• Journal of Plant Biotechnology
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• v.42 no.4
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• pp.388-395
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• 2015

Somatic embryogenesis is as an excellent technology for potential use in plant mass production, germplasm conservation, or genetic engineering. We examined the effect of cold storage using 3 embryogenic callus lines with different levels of embryogenesis competence derived from immature zygotic embryo cultures of Kalopanax setemlobus. Somatic embryo induction, germination and plant conversion were evaluated after 1, 3 and 6 months storage at $4^{\circ}C$ in the dark. Most cold-stored embryogenic calli formed somatic embryos normally even after 6 months; however, the induction rate was gradually decreased by increasing the storage period. The most competent line tended to show a slight decline in somatic embryo induction rate, as compared with other lines after cold storage. In general, cold storage resulted in reduced somatic embryo germination and plant regeneration, although 93% somatic embryo germination and 91% plant conversion were achieved regardless of the storage period. Cold storage led to cell browning and degradation. Additionally, the cell structures were confirmed by the aceto-carmine and evans blue dye evaluation. Collectively, our results showed that embryogenic callus of K. septemlobus could be preserved at $4^{\circ}C$ without subculture for 6 months, and suggested the need for storage of relatively more competent embryogenic calli lines to support somatic embryo induction.

• Korean Journal of Plant Resources
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• v.8 no.3
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• pp.237-246
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• 1995

In this study, the callus was induced and regenerated from the immature embryo and ultrastructural characteristics of developmental stages in Citrus junos SIEB, were investigated. The yellowish callus was induced by 5 to 6 week of culture of citrus. In proliferation callus after 6 weeks of culture, large vacuole was formed by fusion between adjacent small ones. In the non-embryogenic callus cultured for 12weeks, re-differentiated cells of callus showed the large nucleus with globular nucleus and amyloplast with large size of starches. In the embryogenic callus cltured for 14-16 weeks, the active exocytosis occurred in cells, secretory vesicles appeared on cell membrane and small particles from cytoplasm were released to intercelluar space. In the embryogenic callus cultured for 24 weeks, a sperical type of chloroplast bounded on cytoplasm by double membrane and typical grana was dispersed equally among matrix. In the normal plantlet after 26 weeks of culture, a lot of vessels and companion cells apperaed in the leaf cell of plantlet. In the normal plantlet after 30 weeks of culture, the immature leaf showed many small companion cells, sieve tubes and central vacuole. Also, the secondary vacuole protruded into the central vacuole and elongated chloroplasts near plasma membrane. In the matured plant habituated on the soil, palisada tissue composed of orderly arranged cells contained the nucleus in the center of the cell and large vacuoles on either side of the nucleus.

• Korean Journal of Plant Tissue Culture
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• v.26 no.2
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• pp.121-126
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• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

• Journal of Plant Biotechnology
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• v.29 no.3
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• pp.185-188
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• 2002

Off-white, friable embryogenic calluses were formed on the internal integument of mature seeds of yuzu (Citrus junos) cultured on Murashige and Skoog's basal medium at a frequency of 1.2%. Embryogenic calluses were proliferated when cultured on medium with 1 mg/L 2,4-D. Upon transfer to medium with 0.1 mg/L kinetin, embryogenic calluses produced numerous somatic embryos. Embryogenic suspension cultures were established by placing embryogenic calluses into liquid medium with 1 mg/L 2,4-D. When plated onto medium with 0.5 mg/L ABA, embryogenic cells developed into somatic embryos at a high frequency, and then regenerated into plantlets. Plantlets were successfully transplanted to potting soil and grown in a greenhouse.

• Journal of Ginseng Research
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• v.23 no.2 s.54
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• pp.74-80
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• 1999

Excised cotyledon segments of ginseng zygotic embryos cultured on MS basal medium without growth regulators produced somatic embryos near the basal excised portion at a high frequency. The frequency of somatic embryo formation on the segments declined along with advancing zygotic embryo maturity. In immature cotyledons, all the cells of the epidermis and subepidermis were smaller and more densely cytoplasmic than those in mature cotyledons, and from which multiple cells participated in embryogenic division to form somatic embryos with multiple cotyledons and fasciated radicles (poly-embryos). But in germinating cotyledons, only the epidermal cells were densely cytoplasmic and singularly competent to develop into somatic embryos resulting in single-embryos with closed radicles. This result means that the origin and development of somatic embryos is determined according to whether the cells participating in embryonic division are in a single state or a massive state relative to cotyledon maturity.

• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.335-339
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• 1998

The factors affecting the isolation and culture of the protoplast of embryogenic callus, derived from immature ovule in Citrus junos, were examined. An incubation time in enzyme solution of 16 hrs was preferable for protoplast isolation. Efficient protoplast yields were obtained from the treatment of equal concentration of 0.7 M $\textrm{BH}_{3}$ to the enzyme solution containing 1.0% cellulase, 1.0% macerozyme and 0.2% pectolyase. Protoplast cultured in MT medium with 0.1 mg/L 2,4-D showed vigorous division and some of them formed callus. Induced callus was subcultured on solid MT medium but the callus showed very slow growth. The above results show the possibility to culture from protoplast fusion in Citrus genera.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.287-291
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• 1994

In order to elucidate the effect of abscisic acid (ABA) on the abnormality of somatic embryos, somatic embryos were induced from embryogenic cell clumps derived from cotyledon segment of Aralia cordata. When embryogenic cell clumps were pretreated medium containing 0.2 mg/L ABA for 3 weeks before transferring to MS basal medium, the frequency of embryos with normal cotyledons enhanced 68% as compared with control. However when clumps pretreated in medium containing 0.2 mg/L ABA were transferred to medium containing 0.1 mg/L ABA, the Sequency decreased to about 29%. In the case of globular embryos cultures in medium containing various concentrations of ABA (0.01 to 1.0mg/L), the frequency of dicotyledonary embryo formation decreased propotionally to ABA concentration. Also, when somatic embryos at various stages were cultured in medium containing ABA, those with polycotyledons appeared at higher frequency.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.65-65
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• 2001

본 연구는 칡소 귀세포를 공여핵으로 이용한 체세포 복제송아지 생산에 있어서 배양방법이 배발생 및 배반포 발달율에 미치는 영향과 체세포 복제란의 이식후 수태율에 미치는 영향을조사하여 복제송아지의 생산 효율을 제고하고자 실시하였다. 실험에 공시된 공여핵은 칡소 의 귀세포를 회수하여 10%FBS가 첨가된 DMEM배지에서 3-4일 동안 배양하여 monolayar Confluent 형성 후 0.25% trypsin을 처리하여 준비하였으며 공여세포는 적어도 passage가 5회 이상의 세포만을 사용하였다. 복제수정란의 생산은 18-20시간 동안 체외성숙 된 난자의 핵을 제거하고 공여핵을 주입하여 2.2kv/cm, 10$\mu\textrm{s}$의 전압으로 2회 자극함으로 융합하였으며, 융합된 난자는 5$\mu\textrm{g}$/$m\ell$ ionomycin에서 4분간, 1.9mM 6-dimethyl aminopurine에서 4시간동안 배양하여 활성화처리를 하였다. 핵이식수정란의 배양은 39$^{\circ}C$, 5%$CO_2$ incubator에서 처리구 I은 CRlaa에서 4일간 배양 후 CR2aa배지에서 배양, 처리구II는 CRlaa에 4일간 배양후 CR2aa배지에 cumulus cell과 공배양, 처리구III은 CR2aa 배지에 camulus cell과 함께 배양하였다. 수정란이식은 발정발현 7일째에 비외과적 방법으로 젖소 미경산우에 이식하였으며 이식란수는 2~4개의 핵이식된 수정란을 이식하였다. 임신진단은 45~60일 사이에 직장검사 및 초음파 진단기를 이용하여 실시하였다. 배양방법에 따른 배발생율은 처리구 I에서 92.2 %(83/90)으로 처리구II와 III의 62.4%(63/101)와 77.8%(144/185)에 비하여 높게 나타났으나 배반포 발달율은 처리구II와III에서 65.1%(41/63)와 50.0%(72/144)로 처리구 I의 30.1%(25/83)보다 높게 나타났다. 각 처리구에 따른 수정란 이식후 수태율은 처리구II와 III에서 공히 20%의 수태율을 나타낸 반면 처리구 I에서는 수태가 되지 않았다. 따라서 체세포 복제수정란의 생산에 있어서 배반포 발달율과 수태율을 높이기 위해서는 단순배양보다 공배양이 더 효과적인 것으로 사료되지만 이런 결과가 복제송아지 생산효율에 있어서도 효과적일지는 향후 더 많은 연구가 있어야 할 것으로 사료된다.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2003.05a
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• pp.275-276
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• 2003

인간과 같은 척삭동물문에 속하는 우렁쉥이 (Halocynthia roretzi)의 유생은 척추동물의 체제를 매우 단순화시킨 구조를 하고 있다 (Nishida, 1997). 유생은 약 10여종의 세포들로 구성되며, 미부 중앙에 척삭과 그 위에 신경관의 원형 (prototype)을 갖는다. 모든 배(embryo)들은 초기 발생과정에서 언제나 동일한 세포분열과 발생운명 양식을 나타낸다. 또한, 해부현미경을 이용하여 수정란으로부터 초기 원장배에 이르기까지 원하는 하나의 할구만을 분리, 제거 및 재결합하는 등의 micromanipulation실험이 가능하다. (중략)

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.203-211
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• 1999

목적: 일산화질소 (nitric oxide; NO)는 생식계를 비롯한 여러 생체내 기관에서 다양하고도 중요한 작용을 하는 것으로 알려져 있으며, 복강액은 난관내강과 연결되어 복강액 내의 세포 성분의 변화는 난관의 미세환경을 변화시켜 수정과 초기 배아 발생에 영향을 줄 수 있다. 본 연구는 배아 발생에 있어서 일산화질소의 역할을 이해하고 복강액 내의 NO농도 변화가 배아 발생에 미치는 역할을 조사하기 위해 수행되었다. 방법: 과배란시킨 1세대 잡종 암컷 생쥐 (C57BL${\times}$CBA/Ca)로부터 1세포기 배아를 얻어 10% synthetic serum substitute가 첨가된 modified human tubal fluid 배양액에서 4일 동안 체외배양하였다(대조군). 실험을 위해 이러한 배양조건에 sodium nitroprusside (SNP)를 $0{\sim}1mM$의 다양한 농도로 배양초기부터 첨가하거나, $200{\mu}M$ SNP를 2-, 4-, 8-세포기의 각기 다른 배아시기에 첨가하였으며, 복강경수술을 받는 42명의 여성으로부터 채취한 복강액을 SSS대신 단백질원으로 사용하여 포배아까지의 배아 발달율을 관찰하였으며, 복강액 내의 NO농도를 Griess방법에 의해 측정하였다. 배아의 apoptotic body는 H33342 염색법으로 조사하였으며 배아 발달율은 3회 이상 반복 실험한 결과의 mean${\pm}$SEM으로 나타내었다. 결과: SNP는 농도에 의존적으로 배발생을 억제하였으나 배아 단계에 대한 특이성은 관찰할 수 없었으며, 특히 $100{\mu}M$ 이상의 고농도의 SNP는 2-세포기 단계에서 배아 발생을 정지시켰다. 또한 단백질원으로 복강액 이용시 배 발생율은 복강액 내의 NO 농도에 따라 현저한 차이가 발견되었으며, $2.5{\mu}M$이상의 NO를 함유한 복강액에서 배양한 배아의 발생율은 현저하게 감소하였다. cGMP analogue인 8-bromo-cGMP를 배양액에 첨가시 배아 발생에는 변화가 없었으며, SNP에 의해 배발생이 정지된 2-세포기 배아에서 apoptotic body를 발견할 수 없었다. 결론: 이상의 결과로 보아 NO는 고농도에서 배아 발생을 저해하며, 복강액 내의 NO와 같은 성분의 변화는 배아 발생에 유해한 효과를 유발할 것으로 사료된다. 이러한 NO의 배아 발생 억제효과는 cGMP로 중재되는 경로나 apoptosis유발과는 관계가 없는 것 같다.