RNA Sendai virus (SeV) vector system has no risk of being integrated into the host genome. Sendai virus (SeV) vectors expressing pluripotent factors have been used to produce integration-free induced pluripotent stem cells (iPSCs) with high efficiency from various cell types in human and mouse. In this study, we generated iPSCs from pig ear fibroblast cells using the SeV vector expressing 4 human factors (POU5F1, SOX2, C-MYC, and KLF4). Colonies were emerged at Day 14 of transduction and expressed the classical pluripotency markers (POU5F1, NANOG, and SOX2) and surface marker (SSEA1). Furthermore, they showed a domed shape and could passage over 40 times under 2i (CHIR99021 and PD0325901)-LIF and MEF feeder culture condition having in vitro differentiation ability into 3 germ layers. Next, we examined the ability of six feeder free culture conditions to maintain piPSCs in a pluripotent state. piPSCs were plated on Matrigel coated dishes in different media: 1. CM: control media (LIF culture media); 2. CM-F: CM+100 ng Fetuin-A; 3. CM-N: CM+100 ng Nanog-TAT; 4. CM-2i: CM+3 uM CHIR99021+1 uM PD0325901; 5. CM-2iN: CM-2i+100 ng Nanog-TAT; 6. CM-2iN+100 ng Fetuin-A. However, piPSC could not maintain the typical self-renewal morphology on feeder free conditions regardless of culture media tested here. Further, expression of pluripotency-related genes (Oct4, Nanog and Klf4) of piPSCs cultured on feeder free conditions could not be compared with that of iPSCs cultured on MEF feeder plate. Our results suggest that integration free pluripotent stem cell from pigs could be generated by SeV vector system and maintained their pluripotency under 2i-LIF and MEF feeder culture condition, but further optimization of culture conditions may be required.

Carbon source, an essential nutrient for plant growth, mainly includes exogenous sugar and CO2 of the environment in vitro. Therefore, the exogenous sugar and CO2 of the environment make the important roles in tissue culture. The aim of this study is to investigate the effects of different sugar concentrations (0, 10, 15 and 30 g·L-1) on the growth of colored Zantedeschia in vitro under certain CO2 concentration and explore the optimal sugar concentration. The plantlets in vitro of colored Zantedeschia had the largest root number, root weight, and root vigor under 0 g·L-1 (sugar-free culture) treatment. And they had the largest plant height, leaf length and leaf chlorophyll content, but p oor r oot v igor under 3 0 g·L-1 sugar. This study indicated that the optimal condition for proliferation and seedling culture of colored Zantedeschia plantlets in vitro was MS medium with 30 g·L-1 sugar, and the suitable medium for rooting culture and transplanting of colored Zantedeschia was MS medium with sugar-free culture under CO2 enrichment condition.

This study was carried out to determine the basic mycelial culture conditions for Poria cocos growth. According to colony diameter and mycelial density, suitable media for mycelial growth were Malt yeast extract, Potato dextrose agar, Yeast extract agar, and Yeast malt agar. The optimum temperature for mycelial growth was between 25 and 35oC, and the optimum pH value was between 4 and 7. Carbon and nitrogen sources were fructose and yeast extract. The optimum C/N ratio was about 10 to 1 with 2% glucose. Other minor components for optimal growth were thiamine-HCl and nicotinamide as vitamins, acetic and lactic acid as organic acids, and MgSO4·7H2O and FeSO4·7H2O as mineral salts.

Ochratoxin A와 aflatoxin은 곰팡이 독소 생산 균주가 자연 발효되는 식품에 오염되거나 발표식품의 종균을 분별없이 선택했을 때 검출 될 수 있다. 본 연구에서는 다양한 배양 환경이 ochratoxin A와 aflatoxin의 생산에 주는 영향에 대해 연구하였다. Aspergillus usamii KFRI 999와 A. awamori KFRI 983의 배양 온도, 배양 배지, 배양 시간을 달리하여 ochratoxin A생산 정도를 분석하였다. 또한 초기 pH, 온도, 배양 시간, 배양 배지를 다르게 하였을 때 A. flavus KACC 41403와 A. oryzae KACC 46471가 생산하는 aflatoxin의 양을 평가하였다. Ochratoxin A와 aflatoxin의 양은 HPLC로 분석하였다. 연구 결과, 곰팡이 독소는 배양 온도에 큰 영향을 받는 것으로 나타났다. A. oryzae KACC 46471는 aflatoxin을 생산하지 않았다. 곰팡이 독소를 생산하는 균주는 모두 30℃에서 가장 높은 독소 생산량을 보였다. A. awamori KFRI 983은 PDA 배지에서 가장 적은 ochratoxin A 생산량을 보였다. 본 연구 결과는 다양한 식품의 ochratoxin A와 aflatoxin 저감화에 유용하게 활용 될 수 있다.

Despite that porcine spermatogonial stem cells (pSSCs) have been regarded as a practical tool for preserving eternally genetic backgrounds derived from pigs with high performance in the economic traits or phenotypes of specific human diseases, there were no reports about precise definition of niche conditions promoting proliferation and maintenance of pSSCs. Accordingly, we tried to determine niche conditions supporting proliferation and maintenance of undifferentiated pSSCs for short-term. For these, undifferentiated pSSCs were progressively cultured in different composition of culture medium, seeding density of pSSCs, type of feeder cells and concentration of growth factors, and then total number of and alkaline phosphatase (AP) activity of pSSCs were investigated at post-6 day culture. As the results, the culture of 4x105 pSSCs on mitotically in activated 2x105 STO cells in the mouse embryonic stem cell culture medium (mESCCM) supplemented with 30 ng/ml glial cell line-derived neurotrophic factor (GDNF) was identified as the best niche condition supporting effectively the short-term maintenance of undifferentiated pSSCs. Moreover, the optimized short-term culture system will be a basis for developing long-term culture system of pSSCs in the following researches.

Auricularia auricula-judae is an edible mushroom, which is known as wood ear, free ear, black ear mushroom, and free jelly fish. This study was carried out to obtain the basic information for mycelial culture conditions of Auricularia auriculajudae. According to colony diameter and mycelial density, the media for suitable mycelial growth were PDA and MCM. The optimum temperature for mycelial growth was 25~30oC. Carbon and nitrogen sources were mannose and malt extract, respectively. The optimum C/N ratio was in the range of 10 to 1 with 2% glucose. Other minor components for the optimal growth were thiamine-HCl and biotin as vitamins, succinic acid and lactic acid as organic acids, and MgSO4·7H2O and KH2PO4 as mineral salts.

Polyporus umbellatus (Syn. Grifola umbellata) is a sclerotium forming mushroom belongs to family Poly-poraceae of Polyphorales, Basidiomycota. The sclerotia of P. umbellatus have long been used for traditional medicinesin China, Korea and Japan. This study was initiated to obtain the basic data for artificial sclerotial production of P. umbel-latus. Here, we investigated the favorable conditions for mycelial growth of P. umbellatus and its symbiotic fungus Armill-aria mellea. We also evaluate the favorable carbon and nitrogen sources for sclerotial formation in dual culture betweenP. umbellatus and A. mellea. The favorable conditions for mycelial growth of P. umbellatus were 20°C and pH 4, whileoptimal conditions for mycelial growth of A. mellea were 25°C and pH 6. The carbon sources for optimal mycelial growthof P. umbellatus were fructose and glucose, while carbon sources for favorable mycelial growth of A. mellea were alsofructose and glucose. The nitrogen sources for favorable mycelial growth P. umbellatus were peptone and yeast extract,while optimal mycelial growth of A. mellea were obtained in peptone and yeast extract. When P. umbellatus and A. melleawere dual cultured on carbon sources, sclerotia were induced on basal media supplemented with glucose, fructose andmaltose at pH 4~6, while nitrogen sources inducing sclerotia were basal media supplemented with peptone and yeastextract for 60 days at 20°C under dark condition.

1995년이후 부터 재배되어온 큰느타리버섯은 배양중 오염율 증가, 발이상태불량, 기형버섯의 발생, 수량 격감 등 이른바 연작장해로 불리어지는 재배상 문제점들에 대한 원인파악과 해결책이 요구되어, 발이상태 불량과 기형버섯 발생원인이 병원균과 발이 및 생육환경 관리방법에 있을 것으로 판단하고 병원균 종류 및 접종시기와 발이유기시 환기량에 따른 기형버섯 발생양상과 발이특징를 조사한 결과는 다음과 같다. 병원균 종류 및 접종시기별 배양율은 병원균 접종시기를 버섯종균접종과 동시에 접종할 때 44~63%로 낮았고, 병원균 종류별로는 Erwinia sp.+Peudomonas sp. 혼합 처리구에서 낮았다. 초발이소요일수는 발이유기시 환기를 충분히 시켰을 때(1,000±250ppm) 병원균을 종균접종시 접종한 처리구에서 12~15일이 소요되었고, 나머지 다른 처리구에서는 10일로 동일하였으나, 환기량이 충분하지 않을 경우(2,000±250ppm)모든 처리구에서 12~14일로 지연되는 경향이었다. 발이율은 발이유기시 환기량이 충분할 때, 종균접종시 병원균이 접종된 처리구에서 정상발이율이 29~56%로 낮았고, 나머지 처리구에서는 77~86%로 병원균 무접종 처리구 89%와 큰 차이가 없었으며, 환기량이 부족할 경우 병원균종류와 시기에 관계없이 병원균 무처리구를 포함한 모든 처리구에서 32~57%정도로 낮았다. 수량은 환기량이 충분할 때 병원균 무접종 처리구에서 83.8g/병으로 높았고, 종균접종시 처리구에서 10.9~28.2g으로 낮았으며, 병원균접종시기가 배양완료10일전, 발이유기시 처리구에서는 68.1~79.6g/병으로 병원균 무접종 처리구와 비슷하였다.

Osmolarity of culture media is one of the most important factors affecting in vitro development. This study was conducted to investigate the DNA methylation status of Pre-1 and satellite sequence in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaCl or 0.05M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. Previous our studies have reported that pNT embryos cultured in both hypertonic media showed significantly higher blastocyst formation rate than that of control. The DNA methylation status of the satellite sequences in blastocyst was characterized using bisulfite-sequencing technology. The satellite region had a similar methylation pattern of in vivo blastocyst among two culture groups excepting the control group. Each level of methylation is that the satellite DNA moderately methylated (43.10% of PZM-3; 56.12% of NaCl; 55.06% of sucrose; 60.00% of in vivo embryos). As a result of the sequence of PRE-1, CpG methylation pattern was similar to three groups, including in vivo group. In case of the satellite DNA region, the osmolarity conditions were affected CpG DNA methylation status while PRE-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. These results indicate that the modification of osmolarity in a culture media may influence to spatially change of DNA methylation of repetitive sequence for pNT embryo development.

본 연구에서는 우단일엽(Pyrrosia linearifolia)의 전 엽체 및 포자체의 생활환을 이용한 주년 대량번식체계의 기초를 제공하기 위하여 전엽체 증식 및 포자체를 이용한 식물체 재생에 미치는 기내 환경요소를 구명하였다. 전엽 체 증식은 2MS배지에서 가장 왕성하였다. 질소급원 또한 첨가량이 많아질수록 증식율과 생육이 우수하였으며, 120mM 첨가구에서 생육이 가장 왕성하였다. 전엽체 증 식에 가장 적합한 sucrose의 첨가농도는 3%였다. IAA, NAA, BA 및 kinetin을 5~20 μM정도 첨가하는 것은 전엽체의 생육을 촉진하였으나, 2iP는 전엽체의 생육을 억제하였다. 액체배지에서는 전엽체의 생육이 억제되었 으나, 진탕배양했을 때에는 고체배지와 증식량이 유사하 였으며, 생식기관 또한 정상적으로 형성되었다. 포자체의 엽신과 근경의 절편을 다져서 배양한 결과, 근경 절편 에서만 포자체가 재생되었다. 곱게 다진 근경의 절편은 1/8MS배지에서 포자체 재생이 가장 왕성하였으나, 재 생된 포자체의 생육은 1/2MS배지에서 가장 우수하였다. 1/8MS배지에서 포자체 재생 및 생육을 촉진시킬 수 있는 적정 sucrose의 첨가 농도는 1%이며, NaH2PO4를 50~ 100mg·L−1 첨가하는 것은 균질배양한 근경의 절편에서 포자체가 형성되는 것을 촉진하였다.

파김치에서 분리한 마늘 내성 유산균인 Lactobacillus plantarum TJ-LP-002 균주의 균체생육과 항균활성에 영향을 미치는 배양조건 및 배지조건을 조사하였다. 선정 유산균의 배양 상등액 내에는 acetic acid, citric acid, lactic acid, tartaric acid와 같은 유기산이 존재하였고, 배양 중에 lactic acid와 acetic acid의 생성이 크게 증가하는 것으로 확인되었다. 단백질분해효소를 비롯한 각종 효소 처리에 의해 항균활성이 소실되지 않아, 선정 유산균이 생산하는 항균활성은 단백질성 물질이 아닌 산 생성에 의한 작용일 것으로 판단되었다. 항생제와 생균제의 병용 가능성을 확인하기 위하여 선정 유산균의 항생제 감수성을 조사한 결과, neomycin sulfate, spectinomycin dihydrochloride, lincomycin hydrochloride에 내성을 나타내었고, streptomycin sulfate에는 감수성을 나타내었으며, ampicillin trihydrate, chloramphenicol, erythromycin, tetracycline hydrochloride, kanamycin sulfate에는 중간 내성을 나타내었다. L. plantarum TJ-LP-002는 배양온도 30oC, 초기 pH 7.0, 24시간의 배양 조건에서 최적의 균체생육과 항균활성을 나타내었으며, 탄소원은 glucose 3%(w/v), 질소원은 yeast extract 3%(w/v) 첨가 시에 균체생육과 항균활성이 높게 나타났다. 무기염류는 manganese sulfate와 ammonium citrate가 항균활성에 많은 영향을 주는 것으로 나타났으며, 각 성분을 단독 첨가하는 것보다 혼합 첨가하는 것이 더 우수한 영향을 나타내는 것으로 확인되었다.

The objective of this study was to determine effects of different culture media. Preantral follicles were mechanically extracted from bovine ovaries and cultured for 16 days in tissue culture medium (TCM)‐199, DMEM or alphaminimal essential medium (α‐MEM) + 10% FBS + 0.1 mg/ml sodium pyruvate + 100 mIU/ml FSH. The collected primary follicles from ovary were higher than the primary and secondary follicles. The survival rates of the follicles in TCM‐199 were significantly higher (p<0.05) than those in DMEM and α‐MEM. The diameter of the follicles progressively increased during 12 days of culture. The maximum size (139.1±5.4 μm) reached on Day 12 of the in vitro culture and decreased on Day 16. These results suggest that in a culture of bovine preantral follicles, TCM‐199 is an optimal medium and a longer‐term culture of preantral follicles (>12 days) may be needed to form antra.

This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture (, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.

새로운 항균물질의 탐색이 활발하다. 선행연구에서 새로운 항균활성물질을 생산하는 세균이 한국토양에서 분리되어 Paenibacillus polymyxa DY1으로 동정 및 명명되었으며, 다제내성 장내세균들에 대한 항균활성 특성이 규명되어 새로운 항생물질로서 잠재력을 보여주었다. 본 연구에서 배지의 탄소원, 무기질소원, 유기질소원, 아미노산, 무기염류 등의 영양 조성물과 물리화학적 생장조건이 P. polymyxa DY1 균체의 생장과 항균활성 생산에 미치는

호접란 조직배양시 발생하는 체세포변이의 발생을 줄 이기 위한 적절한 다신초 유기배양 조건과 계대배양 조 건에 관한 연구를 실시하였다. 다신초 유기 및 증식의 적 정 배지로서는 1/2 MS 배지가 VW 배지와 Hyponex 배 지보다 더 효율적이었다. 다신초 유기에 적절한 생장조 절제 농도는 BA 5mg · L-1와 TDZ 1mg · L-1가 적절하였 다. 계대 배양의 횟수가 증가할수록 기형 신초수의 발생 이 증가하였다. 그러나 2개월마다 3회 계대배양할 때까 지는 기형 신초가 거의 발생하지 않았다. 특히 핑크계 호 접란 품종에서 6회 이상 계대할 경우 기형 신초 발생율 이 30%에 육박하였고 배지내 페놀 물질의 집적이 더 많 이 관찰되었다. 식물체를 배지에 치상한 후 2개월이 지 나 처음으로 계대 배양하였을 때 모든 품종에서 기형 신 초 발생이 거의 없었지만, 이후 계대배양 기간이 증가함 에 따라 기형 신초 발생율과 고사율이 증가하였다. 그러 므로 적어도 2개월에 한 번씩 계대 배양하는 것이 바람 직한 것으로 판단되었다.

Cyclin B1 is known to reflect the M-phase promoting factor (MPF), a universal regulator of G2/M-phase transition, activity during the process of oocytes maturation. To investigate whether culture condition affects the maturation rate and the expression of cyclin B1 protein, bovine immature oocytes are stimulated and cultured according to the following protocols: Experiment 1: denuded oocytes (denude) only, COC only, denuded oocytes + granulosa cells (denude + GCs) and COC + GCs; Experiment 2: no-activation (control), 7% ethanol for 5 min and 10 l/ml ionomycin for 5 min at immediately before maturation. The maturation rates of denude and no-activation group were significantly lower in both experiments (P<0.05), respectively. Co-culture or stimulation method in bovine immature oocytes culture increases the cyclin B1 expression significantly in both experiments (P<0.05). Based on these results, culture condition affects the maturation rate and the expression of cyclin B1 protein during the first meiotic maturation in bovine immature oocytes.