• Journal of Plant Biotechnology
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• 제4권4호
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• pp.143-150
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• 2002

A strong oxidative stress-inducible peroxidase promoter (referred to as SWPA2 promoter) was cloned from tell cultures of sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco cultured cells in terms of biotechnological applications. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the $\beta$-glucuronidase (GUS) reporter gene, the 1314 bp deletion mutant showed approximately 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed following 15 days of subculture compared to other deletion mutants, suggesting that the 1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic cell lines engineered to produce key pharmaceutical proteins. In this respect, we developed transgenic cell lines such as tobacco (Nicotiana tabacum L. BY-2), ginseng (Panax ginseng) and Siberian ginseng (Acanthopanax senticosus) using a SWPA2 promoter to produce a human lactoferrin (hLf) and characterized the hLf production in cultured cells. The hLf production monitored by ELISA analysis in transgenic BY-2 cells was directly increased proportional to cell growth and reached a maximal level (up to 4.3% of total soluble protein) at the stationary phase in suspension cultures. The SWPA2 promoter should result in higher productivity and increased applications of plant cultured cells for the production of high-value recombinant proteins.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.112-119
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• 2005

Efficient plant regenerationsystem from cell suspension cultures was established in D. acicularis (2n = 90) by monitoring ploidy level and visual selection of the cultures. The highly regenerable cell lines selected maintained original ploidy level and consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.

• Journal of Plant Biotechnology
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• 제30권2호
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• pp.195-199
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• 2003

Extracts of Escherichia coli as a elicit were treated to suspension cultures of Streptanthus tostus in order to observe the effect on the pholem development. By the elicit treatment, cell wall, sieve endoplasmic reticulum (SER) and p-protein were normally synthesized, but the structure of amyloplast was changed from a round form to irregular and swollen unhalthy form with a tiny starch granular. Oil drops were new synthesized and accumulated in a large oleoplast and proteins were also accumulated in a single membrane. The concentration of sucrose in the phloem, which was induced during the elicit treatment, was higher than normally developed phloem cells. These results suggest that phloem cells might be changed in the normal cycles of metabolism of lipids, carbohydrates and proteins to overcome during the eilicit stress.

• 식물조직배양학회지
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• 제21권1호
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• pp.47-53
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• 1994

본 실험은 포도와 미국자리공의 세포현탁배양계에서 세포생장과 athocyaninn 및 betacyanin 색소생성에 미치는 광의 영향을 조사하였다. 그 결과, 포도의 세포현탁배양계에서의 세포증식 패턴은 광의 효과가 미약하였으나, 미국자리공의 경우는 암배양에 비하여 광조사구에서 뚜렷한 세포증식 패턴을 나타내었다. 포도세포는 12일간의 배양기간중 한번의 생장 피크를 보여주는데 비하여 미국자리공의 배양세포는 두 번의 증식 피크를 보여 광이 세포의 생장 및 분열 주기를 촉진시킬 뿐만 아니라 색소의 축적도 촉진시키는 것으로 나타났으며, 미국자리공 배양세포는 배양 후 4일째에 액포화에 따른 색소의 축적이 확인되었다. 한편, 색소생성에 미치는 광의 영향은 포도 현탁배양계에서는 배양후 6일째부터 생성되기 시작하여 배양 후 10일 전후에 최고치를 보이는데 비하여, 미국자리공의 현탁배양계에서는 세포증식 패턴에 따라 배양 후 4일째와 8일째에 두 번의 피크를 보였다. 그러나 이들 두 가지 모두 암배양하에서는 거의 색소 생합성이 이루어지지 않고 광상태하에서만 색소의 축적을 볼 수 있었다. 한편, 포도 세포배양계에서는 배양 후 4일째에 처리한 광은 약간의 효과가 있었으나, 배양 후 7일째 12시간 이상의 광에 의하여 색소생성이 크게 촉진되는 것을 알 수 있었다. 미국자리공의 경우에는 배양 후 7일 째보다는 오히려 배양 후 4일째에 광이 더욱 효과적이었다. 이상의 사실은 이들 두 가지 색소의 생합성과정에는 광이 절대적으로 필수적이지만 광을 필요로 하는 시기 및 요구하는 광량 등이 서로 다르다는 것을 시사해 준다.

• Journal of Plant Biology
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• 제36권3호
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• pp.211-218
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• 1993

Plant regeneration was accomplished from protoplast culture of rice (Oryza sativa L. cv. Taebaeg). Embryogenic callus was induced from mature seed on MS medium containing 5 mM proline, 2.5 mg/L 2,4-D, 30 g/L sucrose in the dark at 28$^{\circ}C$ and used to establish embryogenic cell suspension culture. Suspension cells were subcultured every one week in N6 medium supplemented with 5 mM proline, 200 mg/L casein hydrolysate, 2.5 mg/L 2,4-D and amino acids of AA medium. Suspension cultures were composed of cells that were densely cytoplasmic, potentially embryogenic and were at least maintained for more than 6 months in liquid medium. Protoplasts were isolated from fast-growing suspension culture cells and cultured in a slightly modified KpR medium by mixed nurse culture. Isolated protoplasts began to divide within 5~7 days and thereafter, protoplast-derived calli were sequentially transferred to callus proliferating medium that soft agar MS medium contained 2 mg/L 2,4-D and produced distinct embryogenic cells. Microcolonies were then transferred to solid medium which consisted of MS medium containing 5 mg/L kinetin, 1 mg/L NAA, 1 mg/L ABA, 30 g/L sucrose and 10 g/L sorbitol under fluorescent light. Mulitple shoots of 4~5 per callus emerged and were transferred to hormone-free MS medium for root initiation. Thereafter, The plantlets were transferred to pots of soil to mature in the culture room.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.442-448
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• 2006

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducible SWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
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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.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2003년도 식물바이오벤처 페스티발
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• pp.189-189
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• 2003

• Journal of Plant Biotechnology
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• 제29권3호
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• pp.185-188
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• 2002

유자의 성숙종자를 MS 기본배지에 치상하여 종자의 내피에서 주심조직 유래의 희고 부서지기 쉬운 배발생캘러스가 1.2%의 빈도로 형성되었다. 이 캘러스는 1 mg/L 2,4-D를 첨가한 MS배지에서 증식되었다. 증식된 캘러스를 0.1 mg/L kinetin을 첨가한 MS 배지에 옮겼을 때 많은 수의 체세포배가 형성되었다. 배발생캘러스를 1 mg/L 2,4-D를 첨가한 액체 배지에 넣어 배발생 현탁배양계를 확립하였다. 배양된 현탁배양세포를 0.5 mg/L ABA를 첨가한 고체배지에 평판하였을 때 높은 빈도로 체세포배로 발달하였으며 MS 기본배지 혹은 1 mg/L kinetin 첨가배지에서 소식물체로 발달하였다. 소식물체는 성공적으로 토양으로 옮겨서 온실에서 육성되었다.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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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.