• Journal of Life Science
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• v.31 no.4
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• pp.385-388
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• 2021

Anther cultivation for crop breeding is a method of rapid production of homozygosities by greatly reducing the time required for at least six generations to develop new varieties using conventional breeding methods. This technique of producing anther culture provides an opportunity to obtain more green plants from a methodological point of view, and the techniques that save time and effort in anther culture are also important because they increase the efficiency of culture. This study compared the callus induction rate and green plant regeneration rate of a one-step and a two-step culture that differ in their culture media and culture methods. One-step culture allows callus induction and plant regeneration in one medium, whereas two-step culture requires induction and plant regeneration in two different media. In this study, we compared the callus induction and plant regeneration rates of rice anthers as one-step and two-step cultures. The callus formation rate was 13.0% for one-step cultures and 8.6% for two-step cultures, so the rate was 4.4% higher for one-step cultures than for two-step cultures. The plant regeneration rate was 1.0% in one-step cultures and 3.0% in two-step cultures, so the regeneration rate was three times higher for the two-step cultures than for one-step cultures. This suggests that the two-step cultures are more efficient than the one-step cultures for haploid production.

• Journal of Embryo Transfer
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• v.6 no.1
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• pp.1-11
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• 1991

가축의 일란성 쌍태를 생산하기 위한 기술 개발을 확립하고자 상실배 및 포배기에 있는 BALB/c 계통의 생쥐 수정란을 micromanipulator로 분할 수정란을 작출하고 이를 체외배양을 실시하여 발달성적을 조사하였으며, 외과적 및 비외과적 이식을 실시하여 착상율 및 산자생산 성적을 조사한 결과는 다음과 같다. 1. 상실배 및 포기배에 있는 총 811개의 정상적인 수정란을 분할하여 이중에서 666(82.1%)개가 분할시의 물리적인 손상이 없이 분할되었고, 이때 분할 성공율은 발달단계 간에 유의적(P<0.05)인 차이가 없었다. 2. 분할 수정란중 상실배는 30-36시간, 초기 배반포 및 확장 배반포는 3-6시간 배양을 실시한 결과 분할 수정란중 한쌍이 모두 정상적으로 배양된 것은 각각 70.0%, 80.4% 및 73.1%로써 이들 발달단계 간에 유의적(P<0.05)인 차이가 없었다. 3. 분할된 상실배와 정상적인 수정란의 이식후 수태율은 각각 63.6% 및 61.3%로써 유의적(P<0.05)인 차이가 없었다. 그러나 분할된 상실배에 있어서 배양을 하지 않고 이식한 경우에는 전혀 수태되지 않았다(P<0.05). 4. 분할된 포배기 수정란을 체외 배양후 이식한 수태율(55.5%)과 배양과정을 거치지 않고 이식한 성적(43.8%) 그리고 정상적인 포배기 수정란을 이식한 수태율(55.4%) 간에는 유의적(P<0.05)인 차이가 없었다. 5. 분할 수정란을 외과적 방법으로 이식한 경우는 52.8%의 수태율을 얻었으나, 비외과적 방법으로 이식한 경우 27.5%로써 외과적 방법으로 이식한 경우보다 수태율이 유의적(P<0.05)으로 낮았다.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.196-202
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• 1992

Zoogloea ramigera 115 was selected for the production of viscous microbial polysaccharide for bioflocculants usage. Batch, fed-batch, and continuous culture processes were examined with regard to the high biopolymer production. Several carbon sources were tested, including glucose, lactose, molasses, and cheese whey. The C/N ratio of 90 was most effective for biopolymer production from glucose, while the C/N ratios of 30 for lactose and 60 for both molasses and cheese whey substrate gave a maximum production. Fed batch culture proved more effective to increase final biopolymer concentration than batch culture. Continuous fermentation with two stages modifying C/N ratio increased the productivity. The production rates were a maximum at dilution rate of 0.048 $hr^{-1}$ for molasses and at 0.096 $hr^{-1}$for cheese whey.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.43-52
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• 1996

This study was carried out to establish the in vitro short-term culture system of developing male germ cells by purifing germ cells of various stages. The decapulated testicular cells were incubated with collagenase (lmg/ml) and try psin (2.5mg/ml) in HBSS. After separating male germ cell, the separated germ cells were stained with heamatoxylin/eosin and determined developing stages under light microscopy. The purity of pachtene spermatocytes a and round spermatid were 85%, respectively. Yield of total male germ cells was highly variable between individuals, with a mean value of 3.5 to 4.5 ${\times}$ 10$^7$ cells/testis. Viability of the cell was over 97% after separation. In DMEM medium, the optimal cell number for culture is approximately 1 x 10$^5$ cells/dish, but low cell den-sities than 1 ${\times}$ 10$^5$ cell/dish showed a decreased cell viability. Furthermore, about :36.8% of pac-hytene cells was successfully cultured for 6 days and some of cells were developed to secondary spermatids and round spermatids. Therefore, our data suggested that this culture conditions will be utilize as a feasible tools to produce tran-sgenic livestock using techniques such as intrac-ytoplasmic injection and cell fusion.

• Journal of Plant Biology
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• v.39 no.1
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• pp.63-69
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• 1996

A hairy root clone of Panax ginseng C.A. Meyer, HRB-15 was cultured iu various conditions with 3 L bubble type bioreactor to enhance both growth and ginsenoside production. The hairy roots were more rapidly grown under the dark condition than under the light condition. However, total amount of ginsenoside of hairy roots cultured under the light for 30 days increased 2 folds as compared with the dark condition and was 1.10% based on 6 ginsenosides. Especially, ginsenoside-Re was significantly increased and some ginsenosides except for ginsenoside-Re was slightly reduced. Also, the growth of hairy roots decreased about 30% as compared with the dark condition. In contrast, addition of sodium acetate led to decreased production of ginsenoside and growth of hairy roots under light condition. The influence of potassium dihydrogenphosphate concentration was examined in MS medium and a 1.25 mM concentration was found to be the most appropriate for growth and ginsenoside production under light condition. Two-step process of hairy roots culture with yeast elicitation or without ammonia in culture medium was developed to enhance growth and giusenoside synthesis. $50\;\mu\textrm{g}$ of yeast elicitor per g of fresh weight showed a synergistic effect on the ginsenoside synthesis of hairy roots on 20 days after culture. At that time, the content of total ginsenoside was 1.15%, while the growth of hairy roots decreased 21 % as compared with the dark condition. In addition, when elimination of ammonia on 20 days after culture, the content of total ginsenoside was 1.26% with significant increment of ginsenoside-Rd (0.27%) in addition to ginsenoside-Re and the growth of hairy roots decreased 10% as compared with the dark condition. In this system, we have demonstrated a unique two-step process of hairy root cultures to maximize biomass and secondary metabolites. It has found possibility to enhance ginsenosides production by growing hairy roots in this method.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.41-49
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• 1996

본 연구는 체외수정에 의해 생산된 생쥐 배반포기배를 vitrification 방법으로 동결보존하였을때 높은 생존율을 얻기 위한 적정조건을 검토하고자 실시하였다. 배반포기배를 생산하기 위하여, B6CBA F1 (C57BL/6, (표현불가)${\times}CBA/N$, (표현불가)) 계통의 생쥐 미수정란에 $1{\times}10^6$ spermatozoa/ml 농도의 정자로서 수정을 유도하였으며, 이후 $37^{\circ}C$, 5% $CO_2$배양기내에서 96시간동안 체외배양하였다. 배양 4일째의 배반포기배는 발달상태에 따라 early, middle 그리고 hatching blastocysts로 구분하였다. 본 실험에 사용된 동결보존액은 30% Ficoll과 0.5mol의 sucrose가 첨가된 mDPBS 용액에 40%의 ethylene glycol를 첨가한 EFS 40 (Zhu et al., 1993) 이었고, 수정란은 $25^{\circ}C$의 상온에서 먼저 20% ethylene glycol에 노출된 후 EFS 40 용액으로 옮겨 액체질소에 침지하는 2단계 동결법에 의해 동결보존되었으며, 급속융해하여 다음과 같은 결과를 얻었다. 1. 체외수정율과 배양 4일째 배반포기까지의 배발달율은 각각 89.4%와 86.1%였다. 2. 20% ethylene glycol에서 5분간 평형된 후 EFS 40 용액에 냉동보존된 후 융해된 난자의 생존율은 20% ethylene glycol에 0, 1, 3분간 평형된 난자의 생존율에 비해 유의하게 높았다. 3. 배반포기배를 20% ethylene glycol에서 5분간, EFS 40 용액에 1분간 차례로 노출한 다음 체와배양하였던 바, 배양 24시간째 생존율은 $82.9%{\sim}88.4%$ 였다. 본 연구 결과, 체외수정, 배양된 생쥐 배반포기배는 20% ethylene glycol과 EFS40에 대한 노출만으로는 난자의 생존성에 나쁜 영향을 미치지 않는 것으로 미루어 보아 배반포기배의 초자화 동결이 가능함을 시사하였다. 따라서 동결 융해 후 높은 생존율은 상온에서 난자를 2단계 즉, 20% ethylene glycol에 5분간 평형시킨 후 EFS 40 용액에 노출하여 1분내에 LN2에 직접 침지하는 간편한 동결방법으로 얻을 수 있었다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.231-231
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• 1994

이 연구는 인삼 성분 약물이 도파민 수용체에 미치는 영향을 연구하기 위한 첫 단계로써 그 기본적인 assay system을 정착 시키기 위함이 목적이었다. 우리는 이 연구를 수행하기 위하여 생후 2-4 일의 쥐를 사용하여 흑질, 선조체, 해마구, nucleus accumbens등의 뇌 부위에서 신경세포 배양을 시도하였다. 흑질로 부터 배양한 신경세포들의 경우엔 면역 세포학적방법을 써서 살펴본 결과 대부분의 신경이 도파민성 신경이나 GABA성 신경들 이었다. 또한 이들 세포들의 전기 생리학적 상태를 알아보기 위하여 흑질에서 신호 전달체계가 잘 확립된 GABA 수용체의 작용을 살펴 본 결과 이 신경세포들은 GABA-A 및 GABA-B 수용체의 발현은 물론 이온 채널에 미치는 신호 전달체계를 완전히 갖추고 있었다. 도파민 수용체의 작용을 전기 생리학적으로 연구하기 위하여 배양한 신경세포에 도파민agonist를 가해서 이온 채널에 미치는 효과를 살펴 보았다. 선조체에서 배양한 신경세포들은 Dl 과 D2 agonist에 대해서 상반되는 반응을 나타냈다. 즉 Dl agonist는 선조체 신경세포를 활성화 시켰으나 D2 agonist는 선조체 신경세포들을 억제 하였다. 한편 해마구의 CAI 과 CA3 부위로 부터 배양한 신경세포에 대한 도파민 agonists의 작용은 선조체의 신경세포에 대한도파민 agonists의 작용과는 상반되는 반응 이었다. Dl agonist는 해마구로 부터 배양한 신경세포의 활성을 억제 하였으나 D2 agonist는 이들 신경세포들의 활성을 증가 시켰다. Nucleus accumbens 에서 배양한 신경세포들은 도파민에 의해서 그 활성이 억제 되었다. 이러한 결과로 미루어 봐서 같은 도파민 수용체라도 분포되어 있는 조직에 따라서 신호전달 에 관여 하고 있는 C-단백이나 이차 전령물질이 달라서 신경세포에 대한 작용이 다르든지. 약리학적으로는 구분되지 않으나 뇌의 조직에 따라서 분포가 다른 도파민 수용체의 아그룹이 존재 한다고 생각된다.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.255-262
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• 2006

The purpose of this study is to investigate the effects of vitrification in open pulled straws (OPS) methods on in vitro survival ability of porcine embryos. For in vitro maturation of immature oocytes, the porcine ovaries were collected from local slaughter-house. The cumulus-oocytes complexes were aspirated from 2 to 6 mm follicles. The collected oocytes were cultured for in vitro maturation in NCSU-23 medium with 5 mM hypotaurine, 0.57 mM cysteine, 10% porcine follicle fluid, 10 IU/ml PMSG and 10 IU/ml hCG for $21{\sim}22$ hrs. Then, the oocytes were more cultured $21{\sim}22$ hrs in vitro maturation in medium removed hormones. The frozen-thawed spermatozoa were washed by centrifugation 2 times for 10 min at 1,500 rpm in D-PBS with 5.56 mM glucose, 0.33 mM Na-pyruvate, 100 IU/ml penicillin, $100 {\mu}g/ml$ streptomycin and 4 mg/ml BSA. The fertilization medium used mTBM with 2 mM caffeine and 2 mg/ml BSA and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to $2.5{\times}10^6$cells/ml motile sperm during fertilization in vitro. At 8 hrs after insemination, the oocytes were transferred into NCSU-23 medium with 5.0 mM hypotaurine, 4 mg/ml BSA and 10 ng/ml EGF and cultured for 7 days. When the blastocysts of different stages were frozen-thawed by OPS methods, the proportions of embryos with normal morphology were significantly (p<0.05) higher in embryos frozen-thawed at expanded blastocyst stage (38.9%) than in early blastocyst stage (28.3%). On the other hand, the proportions of embryos damaged after frozen-thawing were significantly (p<0.05) higher in embryos frozen at early blastocyst stages than in expanded blastocyst stage. In another experiment, the normal embryos morphology after frozen- thawing were further cultured for 48 hrs. After culture, the proportions of embryos hatched were 6.7, 20.0 and 33.3% for embryos frozen-thawed at early blastocyst, mid-blastocyst and expanded blastocyst stages. These finding indicate the possible broader application for OPS methods, as frozen-thawed embryos may be accompanied by developmental stage according to requirements of the survival ability after freezing of blastocyst stage in the pig.

• Journal of Bio-Environment Control
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• v.18 no.1
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• pp.23-28
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• 2009

This study was conducted to evaluate the effect of optimized environment conditions with growth stage in photoautotrophic micropropagation on the growth of potato (Solanum tuberosum L. cv. Dejima) plantlets and energy efficiency. Optimum environment conditions at each stage were decided in our previous study. For the evaluation of optimized environment control, potato plantlets were cultured under four different conditions: photoautotrophic optimum conditions of photosynthetic photon flux density (PPFD) and $CO_2$ levels with growth stage (POG), photoautotrophic constant condition with average PPFD and $CO_2$ levels (PCA), photoauototrophic constant condition with maximum PPFD and $CO_2$ levels (PCM), and photomixotrophic conventional condition with 3% sucrose (PMC) as control. As a result, environment control with growth stage (POG) significantly promoted all the growth characteristics such as the number of nodes and unfolded leaves, shoot height, shoot diameter, and fresh and dry weights of potato grown in vitro. In addition, based on dry weight consumed electricity and $CO_2$ were the lowest in POG suggesting the highest energy efficiency among the treatments. After transferring potato plantlets to greenhouse, the plantlets under POG showed vigorous growth, which was pretty similar with those under PMC. The accumulations of dry matter in POG were 4.7 times in vitro and 3.8 times in greenhouse as much as those in the conventional control (PCM). Thus, we concluded that in vitro environment control with growth stage induced vigorous growth of potato plantlets both in vitro and in greenhouse with less energy consumption.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2014.05a
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• pp.822-825
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• 2014

For in vitro myelination system, Schwann cells and neuronal cells of rat were cocultured. Schwann cells and neuronal cells, respectively, were obtained from dorsal root ganglion of rat embryos (E15). This method includes four steps: first step of suspension of the embryonic dorsal root ganglion cells, second step of addition of anti-mitotic cocktail, third step of purification of dorsal root cells, and fourth step of addition of Schwann cells to dorsal root ganglion cells. We made a highly purified population of myelination in a short period through this procedure and identified myelination basic protein using antibody of myelination basic protein.