• 미생물학회지
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• 제27권2호
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• pp.98-107
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• 1989

Trichoderma koningii ATCC26113으로부터 얻어진 영양요구성 돌연변이주간의 종내 원형질체 융합을 시도하여 생성된 융합체의 유전자형을 완전평판배상에서 분석하였다. 생성된 융합체 중 모균고 다른 독립영양형 균주에서 섬유소 분해능이 증진된 융합체를 얻었으며 이들이 이배체(혹은 이수체)임을 확인하였다. 또한 이들 이배체(혹은 이수체)의 독립영양청 잡종의 원형 질체 융합을 통하여 유전자의 교환 및 재조합에 의하여 자연분리되므로써 형성됨을 확인하였다.

• 미생물학회지
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• 제26권2호
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• pp.82-87
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• 1988

The genetic analysis and characterization of protoplast fusion hybrids between complementary auxotrophic mutants of Candida pseudotropicalis were carried out. Nuclear fusion appeared to occur in fusion hybrids (e.g., F15 and F33), as strongly suggested by isolation of recombinants after mitotic segregation of parental genetic markers. This was confirmed by KNA content, nuclear staining and comparison of survival rate to UV light. After keeping fusion hybrids for approximately one year, the frequency of spontaneous mitotic segregation was $3.0\times 10^{-4}$ - $8.1\times 10^{-4}$ while that of induced mitotic segregation was $1.4\times 10^{-3}$- $1.7\times 10^{-3}$. These results suggested that they maintained stable karyogamy state. It was also found that the production of $\beta$-D-galactosidase from F15, F33 and F158 was somewhat increased when compared with that from either auxotrophic parents or wild type.

• Archives of Pharmacal Research
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• 제20권5호
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• pp.448-453
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• 1997

Protoplast fusion between isoleucine-, argihine- and thymidine-requiring auxotroph $(Ile^{-}, Arg^{-}, Thy^{-})$ of Lentinula edodes and arginine-requiring auxotroph $(Arg^-)$ of Coriolus versicolor has been achieved using 30% polyethylene glycol (M.W.4000) in 10 mM $CaCl_2$-glycine solution (pH 8.0). Fusion hybrids were selected in the 0.6 M sucrose supplemented minimal media on the basis of nutritional complementation with fusion frequency of $7.4{\times}10{-6}$ The hybrids included both parental and non-parental types in colony morphology, growth rate and isozyme patterns. We succeeded inter-order protoplast fusion between the auxotrophs of Lentinula edodes and Coriolus versicolor overcoming the natural barriers of incompatibility. We examined the characteristics of the hybrids and clarified the fusion rocess using electron microscopy.

• 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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• 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.

• Journal of Plant Biotechnology
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• 제3권1호
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• pp.33-38
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• 2001

Protoplasts isolated from inbred lines of Brassica oleracea L. var capitata (cabbage) and Brassica campestris L. ssp. pekinensis (Chinese cabbage) were fused by PEG-mediated method, and somatic hybrid cells were differentiated into plants. for the identification of somatic hybrid plants, ploidy level, plant morphology, and cytological analysis were performed. All of the regenerated plants derived from fused protoplasts were shown to be 2X-4X, or higher ploidy level, presumably due to somatic hybridization or chromosome doubling. The morphology of leaves, petioles, and flowers showed an intermediate phenotype between Chinese cabbage and cabbage. Chromosome numbers in these somatic hybrids ranged mostly from 33 to 38. According to Genomic in situ hybridization (GISH) pattern, signals from both fusion parents of B.campestris or B.oleracea were detected in different colors when chromosomes of putative somatic hybrids were observed.

• 한국균학회지
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• 제21권4호
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• pp.323-331
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• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

• 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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• 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.

• KSBB Journal
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• 제5권3호
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• pp.255-261
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• 1990

Astaxanthin을 생산하는 효모 Phaffia rhodozyma로부터 제조된 상보적 돌연변이 균주간의 세포융합을 통하여 astaxanthin을 대량 생산하는 균주를 얻고자 시도하였다. 이들의 원형질체 융합빈도는 $1.3$\times$10^-^5-6.0$\times$10^-^5$이었고 DNA함량, 핵염색, UV조사에 대한 생존력 비교 그리고 형질분리분석등으로 핵융합을 확인하였다. 융합체 중 F1은 야생형의 모 균주와 비교했을때 astaxanthin생성량이 약 3배 증가하였다.