• KSBB Journal
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• v.13 no.3
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• pp.231-237
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• 1998

An attenuated Japanese encephalitis virus (JEV) clone SA-14-14-2(Vero) was obtained through successive adaptation of a primary cell adapted strain, SA-14-14-2(PDK) to Vero cell, a continuous line of monkey kidney cells. The virus titer reached above the 107 plaque forming unit (pfu) per mL of culture supernatant with 3 passages in Vero cells and was maintained close to this level in the further passages. The optimum temperature for the virus growth was $35^{\circ}C$. Growth pattern of the virus indicated that optimum time for the virus harvest is 4 days post infection and the virus accomplished rapid initial propagation even in medium containing no serum supplement. The roller bottle (RB) system and the spinner flask (SF) system using micro-carrier (Cytodex-1) for the JEV cultivation were explored. When RB, SF, and T-flask culture system were compared, there was no significant difference in virus yield. Furthermore, the results indicated that virus could be harvested multiple times from 3 days to 9 days post infection; neither severe cytopathic effect (CPE) in the infected cells nor the decrease in the titer was observed on duration of 9 days.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• Korean Journal of Plant Resources
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• v.22 no.4
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• pp.337-342
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• 2009

Present studies were conducted to evaluate the effects of medium strength(MS and Hyponex), carbon sources and their concentrations, agar concentrations, and inoculation amounts on prothallus propagation of Pterdium aquilinum var. latiusculum(Desv.) Underw. ex Hell in vitro. The optimum MS medium strength for prothallus propagation was 2MS concentration. Phosphate source was most effective for prothallus growth of P. aquilinum var. latisculum. The addition of 1% sucrose or glucose to MS medium promoted prothallus multiplication. Growth of prothallus was not affected by agar concentration. Propagation of homogenized prothallus was vigorous even in liquid medium. Chopped gametophytes(100 and 200 mg) were inoculated on 250 ml ${\Delta}$flask with 100 mL of 2MS concentration medium and suspension culture was done at 100 rpm for 22 days. After 20 days, prothallus multiplication slowed down, so 100 mg of chopped prothalli is recommended for initial inoculation, since initial amount of inoculum did not affect subsequent prothallus multiplication. Consequently after 20 days of suspension culture, prothallus should be subcultured or transplanted outside of growing vessels.

• Journal of Sericultural and Entomological Science
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• v.53 no.1
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• pp.55-60
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• 2015

Melittin is the main component of Bee Venom and has antibacterial activity against several bacteria. To produce the melittin antimicrobial peptide, we constructed transgenic silkworm that expressed melittin gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 300 eggs of bivoltin silkworms, Baegokjam. In total, 131 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 36 broods, and we selected 4 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 12 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a melittin as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that melittin possesses high antibacterial activities against gramnegative bacteria.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.22-22
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• 2018

실고사리[Lygodium japonicum (Thunb.) Sw.]는 국내 자생하는 덩굴성 양치식물로 유인재배가 용이하여 실내 외 조경 및 관상소재로 활용이 가능하다. 한방에서는 전초를 해금사초, 포자를 해금사, 뿌리와 지하경을 해금사근이라 약재로 사용한다. 본 연구는 관상 및 약재로 이용이 가능한 실고사리의 대량생산을 위한 기내 외 번식방법을 개발하고자 수행되었다. 식물재료는 경상북도 의성군 일대에서 성체를 수집하여 청주의 일반하우스에 식재하여 성숙한 포자를 채취하였다. 포자를 기내 발아시켜 전엽체를 획득한 다음 8주 간격으로 계대하면서 연구의 재료를 확보하였다. 전엽체의 증식과 생육에 적합한 배지를 비교 하고자, 1/4, 1/2, 1, 2MS배지와 Knop배지를 조성하여 배양하였다. 배양방법은 전엽체 300mg을 메스로 균일하게 다져서 배양하는 방법을 이용하였으며, 배양환경은 온도($25{\pm}1.0^{\circ}C$), 광도($30{\pm}1.0{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$), 광주기(16/8h light/dark)로 조절되었다. 연구결과, 모든 처리구의 생체중은 초기접종량 보다 11배 이상 증가하였고, 그 중 1MS배지에서는 전엽체의 생체중이 7.3g으로 가장 많이 증가하였다. 뿐만 아니라, 형태형성도 우수하여 모두 정상적인 전엽체의 형태인 하트형으로 발달이 유도되었다. 전엽체로부터 포자체 형성을 유도하고자, 원예상토, 피트모스, 펄라이트 및 마사토의 혼합비율을 5종류로 달리하여 사각분($7.5{\times}7.5{\times}7.5cm$)에 혼합토양을 충진하였다. 전엽체 1g과 증류수 25mL를 핸드블랜더로 10초간 분쇄하여 사각분의 토양표면에 균일하게 분주하는 방법을 사용하였다. 이후 온도($25{\pm}1.0^{\circ}C$), 광도($43{\pm}2.0{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$), 광주기(16/8h light/dark), 습도($72{\pm}2.0%$)를 유지하면서 10주간 재배되었다. 연구결과, 원예상토 단용, 원예상토와 펄라이트 및 마사토가 2:1(v:v)로 혼합된 토양에서 각 498.0, 402.5, 482.5개의 포자체가 형성되어 사각분 면적대비 7.16개($cm^2$)가 생산되었다. 한편 포자체의 생육은 원예상토와 펄라이트가 2:1(v:v)로 혼합된 토양에서 생체중, 엽장, 엽폭, 근장 및 SPAD value 등이 우수하였다.

• Korean Journal of Plant Tissue Culture
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• v.24 no.3
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• pp.183-188
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• 1997

Callus from scale segments of Lilium longiflorum 'Gelia' was effectively induced and maintained from unorganized tissue on the semi-solid medium by 0.42% Bacto agar with MS basal salts and vitamins of SH medium supplemented with 0.5 mg/L 2, 4-D, 1.0 mg/L NAA, 0.3 mg/L BA, and 3% sucrose. More than 5% of high sucrose level had inhibiting effect on regeneration capacity of formed callus and decreased callus growth. Various combinations of nitrogen did not effective to proliferate the ELC (Embryogenic-like callus), but friability of callus was increased in the medium containing only nitrate as nitrogen source. 5 mL conditioned medium into 30 mL fresh medium was good for cell growth. However friable cell aggregates during suspension culture had to form hard callus which hindered to establish suspention culture system. Addition of 2 g/L casein hydrolysate increased callus growth and friability of the hard callus. As a result of anatomical observation of callus, organogenesis such as shoots, roots and bulblets was independently induced from callus tissue. Somatic embryogenesis from callus tissue could be observed with low frequency.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.57-57
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• 2019

본 연구는 관상 및 조경용으로 개발이 가능한 남방계 양치식물인 점고사리[Hypolepis punctata (Thunb.) Mett.]의 전엽체 증식 및 포자체 형성에 적합한 배양조건을 구명하고자 수행되었다. 무가온 온실에서 성숙한 포자엽을 채집한 후 포자를 기내에서 발아시켜 전엽체를 획득하였으며, 8주 간격으로 계대배양 하여 실험의 재료로 사용하였다. 배지종류에 따른 전엽체의 증식 및 형태형성을 확인하고자, 배양된 전엽체 0.3g을 메스로 잘게 다진 후, 농도를 1/4, 1/2, 1, 2배로 조절한 MS배지에 8주간 배양하였다. 이후 선발된 배지를 기준으로 sucrose, 활성탄, 질소급원의 농도를 조절하여 전엽체의 증식과 형태형성을 확인하였다. 그 결과, 1MS배지에서 전엽체의 생체중이 초기 접종량인 0.3g에 비해 10.7배 증가한 3.2g으로 가장 높은 증가율을 보였다. 형태관찰에서도 장정기의 형성이 관찰되었으며, 전엽체의 쿠션조직이 비교적 잘 발달하였다. 전엽체 증식에 가장 좋은 효과를 보인 1MS배지를 기준으로 sucrose의 농도를 0-4%로 달리하여 실험한 결과, 1%의 처리구에서 6.7g으로 가장 높은 생체중을 보였다. 활성탄의 농도를 0-0.8%로 첨가한 네 처리구 중에서는 0.8%의 처리구에서 14.2g으로 무처리구에 비해 생체중이 2배 이상 증가하였다. 전엽체의 형태 또한 정상적인 발달을 보였다. 질소급원의 비율을 30-120mM로 조절한 배지에서는 60mM의 처리구에서 4.9g으로 가장 높은 생체중을보였다. 이후 포자체 형성을 위한 최적의 토양조건을 구명하고자, 원예상토, 피트모스, 펄라이트 및 마사토의 비율을 달리하여 5종류의 배양토를 조성하였다. 혼합된 토양은 사각분($7.5{\times}7.5{\times}7.5cm$)에 충진 하였으며, 배양된 전엽체 1g을 증류수와 함께 10초간 분쇄한 다음 준비된 토양표면에 분주하여 재배하였다. 12주간의 재배 결과, 원예상토를 단용한 토양에서 포자체의 수가 포트당 250.0개로 가장 많이 형성되었으며, 포자체의 생육 또한 다른 처리구에 비해 우수한 결과를 보였다. 한편 피트모스가 혼합된 토양에서는 포자체가 형성되지 않았다. 따라서 점고사리의 전엽체 대량증식에 적합한 배지는 sucrose 1%와 질소급원의 농도를 60mM, 활성탄을 0.8% 첨가한 1MS배지로 판단되며, 포자체 대량생산을 위해서는 원예상토를 단용한 토양이 적합하다고 판단된다.

• Research in Plant Disease
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• v.24 no.4
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• pp.328-331
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• 2018

A severe disease with leaf spots and necrotic symptoms was observed in cucumber (Cucumis sativus L.) seedlings in April 2018 at a nursery in Kimjae, Korea (35o 47'09.8"N 127o 2'24.3"E). The infected plants initially showed spots on water-soaked cotyledons which, at later stages, enlarged and spread to the leaves, which the lesions becoming dry and chlorotic. The symptomatic samples were collected from cucumber and the isolates were cultured on LB agar. The representative bacterial strain selected for identification showed fluorescent on King's medium B, was potato rot-positive, levan and arginine dihydrolase-negative, oxidase-negative and tobacco hypersensitivity-positive in LOPAT group 2 as determined by LOPAT tests. A pathogenicity test was carried out on a 3-week-old cucumber. After 3 days of inoculation, leaf spots and necrotic symptoms appeared on the cucumber, similar to the originally infected plants. The infecting bacterial strain was identified as Pseudomonas viridiflava, by 16S rDNA sequence analysis. This is the first report of leaf spot diseases on cucumber caused by P. viridiflava.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.56-56
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• 2019

본 연구는 식용과 약용으로 이용되는 국내 자생식물인 야산고비(Onoclea sensibilis var. interrupta Maxim.)를 기내에서 대량증식하기 위한 조건을 구명하기 위하여 수행되었다. 무가온 온실에서 수집한 포자를 기내에서 발아시켜 전엽체를 획득하였으며, 계대배양하며 실험의 재료로 사용하였다. 전엽체의 대량증식을 위해 전엽체 0.3g을 메스로 균일하게 다진 후 증류수 1ml와 함께 배지에 고루 펼쳐서 배양하는 방법을 사용하였으며, 증식에 미치는 배지의 영향을 확인하기 위하여 1/4, 1/2, 1, 2배로 조절한 MS배지를 조성하여 8주간 배양하였다. 이후, 증식이 우수한 배지를 선정하여 sucrose와 질소급원의 농도를 조절하였으며, 활성탄을 첨가하여 증식에 미치는 영향을 확인하였다. 배지종류 실험의 결과, 생체중이 1MS에서 10.2g으로 초기 접종량에 비해 가장 많이 증가하였으며, 현미경을 이용하여 관찰한 결과에서도 정상적인 전엽체의 형태인 심장형으로 발달하였다. 증식이 우수하였던 1MS배지에 sucrose의 농도를 조절하여 배양한 결과에서는 1%의 처리구에서 가장 증식률이 좋았으며, 질소급원의 경우 30mM의 농도로 조절한 처리구가 가장 좋은 결과를 보였다. 배지 내 활성탄의 첨가는 처리구당 증가된 전엽체의 생체중이 유의적인 차이를 보이지 않았다. 포자체 대량 형성을 위한 적정 배양토의 혼합조건을 확인하기 위하여 원예상토, 피트모스, 펄라이트 및 마사토의 혼합비율을 5종류로 달리하여 조성하여 사각분($7.5{\times}7.5{\times}7.5cm$)에 충진하였다. 조성한 배양토에 기내에서 배양한 전엽체 1g을 증류수와 함께 블렌더를 이용하여 10초간 분쇄하여 토양표면에 고루 분주하였다. 이후 12주간 재배한 결과, 모든 토양조합에서 포자체가 형성되었다. 그중 원예상토와 마사토를 2:1(v:v)로 혼합한 토양에서 포트 당 405.0개로 가장 많은 포자체가 형성되었으며, 전체적인 생육 또한 비교적 양호한 결과를 보였다. 따라서 야산 고비의 전엽체 대량증식에 적합한 배지는 경제성과 생육수준을 고려하여 1%의 sucrose와 질소급원의 농도를 30mM로 조절한 1MS 배지가 적합하며, 포자체 대량생산을 위해서는 원예상토, 마사토를 2:1(v:v)로 혼합한 토양이 적합하다고 판단되었다.

• KSBB Journal
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• v.22 no.5
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• pp.297-304
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• 2007

For large and rapid screening of high-yielding mutants of lovastatin produced by filamentous fungal cells of Aspergillus terreus, one of the most important stage is to test as large amounts of mutated strains as possible. For this purpose, we intended to develop a miniaturized cultivation method using $7m{\ell}$ culture tube instead of traditional $250m{\ell}$ flask (working volume $50m{\ell}$). For obtaining large amounts of conidiospores to be used as inoculums for miniaturized cultures, 4 components i.e., glucose, sucrose, yeast extract and $KH_2PO_4$ were intensively investigated, which had been observed to show positive effect on enhancement of spore production through Plackett-Burman design experimet. When optimum concentrations of these components that were determined through application of response surface method (RSM) based on central composite design (CCD) were used, maximum spore numbers amounting to $1.9\times10^{10}$ spores/plate were obtained, resulting in approximately 190 fold increase as compared to the commonly used PDA sporulation medium. Using the miniaturized cultures, intensive strain development programs were carried out for screening of lovastatin high-yielding as well as highly reproducible mutants. It was observed that, for maximum production of lovastatin, the producers should be activated through 'PaB' adaptation process during the early solid culture stage. In addition, they should be proliferated in condensed filamentous forms in miniaturized growth cultures, so that optimum amounts of highly active cells could be transferred to the production culture-tube as reproducible inoculums. Under these highly controlled fermentation conditions, compact-pelleted morphology of optimum size (less than 1 mm in diameter) was successfully induced in the miniaturized production cultures, which proved essential for maximal utilization of the producers' physiology leading to significantly enhanced production of lovastatin. As a result of continuous screening in the miniaturized cultures, lovastatin production levels of the 81% of the daughter cells derived from the high-yielding producers turned out to be in the range of 80%$\sim$120% of the lovastatin production level of the parallel flask cultures. These results demonstrate that the miniaturized cultivation method developed in this study is efficient high throughput system for large and rapid screening of highly stable and productive strains.