파(Alium fistulosum L.)의 회기조직배양으로부터 체세포배 발생

金載焄,蘇雄永 全北大學校 基礎科學硏究所 1994 基礎科學 Vol.16 No.-

파(Allium fistulosum L.)의 화기를 배양하여 화판과 약편으로부터 유래된 캘러스부터 체세포배를 유도하였고, 이 과정중의 조직학적 특징을 관찰하여 수선형 단자엽 식물의 체세포배발생과정을 구명하고자 본연구를 실시하였다. 황색의 배발생캘러스는 2,4-D(특히 1.0 mg/L)가 첨가된 MS배지에서 유도되었다. 구상배는 배발생캘러스를 0.2 mg/L 2,4-D로 옮겨 주었을 때 세포는 작고, 세포질이 농후한 세포군으로부터 기원되었다. 그러나 배발생캘러스를 사이토키닌이 첨가된 배지에 이식하였을 때에는 부정근이 관찰되었다. 구상배는 정단부가 길게 신장되면서 한개의 자엽이되고, 이 자엽의 기저부 측면의 작게 열개된 홈으로부터 정단분열조직이 발생되었다. 파의 체세포배의 초기 발생과정의 특징을 접합자배 발생과정과 비교하였을 때, 원배시기의 세포분열 패턴이 약간 달랐고, 체세포배에서 배병이 뚜렸하지않는 차이점이 관찰되었다. 그러나 그 이후의 배발달의 형태적 특징은 서로 비슷하였다. Yellowish embryogenic calli formed on the petal and the sepal of Allium fistulosum L. cultured on MS medium supplemented with 1.0 mg/L 2,4-D. Upon transfer onto MS medium with 0.2 mg/L 2,4-D, the calli gave rise to numerous globular embryos. Subsequently they were transferred onto MS basal medium, wherein they developed into cotyledonary embryos. A slight depression on one side of cotyledon base revealed the site of the future shoot apical meristem. The pattern of this somatic embryogenesis at the early stage was similar to that of amaryllid zygotic embryogenesis. However, there were some differences including the absence of suspensor development at the proembryo stage.

Water Extracts of Cultured Mountain Ginseng Stimulate Immune Cells and Inhibit Cancer Cell Proliferation

Chan-Ho Oh,Pil-Sung Kang,Jae-Whune Kim,Jin Kwon,Suk-Heung Oh 한국식품과학회 2006 Food Science and Biotechnology Vol.15 No.3

Acanthopanax senticosus Extract Prepared from Cultured Cells Improves Lipid Parameters in Rats Fed with a High Fat Diet

Youn-Soo Cha,Ju-Ryoun Soh,Jae-Whune Kim 한국식품영양과학회 2003 Preventive Nutrition and Food Science Vol.8 No.1

Acanthopanax senticosus was grown by a novel, proprietary method, of culturing isolated cells in a bioreactor. An extract from the cells was evaluated for its effect on lipid metabolism in rats fed a high fat diet. Male Sprague-Dawley rats (n=6) were fed either an AIN-76 diet (control, NDCon), control diet plus Acanthopanax senticosus extract (ND+Ex), a modified AIN-76 diet supplemented with 20% beef tallow (high fat, HFCon), or a high fat diet plus Acanthopanax senticosus extract (HF+Ex), for 5weeks. Body weight gain was significantly higher in the HFCon group than the NDCon group. Feed consumption was significantly lower, but energy intake higher, in the groups fed high fat diets compared with the groups fed control diets. Serum HDL-cholesterol concentrations were significantly increased but serum LDL-cholesterol concentrations were decreased in the groups fed the Acanthopanax senticosus extract. Abdominal fat accumulation, serum leptin levels were significantly higher in the HFCon group than the other groups. Carnitine palmitoyltransferase-I (CPT-I) mRNA levels were increased in the groups fed Acanthopanx senticosus extract. These results suggest that supplementation of cell cultured Acanthopanax senticosus extract regulates CPT-I mRNA levels in liver, has an effect on the normalization of lipids in rats fed a high fat diet.

Poster Session 2 : Plant Biology ; Purification and characterization of recombinant human Lactoferrin protein in cell cultures of Siberian ginseng (Acanthopanax senticosus)

( Seung Hyun Jo ),( Suk Yoon Kwon ),( Jae Whune Kim ),( Ki Teak Lee ),( Sang Soo Kwak ),( Haeng Soon Lee ) 한국생화학분자생물학회 (구 한국생화학회) 2004 생화학분자생물학회 춘계학술발표논문집 Vol.2004 No.-

Acanthopanax senticosus Extract Prepared from Cultured Cells Improves Lipid Parameters in Rats Fed with a High Fat Diet

Cha, Youn-Soo,Soh, Ju-Ryoun,Kim, Jae-Whune The Korean Society of Food Science and Nutrition 2003 Preventive Nutrition and Food Science Vol.8 No.1

Acanthopanax senticosus was grown by a novel, proprietary method, of culturing isolated cells in a bioreactor. An extract from the cells was evaluated for its effect on lipid metabolism in rats fed a high fat diet. Male Sprague-Dawley rats (n=6) were fed either an AIN-76 diet (control, NDCon), control diet plus Acanthopanax senticosus extract (ND+Ex), a modified AIN-76 diet supplemented with 20% beef tallow (high fat, HFCon), or a high fat diet plus Acanthopanax senticosus extract (HF+Ex), for 5weeks. Body weight gain was significantly higher in the HFCon group than the NDCon group. Feed consumption was significantly lower, but energy intake higher, in the groups fed high fat diets compared with the groups fed control diets. Serum HDL-cholesterol concentrations were significantly increased but serum LDL-cholesterol concentrations were decreased in the groups fed the Acanthopanax senticosus extract. Abdominal fat accumulation and serum leptin levels were significantly higher in the HFCon group than the other groups. Carnitine palmitoyltransferase-I (CPT-I) mRNA levels were increased in the groups fed Acanthopanx senticosus extract. These results suggest that supplementation of cell cultured Acanthopanax senticosus extract regulates CPT-I mRNA levels in liver and has an effect on the normalization of lipids in rats fed a high fat diet.

참옻나무(Rhus verniciflua)배발생캘러스로부터 체세포배발생에 의한 식물체 재분화

김재훈,이원석,권기원,안준교,최용익,Kim, Jae-Whune,Lee, Won-Seok,Kwon, Ki-Won,In, Jun-Gyo,Choi, Yong-Eui 한국식물생명공학회 2003 식물생명공학회지 Vol.30 No.3

Excised cotyledons and embryo axises of zygotic embryos of Rhus vemicifera were cultured on Murashige and Skoog(MS) medium with various concentrations of 2,4-D. About 3-5% of explants produced callus. Embryogenic callus was preferentially induced from basal parts of embryo axis of zygotic embryos seeds when they were cultured without removal of seed coats. Somatic embryos were developed from embryogenic callus in growth regulator-free medium after 2-3 subcultures on medium with 1.0mg/L 2,4-D and these embryos were matured to cotyledonary stage. Plantlets with well-developed shoots and roots from embryos were obtained on $\frac{1}{4}$MS medium with GA$_{3}$. After acclimatization of plantlets on artificial soil, they were exposed to soil pots.

A Novel Oxidative Stress-inducible Peroxidase Promoter and Its Applications to Production of Pharmaceutical Proteins in Transgenic Cell Cultures

Lee, Ok-Sun,Park, Sun-Mi,Kwon, Suk-Yoon,Lee, Haeng-Soon,Kim, Kee-Yeun,Kim, Jae-Whune,Kwak, Sang-Soo The Korean Society of Plant Biotechnology 2002 Plant molecular biology and biotechnology research Vol.4 No.4

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.

인체 락토페린 생산 형질전환 가시오갈피 배양세포

조승현,권석윤,김재훈,이기택,곽상수,이행순,Jo Seung-Hyun,Kwon Suk-Yoon,Kim Jae-Whune,Lee Ki-Teak,Kwak Sang-Soo,Lee Haeng-Soon 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.3

락토페린은 철 결합 당단백질로서 항 미생물활성과 면역강화와 같은 생리활성 기능을 가지고 있다. 본 연구는 배양 세포 고 발현 SWPA2 promoter를 이용하여 인체락토페린(hLf)을 생산하는 형질전환 가시오갈피 배양세포주 개발에 관한 것이다. 형질전환에 이용된 벡터는 산화스트레스 유도성 SWPA2 promoter의 조절 하에서 hLf이 소포체로 targeting되도록 제작된 SWPA2pro::ER-hLf/pCAMBIA이다. hLf을 생산하는 각 형질전환 배양세포들은 PCR과 Southern분석을 통해 hLf 유전자가 가시오갈피 게놈내로 성공적으로 도입되었음을 확인하였으며, western blot과 ELISA를 통해 형질전환 가시오갈피 배양 세포주에서 hLf 단백질이 활성이 있음을 확인하였다. 형질전환 가시오갈피 배양세포에서 hLf 단백질의 함량은 세포배양이 진행될수록 증가하여 정지기 때 가장 높았으며 전체 수용성 단백질의 약 3%를 차지하였다. 따라서 본 연구에서 개발된 인체락토페린을 고생산하는 약용식물 가시오갈피 배양 세포주는 산업적으로 이용될 수 있을 것으로 기대된다. Lactoferrin is an iron-binding glycoprotein with many biological roles, including the protection against microbial and virus infection, stimulation of the immune system. We developed the transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing the human lactoferrin (hLf) protein following Agrobacterium tumefaciens-mediated transformation. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to hLf cDNA under the control of an oxidative stress-inducible SWPA2 promoter was engineered. Transgenic Siberian ginseng cultured cells to produce a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analysis. ELISA and western blot analysis showed that full length-hLf protein was synthesized in the transgenic cells. The production of hLf increased proportionally to cell growth and reached a maximal (up to 3% of total soluble proteins) at the stationary phase. These results suggest that the transgenic Siberian ginseng cultured cells in this study will be biotechnologically useful for the commercial production of medicinal plant cell cultures to produce hLf protein.

생물반응기 배양을 통한 두릅나무(Aralia elata)의 체세포배 및 유식물체 대량증식

이원석,최은경,김재훈,Lee, Won-Seok,Choi, Eun-Gyung,Kim, Jae-Whune 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.3

두릅나무의 엽병을 1.0 mg/L 2,4-D가 포함된 MS 고체배지에서 배발생캘러스를 유도하였다. 배발생세포와 배발생세포괴는 1.0 mg/L 2.4-D가 포함된 MS 액체배지에서 배발생캘러스를 2주간 현탁배양하여 대량으로 얻었다. 그물망을 통과한 배발생세포는 1.0 mg/L 2.4-D가 포함된 MS 액체배지에서 배양하면 배발생능이 소실되지 않고 지속적으로 유지 및 증식시킬 수 있었다. 그물망을 통과하지 못한 배발생세포괴를 식물생장조절물질이 첨가되지 않은 1/2 MS 액체배지에 옮겨 2주간 배양하면 구상형의 체세포배로 발달하였다. 구상형의 체세포배는 5 L의 bioreactor를 이용하여 배양하면 심장형, 어뢰형, 자엽형의 배와 유식물체로 발달하였다. Bioreactor 배양을 통해 두릅나무의 체세포배를 효과적으로 대량증식 시킬 수 있었다. Embryogenic calli were induced from petioles of Aralia elata on MS solid medium supplemented with 1.0 mg/L 2,4-D. When embryogenic calli were transferred to MS liquid medium supplemented with 1.0 mg/L 2,4-D, embryogenic cells and embryogenic cell clusters were developed after 2 weeks of culture. Embryogenic cells were filtered through a 250 ${\mu}{\textrm}{m}$ sieve and the passed cells were proliferated and maintained in MS liquid medium supplemented with 1.0 mg/L 2,4-D. Embryogenic cell clusters entrapped on the sieve were transferred to 1/2 MS liquid medium without plant growth regulators, globular-shaped embryos were developed from embryogenic cell clusters after 2 weeks of culture. Numerous early stage somatic embryos could be developed to heart-shaped, torpedo-shaped, cotyledonary embryos and plantlets in 5 L bioreactor. Above results suggest that effective somatic embryo proliferation can be achieved via bioreactor culture systems in Aralia elata.

High-yield Production of Functional Human Lactoferrin in Transgenic Cell Cultures of Siberian Ginseng(Acanthopanax senticosus)

Jo, Seung-Hyun,Kwon, Suk-Yoon,Park, Doo-Sang,Yang, Kyoung-Sil,Kim, Jae-Whune,Lee, Ki-Teak,Kwak, Sang-Soo,Lee, Haeng-Soon The Korean Society for Biotechnology and Bioengine 2006 Biotechnology and Bioprocess Engineering Vol.11 No.5

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.