• 미생물학회지
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• 제24권2호
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• pp.91-97
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• 1986

Intra and interspecfic fusants were produced by the protoplast fusion of auxotrophic mutants from Trichoderma koningii ATCC 26113 and Trichoderma reesei QM 9414. It was found that 0.6M $MgSO_4\;and\;0.6M\;NH_4Cl$ was the best osmotic stabilizer for the preparation of protoplasts from the mycelium of T. koningii and T. reesei respectively. However, $MgSO_4$ was the most suitable one for the regeneration of the protoplasts from both species. The intraspecific protoplast fusion frequencies between the auxotrophic mutants from T. reesei were $1.8{\times}10^{-2}\;to\;5.1{\times}10^{-1}$. Interspecific protoplast fusion frequencies between the auxotrophic mutants from T. koningii and T. reesei were $3.6{\times}10^{-3}$\;to\;8.4{\times}10^{-2}. Interspecific complementing fusants, however, were not alwats produced. Fusants obtained from interspecific potoplast fusion were spontaneously segregated into various strains including parental types, non-parental auxotrophic hybrids, and prototrophic hybrids on complete plate. Interspecific hybrids revealed to have partially enhanced celluloytic activities.

• 미생물학회지
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• 제29권2호
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• pp.123-129
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• 1991

In order to develop the protoplast fusion method of the strains of Fusarium, the interspecific protoplast fusion was attempted between Fusarium poae and F. sporotrichioides. Various auxotrophic mutants were isolated by the treatment of N-Methyl-N'-Nitro-N-Nitrosoguanidine. The optimal conditions for the formation and regeneration of protoplasts were examined and the characteristics of a fusant were studied. As a results, protoplasts were readily obtained from 18 hours cultured mycelia by the treatment of driselase for 3 hours and 0.6 M KCl as a best osmotic stabilizer at pH 6.0 for the formation of protoplast. Sucrose was the most suitable for the regeneration. Polyetylene glycol (M.W. 8,000) in $CaCl_{2}$-glycine solution was used to induce the protoplast fusion. The interspecific fusion frequency between protoplasts among the auxotrophic mutants of the two strains ranged from $2.7*10^{-2}$ to $5.7*10^{-3}$ . DNA content and cellulase activity were rather increased in the interspecific fusant. The lag phase of growth curve was slightly elongated in the fusant.

• 미생물학회지
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• 제28권4호
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• pp.311-317
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• 1990

Intraspecific and interspecific protoplast fusions in/between C. thermocellum and C. thermohydrosulfuricum were studied. Protoplast fusions were well induced in 30-40% PEG solution, however, their fusion frequencies were low level of 1.2*10$^{-7}$ for intraspecific fusion of C. thermocellum, $6.7*10^{-7}$ for C. thermohydrosulfuricum, and 4.2*10$^{-7}$ for interspecific fusion between above two Clostridia, respectively. Most fusants were unstable and segregated after 3 subcultures. Relatively stable intraspecific C. thermocellum fusant FTT17, intraspecific C. thermohydrosulfurecum fusant FSS22 and interspecific fusant FTS3, which were stable after several subcultures, were selected and properties of fusants were further investigated, Phenotypes of the fusants were similar with wild types mostly in cellular morphology, carbon source assimilation and enzyme activities. However they were differed in assimilation of pyruvic acid and sorbitol as carbon source. The DNA contents of fusants were slightly increased compared with wild types. Ethanol production by intraspecific and/or interspecific fusants was not increased, however, acetic acid production as byproduct was decreased or not detected, which indicates that industrial thermophilic anaerobes can be improved by means of protoplast fusion of two strains.

• 약학회지
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• 제38권5호
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• pp.595-601
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• 1994

Acetaminophen, a widely used analgesic, can be formed by N-acetylation and p-hydroxylation of aniline. Interspecific protoplast fusion technique was used to get acetaminophen directly from aniline and to increase the productivity of acetaminophen. Three auxotrophic mutants were obtained from S. griseus(ATCC 13273) and S. hygroscopicus(KCTC 1089) by N-methyl-N'-nitro-N-nitrosoguanidine(NTG) treatment. Regeneration frequencies of S. griseus$(his^-)$, S. griseus$(lys^-)$, S. hygroscopicus$(arg^-)$ were 42%, 45%, and 31%, respectively. Fusion of protoplasts carrying different auxotrophic markers was achieved by treatment with polyethylene glycol. When protoplasts were treated with 50% polyethylene glycol for 3 minutes, the fusion frequency between S. griseus$(his^-)$ and S. hygroscopicus$(arg^-)$ was $3.8{\times}10^{-5}$. The fusion frequency between S. griseus$(lys^-)$ and S. hygroscopicus$(arg^-)$ was $5.6{\times}10^{-4}$. When we checked the production of acetaminophen, thirty-four out of the fifty-six fusants produced larger amounts of acetaminophen than the parent strains did. Nine fusants produced twice more and twenty-five fusants produced one to two times more of acetaminophen than their parents.

• 한국미생물·생명공학회지
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• 제16권4호
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• pp.303-309
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• 1988

Cellulose를 이용할 수 있는 Cellulomonas속 균주의 종간 세포의 융합방법을 확립하기 위해 원형질체 재생 및 융합조건에 대해 검토하였다. Cellulomonas속 균주의 원형질체는 osmotic stabilizer를 포함하는 재생배지에서 유동성 한천배지로 중층하여 재생시켰고 재생 확인은 주사전자현미경으로 하였다. 원형질체 융합은 항생물질 내성과 영양요구성 돌연변이주의 유전자 표지에 확인하였다. Lysozyme을 처리하여 형성된 C. flavigena 원형 질체의 세포벽 재생은 osmotic stabilizer로 0.4M sorbitol을 포함하는 재생배지에서 약 15% 수준이었으며 여기에 Polyvinyl Pyrrolidone(PVP) 3% 첨가로 재생율을 약 3배정도 증가시킬 수 있었다. Cellulomonas속 균주의 변이주간의 원형질체 융합은 융합유도제로 PEG 6000을 취하여 최적농도 40% (w/v), 치적 처리시간 15분, Ca농도 25mM에서 약 2.0$\times$$10^{-4}$ - 4.0$\times$$10^{-4}$의 융합빈도를 얻을 수 있었다.

• 미생물학회지
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• 제29권1호
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• pp.8-15
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• 1991

Interspecific hybrids between the ASpergillus spp., A. awamori, A. usamii and A. oryzae, were obtained by nuclear transfer technique. Nuclei isolated from an auxotrophic mutant strain were transferred into the protoplasts of a recipient strain of different species. The frequency of interspecific hybrid formation by nuclear transfer was $2*10^{-5}$ $-7*10^{-4}$ In contrast, no interspecific hybrid was isolated by protoplast fusion. Among the hybrids tested, 10 strains showed increased activity of some or all components of cellulases, xylanases and amylase up to more than two times. Isozyme pattern of the hybrids were analyzed by polyacrylamide gel electrophoresis and isoelectric focusing followed by activity staining, which showed that some of the hybrids have isozyme patterns unidentical to either of the two parents. By measuring the DNA contents and the sizes ofthe conidia, the karyotypes of the hybrids were estimated to be aneuploid near to haploid, diploid or triploid. It was concluded that the unclear transfer technique is much more efficient in the formation of interspecific hybrids than protoplast fusion and is very useful for the improvement of Aspergillus strains.

• 한국균학회지
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• 제16권1호
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• pp.16-20
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• 1988

영지(靈芝)의 항암물질개량(抗癌物質改良)과 신품종(新品種)의 육성(育成)을 위(爲)한 (육종역법)育種方法으로 제시된 원형질체융합(原形質體融合)의 기초자료를 얻고자 영양요구성균주(營養要求性菌株)의 선발(選拔), 종간원형질체융합(種間原形質體融合)과 융합균주(融合菌株)의 선발(選拔)을 실험(實驗)하였다. 1. 돌연변이(突然變異) 대상체(對相體)로는 균사체(菌絲體), mutagen으로는 자외선(紫外線)을 사용하였는데 영양요구성균주(營養要求性菌株)의 유기율(誘起率)은5.78%이었다. 2. 영지(靈芝)와 잔나비걸상버섯간(間) 원형질체융합률(原形質體融合率)은 2.03%이었다. 3. 융합균주(融合菌株)는 환원균총(還元菌叢)의 생장속도(生長速度)와 형태(形態)를 비교(比較)하여 선발(選拔)하였고, 계대배양후(繼代培養後)의 형태(形態)와 esterase isozyme pattern의 분석(分析)에 의해서 확인(確認)하였으며, 참나무톱밥배지(培地)에서 재배(栽培)하여 자실체(子實體)를 관제(觀祭)하였다.

• 한국균학회지
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• 제12권4호
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• pp.164-169
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• 1984

식용버섯의 재배와 소비가 증가해가는 중요성에 비추어 새로운 품종 육성을 위하여 국내의 주요 재배 버섯인 느타리버섯과 사철느타리버섯의 원형질체를 융합하여 종간 체세포 잡종을 선발하였으며, 그 특성을 조사한 결과를 요약하면 다음과 같다. 1. 느타리버섯과 사철느타리버섯의 종간 원형질체 융합율은 0.18-3.80%였다. 2. 융합된 균총의 생장은 빠른 것, 중간 그리고 아주 느린 것으로 나눌 수 있었으며 그 형태는 불규칙 하였다. 3. 선발한 40개의 체세포 잡종 모두가 정상적인 자실체를 맺었으며, 자실체의 형태가 모본과는 달리 아주 다양하게 나타났다. 4. 체세포 잡종의 자실체색은 짙은 회색을 가진 느타리버섯과 연한 백색을 가진 사철느타리버섯의 중간색을 나타내었다. 5. 무포자 또는 극히 적은량의 포자를 지닌 균주를 선발하였으며 이들의 자실체 형태는 정상이었다. 6. 수량이 다소 낮은 무포자 균주를 제외하고는 대체적으로 체세포 잡종의 자실체 수량이 모본보다 증가했다.

• 한국균학회지
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• 제15권3호
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• pp.135-141
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• 1987

원형질체 융합에 의한 식용버섯의 유전육종연구는 종래의 전통적 육종방법인 균사융합으로는 해결할 수 없는 균주간의 불화합성 장벽을 능가하여 새로운 품중육성에 그 기대가 크다. 한국의 주요재배 버섯이며, 날로 세계의 재배면적이 증가하는 느타리버섯류의 유전육종을 위하여 몇가지 종간의 체세포 잡종의 선발과 이들의 유성에 대하여 조사한 결과는 다음과 같다. 1. 이 종간 원형질체 융합으로 체세포 잡종을 선발하였는데, 느타리+노랑느타리, 느타리+사철느타리, 느타리+여름느타리, 사철느타리+노랑느타리, 사철느타리+여름느타리 그리고 여름느타리+노랑느타리의 원형질체 융합율은 0.002-3.25%였으며, MCM과 MCM+benomyl에서 융합주의 균총분리가 일어나지 않았다. 2. 화합성 균주간에는 균사융합 및 원형질체 융합에 의하여 모두 균사체에 clamp connection을 형성하였으며, 자실체도 형성하였다. 그러나 불화합성 균주간에는 균사 및 원형질체 융합에 의하여 clamp connection을 확인할 수 없었으며, 자실체도 형성하지 않았다. 3. clamp connection이 없고 자실체도 형성하지 않는 원형질체 융합주의 임성을 유도하기 위하여, 이들 모균주와 균사융합한 결과 단핵균주와는 드물게 clamp connection을 형성하였으며, 이 핵균주와는 모두 clamp connetion을 형성하였다. 4. 불화합성 균주간의 원형질체 융합주는 새로운 불화합성을 형성하였는데, ASI 2-3+ASI 2-53의 P148은 ASI 2-3과 화합성인 ASI 2-1, ASI 2-2와 불화합성을 나타냈다. 또한 ASI 2-3+ASI 2-44의 P150은 ASI 2-3과 화합성인 ASI 2-1, ASI 2-2와 불화합성이었다.

• 미생물학회지
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• 제27권1호
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• pp.27-34
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• 1989

In order to evaluate the applicability of enzyme electrophoresis for the identification of intra/interspecific hybride obtained by the protoplast fusion in Trichoderma, soluble proteins, intracellular soluble enzymes and extracellular $\beta$-glucosidase were analyzed by polyacrylamide gel electrophorsis. As the results, patterns of soluble protein, and isozyme patterns of peroxidase, malate dehydrogenase, and $\beta$-glucosidase in hydrids were defferent from those in parental and wild type strains. Therefore, it was established that the analysis of protein pattern by electrophoresis could be applied to isolate and identify the hybrids from the protoplast fusion.