• Journal of Plant Biotechnology
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• 제39권1호
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• pp.13-22
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• 2012

Plant regeneration has been optimized increasingly by organogenesis and somatic embryogenesis using a range of explants with tissue culture improvements focusing on factors, such as the age of the explant, genotype, media supplements and $Agrobacterium$ co-cultivation. The production of haploids and doubled haploids using microspores has accelerated the production of homozygous lines in Brassicaceae crop plants. Somatic cell fusion has facilitated the development of interspecific and intergeneric hybrids in sexually incompatible species of $Brassica$. Crop improvement using somaclonal variation has also been achieved. Transformation technologies are being exploited routinely to elucidate the gene function and contribute to the development of novel enhanced crops. The $Agrobacterium$-mediated transformation is the most widely used approach for the introduction of transgenes into Brassicaceae, and $in$ $vitro$ regeneration is a key factor in developing an efficient transformation method in plants. Although many other Brassicaceae are used as model species for improving plant regeneration and transformation systems, this paper focuses on the recent technologies used to regenerate the most important Brassicaceae crop plants.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2002년도 제42차 춘계학술대회
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• pp.11-17
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• 2002

Animal clones derived from somatic cells have been successfully produced in a variety of mammalian species such as sheep, cattle, mice, goats, pigs, cat and rabbits. However, there are still many unsolved problems in the present cloning technology. Somatic cell nuclear transfer has shown several developmental aberrancies including high rate of abortion in early gestation and increased perinatal death. These developmental failures of cloned embryos may arise from abnormal reprogramming of donor genome and/or incomplete cloning procedure. We have found that overall genomic methylation status of cloned bovine embryos is quite different from that of normal embryos in various genomic regions, suggesting that the developmental failures of cloned embryos may be due to incomplete reprogramming of donor genomic DNA. Many of the advances in understanding the molecular events for reprogramming of donor genome will more clarify the developmental defects of cloned embryos.

• Reproductive and Developmental Biology
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• 제34권4호
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• pp.283-288
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• 2010

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked. changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

• 식물조직배양학회지
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• 제24권2호
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• pp.119-124
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• 1997

십자화과 향신료 작물인 서양고추냉이 (Armoracia rusticana (Lam.) Gaerth., May. et Scherbs)의 재분화에 미치는 제 요인을 조사하였다. 서양고추냉이의 callus로부터 식물체 재분화는 IAA 0.5 mg/L, BA 5.0 mg/L, spermine 10 mg/L이 첨가된 MS배지에서 가장 높았다. BA 2.0 mg/L과 NAA 2.0 mg/L가 첨가된 MS배지에 엽, 엽병, 뿌리의 조직을 치상한 결과 각각의 조직으로부터 체세포배가 발생하였으며, 기관발생 경로를 거쳐서 식물체가 재분화 되었으며, 빈도는 낮았지만 생장조절물질이 포함되지 않은 MS배지에서도 각 조직으로부터 식물체가 분화되었다. 수소이온 농도는 pH 4.0부터 8.0의 비교적 넓은 범위에서 multiple shoot가 형성되었으며, polyamine류의 첨가는 shoot와 뿌리의 형성을 촉진시켰다. Callus로부터 뿌리 또는 잎이 분화 중인 조직에서 특이적인 단백질이 생성되는 것이 확인되었다. 이상의 결과, 서양고추냉이는 생장점 배양, 체세포배발생, 기관발생과정을 각기 거쳐 식물체가 재생될 수 있는 식물체로 확인되었다. 서양고추냉이의 이러한 특성은 세포, 조직, 기관의 형태형성 연구 및 형질전환 연구에도 좋은 재료로 이용되리라 생각된다.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1993년도 제7회 식물생명공학 심포지움 식물 세포 분화의 분자적 접근 Seventh Symposium on Plant Biotechnology -Approach to Plant Cell Differentiation-
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• pp.1-36
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• 1993

It is possible to regenerate plants from calli, single cells and protoplasts of numerous species via organogenasis or embryogenesis in cell and tissue culture systems. Also such regeneration of plants can directly occur from cells of explants. However certain plant species has not been yet provided cultures suitable for plant regeneration from cells or tissues. For example, we have to confirm the regenerability of plant from cells before preparing transformed cells for application. Even more, it is very important to notice that regenerated plants in cell and tissue cultures often show structural abnormality. The mojority of those plants is functionally disordered and eventually cases degenerated. One of such examples is vitreous plants which are manifested mainly in the leaves and manifesteds to a lesser extent in the stems and roots. Regenerants in suspension cultures show more frequent vitrification than on gelled media so that relative humidity and water potential are the key factors involved in abnormal morphogenesis in vitro. The other is that somatic embryos formed in media containing BAP or high concentration of sucrose show frequently cotyledon aberrancy such as polycotyledon and born type cotyledon. The embryos with aberrant cotyledon of Codonopsis lanceolata could not germinate or regenerate into plants in many cases. In contrast, the polycotyledon embryos of Aralia cordata germinated in higher percentage than two cotyledonary embryos, but horn type cotyledonary embryos rarely germinated. The major cause of poor germination is the abnormal development of plumule apex meristem.

• Journal of Plant Biotechnology
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• 제32권1호
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• pp.51-55
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• 2005

양초(Leymus chinensis Trin.)의 성숙종자로부터 캘러스 유도조건 및 식물체 재분화 체계를 확립하였다. 성숙종자로부터 $1.5\;\cal{mg/L}$ 2,4-D가 포함된 MS 배지로부터 배양 6주 후, 캘러스가 높은 빈도 (약 $70\%$)로 유도되었다. 배발생 캘러스는 $2.0\;\cal{mg/L}$ kinetin과 $0.5\;\cal{mg/L}$ NAA가 첨가된 M배지에서 배양 3주 후부터 다양한 단계의 체세포배로 발달하였다. 식물 생장조절제가 포함되지 않은 MS 배지에서 배양4주부터 소식물체로 재분화되었다 (재분화율 $36\%$). 재분화된 소식물체를 1/2 MS 배지에서 1주 동안 배양하여 뿌리가 발달한 완전한 식물체로 성장하였으며 토양에 이식하여 온실에서 정상적인 식물체로 재배할 수 있었다. 본 연구를 통하여 확립한 재분화시스템은 분자육종을 통한 복합환경스트레스 양초의 개발에 유용하게 응용되어질 수 있을 것이다.

• 식물조직배양학회지
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• 제22권6호
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• pp.323-327
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• 1995

미나리의 체세포 배발생 과정을 해부학적으로 구명하기 위하여 배발생 기원세포와 캘러스를 광학현미경 및 전자현미경으로 관찰한 결과, 배발생 세포는 hematoxylin에 짙은 보라색으로, 비배발생 세포는 safranin에 적색으로 염색되어 광학현미경하에서 쉽게 구별할 수 있었다. 배발생 캘러스는 많은 수의 원배 및 발육중인 배, 비배발생 세포 등으로 구성되어 있었다. 체세포 배발생은 발육 중인 배나 세포괴의 표피세포에 위치한 배발생 세포의 하나가 분열하거나 세포괴내의 비배발생 세포속에 묻혀 있는 배발생 세포가 분열하여 일어났다. 배발생 과정은 항상 일정한 형태는 아니지만 단세포로부터 일정한 segmentation 과정를 거쳐서 배발생이 진행되는 것으로 나타났다. 투과전자현미경에 의한 관찰에서 배발생 세포는 비배발생 세포에 비하여 세포질이 조밀하고 핵이 대형이며 amyloplast, 인지질체 및 세포소기 관들이 많으며 액포가 없거나 매우 작았다. 이들 세포들은 두터운 세포벽에 의하여 주위의 비배발생 세포와 분리되어 있으며 세포윤곽은 둥글었다. 주사전자현미경으로 관찰한 배발생캘러스는 외부가 그물이 씌워진 형태의 구형의 다양한 크기의 배들과 비교적 크기가 큰 비배발생 세포들이 혼재하였다. 한편 비배발생능 캘러스는 구성세포가 크고 외부에는 gelatin같은 물질로 덮여 있었다.

• Molecules and Cells
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• 제20권2호
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• pp.228-234
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• 2005

Caenorhabditis elegans him-6 mutants, which show a high incidence of males and partial embryonic lethality, are defective in the orthologue of human Bloom's syndrome protein (BLM). When strain him-6(e1104) containing a missense him-6 mutation was irradiated with ${\gamma}$-rays during germ cell development or embryogenesis, embryonic lethality was higher than in the wild type, suggesting a critical function of the wild type gene in mitotic and pachytene stage germ cells as well as in early embryos. Even in the absence of ${\gamma}$-irradiation, apoptosis was elevated in the germ cells of the him-6 strain and this increase was dependent on a functional p53 homologue (CEP-1), suggesting that spontaneous DNA damage accumulates due to him-6 deficiency. However, induction of germline apoptosis by ionizing radiation was not significantly affected by the deficiency, indicating that HIM-6 has no role in the induction of apoptosis by exogenous DNA damage. We conclude that the C. elegans BLM orthologue is involved in DNA repair in promeiotic cells undergoing homologous recombination, as well as in actively dividing germline and somatic cells.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 춘계 학술발표대회
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• pp.61-62
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• 2003

Clonal propagation of high-value forest trees through somatic embryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Several factors limit commercialization of SE for Corsican pine, including low initiation rates, low culture survival, culture decline causing low or no embryo production, and inability of somatic embryos to fully mature, resulting in low germination and reduced vigour of somatic seedlings. The objective was to develop a Corsican pine maturation medium that would produce cotyledonary embryos capable of germination. Treatments were arranged in a completely randomized design. Data were analyzed by analysis of variance, and significant differences between treatments determined by multiple range test at P=0.05. Corsican pine (Pinus nigra var. maritima) cultures were initiated on modified !P6 medium. Modifications of the same media were used for culture multiplication and maintenance. Embryogenic cultures were maintained on the same medium semi solidified with 2.5 g/l Gelrite. A maturation medium, capable of promoting the development of Corsican pine somatic embryos that can germinate, is a combination of iP6 modified salts, 2% maltose, 13% polyethylene glycol (PEG), 5 mg!l abscisic acid (ABA), and 2.5 g/l Gelrite. After initiation and once enough tissue developed they were grown in liquid medium. Embryogenic cell suspensions were established by adding 0.951.05 g of 10- to 14-day-old semisolid-grown embryogenic tissue to 9 ml of liquid maintenance media in a 250ml Erlenmeyer flask. Cultures were then incubated in the dark at 2022$^{\circ}$C and rotated at 120 rpm. After 2.53 months on maturation medium, somatic embryos were selected that exhibited normal embryo shape. Ten embryos were placed horizontally on 20 ml of either germination medium ($\frac{2}{1}$strength Murashige and Skoog (1962) salts with 2.5 g/l activated charcoal) or same medium with copper sulphate adjusted to 0.25 mg/1 to compensate for copper adsorption by activated carbon. 2% and 4% maltose was substituted by 7.5% and 13% PEG respectively to improve the yield of the embryos. Substitution of' maltose with PEG was clearly beneficial to embryo development. When 2% of the maltose was replaced with 7.5% PEG, many embryos developed to large bullet-shaped embryos. At latter stages of development most embryos callused and stopped development. A few short, barrel-shaped cotyledonary embryos formed that were covered by callus on the sides and base. When 4% of the maltose was removed and substituted with 13% PEG, the embryos developed further, emerging from the callus and increasing yield slightly. Microscopic examination of the cultures showed differing morphologies, varying from mostly single cells or clumps to well-formed somatic embryos that resembled early zygotic embryos only liquid cultures with organized early-stag. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also tested. Seedling conversion and growth were highly related to the quality of the germinant at the time of planting. Germinants with larger shoots, longer, straighter hypocotyls and longer roots performed best. When mature zygotic embryos germinate the root emerges, before or coincident with the shoot. In contrast, somatic embryos germinate in reverse sequence, with the cotyledons greening first, then shoot emergence and then, much later, if at all, the appearance of the root. Somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.263-268
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• 2005

Porcine epidemic diarrhea virus (PEDV)는 돼지의 급성장염을 유발하여 설사 증상을 일으키는 바이러스이다. 본 연구에서는 고구마 저장뿌리 고발현 sporamin 프로모터 및 CaMV 35S promoter를 이용하여 PEDV 항원단백질을 생산하는 고구마 식물체를 개발하고자 하였다. 형질전환 벡터를 제작하기 위하여 PEDV에서 항원성이 알려진 스파이크 단백질의 일부분을 암호화하는 유전자를 PCR로 합성하였다. 고구마 [Ipomoea batatas (L.) Lam] 율미 품종의 배발생 캘러스를 재료로 하여 Agrobacterium tumefaciens을 매개로 형질전환하였다. 선발배지 (MS medium, 1 mg/L 2,4-D, 100 mg/L kanamycin, and 400 mg/L claforan)에서 배발생 캘러스를 3주 간격으로 4개월 동안 계대배양하여 카나마이신 저항성 캘러스를 선발하였다. 선발된 배발생 캘러스를 호르몬을 제거한 배지로 옮겨 체세포배를 유도하였으며 이후 shoot과 뿌리가 형성되었다. 재분화된 소식물체를 대상으로 PCR 분석으로 카나마이신 저항성 재분화 개체의 50% 이상이 도입 유전자를 가지고 있었으며 이들 식물체를 대상으로 Southern blot 분석하여 PEDV 유전자가 고구마 식물체의 게놈으로 안정적으로 도입되었음을 확인하였다. 형질전환 식물체에서 도입유전자의 발현을 RT-PCR로 분석한 결과 PEDV의 spike 유전자가 높은 수준으로 발현하였다.