• Korean Journal of Plant Resources
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• v.24 no.3
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• pp.280-285
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• 2011

In this study, we established a novel somatic embryogenesis and plant regeneration system through cell suspension culture of white dandelion (Taraxacum coreanum NAKAI.). Embryogenic calli could be initiated from leaf and root explants of sterile seedlings on solid Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 3-week cultures. To proliferate embryogenic calli rapidly, cell suspension culture was performed with transferred to liquid MS medium with various combinations of plant growth regulators (PGRs) including 2,4-D, ${\alpha}$-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), $N^6$-benzylamino purine (BAP), thidiazuron (TDZ), and kinetin. During suspension cultures, embryogenic calli not only greatly proliferated, but shoot organogenesis also simultaneously occurred from the surface of somatic embryos. Among them, TDZ at lower concentration, 0.1 mg/L produced the highest efficiency of somatic embryo formation and shoot organogenesis. Rooting of embryogenic calli with adventitious shoots was done on solid MS medium containing 0.1 mg/L NAA and 0.3% activated carbon. Nearly 80% of embryogenic calli with shoot organogenesis could be rooted normal. Well-rooted plantlets were transferred into pots under a greenhouse condition, and plants derived from this system appeared phenotypically normal.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.564-570
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• 2005

In the present study, the protective effects of Bcl-2 over-expression in a suspension culture (without any adaptation) and spent medium (low nutrient and high toxic metabolite conditions) were investigated. In the suspension culture without prior adaptation, the viability of the control cell line fall to 0% by day 7, whereas the Bcl-2 cell line had a viability of 65%. The difference in the viability and viable cell density between the Bcl-2 and control cell lines was more apparent in the suspension culture than the static culture, and became even more apparent on day 6. Fluorescence microscopic counting revealed that the major mechanism of cell death in the control cell line in both the static and suspension cultures was apoptosis. For the Bcl-2 cell lines, necrosis was the major mode of cell death in the static culture, but apoptosis became equally important in the suspension culture. When the NS0 6A1 cell line was cultured in spent medium taken from a 14 day batch culture, the control cell line almost completely lost its viability by day 5, whereas, the Bcl-2 still had a viability of 73%. The viable cell density and viability of the Bcl-2 cell line cultivated in fresh medium were 2.2 and 2.7 fold higher, respectively, than those of the control cultures. However, the viable cell density and viability of the Bcl-2 cultivated in the spent medium were 8.7 and 7.8 fold higher, respectively, than those of the control cultures. Most of the dead cells in the control cell line were apoptotic; whereas, the major cell death mechanisms in the Bcl-2 cell line were necrotic.

• Journal of Plant Biotechnology
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• v.47 no.3
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• pp.227-234
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• 2020

To produce the miraculin protein in suspension cultures, rice (Oryza sativa L.) was transformed with Agrobacterium tumefacience EHA105 containing the miraculin AB512278 gene. The cell suspension cultures were established using cell lines selected from transgenic rice callus. The integration of the miraculin gene into the rice chromosome was confirmed using genomic PCR analysis. In addition, RT-PCR analysis indicated that the miraculin gene is expressed in the selected suspension cell lines. Thus, the recombinant miraculin was expressed in the transgenic suspension cell line, HK-2. Therefore, we have successfully developed a HK-2 line that produces miraculin. These results demonstrate that transformed cell suspension cultures can be used to produce a taste-modifying protein such as miraculin.

• KSBB Journal
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• v.19 no.5
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• pp.374-380
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• 2004

Production of therapeutic proteins by transgenic plant cell suspension cultures is an attractive system alternative to the other expression system. However, plant cell cultures have shown low expression level of foreign proteins and decreased cell viability by the changes of culture conditions. Therefore, it is necessary to enhance cell viability during the culture period. In this study, a quantitative analysis technique was designed to measure relative cell viability for plant suspension cells which have cell wall and aggregates. It was found that the programmed cell death of plant cells by apoptosis was essentially linked with the apoptotic pathway of animal cells. Therefore, effects of nicotinamide, 3-aminobenzamide and antioxidants on cell viability and apoptosis were examined in transgenic Nicotiana tabacum cells producing hGM-CSF. With those additives, cell viability could be maintained and apoptosis could be redued. In the result, the extracellular production of hGM-CSF could be enhanced 2.5 fold. It was also found that the supplementation of glutathione and ascorbic acid suppressed both the cold stress-induced decrease in cell viability and the increase of total genomic DNA fragmentation.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.33-39
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• 2008

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with $0.5mg\;l^{-1}$ 2,4-D and $1.0mg\;l^{-1}$ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin ($0.0-2.0mg\;l^{-1}$), $GA_3$ ($0.0-2.0mg\;l^{-1}$) and AS ($80.0mg\;l^{-1}$). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on $GA_3$-free medium, but the best morphological quality of embryos was observed in the presence of $0.5-1.0mg\;l^{-1}$ Kin, $0.5mg\;l^{-1}$ $GA_3$ and $80.0mg\;l^{-1}$ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.

• Journal of Plant Biotechnology
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• v.5 no.2
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• pp.131-135
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• 2003

Callus and cell suspension cultures of Eurycoma longifolia Jack were initiated from leaves of different trees. The leaf explant of tree Eu9 produced the most calli and also induced high cell biomass in the cell suspension culture. Optimum production of cell biomass could be initiated in proliferating culture medium with a pH of 5.75 prior to autoclaving. The effects of macronutrient inorganic salts of Murashige and Skoog (MS) liquid medium supplemented with X on production of cell biomass of Eurycoma longifolia were also investigated. The highest cell biomass was produced in MS medium containing macronutrients of $21\;mM\;NH_4NO_3,\;12.25\;mM\;KNO_3,\;3.00\;mM\;CaCl_2.2H_2O,\;0.575\;mM\;MgSO_4.7H_2O$, and $1.83\;mM\;KH_2PO_4$. A new medium labeled as TAM was formulated for the production of Eurycoma longifolia cell biomass in the cell suspension culture.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.87-92
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• 2008

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on halfstrength MS medium supplemented with $0.3mg\;l^{-1}$ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to $3mg\;l^{-1}$, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryoderived white friable callus were established using half-strength MS medium supplemented with $0.3mg\;l^{-1}$ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of water-shield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.

• Journal of Plant Biotechnology
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• v.36 no.4
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• pp.309-319
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• 2009

Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effectiveness, scalability and possibility of complex post-translational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.95-98
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• 2001

The effect of stabilizing polymer on hGM-CSF production was investigated in suspension cell cultures of transgenic tobacco. Secreted human GM -CSF from cell suspension cultures was detected in the medium at a maximum concentration of 180 ${\mu}g/L$ by ELISA. However, the secreted hGM -CSF was unstable in the medium, and rapidly degraded after day 5. In order to stabilize the secreted hGM-CSF, three stabilizing polymers were tested, polyethylene glycol, polyvinylpyrrolidone and gelatin. Gelatin was the most effective in stabilizing the secreted GM-CSF. Following the addition of 5% (w/v) gelatin, the maximum GM -CSF concentration reached 783 ${\mu}g/L$, a 4.6-fold increase over control.

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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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.