• 한국자원식물학회지
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• 제32권1호
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• pp.19-28
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• 2019

An efficient shoot regeneration condition for pea cv. 'Sparkle' was developed by using optimum explant, plant growth regulator concentrations, and pretreatment of BA onto explant. The average shoot number per explant showed the highest on two kinds of shoot induction media (MSB5 media containing 2 mg/L BA and a combination of 2 mg/L BA and 1 mg/L TDZ) when cotyledonary node explants were cultured. Moreover, the pretreatment of explant in 200 mg/L BA solution was found to be more effective in shoot induction than that of non-pretreatment. By histological study, cell division and proto-meristem were formed near the surface of the sub-epidermal and epidermal cell layers of cotyledonary node in earlier than 3 days after culture. The analysis of genetic stability of regenerants by using thirteen ISSR markers showed that in vitro regenerated plants showed polymorphism with 8.3% compared with their mother plants.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.3-7
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• 2002

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.

• 한국가축번식학회지
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• 제16권4호
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• pp.347-352
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• 1993

This study was designed to develop the in vitro culture systemof mammalian preimplantation embryos. We proposed mouse fetal fibroblast cells (MFFC) from 14∼15 day mouse fetus. Zygotes from superovulated female ICR mice were cultured 96 hrs in simple defined media (T6) or on the monolayer of MFFC. In addition, to evaluate the action of the co-culture of MFFC, various diluted superoxide dismutase antibody (SOD-Ab) was supplemented into the monolayer of MFFC and zygotes were cultrued in presence or absence of SOD-Ab. The developmental rates of zygotes were significantly increased in co-culture with MFFC compared to the control. The rates of zygotes to the 4-cell stage in media treated with EDTA were higher than those cultured in MFFC but the proportions of morula and blastocyst were not differ between EDTA and MFFC. Interestingly blastocysts in co-culture with MFFC possessed as many as blastomere as those developing in vivo, but blastocysts cultured with EDTA had significantly fewer blastomeres. In addition, the treatment of SOD-Ab suppressed the beneficial effect of MFFC. Therefore, our findings suggest that co-cultrue system using MFFC may have an advantage in the development of mouse zygotes as well as embryonic differentiation.

• Clinical and Experimental Reproductive Medicine
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• 제41권1호
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• pp.1-8
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• 2014

Objective: Estrogen related receptor ${\beta}$ (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotencyrelated genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. Methods: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without $2{\mu}g/mL$ CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. Results: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. Conclusion: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

• 한국임상수의학회지
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• 제9권1호
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.157-164
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• 1997

To study the regulation of porcine follicular cell apostosis by gonadotropin, steroid, and nitric oxide, we analyzed DNA fragmentation, the hallmark of apoptosis, and nitrite production of porcine granulosa cells. Dissected indiidual follicles from ovary were separated in size (small, 2-3 mm; medium, 5-6 mm; large, 7-8 mm) and isolated granulosa cells were classified morpholocally as atretic or nonatretic. Nitrite concentration was measured by mixing follicular fluids with an equal volume of Griess reagent. Follicular nitric oxide (NO) concentration of healthy follicles was higher than that of atretic follicles. Apoptotic DNA fragmentation was suppressed in non-apoptotic granulosa cells. Follicular apoptosis was induced by androgen but prevented by gonadotropin in vitro. Apoptosis was confined to the granulosa cells. But it was not clear whether apoptosis of granulosa cells were isolated, incubated with or without gonadotropin, androgen and sodium nitroprusside (SNP), respectively at $37^{\circ}C$ for 24 hrs. Cultured granulosa cells were used to extract genomic DNA and culture media was asssayed for nitrite concentration. Nitrite production of culture media was increased, while apoptotic DNA fragmentation was suppressed in PMSG, hCG, testosterone+SNP and SNP treated groups. Nitrite concentration in culture media was decreased, but apoptotic DNA fragmentation was induced in testosterone treated group. These data suggest that NO production and apoptosis may be involved of granulosa cell apoptosis induced by testosterone.

• 한국약용작물학회지
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• 제15권3호
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• pp.210-214
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• 2007

반하의 기내 조직배양을 통해 소괴경 생산을 하고자 적합한 식물생장조절제 처리조건, 배지와 처리 농도, 고형지지물의 종류와 농도를 조사하였다. 기내 소괴경 형성에 적합한 식물생장조절물질과 농도 조건을 연구 한 결과 0.1mg/l 2,4-D와 0.5 mg/l BAP 혼합처리에서 잎 (3.9개)과 엽병 (4.7개) 배양모두 최고치의 소괴경을 생산할 수 있었다. 기내 소괴경 형성에 적합한 배지는 SH배지로 나타났으며 잎과 엽병 배양에서 4.5와 5.2개의 소괴경을 생산하였으며, 최적 농도는 SH 배지의 무기염류를 반으로 줄인 1/2 SH배지가 가장 효과적이었다. 기내 소괴경 생산에 적합한 고형지지물과 그 농도를 조사한 결과, 잎과 엽병 배양 모두 agar보다 gelrite에서 소괴경 형성이 잘 되었으며, 농도별로는 agar 6%, Gelrite는 4% 처리에서최고치의 소괴경을 생산할 수 있었다. 기내에서 생산된 소괴경은 5$^{\circ}$C에서 1개월간 냉장 보관 후에 생장상에서 MS 고형배지가 든 배양용기와 멸균 소독한 상토에 옮겨 발아율을 조사한 결과, MS고형 배지에서 자란 소괴경은 86%의 발아율을 보였으며, 멸균 소독한 상토에서는 43%의 발아율을 보였다. 추후 반하 소괴경의 발아율과 토양 적응성을 높이는 연구보강으로 소경을 번식용 종자로 활용할 수 있는 방안을 모색할 예정이다.

• 한국자원식물학회지
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• 제27권5호
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• pp.540-545
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• 2014

경상남도 농업기술원에서 육성된 둥굴레 '건강백세(Polygonatum odoratum cv. Gungangbeaksea)'를 분양받아 배양하였다. 둥굴레 지하경 절편체를 MS 배지에 BA 3.0 mg/L가 첨가된 배지에서 배양하면서 증식된 부정신초괴를 실험재료로 사용하였다. 배양 8주후, 신초의 증식은 TDZ 3.0 mg/L 첨가배지에서 2.8개로 가장 많았으며 생체중도 절편체당 2.32 g으로 가장 높게 나타났다. TDZ와 NAA가 첨가된 배지에서는 신초 및 부정신초괴의 증식이 다소 증가하였다. TDZ 3.0 mg/L와 NAA 5.0 mg/L 첨가배지, TDZ 5.0 mg/L와 NAA 3.0 mg/L 첨가배지에서 신초수 3.7개, 생체중 2.9 g으로 다소 증가하였다. MS 염류농도가 높아질수록 신초 및 부정신초괴의 증식은 저조하였다. 그러나 sucrose 농도가 3%에서 7%로 증가함에 따라 신초수, 생체중 및 부정신초괴의 형성이 증가하였다. 뿐 만 아니라 둥굴레 부정신초절편체에서 신초 및 부정신초괴의 증식은 MS 배지에 TDZ와 NAA 3.0~5.0 mg/L와 sucrose 7%가 첨가된 배지가 적합하였다. 발근율은 IBA 3.0 mg/L 또는 NAA 2.0 mg/L 첨가배지에서 80% 이상으로 높았으며, 발근된 식물체의 순화는 vermiculite 단용 또는 perlite, vermiculite 1:1 혼용처리에서 순화율 100%로 적합하였다.

• Journal of Plant Biotechnology
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• 제8권1호
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• pp.15-19
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• 2006

An efficient protocol for in vitro multiple shoot bud induction and plant regeneration from mature green cotyledon derived callus tissues of Acacia sinuata has been developed. Callus formation occurs at all the concentrations of thidiazuron (TDZ) in Murashige and Skoog's (MS) medium, but 0.6 ${\mu}M$ proved to be the best with maximum callus formation frequency. Supplementation of TDZ in combination with indole-acetic acid (IAA) in MS media accelerates shoot bud organogenesis in differentiating callus tissues with 60-70% conversion of shoot buds into shoot Most efficient shoot organogenesis was recorded when TDZ induced calli were subcultured at different concentrations of 6-benzyla-denine (BA). Optimum shoot bud induction and plant regeneration from callus was achieved when 0.6 ${\mu}M$ (TDZ) induced calli were subcultured at 3.0 ${\mu}M$ (BA) where $16.6{\pm}0.74$ shoots/unit callus on obtained. Rooting in in vitro differentiated shoots was achieved when transferred to medium containing different concentration of indole-3-butyric acid (IBA) in full & half strength MS medium. The well rooted plantlets were hardened and transferred to net house with 90% survival rate.

• 한국가축번식학회지
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• 제26권1호
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• pp.17-23
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• 2002

본 연구는 지금까지 돼지 난포란 성숙을 위해서 많이 사용하고 있는 mTCM-199, mWaymouth MB 752/1 그리고 NCSU-23 성숙배지를 비교하고 액상정액을 이용한 체외수정 방법을 개발하고자 실시하였다. 미성숙 난포란은 0.5 $m\ell$의 성숙배지에 각 well 당 30~40개씩 적하하였고, 38.5$^{\circ}C$, 5% CO2, 95% 공기로 조절된 CO2 배양기에서 44시간 성숙 시켰다. 미성숙 난포란을 mTCM-199, mWaymouth MB 752/l 그리고 NCSU-23 성숙배지에서 44시간 배양한 결과 CVBD 발생율은 각각 95.6, 94.1 그리고 94.9%였으며, MH단계까지의 성숙율은 각각 92.5, 90.1 그리고 91.1%였다. 성숙배지별, GVBD 발생율과 MH까지의 성숙율간에 유의성은 인정되지 않았다. 액상정액의 제조용 정액은 90% 이상의 운동성을 가진 농후정자부분을 사용하였으며 정액 채취 후 2시간 동안에 22~24$^{\circ}C$의 실온까지 냉각시켰다.실온까지 냉각한 정액은 BTS 희석액으로 2$\times$$10^{8}$ $m\ell$ 정자농도로 조정하여 100 $m\ell$ 플라스틱병에 30 $m\ell$씩 주입하여 17$^{\circ}C$에서 5일간 보관하였다. 5일 보관 후 운동성이 70% 이상인 정자를 체외수정에 이용하였다. 성숙 후 cumulus cell들이 제거된 성숙난포란은 0.5 $m\ell$의 mTCM-199 또는mTBM 수정배지에 30~40개씩 적하하고, 최종정자농도를 2$\times$$10^{6}$$m\ell$되도록하여 6시간 동안 수정시켰다. 체외수정시킨 수정란들은 0.5 $m\ell$의 NCSU-23 배양배지에서 수정 후 6시간 배양하여 정자침입율, 다정자침입율 그리고 웅성전핵형성율을 조사하였고, 수정 후 45시간 배양하여 난할율을 조사하였다. NC-SU-23 성숙배지와 mTBM 수정배지를 이용하였을때 웅성전핵형성율이 48.0%로써 mTCM-199 성숙배지와 수정배지 또는 mWaymouth MB 752/1 성숙배지와 mTCM-199 수정배지를 이용하였을 때보다 웅성 전핵 형성율이 높았다. 2~4세포기까지의 난할율은 mTCM-199 성숙, 수정 및 배양배지에서 24.1%, mWaymouth 752/1 성숙배지, mTCM-199 수정 및 배양배지에서 43.6%, 그리고 NCSU-23 성숙배지, mTBM 수정배지 및 WCSU-23 배양배지를 이용한 것이 71.2%였다. 이상의 결과를 종합하면 BTS 희석액으로 17$^{\circ}C$에서 5일 보존한 액상정액으로 체외수정이 가능함을 입증하였고, NCSU-23 성숙배지, mTBM 수정배지 및 NCSU-223 배지가 미성숙 난포란의 성숙, 수정 및 배양에 우수한 배지임을 입증하였다.