• KSBB Journal
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• v.13 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.255-262
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• 2008

Methyl jasmonate (MJ) and yeast extract (YE) induce protein expression and benzophenanthridine alkaloid accumulation in Eschscholtzia californica suspension cell cultures. One hundred ${\mu}M$ MJ primarily induced dihydrosanguinarine $(509.0{\pm}7.4mg/l)$ ; 0.2g/l YE induced sanguinarine $(146.8{\pm}3.8mg/l)$ and an unknown compound. These results occur because dihydrobenzophenanthridine oxidase (DHBO) is induced by YE and not by MJ. YE and chitin (CHI) had similar effects on sanguinarine production and DHBO expression. Differential induction of secondary metabolites was shown in E. californica suspension cultures and the expression of proteins confirmed the metabolite results. Furthermore, treatment by various oligosaccharides helped us to understand the elicitation effect of YE in signal transduction pathways.

• KSBB Journal
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• v.6 no.1
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• pp.1-7
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• 1991

To produce economically important indole alkaloids by cell cultures, we selected protoplastsderived clones (protoclones) of vinca (Catharanthus roseus) for high yields of catharanthine and ajmalicine. Protoplasts were enzymatically isolated from suspension-cultured cells. The highest plating efficiency (1%) was obtained when protoplasts were plated at a density of 1$\times$105 protoplasts/ml in a culture medium solidified with 0.4% Seaplaque agarose. The growth rates of 40 protoclones subcultured on a solid medium varied over a wide range. Protoclone VPC-6, which had the highest growth rate, was observed to produce relatively high yields of catharanthine and ajmalicine when cultured in a liquid medium. Although the original cell line did not produce catharanthine at a detectable level by HPLC, protoclone VPC-10 produced it at a level of 5.9$\mu\textrm{g}$/g fresh weight of cells for 10 days of culture. Under the same conditions, protoclone VPC-15 produced ajmalicine at a level of 133.6$\mu\textrm{g}$/g, of which productivity was improved about ,3 times than that of the original cell line. The results indicate that differences in the growth rate and indole alkaloid yield among the protoclones reflect the somaclonal variation in suspnsion-cultured cells.

• KSBB Journal
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• v.30 no.3
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• Journal of Plant Biology
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• v.38 no.1
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• pp.55-62
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• 1995

Embryogenic cell (EC) suspension cultures derived from mature seed-embryo of rice (Oryza sativa L cv. Kye Hwa) were used for the expression patterns of isozyme and enzyme activity. EC suspension cultures were composed of cells that were densely cytoplasmic, potentially embryogenic. However, nonembryogenic cell (NEC) cultures were composed of large, elongated and vacuolated cells. These cells were analyzed for the isozyme pattern and enzyme activity of EC and NEC. Isozyme patterns of peroxidase, esterase, acid phosphatase and malate dehydrogenase exhibited striking difference in the total number of bands, specificity and intensity of band. Also, these isozymes showed very high activity in the EC. Specific band, band activity and higher enzyme activity of isozyme in EC was absent or low in NEC, which may indicate an association of these specific isozymes with morphological characterization and totipotency of embryogenic cells. These results indicate that specific pattern and activity of enzyme in EC could probably be used as a biochemical marker of EC in rice.n rice.

• Journal of Plant Biotechnology
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• v.34 no.3
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• pp.207-212
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• 2007

This study describes a simple and rapid method for quantitative determination of $\gamma$-aminobutyric acid (GABA) using $^1H$ NMR spectroscopy from whole cell extracts of plant suspension cultures. When 9 cell lines derived from 8 species of higher plants maintained in liquid Marashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) were subjected to $^1H$ NMR, a cell line of Coriandrum sativum L. exhibited the highest level of GABA. The level reached up to 16.9 mg/dry wt when cells were cultured in MS medium supplemented with 0.5 mg/L 2,4-D after 3 weeks of incubation. The method for quantitative determination of GABA using $^1H$ NMR established in this study could be applied to high-throughput screening of various plant resources for GABA production and the cell suspension culture system of C. sativum could be further developed for commercial production of GABA.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.23-33
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• 1985

Plant cell culture is emerging to express bioactive foreign proteins because it has several advantages in that it is safe, economical, genetically stable and eukaryotic expression system comparing with other expression systems. However several limitations such as slow growth rate, low expression level and lack of well established down stream process need to be answered. As a preliminary approach to produce the immunologically interested molecules through the plant cell culture, we tested if granulocyte-macrophage colony stimulating factors (GM-CSFs) from both murine (mGM-CSF) and human (hGM-CSF) are produced as a biologically active form through plant cell culture. The murine and human GM-CSF genes were cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring both recombinant GM-CSF (rGM-CSF) genes. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activities of both murine and human GM-CSF from plant cell culture were confirmed by measuring the proliferation of the GM-CSF dependent FDC-PI and TF-1 cells, respectively.

• Proceedings of the Botanical Society of Korea Conference
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• 1995.06a
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• pp.40-56
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• 1995

During the past two decades, China has seen her great progress in plant biotechnology. Since the Chinese market of herb medicine is huge, while the plant resources are shrinking, particular emphasis has been placed in plant tissue and cell cultures of medicinal plants, this includes fast propagation, protoplast isolation and regeneration, cell suspension cultures and large scale fermentation. To optimize culture conditions for producing secondary compounds in vitro, various media, additives and elicitors have been tested. Successful examples of large scale culture for the secondary metabolite biosynthesis are quite limited : Lithospermum ery throrhizon and Arnebia euchroma for shikonin derivatives, Panax ginseng, P. notoginseng, P. quinquefolium for saponins, and a few other medicinal plants. Recent development of genetic transformation systems of plant cells offered a new approach to in vitro production of secondary compounds. Hairy root induction and cultures, by using Ri-plasmid, have been reported from a number of medicinal plant species, such as Artemisia annua that produces little artemisinin in normal cultured cells, and from Glycyrrhiza uralensis. In the coming five years, Chinese scientists will continue their work on large scale cell cultures of a few of selected plant species, including Taxus spp. and A. annua, for the production of secondary metabolites with medicinal interests, one or two groups of scientists will be engaged in molecular cloning of the key enzymes in plant secondary metabolism.

• Korean Journal of Pharmacognosy
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• v.16 no.3
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• pp.171-174
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• 1985

Panax ginseng root has been widely used as an important drug for thousands years in China, Korea and Japan. The main effective components of ginseng have been believed to be saponins. However, ginseng cultivation is very difficult and needs many years for growth. It has already been shown that Panax ginseng callus produces a considerable amount of the same kinds of saponins as in intact plants. Various culture conditions were examined for increased production of ginseng saponins by cell culture. The saponin contents and the growth rates in two cell lines of ginseng callus were compared in static and suspension cultures, rotary and reciprocal shaking cultures. It was shown that the growth rate in rotary shaking cultures of D5-B2K-B2K callus was the highest and ginseng saponin production was most effective in reciprocal cultures of D5-B2K-B2K callus. The saponin content per fresh weight of the culture was 1.03 times higher than that of the fresh ginseng root.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.