. velutipes were second developed in the world after Japan. We also developed the new A. bisporus cultivar ‘Saeah’, which is easy to grow in Korea. In hopes to advance the mushroom industry, we will continue to develop cultivars with international competitive power and to improve cultivation techniques.