• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2003년도 제10차 국제학술회의 및 추계정기 학술발표회
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• pp.20-20
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• 2003

Isolation and culture of leaf protoplasts from two chilli cultivars (Capsicum annuum var. accumnatum and Bird chilli) were developed to enhance selection process in the somatic hybridization programmes. In order to isolate the protoplasts from leaves of these two chilli cultivars different incubation periods (3, 5 and 10 hours) were tested with combinations of enzyme mixtures containing cellulase and macerozyme. Leaves were incubated on three enzyme mixtures (2% cellulase +0.4% macerozyme, 1% cellulase +0.2% macerozyme and 0.5% cellulase +0.1% macerozyme in 13% mannitol) at 251oC in the dark. Three hours of incubation using 2% cellulase and 0.4% macerozyme was the best for the protoplast isolation of both chilli cultivars tested. The yield was 5${\times}$108protoplasts/m1/g leaf tissue in both chilli varieties. It was found that in the mixed nurse method using Nagata and Takebe (NT) medium supplemented with 1.0mg/12,4-D, NAA and BAP with 0.5M mannitol and 1.2% Sea Plaque agarose is the best medium for protoplast culture. Protoplasts of Capsicum annum var. accumnatum were alive for 14 days forming cell walls and initiating cell division.

• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.46-49
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• 1992

The availability of Phaffia rhodozyma as an astaxanthin sources in the aquaculture industry is limited because of the low carotenoid content of natural isolate. In this study, we have used the protoplast fusion technique to construct cell hybrids with an increased content of astaxanthin from P. rhodozyma. Cell hybrids (F307 and F406) obtained were very stable and produced considerably more astaxanthin (> 1 mg/g yeast) than the wild parent. Karyogamy was confirmed by the isolation of recombinants after mitotic segregation of parental auxotrophic genetic markers, the increased amount of chromosomal DNA/cell and the presence of single nucleus/cell.

• 미생물학회지
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• 제28권3호
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• pp.274-277
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• 1990

새로운 고체배지 응고제인 Pluronic Polyol F127을 한천대신 사용하여 C. congregatus를 배양하였고 균사 끝(hyphaltip)에 있는 phenoloxidase들의 세포내에서의 존재위치를 확인하였다. 고체배지의 균사에서 원형질체를 얻었고 concanavalin A를 처리하여 세포막을 분리하였다. 세포막 분획에 phenoloxidase들이 존재하였고 이는 이 효소가 light receptor complex의 한 부분임을 시사한다.

• Journal of Plant Biotechnology
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• 제3권1호
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• pp.27-31
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• 2001

New types of cytoplasmic male sterility in Brassica species would be very useful for the production of F$_1$, hybrid seeds. Leaves and stems of rapid cycling stock of B.juncea (CrGC4-3) containing Anand CMS were used as experimental materials for plant regeneration from protoplast culture. Very high plant regeneration rate (85%) was found in the Kao & Michayluk medium supplemented with 2 mg/L zeatin, 0.5 mg/L BAP, and 1 mg/L NAA when only leaf, not stem, segments were cultured. Protoplasts were isolated from leaves using mixtures of enzymes (1% Cellulycin, 0.5% Macerozyme) in 0.4 M mannitol and 50 mM $CaCl_2$.$2H_2$O. Mcrocalli induced from protoplasts were transferred to the shoot regeneration medium containing 2 mg/L BAP, 2 mg/L zeatin, and 0.5 mg/L NAA. After 60 days of initial protoplast culture, regenerated plantlets were obtained, acclimatized, transplanted into the pots, and grown up to the flowering stage.

• 한국연초학회지
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• 제20권1호
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• pp.40-49
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• 1998

Protoplasts were isolated from mesophyll of tobacco(Nicotiana glauca) transformed with kanamycin-resistant gene (NPT II gene) and potato hairy root callus containing Ri plasmid of Agrobacterium rhiEogenes, and protoplasm fusion was made between the isolated protoplasts. The transgenic tobacco leaf tissue could grow on the media containing high concentrations of kanamycin, but not on the phytohormone-free media. On the other hand, the potato hairy root calli could be cultured on the phytohormone-free media but not on media containing more than 40 ㎍/ml kanamycin. In these conditions, the viability of both protoplasts were above 90%, These selection markers were used for the selection of protoplasts fused between the two, i.e. protoplast fusion was detected using selection media containing 100㎍/ml kanamycin and with no phytohormone. The mixture of 1.0% cellulase, 0.3% macerozyme, and 0.7M mannitol was best for the maximum protoplast production for tobacco, and that of 2.0% cellulase, 2.0% macerozyme, 1.0% dricelase, and 0.5M mannitol for potato. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution on the selectable medium. Cell walls were regenerated after 5 days in this medium, and colonies were alive until 4 weeks after cultural, but died after 6 weeks.

• 한국산림과학회지
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• 제71권1호
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• pp.66-73
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• 1985

본(本) 연구(硏究)는 국내(國內) 삼림생태계(森林生態係)의 우점수종(優占樹種)으로서 생태적(生態的) 및 경제적(經濟的)으로 유망(有望)한 참나무류(類)의 육종(育種) 및 우량(優良)크론의 대량증식(大量增殖)을 저해(低害)하고 있는 인공교잡(人工交雜)과 무성번식(無性繁殖)의 난점(難點)을 타개하기 위한 세포배양(細胞培養) 및 체세포(體細胞) 교잡(交雜)에 필요(必要)한 기초자료(基礎資料)를 획득하기 위하여 실시(實施)되었다. 주요시험결과(主要試驗結果)를 요약(要約)하면 다음과 같다. 1) 미경화엽(未硬化葉)과 왕성(旺盛)한 성장(成長)을 보이는 Callus가 지속적인 원형질체(原形質體) 공급원(供給源)으로 적합(適合)하며 2) 원형질체(原形質體) 라출(裸出)을 위(爲)한 효소용액(酵素溶液)의 조성(組成)은 Cellulase Onozuka R-10($20g/{\ell}$), Macerozyme R-10($10g/{\ell}$), Pectinase($250units/{\ell}$), $CaCl_2$, $2H_2O$(14mM), $MgSO_4{\cdot}7H_2O$(1.8mM), $KNO_3$(1.0mM), $H_3BO_3$(1.0mM), $KH_2PO_4$(0.2mM), KI($1.0{\mu}M$), 1.4-dithiothreitol(0.1mM), mannitol(0.6M)을 조합(組合)한 것의 성적(成績)이 가장 좋았다. 3) 세포융합율(細胞融合率)이 가장 높았던 원형질체(原形質體)의 밀도(密度)는 $2{\times}10^5/ml$이었다. 이는 본(本) 시험(試驗)에 채용(採用)된 원형질체밀도중(原形質體密度中) 가장 높은 밀도(密度)였다. 4) 세포융합유도(細胞融合誘導)를 위(爲)한 polyethylene glycol 처리(處理)의 적정농도(適正濃度)와 처리시간(處理時間)은 각각(各各) 29%(w/v)와 5~10분(分)으로 나타났다.

• 한국미생물·생명공학회지
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• 제14권5호
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• pp.359-363
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• 1986

본 실험의 목적은 starch의 alcohol발효에 있어서 전분의 $\alpha$-1.4 linkage을 분해하여 alcohol발효를 하는 S diastaticus와 전분 중의 $\alpha$-1.4, $\alpha$-1.6 linkage를 모두 분해할 수 있는 C. tropicalis 간의 이속간의 protoplast fusion을 시켰다. 이때 세포 융합의 빈도는 $10^{-5}$-$10^{-6}$이였으며, 이틀 융합체중 amylase 생성능과 유전적으로 안정한 융합체를 분리하였다. fusant의 성질을 조사하기 위하여 탄소원 자화능을 조사한바 parent와 달리 탄소원 자화능이 일부 보완됨을 보였고 또한 fusant는 원 parent보다 세포의 크기가 클 뿐만아니라, DNA함량도 많았다. spore형성능은 S. diastaticus A4는 spore를 형성하나 C. tropicalis는 형성할 수 없는 반면에 fusant는 형성하였고, Cu$^{++}$ 내성과 NaCl 내염성도 조사하였는데 fusant는 parent의 중간 성질을 가졌다. Fusant의 증식율을 조사하기 위하여 생육도를 조사한 결과 Parent보다 유도기가 길었음을 알았다.

• 한국작물학회지
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• 제42권3호
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• pp.306-316
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• 1997

약배양을 통해 유도된 벼의 품종 Zhonghua 8의 종자로부터 배발생 캘러스를 유기한 캘러스로부터 현탁배양을 실시하였다. 원형질체 분리 는 이러한 현탁배양된 캘러스를 사용하였으며, 일반적으로, 오래되고 미세한 현탁배양세포를 사용했을 때 어린 현탁배양세포보다 원형질체 나출율이 증가되었다. 원형질체는 feeder cell 없이 agarose embedding 방법에 의해 0.5 mg $l^{01}$ 2,4-D, 1.0mg $l^{-1}$ NAA와 0.5 mg $l^{-1}$zeatin이 첨가된 KPR 배지에서 배양하였을 때 세포분열이 일어났으며 microcalli가 형성되었다. 원형질체의 plating 효율은 0.20~0.54% 범위로 나타났으며, 원형질체로부터 유도된 microcalli는 식물체 재분화를 위해 2.0 mg $l^{-1}$ kinetin과 0.5 mg $l^{-1}$ NAA가 첨가된 MS 배지에 옮겨 주었다 실물체 재분화 빈도는 현탁배양의 line에 따라 2~l2%였다. 원형질체로부터 재분화된 식물체들은 온실에서 종자를 맺었다었다

• 생명과학회지
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• 제6권2호
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• pp.94-103
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• 1996

To understand effect of inoculum size, cell density, sucrose concentration and concentrations of MS basal on suspension culture and protoplast isolation of wild viola(Viola patrinii DC.) callus from petiole segments this experiment was conducted. In the lot of 30 mesh inoculum size, two observations were; One was that a considerable increase in the fresh and dry weight of callus was determined. Another was that the callus mass was relatively compact compared with others. A recommendable cell density was 0.4g for 20ml culture medium and the higher sucrose concentration, the higher fresh and dry weight were obtained. The dilution of MS basal salt was differently affected on fresh and dry weight; the highest fresh weight was found in 1x MS salt, while the higest dry weight was in 1/3x dilution.The addition of casein hydrolysate(3g/L) was more effective to increase of both fresh and dry weight. THe contents of protein was great in the inoculum lots with larger inoculum sizeand higher concentration of MS basal salts contenting 3g/L of casein hydrolysate and higher sucrose compared with others. The greatest protoplasts were isolated from the lot of 10 mesh size treated with 1%pectinase SE-150 and 2% cellulase YC. In general, for optimal protoplast isolation the followingconditions were recommended; 1) Use of smaller cell size cultured for 2-5 weeks, and 2) more than 5 hours incubation using the combined mixture of the enzymes with proper concentrations.

• 한국양식학회지
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• 제12권1호
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• pp.7-14
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• 1999

갈래곰보의 원혈질체 분리와 배양 조건을 확립하여 생명공학기술에 의한 유전적 형질개량의 기초조건을 마련코자 하였다. Abalone acetone powder,소라의 내장조효소, Pseudomanas와 Vibrio의 조효소, Cellulas R-10, Macerozyme R-10, Hemicellulase, Pectinase, Driselase, Protease 등을 0.6M mannitol과 0.5% potassium dextran sulfate를 함유한 50mM MES 해수완충액(pH 6.0)에 단독 또는 조합하여 조제한 효소액을 갈래곰보의엽체에 처리하였을 때 Abalone acetone powder 4.0% + Macerozyme R-10 4.0% + Hemicellulase 4.0% 효소조합액의 처리로서 생체조직 1g당 $107.6{\times}10^4$개의 원혈질체를 분리시킬 수 있었다. 갓 분리한 원형질체는 투명한 타원형으로 $7{\mu} m$~ $24{\mu} m$의 범위에 분포하였다. 0.2M mannitol을 첨가한 $ASP_{12}$배지에 배양한 세포는 9일 후 분열되고 25일 후 발아하였다. $ASP_{12}$배지는 f/2배지보다 갈래 곰보의 원혈질체 배양에 효과적이었으며 Guillard 항생물질조합액의 처리는 분화에 장애가 되었다.