• Applied Biological Chemistry
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• 제43권4호
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• pp.231-235
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• 2000

담배 배양 세포의 성장과정 중의 칼모듈린 농도변화 및 칼모듈린 결합 단백질의 종류에 대하여 조사하고 이들 단백질들 중 글루탐산 탈탄산효소를 immunodetection과 활성측정으로 확인하였다. 담배세포는 유도기(초기 $1{\sim}2$일간), 대수증식기($3{\sim}5$일), 정지기 등의 전형적인 성장 패턴을 보였다. 칼모듈린의 농도는 비록 대수증식기에 약간 감소하는 경향을 보이다 정지기에 이르면서 유도기의 수준을 회복하는 것으로 나타났지만 전체적으로는 성장단계에 관계없이 유사한 수준을 유지하는 것으로 나타났다. 주요 칼슘-의존형 칼모듈린 결합단백질은 56, 46, 36, 32-kDa의 4종류인 것으로 조사되었고, 모노클로날 항체를 이용하여 immunodetection을 실시해 본 결과 56-kDa 단백질이 담배 글루탐산 탈탄산효소로 확인되었다. 56-kDa의 글루탐산 탈탄산효소는 대수증식기에 수확한 세포에서 가장 많이 검출되었고, 이와같은 패턴은 효소활성 측정에서도 확인되었다. 이러한 결과들은 담배세포의 성장과정 중에 칼슘/칼모듈린-의존형 글루탐산 탈탄산효소 농도가 조절되고 있음을 제안해 주는 것이다.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2002년도 제38회 학술심포지움
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• pp.48-60
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• 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the production of granulocytes, macrophages, and white blood cells. The effects of osmotic pressure on secretion of human GM-CSF into the culture medium were investigated in suspension cultures of transgenic tobacco cells. An increase in osmotic pressure caused by the addition of mannitol decreased the cell size index, with the effect being more pronounced when cells were measured wet rather than dry. Increased osmotic pressure enhanced the secretion of hGM-CSF. At 90 g/L mannitol, the maximum concentration tested, hGM-CSF was present in the culture medium at 980 ug/L. As the concentration of mannitol increased, the total amount of protein secreted also increased, but was disproportionately enriched in GM-CSF NaCl, another osmoticum, had very similar effects on cell growth and hGM-CSF production, but did not cause enrichment for hGM-CSF Additionally, protein-stabilizing polymer was added to culture broth to enhance stability of secreted recombinant protein. Finally, above two method were applied together to maximize the productivity.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 1993년도 학술대회지
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• pp.143-149
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• 1993

Ginseng root explants and calli induced from selected cell lines were cultured on modified Murashige and Skoog's media supplemented with different concentrations of organic or inorganic compounds and plant growth requlators to clarify the effects of chemical composition and plant growth regulators in the medium on the growth of ginseng calli and the production of ginseng saponin. For optimum growth of calli, the concentrations of 2, 4-D and sucrose were the range of 1 to 3 mg/${\ell}$l and 1 to $3\%,$ respectively. And it was clarified that sucrose, nitrogen, phosphate, calcium, magmesian plant growth regulators and their concentrations influcenced the relative biosynthesis of saponin in tissue cultures of Panax ginseng. The patterns of ginsenosides, pharmacologically useful component, were different among the cell lines and contents of ginsenosides were much higher in selected cell lines than in original cell line.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.447-448
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• 2001

Human garnulocyte-colony stimulating factor(hG-CSF), a hematopoietic growth factor$^{1)}$, was produced and secreted from tobacco cell suspension. hG-CSF produced from tobacco cell suspension culture is biologically active form. The produced amount of hG-CSF is about 100${\mu}g/L$ in 9 days after inoculation.

• 한국연초학회지
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• 제17권1호
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• pp.33-40
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• 1995

Structures of the adventitious root meristem induced from callus culture of tobaco (Nicotiana tabacum cv. NC 82) leaves were investigated by light and transmission electron microscopy. Structural differences between in vitro root and callus cells were also examined by the microscopy. The submicroscopic features of the in vitro root cells were as follows. Intercellular spaces were not developed and nuclei with two nucleoli were observed occasionally. Plasmodesmate were found in groups or sing1y on transverse and longitudinal walls. Amyloplast solely filled with starch grains, with one to five electron - dense bands, was surrounded by single membrane. in the callus cells, vacuolization of central part in the cytoplasm having mitochondria with swollen cristae and starch grains like those of in vitro root cells was a distinct feature. Vesicles which were found between cell wall and plasma membrane may be arisen by a process of protoplasmic invagination. By comparing of ultrastructures between the cells of callus and in vitro roots we found that the distinct differences lied on thickened cell walls and hypertrophed vacuoles in the former, and less thickened cell walls and several small vacuoles in the later.

• Journal of Plant Biology
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• 제30권1호
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• pp.69-77
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• 1987

From the single cell cultures of haploid tobacco (Nicotiana tabacum L. cv. NC 2326) glyphosate-resistant plants were regenerated. After treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then inoculated onto the LS medium supplemented with 1 mM glyphosate, the single cells survived formed colonies and calluses in 65 and 95 days after culture, respectively, and then whole plants were regenerated in 0.1 mM glyphosate-containing medium from the selected calluses. There was no difference in fresh weight and shikimate content between the selected and normal haploid calluses. When sprayed with 0.1 mM glyphosate, the shikimate contents in the regenerated and normal plants were 0.659 and 20.816 mol/g fr. wt., while that in other normal plants which were not sprayed was 0.921. In addition, the calluses induced from the regenerated plants grew without showing any retardation when treated with glyphosate. These results indicate that the secelcted calluses and regenerated plants are resistant to glyphosate.

• Journal of Ginseng Research
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• 제43권2호
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• pp.261-271
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• 2019

Background: Protopanaxatriol (PPT) is an aglycone of ginsenosides, which has high medicinal values. Production of PPT from natural ginseng plants requires artificial deglycosylation procedures of ginsenosides via enzymatic or physicochemical treatments. Metabolic engineering could be an efficient technology for production of ginsenoside sapogenin. For PPT biosynthesis in Panax ginseng, damarenediol-II synthase (PgDDS) and two cytochrome P450 enzymes (CYP716A47 and CYP716A53v2) are essentially required. Methods: Transgenic tobacco co-overexpressing P. ginseng PgDDS, CYP716A47, and CYP716A53v2 was constructed via Agrobacterium-mediated transformation. Results: Expression of the three introduced genes in transgenic tobacco lines was confirmed by Reverse transcription-polymerase chain reaction (RT-PCR). Analysis of liquid chromatography showed three new peaks, dammarenediol-II (DD), protopanaxadiol (PPD), and PPT, in leaves of transgenic tobacco. Transgenic tobacco (line 6) contained $2.8{\mu}g/g$ dry weight (DW), $7.3{\mu}g/g$ DW, and $11.6{\mu}g/g$ DW of PPT, PPD, and DD in leaves, respectively. Production of PPT was achieved via cell suspension culture and was highly affected by auxin treatment. The content of PPT in cell suspension was increased 37.25-fold compared with that of leaves of the transgenic tobacco. Transgenic tobacco was not able to set seeds because of microspore degeneration in anthers. Transmission electron microscopy analysis revealed that cells of phloem tissue situated in the center of the anther showed an abnormally condensed nuclei and degenerated mitochondria. Conclusion: We successfully achieved the production of PPT in transgenic tobacco. The possible factors deriving male sterility in transgenic tobacco are discussed.

• 한국연초학회지
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• 제1권1호
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• pp.1-8
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• 1979

담배 (Va 115) 細胞의 液 培養에서 담배細胞의 多量生産을 할 수 있는 Tank 培養에 關한 基礎調査로 培地造成의 果를 調査하였으며 細胞增殖率에 큰 影響을 미쳤던 2,4-D와 無機燐酸의 果에 關하여 調査하였다. 1. 培地造成에서 細胞增殖率에 많은 影響을 미쳤던 要素는 무糖, 無機燐酸의 濃度,窒素源의 形態 및 植物홀몬,特히 2, 4-D의 有無였다. 2. 무糖의 最適濃度는 3%였으며 3%以上의 濃度에서는 多少 細胞增殖率이 좋은 듯 하였으나 그 差異는 크지 않아 3% 程度로 足하였다. 無機燐酸濃度는 LS 培地內의 無機燐酸의 約 2.5 培인 0.30mg/ml 일 때 細胞增殖率이 가장 좋았다. 3. 液 培地의 窒素源은 암모니아態窒素와 硝酸態窒素가 1 : 2 일 때가 가장 좋았고 窒素源이 암모니아태 만으로 使用하였을 때 細胞增殖率이 가장 낮았으며 硝酸態만 使用되었을 때는 암모니아태만 쓰였을 때 보다는 좋았으나 암모니아태와 硝酸態의 比가 1 : 2 일 때 보다는 떨어졌다. 4. 液 培地에 2,4-D 添加와 無機燐酸濃度를 높이면 細胞增殖率이 增加되는 機作을 調査하기 爲하여 呼吸率과 14C - glucose 吸收利用을 調査하였다. 細胞의 吸收率은 2, 4-D를 添加하면 增加되며 14C-glucose의 吸收는 培地內에 2, 4-D가 包含되거나 (0.2 ppm) 燐酸濃度가 높아지면(對照의 2.5培) 더욱 많았고, 吸收된 14C-glucose는 糖 상태보다 다른 形態,特히 amino 酸이나 有機酸으로 많이 变하였다.

• 식물조직배양학회지
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• 제27권2호
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• pp.133-136
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• 2000

미생물에서 분리한 dykellic acid가 식물세포에 미치는 생리적 영향을 평가하기 위하여, 담배 광혼용영양배양세포(photomixotrophic cultured cells, PM세포)에 dykellic acid를 다양한 농도로 처리하여 세포의 생장과 단백질 함량 및 superoxide diamutase (SOD)활성에 미치는 영향을 조사하였다. 담배 PM세포는 0.7 mg/L 2,4-D, 0.3 mg/L kinetin, 30g/L sucrose, 200 mM NaCl를 함유한 MS배지에서 $25^{\circ}C$, 광조건에서 현탁배양 (100 rpm) 하였다. 계대배양시 화합물을 처리한 후 12일째의 세포생장 억제와 배지의 이온 전도도를 측정한 세포막손상의 결과는 일치하였으며, 화합물은 세포생장을 강하게 억제시켰다 ($IC_{50}$/, 약 20 $\mu$M). Dykellic acid 처리농도가 증가함에 따라 단위세포 무게당 단백질 함량과 SOD 비활성도를 현저히 증가시켰다. 이상의 결과로 dykellic acid는 식물세포 생장을 저해하는 활성을 가지고 있으며, 담배 PM세포는 적은 양으로도 천연물의 생리활성을 평가할 수 있는 유용한 생물소재임이 확인되었다.

• Journal of Plant Biology
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• 제31권2호
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• pp.121-130
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• 1988

In order to clarify effects of phytohormones on the viability and the cell wall regeneration of protoplasts isolated from Nicotiana tobacum L. var. BY4, protoplasts isolated from mesophyll tissue were cultured on the Murashige-Skoog liquid media supplemented with auxin(2, 4-D, NAA, IAA) and/or cytokinin (kinetin, BAP, 2ip). Viability of protopplasts was higher in the culture medium containing auxin and cytokinin, especially in the combination of 2, 4-D and BAP. The effectual cell wall regeneration of protolasts was observed when theprotoplasts were cultrued on the medium supplemented with auxin alone, especially with IAA. Cell wall regernation started from 2-3 days after culture and was not detected at budding regions. When the protoplasts were cultured on the phytohormone-free medium, the viability of protoplasts dramatically decreased 4 days after culture.