• 한국산림과학회지
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• 제87권1호
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• pp.57-61
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• 1998

10년생 두릅나무의 동아(冬芽)를 절편으로 체세포배(體細胞胚)를 유도하고 식물체(植物體)를 재생(再生)시켜서 단지(團地)에 활착시키는 실험을 수행하였다. 배발생(胚發生) 캘러스는 MS 배지(培地)에 2, 4-D를 첨가하여 유도(誘導)되었다. 다량의 체세포배가 MS 배지에 2, 4-D와 BA를 동시에 첨가한 조건에서 발생(發生)되었다. 배발생 캘러스에서는 기형의 체세포배가 흔히 관찰되었으나 대부분은 전형적(全形的)인 배(胚) 형태(形態)를 보였다. 어뢰형에서 자엽(子葉) 단계(段階)에 이르는 성숙(成熟)한 체세포(體細胞) 배(胚)는 모두 발아(發芽)가 가능하였으나 완전한 식물체로의 재분화(再分化)는 MS 기본 배지에서 평균(平均) 14%이었다. 재분화된 식물체는 온실(溫室)에서 순화(馴化) 후(後) 단지(團地)에서 95% 이상 활착(活着)되었고, 이식(移植) 후(後) 3년까지 정상적인 생장(生長)을 보였다.

• 식물조직배양학회지
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• 제22권2호
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• pp.77-81
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• 1995

노각나무의 미숙배를 절편체로 사용하여 배양한 결과, 0.5 mg/L NAA 단독처리와 1.0 mg/L 2,4-D와 0.5 mg/L BA 혼용처리구에서 배발생캘러스 및 체세포배를 관찰할 수 있었으며 특히 0.5 mg/L NAA처리가 체세포배 발생을 더욱 촉진하였고 얻어진 배발생캘러스를 MS 기본배지에 계대배양 하여 배양 2주후 정상적인 식물체를 관찰할 수 있었다. Sucrose 농도에 따른 배발생캘러스로부터 체세포배 발생율은 5% sucrose 처리구에서 가장 높았고 9% sucrose 첨가는 체세포배 발생을 오히려 억제하였고 백색체 발생을 증가시켰다. 식물체 재분화를 위하여 MS, 1/2MS와 1/2MS에 0.1 % charcol를 첨가한 배지에서 배양하였던 바, 3, 6% sucrose 처리구 모두 MS, 1/2MS 기본배지에서는 자엽의 발달은 억제되었고 뿌리 발달만 왕성하였으나 1/2MS에 0.1% charcol 처리구에서는 정상적인 줄기와 뿌리를 가진 식물체를 얻을 수 있었다.

• 한국수정란이식학회지
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• 제17권2호
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• pp.109-115
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• 2002

The present study was conducted for the production of transgenic cloned cows those secrete human lactoferricin into milk by somatic cell nuclear transfer (NT). To estimate detrimental effects of gene transfection on transgenic cloned embryo production, development rates of NT embryos were compared between transfected and non-transfected cumulus and ear fibroblast cells. An expression plasmid for human lactofericin (pbeta-LFC) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human lactoferricin target gene into a pcDNA3 plasmid. Two bovine somatic cell lines (cumulus cell and ear fibroblast) were established and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6 as a carrier. Cumulus cell and ear fibroblast were transfected at the passage of 2 to 4, trypsinized and GFP-expressing cells were randomly selected and used for somatic cell NT. Developmental competences (rates of fusion, cleavage, and blastocyst formation) in bovine transgenic somatic cell NT embryos reconstructed with non-transfectecd cells were significantly higher than those from transfected cells in cumulus cell and ear fibroblast (P＜0.05). This study indicated that transfection of done. cell has detrimental effect on embryo development in bovine transgenic NT.

• 식물조직배양학회지
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• 제28권1호
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• pp.21-24
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• 2001

식용 및 약용으로 사용되는 갯기름나물의 기내배양을 통한 재분화율을 조사하였다. 캘러스는 잎과 엽병의 절편체에서 발생하였고 0∼5 mg/L의 NAA와 0∼10 mg/L BA가 첨가된 MS 기본배지에 치상하여 암배양 결과 자발적인 배 분화와 기관 분화를 보여주었으며 2.5 mg/L NAA와 10 mg/L BA의 조건에서 재분화율이 가장 높았다. 체세포 배의 발생양상은 구형, 심장형, 어뢰형을 거쳐 자엽 단계로 발달하여 전형적인 접합자 배의 모습으로 발달하였다. 배 발생한 일차 체세포배를 0.2 mg/L의 NAA가 첨가된 배지에서 배양하였을 때, 이차 체세포배가 발생하였으며 주로 일차 체세포배의 배축과 하배축에서 많이 발생하였고 그 다음으로 자엽이나 뿌리 부분에서 발생하였다.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• 한국약용작물학회지
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• 제7권1호
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• pp.1-6
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• 1999

지황(Rehmannja glutjnosa)에서 종근 저장시 나타나는 병원균의 오염을 배제하면서 종묘를 대량 증식시키는 방법을 개발하기 위하여 액체 배양을 통해 직접 체세포배를 형성하고자 할 때 요구되는 배지, 질소원, 탄소원 및 pH와 배양 기간 중 배지의 성분 변화를 조사하였다. 줄기나 엽병을 접종조직으로 할 경우, 배지내 질소원인 $NH_4NO_3$ 와 $KNO_3$의 적정비율은 825 mg/1 : 1900 mg/l 이었고, 탄소원으로는 자당이 적합하였으며, 적정 농도는 3% 이었다. 배지로는 1X MS 배지가 적합하였고, 적정 BA 농도는 2.0 mg/l 였는데, NAA와 조합하였을 경우에는 BA 1 mg/l에 NAA를 0.5 mg/l 를 조합한 배지 와 BA 2.0 mg/l 에 NAA를 0.1 mg/l로 첨가한 배지가 체세포배 발생에 효과적이었다. 멸균 전에 pH를 5.7로 조정한 경우에 직접 체세포배의 형성에 효과적이었다. 배양기간 중 배지 내 $Ca^{++}$와 $Mg^{++}$이온은 거의 변화가 없었으나, $K^{+}$이온은 많이 흡수되었다. 배양 3주 째에 자당은 거의 고갈되었고, 과당과 포도당은 3주 째부터 흡수되기 시작하였다.

• Plant Biotechnology Reports
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• 제2권1호
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• pp.87-92
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• 2008

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on halfstrength MS medium supplemented with $0.3mg\;l^{-1}$ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to $3mg\;l^{-1}$, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryoderived white friable callus were established using half-strength MS medium supplemented with $0.3mg\;l^{-1}$ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of water-shield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.

• 한국수정란이식학회지
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• 제26권1호
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• pp.85-89
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• 2011

Somatic cell nuclear transfer (SCNT) is a useful tool for reproducing genetically identical animals or producing transgenic animals. Many reports have demonstrated that the efficiency of animal cloning by SCNT requires reprogramming of the somatic nucleus to a totipotent like-state. The SCNT-related reprogramming might mimic the natural reprogramming process that occurs during normal mammalian development. However, recent evidence indicates that the reprogramming event by SCNT is incomplete. In this study, the traditional SCNT procedure (TNT) was modified by injecting donor nuclei into recipient cytoplasm prior to the enucleation process to expose the donor nucleus before removing the karyoplast containing the chromosomes of the oocytes which might possess additional reprogramming factors, and this modified technique was named as reversing the usual order of SCNT (RONT). Other procedures including activation and in vitro culture were the same as TNT. Contrary to expectations, the rate of blastocyst development was not different significantly between RONT and TNT (8.6% and 7.9%, respectively). However, duration of micromanipulation performed by the same technician and equipments was remarkably reduced because the ruptured oocytes after nuclear injection were excluded from the enucleation process. This study suggests that RONT, a simplified SCNT protocol, shortens the duration of SCNT procedure and this less time-costing protocol may enable the researchers to perform murine SCNT easier.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.43-55
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• 2002

Cloning by nuclear transfer in mammals using somatic cells has enormous potential applications. However, somatic cloning has been inefficient in all species in which NT is successful. High abortion and fetal death rates have been observed. These developmental defects have been attributed to incomplete nuclear reprogramming by the somatic cloning process. In this review, we will discuss studies conducted in our labs to understand the nuclear reprogramming process.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2021년도 춘계학술대회
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• pp.51-51
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• 2021

This study was conducted to develop a somatic embryogenesis protocol for the Cnidium officinale Makino difficult to seed propagation. The immature flowers were used as explants. The concentration of a 2,4-D 1.0mg/L was found to be optimal concentration for induction of embryogenic callus and somatic embryos. Addition of 0.3mg/L, 0.5mg/L and 1.0mg/L to the embryo germination medium promoted somatic embryo germination. Among four concentrations, GA₃ 1.0mg/L were superior to others. Shoots were transferred to hormone-free MS medium after 2 months of culture in the dark. We obtained an optimized protocol for the regeneration of C. officinale.