• Fisheries and Aquatic Sciences
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• 제19권2호
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• pp.9.1-9.7
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• 2016

This study was conducted to identify the general conditions for the isolation and in vitro culture of ovary-derived cells in rockfish (Sebastes schlegeli). The effects of three different enzymes on cell retrieval from ovarian tissues were evaluated first, and then the ovary-dissociated cells were cultured under various culture conditions, with varying basal media and culture temperatures, addition of growth factors, and/or culture types. We found that collagenase type I treatment was effective for cell isolation from ovarian tissues. From a total of 42 trials to evaluate the effects of basal media and culture temperatures on cell culture of ovary-dissociated cells, we observed that Leibovitz's L15 medium was more supportive than Dulbecco's modified Eagle's medium for culture, and the cells could grow at all three temperatures tested, 15, 20, and $25^{\circ}C$, at least up to passage 2. However, growth factor addition did not improve cell growth. Introduction of suspension culture after monolayer culture expanded the culture period significantly more than did monolayer culture alone. Our results may provide a basis for developing an in vitro system for S. schlegeli germline cell culture, which will ultimately lead to improvement of the species.

• 식물조직배양학회지
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• 제22권2호
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• pp.111-114
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• 1995

지치 (Lithospermum erythorhizon SIEB, et ZUCC)의 잎조직으로부터 유도한 캘러스를 현탁배양하여 약리성 지방산인 ${\gamma}$-linolenic acid (GLA)를 생산하는 기술을 확립하고자 탄소원, 질소원, 생장조절제의 영향을 조사하였다. 캘러스 유도는 1.0 mg/L 2,4-D 단용첨가시 가장 왕성하였다. Sucrose 88mM농도에서 세포생육이 가장 왕성하고 총지방산(TFA)중 GLA비율이 높아 단위건물중당 TFA함량이 가장 낮았음에도 flask당 GLA생산량은 가장 많았다. 질소원으로서 potassium nitrate농도증가는 세포생장 및 지방산함 량 및 GLA함량을 증가시켰으며 최고 처리농도인 56.3 mM 에서 최고수준을 나타냈다. IAA 첨가는 배양세포 생장량을 증가시켰고 2,4-D는 배양세포내의 총지방산 함량을 높이는 결과를 나타내었으나, kinetin과 BA는 세포생육에 저해적 영향을 나타냈다. $10^{-7}$ M ABA는 캘러스생장을 유의적으로 증가시켰으며 TFA 및 GLA함량도 증가시켰으나 현탁배양세포의 생육과 지방산 축적에는 저해적으로 작용하였다.

• 한국작물학회지
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• 제42권3호
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• pp.306-316
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• 1997

약배양을 통해 유도된 벼의 품종 Zhonghua 8의 종자로부터 배발생 캘러스를 유기한 캘러스로부터 현탁배양을 실시하였다. 원형질체 분리 는 이러한 현탁배양된 캘러스를 사용하였으며, 일반적으로, 오래되고 미세한 현탁배양세포를 사용했을 때 어린 현탁배양세포보다 원형질체 나출율이 증가되었다. 원형질체는 feeder cell 없이 agarose embedding 방법에 의해 0.5 mg $l^{01}$ 2,4-D, 1.0mg $l^{-1}$ NAA와 0.5 mg $l^{-1}$zeatin이 첨가된 KPR 배지에서 배양하였을 때 세포분열이 일어났으며 microcalli가 형성되었다. 원형질체의 plating 효율은 0.20~0.54% 범위로 나타났으며, 원형질체로부터 유도된 microcalli는 식물체 재분화를 위해 2.0 mg $l^{-1}$ kinetin과 0.5 mg $l^{-1}$ NAA가 첨가된 MS 배지에 옮겨 주었다 실물체 재분화 빈도는 현탁배양의 line에 따라 2~l2%였다. 원형질체로부터 재분화된 식물체들은 온실에서 종자를 맺었다었다

• Journal of Plant Biotechnology
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• 제4권2호
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• pp.83-89
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• 2002

To establish in vitro mass production system of grape anthocyanin pigments through callus and cell suspension culture, the effects of nitrogen amount and the ratio of $NO_3^-$/$NH_4^+$ in the medium on cell growth and anthocyanin production were investigated. Total nitrogen amount and the ratio of $NO_3^-$/$NH_4^+$ in the medium strongly affected anthocyanin production and cell growth. When $NH_4^+$ was fixed, the cell growth was promoted by 50 mM total nitrogen (20 mM $NO_3^-$ : 30 mM $NH_4^+$ ) than other nitrogen combinations, and was strongly inhibited when $NO_3^-$ was lacking (0 mM $NO_3^-$ : 60 mM $NH_4^+$ ) while anthocyanin production was increased. When $NO_3^-$ was fixed, the cell growth was promoted by 70 mM total nitrogen (40 mM $NO_3^-$ : 30 mM $NH_4^+$) than other nitrogen combinations, and was strongly inhibited when $NO_3^-$ was lacking (0 mM $NO_3^-$ : 60 mM $NH_4^+$ ) while anthocyanin production was increased. Cell growth was gradually increased by all nitrogen combinations, but anthocyanin production reached its peak on day 4 in culture. Anthocyanin content increased with decreasing cell density. Sucrose was rapidly hydrolyzed to fructose and glucose within 4 days. Glucose and fructose concentrations in the medium increased and peaked at the 4th day. The anthocyanin content of $NH_4^+$-free 2% sucrose media was 2 times (200 $\mu\textrm{g}$/g) higher than that of 1% sucrose. When $NO_3^-$ was lacking, the highest anthocyanin production was observed at 4% sucrose after 12 days of culture, and increased along with the sucrose concentration.

• 한국산업융합학회 논문집
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• 제2권2호
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• pp.73-81
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• 1999

Shake mixing has been widely used in cell culture. The mixing performance for shake mixing, however, has not been reported quantitatively. The critical circulating frequency and the power consumption for complete suspension of particles, based on the definition of Zwietering, were measured in a shaking vessel containing a solid-liquid system. The critical suspension frequency was correlated by the equation from Baldi's particle suspension model modified with the physical properties of the particles. Critical suspension frequency was correlated as following ; $$N_{JS}={\frac{0.58\;d{_p}^{0.06}(g{\Delta}{\rho}/{\rho}_L)^{0.004}X^{0.03}}{D^{0.35}d^{0.17}{\upsilon}^{0.04}}}$$ The power consumption at the critical suspension condition in the shaking vessel was less than that in an agitated vessel with impeller.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1944-1948
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• 2007

Silkworm hemolymph (SH), prepared from fifth-instar larvae of Bombyx mori and heat-treated at $60^{\circ}C$ for 30 min, was used to improve cell viability and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic Oryza sativa L. cell suspension cultures. Even though SH could not elevate cell viability at the concentrations up to 3% (v/v), addition of 0.3% (v/v) SH to a culture medium enhanced the production of hCTLA4Ig by 36.8% over an SH-free medium. Moreover, the production period of hCTLA4Ig could be shortened in a 0.3% (v/v) SH-added medium compared with that in an SH-free culture. As a result, addition of 0.3% (v/v) SH improved the productivity of hCTLA4Ig significantly in transgenic rice cell cultures.

• KSBB Journal
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• 제13권4호
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• pp.438-443
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• 1998

Effects of various environmental factors on cell size index(FCW/DCW) in Thalictrum rugosum. Lithospermum erythrorhizon and Taxus cuspidata plant cell suspension cultures were investigated. Time course change of cell size index were also observed. In batch cultures, FCW/DCW increased according to the decrease of sugar concentration. For short-term experiment within 24 hr, FCW/DCW value could be reduced significantly by increasing sugar concentration. When an osmoticum such as mannitol was added, FCW/DCW converged to a low value. Therefore, it was confirmed that osmolality of the medium was important in determining cell size or water content of the cells. Inorganic salts or treatment with organic solvent also exhibited some effect on the cell size index. However, pH and centrifugal force did not show any influences. On the other hand, it was found that the addition of Pluronic F-68 reduced FCW/DCW. By combining these results effectively, it may be possible to increase the cell concentration in high density culture to a higher extent.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2007년도 춘계학술발표회
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• pp.65-73
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• 2007

The objective of this study is to understand how regulatory mechanisms respond to sugar status for more efficient carbon utilization and source-sink regulation in plants. So, we need to identify and characterize many components of sugar-response pathways for a better understanding of sugar responses. For this end, genes responding change of sugar status were screened using Arabidpsis cDNA arrays, and confirmed thirty-six genes to be regulated by sucrose supply in detached leaves by RNA blot analysis. Eleven of them encoding proteins for amino acid metabolism and carbohydrate metabolism were repressed by sugars. The remaining genes induced by sugar supply were for protein synthesis including ribosomal proteins and elongation factors. Among them, I focused on three hydrolase genes encoding putative $\beta$-galactosidase, $\beta$-xylosidase, and $\beta$-glucosidase that were transcriptionally induced in sugar starvation. Homology search indicated that these enzymes were involved in hydrolysis of cell wall polysaccharides. In addition to my results, recent transcriptome analysis suggested multiple genes for cell wall degradation were induced by sugar starvation. Thus, I hypothesized that enzyme for cell wall degradation were synthesized and secreted to hydrolyze cell wall polysaccharides producing carbon source under sugar-starved conditions. In fact, the enzymatic activities of these three enzymes increased in culture medium of Arabidopsis suspension cells under sugar starvation. The $\beta$-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved condition with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. Further, contents of cell wall polysaccharides especially pectin and hemicellulose were markedly decreased associating with sugar starvation in detached leaves. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These results supported my idea that cell wall has one of function to supply carbon source in addition to determination of cell shape and physical support of plant bodies.

• 한국산림과학회지
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• 제78권2호
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• pp.198-208
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• 1989

버드나무의 잎으로부터 유도(誘導)된 callus의 현탁배양(懸濁培養)은 MS 기본배지(基本培地)에 2,4-D 1.0mg/l와 zeatin 0.1mg/l를 첨가(添加)한 액체배지(液體培地)에서 생장(生長)이 가장 좋았다. 액체배지(液體培地)에서 24일간(日間) 세포(細胞)를 배양(培養)한 후(後) 분리(分離)된 배양액(培養液)은 상치, 벼 그리고 피 종자(種子)의 발아(發芽)를 강(强)하게 억제(抑制)하여 배양중(培養中)에 생리활성(生理活性) 물질(物質)이 형성(形成)되는 것으로 추정(推定)되었다. 배양세포(培養細胞)에서는 pyrogallol, sinapic acid, cinnamic acid, tannic+gallic acid 그리고 P-chlorobenzoic acid 등(等)과 같은 여러 종류(種類)의 phenol 화합물(化合物)이 검출(檢出)되어 이들이 강(强)한 발아(發芽) 억제작용(抑制作用)을 하는 것으로 사료(思料)되었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.396-401
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• 2006

The objective of this study was to investigate the antitumor activity of suspension cultures of tea callus cells grown in the presence of different concentrations of the growth regulator 2,4-dichlorophenoxy acetic acid (2,4-D) with or without light irradiation. The methanol and ethanol extracts of precipitated cells (MEP, EEP) exhibited stronger inhibitory effects on the growth of tumor cell lines than the water extract of precipitated cells (WEP) or the supernatant Compared to culture under dark conditions, exposure to light irradiation led to significantly higher antitumor activity. The MEP from light irradiated cells at $250{\mu}g/mL$ with 2.0mg/L 2,4-D displayed more than 64% growth inhibition of HEP-2 cells, whereas normal cells showed less than 25% growth inhibition. The some fractions of MEP obtained from Diaion HP-20 column chromatography displayed the majority of inhibitory activity against the HEP-2 cell line. These results show that 2,4-D, and light stimulated the synthesis of antitumor compounds.