• Archives of Pharmacal Research
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• v.20 no.5
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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.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.163-167
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• 1988

The conditions for the protoplast fusion of auxotrophic mutants of Penicillium verruculosum were determined. A preparation of commercial enzyme Novozym 234 was used to successfully isolate protoplast from the 20hr old mycelium of P. verruculosum. Under optimal condition, the protoplast yield ranged from 2.4$\times$10$^7$ to 3.0$\times$10$^7$ protoplasts from 400mg of damp mycelia of various auxotrophic mutant strains. The regeneration frequency ranged from 26.6 to 42.4% and the spontaneous reversion frequency of the protoplasts on the regeneration minimal medium was less than 10$^7$. The optimal concentration of PEG 6000 was 20%, and exposure of protoplasts to PEG for 10 min was found to be sufficient for protoplast fusion. Optimal pH of fusion mixture was deter-mined as 5.5 and l0mM of calcium chloride in fusion mixture effectively enhanced the protoplast fusion frequency. Under optimal condition, the fusion frequency between various auxotrophs ranged from 1.8$\times$10$^{-3}$ to 3.5$\times$0$^{-3}$.

• Journal of Plant Biotechnology
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• v.41 no.2
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• pp.65-72
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• 2014

Plant cells from which the cell walls have been enzymatically or mechanically removed are called protoplasts. The protoplasts are theoretically totipotent and can be used as sources of somatic cell fusion in practical breeding programs. Wild Solanum species have often been used as sources of important agricultural traits including diverse disease resistance. However, they cannot often be directly applied to breeding programs due to their sexual incompatibility with S. tuberosum. Somatic hybridization via protoplast fusion is one of the ideal methods to overcome this limitation and to introgress certain traits into S. tuberosum. This technique has still widely been used in potato since the first fusion was reported in 1970s. Therefore, this review highlights general perspectives of protoplast fusion and discusses the application of protoplast fusion in potato breeding.

• 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.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.391-395
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• 1985

In order to develope a protoplast fusion system for citric acid and SCP producing Candida lipolytica, the optimal conditions for the formation and regeneration of protoplast were examined and the protoplast fusion was performed. At the optimal conditions of growth phase and Zymolyase treatment, frequencies of protoplast formation were 98%. Approximately 20-30% of protoplasts were regenerated on the regeneration minimal medium containing 3% agar and 30mM $CaCl_2$ with the overlay of the same medium. The fusion frequencies, 4-5${\pm}$10$^{-4}$, were accomplished by the treatment of two nutritionally complementary auxotrophic protoplasts, L-14 ($lys^-$) and T-24 (X$30^-$), with 30% PEG 6000 containing 100mM $CaCl_2$ at $30^{\circ}C$ for 20 minutes.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.303-309
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• 1988

In order to establish the process of interspecific protoplast fusion of the genus Cellulomonas capable of utilizing of cellulose, C. flavigena NCIB 12901 and Cellulomonas sp. CSI-1, the optimum conditions for the regeneration and fusion were examined. The condition of suitable osmotic stabilizer for the protoplast regeneration of C. flavigena was established by using 0.4M sorbitol. And then, by addition of 3% po]yvinyl pyrrolidone (PVP) to cell wall regeneration medium, regeneration frequency was increased 3 times higher than that without PVP addition. The optimum conditions for the interspecific protoplast fusion between auxotrophic and antibiotics resistant mutants were obtained with 40%(W/V) of PEG (polyethylene glycol) 6000 as the fusogenic agent and 25mM of CaCl$_2$on treating time for 15 min. The fusion frequency between mutants was from 2.0$\times$10$^{-4}$ to 4.0$\times$10$^{-4}$ under the optimum conditions. The fusants were confirmed to revert from protoplast to cells of rod type during regeneration process and the aggregation of protoplast by PEG was observed. Also the progress of fusion was observed by scanning electron microscopy, Many isolated fusants were shown to be complement clones of both parents which occured at a high frequency among the isolated clones.

• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.151-156
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• 1991

In order to construct a yeast strain having high ethanol tolerance together with good lactose fermentation ability, the protoplast fusion using Saccharomyces cerevisiae STV 89 and Kluyveromyces fragilis CBS 397 was carried out. Auxotrophic mutants of K. fragilis were obtained as a selection marker by treatment of ethylmethane sulfonate. The best mutant for protoplast fusion was selected based on the capabilities of ${\beta}-galactosidase$ production and lactose fermentation. The protoplast fusion using polyethylene glycol and calcium chloride solution led to the fusion frequence of $3{\times}10^{-6}$ and a number of fusants were obtained. Among these fusants, a fusant F-3-19 showed the best results in terms of ethanol tolerance, ${\beta}-galactosidase$ activity and lactose fermentation. The performance of lactose fermentation and ethanol tolerance by this fusant were better than those of K. fragilis. Study on the ethanol tolerance having relation to fatty acid composition and intracellular ethanol concentration revealed that the fusant F-3-19 had a higher unsaturated fatty acids content and accumulated less amount of intracellular ethanol compared with a parent of K. fragilis.

• YAKHAK HOEJI
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• v.38 no.5
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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.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.47-54
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• 1990

In order to investigate physiological characteristics of fusants by interspecific protoplast fusion of the genus Cellulomonas, protoplasts of Cellulomonas flavigena NCIB 12901 and Cellulomonas bibula NCIB 8142 were fused and cell wall regenerated. To give gene maker, C. bibula was treated with 500 ug/ml NTG for 1 hr and arginine requiring auxotrophic mutants were isolated. Protoplasts of the genus Cellulomonas were obtained by treatment with $600{\mu}{\textrm{g}}$/ml lysozyme, and 0.5M sorbitol was optimal for osmotic stabilizer on protoplast fromation. Protoplast fusion was enhanced by 40% PEG)M.W.6,000) containing 25 mM $CaCl_{2}$ at $30^{\circ}C$ for 30 min and fusion frequency between C. bibula and C. flavigena was $5\times 10^{-4}$. Processes of protoplast formation, cell wall regeneration and protoplast fusion were obsdrved by scanning electron microscope. By comparing enzyme activities of cellulase, exocellobiohydrolase, .betha.-glucosidase of the parent strains of Cellulomonas with those of thier mutants and fusants, fusants with increased enzyme activity were obtained. By the studies on nutritional requirement, antibiotic resistance, cellulolytic enzyme activities, type of peptidoglycan and motility of two mutants and fusants, fusants were proved to be recombinant of both mutant strains.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.163-167
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• 1985

To investigate the condition for the protoplast fusion of Streptococcus lactis, streptomycin(200 $\mu\textrm{g}$/$m\ell$) resistant and rifampicin (200$\mu\textrm{g}$/$m\ell$) resistant mutants were isolated. By using these markers, protoplast fusion was carried out in the presence of CaCl$_2$ and polyethylene glycol. The optimal conditions for the protoplast fusion were obtained by treatment of protoplasts with 150 mM CaCl$_2$ (final concentration; 25 mM) and 40％ (w/v) PEG 4, 000 for 2 min. At the optimal conditions, the fusion frequency was 6.26$\times$10$^{-5}$ . On the other hand, genetic recombination between the antibiotic resistant mutants by mating was not observed.