• The Korean Journal of Mycology
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• v.21 no.4
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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.

• Archives of Pharmacal Research
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• v.14 no.3
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• pp.282-283
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• 1991

Stable intergeneric hybrids involving allodiploid were obtained through protoplast fusion and nuclear transfer between the auxotrophic mutans of two basidiomycetes. Ganoderma lucidum and Corolus versicolor.

• Journal of the East Asian Society of Dietary Life
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• v.5 no.1
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• pp.93-99
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• 1995

For the improvement of the L-lysine productivity of Brevibacterium flavum and Corynebacterium glutamicum, fusants were induced by interspecific protoplast fusion of Bacillus subtilis with C. glutamicum and B. flavum. The following results were obtained through protoplast formation of strains condition of protoplast fusion, characteristics of the fusants, and the productivity of lysine form starch. B. flavum BF-5 and C. glutamicum protoplasts were made by the treatment of 0.3unit/$m\ell$ of penicillin G at the early stationary growth phase for 2 hours followed by incubation with 10mg/$m\ell$ of lysozyme at 37$^{\circ}C$ for 120 min. When a mixture of the protoplast was treated with 30% PEG(M.W.6,000) solution containing 50mM CaCl2 at optimal conditions, the intergeneric fusion frequency between protoplasts of C. glutamicum CG-2 and B. subtilis BD 224 was 7.1${\times}$105. The genetic properties on the L-lysine producing fusants were compared with those of parental strains. As a results, the intergeneric fusants were completed in each auxotrophic requirement, resistances for S-(2-amino-ethyl)-L-cysteine and kanamycine were confirmed. And one of fusants selected, FBB-41 were found to be genetically stable fusants. The aspartokinase activity of FBB-41 strain increased than that of the parent strain.

• Journal of Plant Biology
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• v.30 no.4
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• pp.287-298
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• 1987

The regenerative capacities of protoplasts isolated from potato (Solamum tuberosum L.) tubers and tobacco (Nicotiana tabacum L.) mesophyll tissues were examined, and then their intergeneric protoplast fusion was carried out. The potato tuber-derived protoplasts proliferated into the calli some of which showed rudimentary shoot-like structures, which had not been attempted before from tubers, while the tobacco protoplasts were regenerated into the whole plants. Intergeneric protoplast fusion between potato and tobacco was carried out and the heteroplasmic fusion products were formed. The first cell division of some of them was observed after 5 days of culture.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.528.1-528
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• 1986

Cellulose utilising hybrids between Cellulomonas fimi and Bacillus pumilus were isolated after PEG mediated protoplast fusion. 33% (w/v) PEG #6, 000 and 50mM $Ca^{++}$were optimum concentration. The intergeneric fusion frequency was 3.2$\times$10$^{-7}$ xtracellular CMCase and $\beta$-glucosidase activities were detected from one hydrid unlike only CMCase was detected from Cellulomonas fimi.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.232-237
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• 1993

A strain of yeast which can convert starch directly to ethanol was developed by the intergeneric protoplast fusion between Schwanniomyces alluvius possessing $\alpha$ amylase as well as glucoamylase with debranching activity and FSC-14-75 which previously had been formed from a heterologous transformation and subsequent intergeneric protoplast fusion. Fusants were selected on minimal medium after protoplasts of auxotrophic mutant of S. alluvius fused with heat-treated protoplasts of FSC-14-75 in the presence of 30%(w/v) PEG and 20 mM $CaCl_2$. The fusion frequency was in the range of $10^{-6}$ order. All fusants tested were intermediate types of parental strains for carbon compound assimilation, and their cell volumes were approximately 1.1 times larger than FSC-14-75 and 1.8 times larger than S. alluvius. The fusants were unable to sporulate like FSC-14-75, while S. alluvius could sporulate. In flask scale the most promising fusant, FSCSa-R10-6, produced 7.83%(v/v) and 10.17%(v/v) ethanol from 15% and 20% of liquefied potato starch, respectively, indicating that the fermetation efficiency of each case increased 1.2 times and 1.6 times than that of FSC-14-75. The elution pattern on DEAE-cellulose chromatography showed that FSCSa-R10-6 has four distinct amylase peaks of which two peaks originated from S. alluvius and the other two from FSC-14-75. These results suggest that the enhanced fermentation efficiency of the fusant might be due to almost-complemented parental amylases.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.218-223
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• 1993

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

• Journal of Microbiology and Biotechnology
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• v.6 no.4
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• pp.286-291
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• 1996

A study was conducted to investigate the characteristics of segregation and alcohol fermentation of intergeneric fusants. The protoplast fusion of both Pichia stipitis CBS 5776 and Saccharomycess cerevisiae STV 89 was carried out. The fusion frequency was $5\times10^{-8}$ and among fusants selected, a fusant F5 showed the best results in ethanol production by sucrose and xylose fermentations. The performance of xylose fermentation by this fusant was better than that of P. stipitis CBS 5776 and fusant F5 exhibited sucrose fermentation patterns intermediate to the two parent strains. The fusant F5 was segregated into a pair of parental strains during the several culture passages. In the average, 91$%$ of colonies had a similar characteristics of P. stipitis while 7$%$ of colonies resembled S. cerevisiae. Only 2$%$ of colonies had the characteristics of the original fusants. At the sixth passage, all segregants resembled P. stipitis. From these results it is suggested that intergeneric protoplast fusion led to an integration of S. cerevisiae genes, rather than whole chromosomes, within the entire genome of P. stipitis.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.175-181
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• 1988

To produce L-lysine from cellulosic substrate, the intergeneric protoplast fusion between Cellulomonas flavigena and Corynebacterium glutamicum, Cellulomonas flavigena and Brevibacterium flavum was performed. The fusion frequencies were 1.9$\times$10$^{-6}$ to 2.1$\times$10$^{-6}$ for the regenerated protoplasts when two parental strains were treated with 30% of polyethyleneglycol (M.W.6000) containing 5 mM EDTA at 3$0^{\circ}C$ for 30 min. Two fusants, FCB3 and FCC 19 were finally selected by comparision of their genetic stability and L-lysine productivity. The properties of fusants-DNA con-tent, G＋C content and L-lysine productivity-were investigated. The DNA content of fusants was greater than those of the parental strain and their G＋C contents are equal to half of total G＋C con-tent of two parental strains. The fusants showed high productivity of L-lysine from carboxy methyl cellulose as substrate.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.104-111
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• 1987

As a trial method of breeding L-lysine producing strains, the intraspecific protoplast fusion bet-ween Brevibacterium flavum ATCC 21528R and Brevibacterium flavum ATCC 21529S and the intergeneric protoplast fusion between Brevibacterium flavum ATCC 21528R and Corynebacterium glutamicum ATCC 13058S were performed. The optimum conditions for protoplast formation of these strains were examined and the effect of plasma expander on regeneration and/or fusion was also observed. Both fusants No. CH23 and No. CH4l showed higher productivity of L-lysine than those of parental cells under the optimum cultural conditions at a rate of 21％ and 8.9％, respectively. And, activity of several enzymes in L-lysine biosynthetic pathway including aspartokinase, a rate-limiting enzyme, was determined. Besides, metabolic control mechanism of L-lysine biosynthesis in fusant No. CH23 and in No. CH41 was investigated to compare with that of parental strains.