• Korean Journal of Microbiology
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• v.28 no.2
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• pp.98-103
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• 1990

Brevibacterium lactofermentum SWA (arg trp) and B. lactofermentum SWB (met ser) were obtained from UV and NTG treatment. The rates of protoplast formation by B. lactofermentum SWA and SWB were 99.93% and 99.98%, respectively when each strain was treated with penicillin G in mid exponential growth phase, followed by incubation with 400 $\mu\textrm{g}$/ml of lysozyme in lysis fluid supplemented with 0.4M sucrose. Frequencies of protoplast regeneration in B. lactofermentum SWA and B. lactofermentum SWB were 9.27% and 10.32% respectively, on regeneration medium containing 0.5M sodium succinate, 50 mM $Mg^{2+}$, and 3% PVP. In intraspecific protoplast fusion between B. lactofermentum SWA and B. lactofermentum SWB, fusion frequency of $2.30\times 10^{-5}$ was observed by using the 100mM $CaCl_{2}$ and 30% PEG 6,000 in fusion fluid. Relative recombinant frequencies in each marker by means of selective media could be used for genetic analysis.

• Applied Biological Chemistry
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• v.36 no.6
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• pp.562-566
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• 1993

The conditions required for plant transformation through the electroporation system and for the electrofusion of the prtoplasts were investigated for geranium (Pelargonium zonale hybrids). The optimum condition for electroporation was 1.77 kV/cm for $40\;{\mu}sec$ under which 70% of the protoplasts were viable and 58% of the viable protoplasts were stained with methylene blue. The pBin19 DNA plasmid used as a carrier vector was isolated from E.coli $DH5{\alpha}$ strain, purified, identified by the electrophoresis on agarose gel and electroporated into the protoplasts. The KM8 liquid medium gave better cell division than any other media. One MHz of AC frequency with 40 V/cm of amplitude for 15 sec followed by 0.5 kV/cm of DC amplitude for $60\;{\mu}sec$ was most efficient for the electrofusion of protoplasts.

• Korean Journal of Plant Tissue Culture
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• v.28 no.1
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• pp.15-20
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• 2001

Protoplast and callus of three rice varieties, CI5309 (salt-tolerant), Dongjinbyeo (moderately tolerant), Nakdongbyeo (salt-sensitive), were cultured on the MS medium containing 0, 0.3, 0.6, and 0.9% of NaCl. The survival rates of protoplasts were decreased according to the hours after NaCl treatment, and the survival rates of protoplast differed among three varieties at 9∼l2 hours after treatment. The contents of proline of CI5309 and Dongjinbyeo were almost the same at the different levels of NaCl, and that of Nakdongbyeo, however, was greatly decreased at the high concentration of NaCl. The contents of Ca, K and Mg were decreased according to the increasing of NaCl concentrations, but the contents of those did not differ among three varieties at the high concentration of NaCl. The contents of Na in each varieties were almost the same at the three levels of NaCl.

• 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.

• The Korean Journal of Mycology
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• v.38 no.1
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• pp.21-24
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• 2010

This study was carried out to select cell wall degrading enzymes for maximizing protoplast yield from Basidiomycetes. The protoplasts were released from spore suspension, mycelia cultured on cellophane membrane, and homogenized mycelia of Flammulina velutipes using commercial cell wall degrading enzymes. The highest yield of protoplasts was obtained from the homogenized mycelia treated with the enzyme combination of $Glucanex^R$ 200G and cellulase onozuka R-10. The protocol was also available for Pleurotus ostreatus, P. eryngii, and Hypsizygus marmoreus.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.30 no.2
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• pp.195-200
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• 1985

This experiment was carried out to know the processes of protoplast isolation, culture and plant regeneration in aims of introducing foreign genes into plant cells through plant gene vector, and cellular selection for plant improvement. The main results indicated that 2% cellulase plus 0.5% macerozyme is proper for isolation of protoplasts from leaf mesophyll cells of N. plumbaginifolia, plating efficiency was higher in 1.4-2.0 x 10$^4$ cells/ml, complete cell wall was regenerated after 2 days culture, cell division and cell mass were observed after 4 days and 2 weeks, respectively, colony was developed after 3 weeks culture, addition of 1-2mg/l BA promoted shoot differentiation while root differentiation did not required hormone and seeds were harvested from more than 100 cell lines for further investigation and study.

• 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.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.150-155
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• 1988

In order to produce L-lysine from cellulosic substrates by the intergeneric protoplast fusion between cellulolytic bacteria, Cellulomonas flavigena KFCC31221 and amino acid producing bacteria, Brevibacterium flavum ATCC14067, Corynebacteriurn glutamicum ATCC13032, conditions for protoplast formation and regeneration of these strains were investigated. After the strains were mutated with 500$\mu\textrm{g}$/$m\ell$ N-methyl-N'-nitro N-nitrosoguanidine for 30 min and the mutants were enriched by treating 300$\mu\textrm{g}$/$m\ell$ penicillin-G for 2 hrs, B. flavum Hse- Str$^{r}$ , C. glutamicum Met$^{-}$Thr$^{-}$ Rif$^{r}$ and Cellulomonas flavigena Thr$^{-}$Val$^{-}$Kan$^{r}$ were isolated. The rate of protoplast formation ranged from 95 to 98% when strains were treated at the concentration of 500$\mu\textrm{g}$/$m\ell$ of lysozyme, pH 6.5, 33$^{\circ}C$, for 6 hrs. in Tris- malate buffer supplemented with 0.4M sucrose as osmotic stabilizer. Approximately 30-33% protoplast was regenerated on the regeneration complete medium(RCM) containing 1.5% agar and 0.5M sodium succinate overlaid with the same medium except 0.7% agar.

• Korean Journal of Agricultural Science
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• v.15 no.1
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• pp.15-22
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• 1988

Factors affecting the regeneration, reversion of protoplasts from mycelium of F. velutipes were investigated and the selection of auxotrophic mutants from protoplasts of F. velutipes was performed. PDP medium stabilized with 0.6M sucrose was suitable for the regeneration of protoplasts, and regeneration frequency was 0.47-1.32. The regeneration frequency of protoplasts was increased when nutrients were added to the regeneration medium. Especially, yeast extract was the most effective to regeneration of protoplasts. Regeneration pattern of protoplasts was formation of germ tubes from bud-like cells. 13-18% of monokaryotic strains was appeared from reverted protoplasts. Five of auxotrophic mutants were isolated from strains showed survival frequency of 1.9-16.

• Journal of Mushroom
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• v.13 no.3
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• pp.270-273
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• 2015

Hypsizigus marmoreus is commercially the most important edible mushroom in Japan. This mushroom is usually cultivated for a longer period (about 85~120 days) than other mushroom. In order to develop a new cultivar that has a shortened cultivation period, the genome analysis of this strain has been considered. This study aims to obtain parental monokaryotic strains reproducing 'Haemi' cultivar in Hypsizigus marmoreus for reference genome sequencing. The mycelia were cultured in MCM and MYG media for various incubation periods. Homogenized mycelia were treated with commercial cell wall degrading enzymes to maximize protoplasts production yield from Hypsizigus marmoreus. The greatest number of protoplasts was obtained from mycelia cultured in MCM media for 3 days using Novozyme enzyme. The isolated protoplasts were grown in regeneration agar media after two weeks. Regenerated colonies were picked and moved on separated dishes for microscopic observation. Neohaplonts regenerated from dikayotic strains were identified by the absence of clamp connections. We confirmed that one of monokaryotic strains is a parental strain by crossing with an original compatible strain of 'Haemi' cultivar. This parental strain will be used for reference genome sequence analysis.