• Archives of Pharmacal Research
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• v.16 no.4
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• pp.300-304
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• 1993

Genetic transformation of streptomyces gaespitosus by plasmid plJ 702 was camied out. Optimal conditions for the protoplast preparation of streptomyces casepitosus, its regeneration, and its transformation by plJ 702 were evaluated. Addition of 2% glycine to the culture broth was optimal for protoplast yield. Formation and regeneration of protoplasts were most efficient when the mycelium were harvested at between late log and stationary growth phase. The regeneration frequency of the protoplasts was 15% when the protoplats were regenerated on R2YE agar media containing 0.5M sucrose. Under the best condition for protoplats (M.W. 4,000) treatment for 2 minutes.

• The Korean Journal of Mycology
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• v.18 no.3
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• pp.115-126
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• 1990

To establish basic techniques for protoplast fusion of Coriolus versicolor several factors affecting protoplast formation and regeneration were investigated. Protoplast isolation was at maximum with 2.5-day cultured mycelia of C. versicolor treated with the combination of two enzymes, Novozym 234 (10 mg/ml) and cellulase Onozuka R-10 (15 mg/ml), for 3-4.5 hours at $30^{\circ}C.$ As an osmotic stabilizer for stabilizing the protoplast, 0.6 M sucrose was the best for formation and regeneration of the protoplast from the mycelia of the fungus and the regeneration frequency was 3.48%. Protoplast fusion was made by a modified method of Peberdy using PEG (M.W. 4,000). The fusion frequency between two mutants of C. versicolor was 1.86% and the fusion products showed differences in growth rate and colony morphology.

• The Korean Journal of Mycology
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• v.22 no.2
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• pp.130-137
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• 1994

This experiment was carried out to investigate proper conditions for protoplast isolation and regeneration from mycelia of Lyophyllum decastes. Novozym 234(10 mg/ml) with 0.6 M $MgSO_4$ in phosphate buffer(pH 4.0) was proper for protoplast isolation. The optimal reaction time of the mycelium with the lytic enzyme was four hours in shaking condition at 120 strokes per min. When the mycelium of L. decastes was cultured at $24^{\circ}C$ for 5 days, the formation of protoplasts was effective. The liquid medium was more effective for protoplast isolation than the solid medium. In the liquid medium, high yields of protoplasts were obtained from 0.6 M $MgSO_4$ osmotic stabilizer. Protoplasts of L. decastes were regenerated to normal hyphal growth and the regeneration frequency of the protoplasts in the complete agar medium containing Triton X-100(0.0025%) was $5.94{\sim}8.32%$. The regeneration medium stabilized with 0.6 M sucrose was the best for regeneration of the protoplasts. In contrast to protoplast formation, regeneration was inhibited by the inorganic salts used as osmotic stabilizer.

• Journal of Plant Biotechnology
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• v.3 no.1
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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.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.1
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• pp.11-18
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• 1997

This study was to provide the basic data in improving protoplast formation and regeneration of antagonistic bacteria against phytopathogenic fungi and pest. The antagonistic rhizobacterium, BS 101, against Rhizoctonia solrmi and Fusurium oxyspomm was isolated and identified as Bacillus subtilis. Another bacterium for protoplast formation and regeneration was B. thuringiensis subsp. kurstcJtiHD-l (BT 37669) which have insectcidal toxin in the orders Coleopteria, Dipteria etc.. Auxotrophic mutants, BS 1013 and BT 69, were isolated by treating with NTG 300 ug/ml for 40 min. at $37^{\circ}C$, and with NTG 300 ug/ml for 30 min. at $37^{\circ}C$, respectively. The BS 1013 and BT 69 were converted to protoplas by treating with lysozyme 300 ugh1 for 30 min. at 37C, and lysozyme 9 mglml for 60 min. at $37^{\circ}C$, respectively. The fequencies of the protoplast formation of BS 1013 and BT 69 were 90.00 and 92.83% respectively, after 1~2 day at $37^{\circ}C$. The regeneration kequencies of the protoplasts BS 1013 and B T 69 were 0.52 and 0.10%, respectively, after 4~6 days at $37^{\circ}C$.

• The Korean Journal of Mycology
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• v.13 no.2
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• pp.79-82
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• 1985

The experiment of protoplast regeneration and reversion were undertaken to provide a basic techniques for protoplast manipulation. Protoplasts of Pleurotus florida and Pleurotus ostreatus were reverted to normal hyphal growth and the reversion frequency of both fungal protoplasts were $0.24{\sim}3.19%$. Reversion medium stabilized with 0.6 M potassium chloride and sucrose was better than the other stabilized one. The protoplast reversion frequency was increased when various amino acid and vitamin compounds were added to the hypertonic mushroom complete agar medium.

• Korean Journal of Microbiology
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• v.29 no.2
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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.

• KSBB Journal
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• v.18 no.4
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• pp.289-293
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• 2003

Doxorubicin is an anthracycline-family polyketide compound with a very potent anti-cancer activity, typically produced by Streptomyces peucetius. In order to increase doxurubicin productivity, a semi-industrial doxorubicin-producing Streptomyces strain named BR-Dox was cultured in a R2YE liquid medium containing CaCO$_3$, and then converted to a cell wall-free protoplast using lysozyme treatment method, followed by PEG-mediated cell wall regeneration. Among several protoplast-regenerated Streptomyces BR-Dox strains, two independent isolates named BR-Dox4 and BR-Dox6 were visually selected using thin layer chromatography (TLC) based on the pigment overproducing phenotype. Comparing with Streptomyces BR-Dox parental strain, two protoplast-regenerated strains, BR-Dox4 and BR-Dox6 exhibited 25.2％ and 12.2％ higher doxorubicin productivity analyzed by high pressure liquid chromatography (HPLC), respectively. This result suggests that a protoplast-regeneration of an antibiotics-producing Streptomyces strain should be a promising strain development approach for antibiotics overproduction in Streptomyces species.

• Korean Journal of Microbiology
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• v.28 no.4
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• pp.304-310
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• 1990

The conditions for protoplasts formation and regeneration of thermophilic anaerobic C. thermocellum and C. thermohydrosulfuricum were determined under the anaerobic growth conditions. The cells of C. thermocellum in initial exponential growth phase were identified to be the most suited for protoplast formation. The optimal conditions for protoplast formation were found to be at $37^{\circ}C$ for 2 hours with 0.5 mg/ml of lysozyme in TMG buffer (pH7.5). On the other hand, C. thermohydro-sulfuricum grown in the same medium but excluding glycine was optimally protoplasted at the same conditions but with 0.2 mg/ml of lysozyme. The protoplasts of both strains only subjected to lysozyme treatment of the short time were satisfactorily regenerated after 7-10 days incubation at $60^{\circ}C$ in regeneration medium containing 0.3-0.4 M sorbitol, 0.5% casamino acid, and high concentration of $CaCl_{2}$ and $MgCl_{2}$. The regeneration frequencies of the protoplasts of C. thermocellum and C. thermohydrosulfuricum were found to be very low level of $4.85{\times}10^{-3}$ and $4.23{\times}10^{-2}$, respectively. The nonregenerated L-form cells were also observed inregeneration medium together with regenerated cells.

• Korean Journal of Microbiology
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• v.23 no.3
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• pp.190-196
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• 1985

For frequency improvement of protoplast fusion in Brevibacterium flavum, Brevibacterium lactofermentum lactofermentum and Corynebacterium glutamicum, the effect of plasma expanders on fusion and cell wall regeneration, compatison between direct and two-step selection method, tendency of fusion frequency according to pH of fusion fluid and polyethylene glycol concentration were examined. By addition of 3% polyvinyl pyrrolidone to cell wall regeneration medium, regeneration frequencies were expressed 23 (Brevibacterium lactofermentum), 10.4 (Brevibacterium flavum) and 2.7 (Corynebacterium glutamicum) times higher than those of none polyvinyl pyrrolidone medium respectively.