• KSBB Journal
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• 제6권1호
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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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• 제15권5호
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• pp.525-528
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• 2000

Taxus chinensis 세포배양 중의 FCW/DCW ratio, 세포내, 외 단백질 생산 및 peroxidase 활성의 변화를 배양시기별로 조사하였다. 조사 결과, 배지내 잔류당이 고갈되기 직전인 배양 14일째에 FCW/DCW ratio 는 12로 가장 낮은 수치를 나타내었고 DCW를 기준으로 한 세포 외에서의 protein 생산도 14일째 가장 낮은 수치인 4.3 mgfg DCW 였다. 배양시기별 P POD 활성도는 단백질 생산 pattern과 유사하였다. 이 결과들 은 flask에서 활발하게 증식 중인 세포들을 이용하여 측정한 것으로 대량배양시, 공정제어를 위한 자료 및 세포가 받는 stress 정도를 나타내는 지표로 활용할 수 있다

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.104.1-104
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• 2003

To screen for potential biocontrol agents against postharvest disease of garlics caused by Penicillium hirsutum, a total of 933 isolates (432 fungi and 501 bacteria) were isolated from the rhizoshere or rhizoplane of garlics. Among them, Pantoea agglomerans isolate 59-4 (Pa 59-4) was selected for a potential biocontrol agent by in vivo wounded garlic bulb assay, When the spore suspension (10$\^$5/ spores/$m\ell$) of Penicillium hirsutum was co-inoculated with spore or cell suspension of each fungal or bacterial isolate on wounded garlics, the isolate highly suppressed disease development. Soaking garlic bulbs in the suspension of Pa 59-4 significantly reduced garlic decay from p. hirsutum. However, Pa 59-4 did not inhibit the mycelial growth of P. hirsutum in dual-culture with P. hirsutum on Tryptic soy agar. In order to elucidate mode of action of Pa 59-4 nutrient competition between Pa 59-4 and P. hirsutum was investigated using tissue culture plates with cylinder inserts containing defusing membrane reported by Janisiewicz et al. The results showed that Pa 59-4 effectively suppressed spore germination and mycelial growth of blue mold in the low concentration (0.5％) of garlic juice, but did not suppress those of blue mold in the higher concentration (5％) of garlic juice. This result suggests that the mechanism in biocontrol of garlic blue mold by Pa 59-4 may involve in nutrient competition with P. hirsutum on garlic bulbs.

• 한국식생활문화학회지
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• 제27권6호
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• pp.743-750
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• 2012

A tissue cultured wild ginseng (TCWG) suspension was inoculated with lactic acid bacteria and fermented to improve the functionality of TCWG. The utilization of TCWG was increased directly using the freeze-dried powder. The optimal ratio of TCWG powder and water for fermentation was 1:19 (5%), which was selected by measuring the fluidity and viable cell count according to concentration. The effects on ADH activation and immune cell activation by each ferments with 10 kinds of Lactobacillus sp. strains were examined. The ferments with the Lactobacillus casei KFRI 692 strain showed 5.4 times higher ADH activity and 1.3 times higher ALDH activity than the non-fermented TCWG powder (control). The level of NO production and cytotoxicity was also measured by Raw 264.7 cells. The ferment with the Lac. casei KFRI 692 strain showed the highest level of NO production and lower cytotoxicity than the others. Therefore, the Lac. casei KFRI 692 strain was selected as a strain for fermentation of a TCWG suspension to maximize its functionality. To identify the optimal fermentation time of the selected Lac. casei KFRI 692 strain on the 5% TCWG suspension, the viable cell count of lactic acid bacterial and the changes in pH were observed for 72 hours. 24-hrs was found to be the optimal fermentation time. In this way, fermented TCWG with lactic acid bacteria showed higher ADH activation efficacy and immune cell activation than non-fermented TCWG.

• Plant Biotechnology Reports
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• 제4권2호
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• pp.125-128
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• 2010

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 $mg\;l^{-1}$ of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 $mg\;l^{-1}$ with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 $mg\;l^{-1}$ of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.

• 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.

• Journal of Plant Biotechnology
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• 제3권2호
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• pp.107-112
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• 2001

The kinetics of Ti-transformed Salvia miltiorrhiza cell cultures was studied in 250-$m\ell$ shake flasks by using B5 medium with addition of 30 gfL of sucrose. In the cell cultures, the maximum cell mass obtained was 11.5 g DW/L on day 15. The highest amount of phenolic compounds - rosmarinic acid (RA) and lithospermic acid B (LAB) reached 871.3 mg/L (day 15) and 121.3 mg/L (day 13), respectively. The total tanshinone production, i.e., intracellular plus extracellular cryptotanshinone, tanshinone 1, and tanshinone IIA, was 5.3 mg/L on day 13. For the cultivations in 2.4-L stirred bioreactors, the residual sugar level and medium conductivity were a little higher in a small turbine impeller reactor ($T_s$) than those in a large turbine impeller reactor ($T_L$), while a higher cell density was obtained in the $T_L$. For the production of tanshinones and phenolics, better results were obtained in the $T_L$ than in the $T_s$. In the $T_L$, similar or even a little higher production titers of tanshinones and phenolic compounds were achieved compared to those in the flasks. The results suggest that the shake flask results could be successfully scaled up to the $T_L$ reactor. Such a large impeller reactor like $T_L$ may be better than a small impeller one for the large-scale production of the valuable metabolites by the suspension cultures of Ti transformed S.miltiorrhiza cells. This is considered due to the beneficial culture environment in the $T_L$, such as low shear rates as estimated theoretically.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.245-254
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• 2011

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing $N^6$-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.256-262
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• 2003

To enhance the production of heavy chain immunoglobulin G (HC IgG) in the suspension cultures of transgenic tobacco cells (Nicotiana tabacum), the effects of adding various cytokinins (i.e., growth regulators) and organic nitrogen sources to culture media were investigated. Four different cytokinins including kinetin, isopentenyladenine (IPA), 6-benzylaminepurine (BA), and zeatin were tested with or without dichlorophenoxyacetic acid (2,4-D), which is a typical growth regulator supplemented in the standard Murashige and Skoog (MS) medium. The productivity of intracellular HC IgG was increased by 36 and $42\%$, compared to the control, especially when IPA (2 mg/l) or BA (0.2 mg/l) was added to the media in the presence of 2,4-D, respectively. In the study of organic nitrogen sources, addition of each casein hydrolysate and tryptone to the culture media at a final concentration of 0.01 and 1 g/l, respectively. increased the productivity or he IgG as much as 68 and $67\%$, respectively, in comparison with the control, which was is MS medium without supplementation of any organic nitrogen sources. This study shows that the optimization of media composition could offer significant improvements in the production of foreign proteins in the suspension cultures of transgenic plants.

• 한국식품영양학회지
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• 제11권6호
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• pp.622-628
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• 1998

Microorganisms isolated from soil were tested for their flocculating activity in kaolin suspension, Identification of the best producing CH-23 strain showed that the strain belonged to the Bacillus megaterium. The maximum production of the flocculating from Bacillus megaterium CH-23 was observed in the culture medium containing 2% sucrose, 3% NaNo3, 0.1% K2HPO4, 0.5% NaCl, 0.5% MgSO4.7H2O and 0.01% tryptone at initial pH 7.0 and 25~3$0^{\circ}C$. Flocculating activity was improved to 57% when the culture medium contained Mn2+(0.01% MnSo4). In the culture medium containing Mg2+(0.01% MgSO4.7H2O) and Ca2+(0.01% CaCO3), flocculating activity were reached to 48% and 33%, respectively.