• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.345-348
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• 1985

Recombinant chimeric plasmid constructed with Xba I digested pUBl10 and -pE194 was transformed by polyethylene glycol induced protoplast transformation system into Bacillus subtilis BR 151 on the mannitol regeneration media, and two genes of antibiotics resistance were expressed simultaneously in the transfromant. Transformation frequency of the recombinant plasmid was 6.5 $\times$ 10$^{-5}$ on the mannitol regeneration agar plate containing neomycin and erythromycin. The replication of recombinant plasmid in the recipient cells was confirmed by the alkaline extraction method and agarose gel electrophoresis.

• 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.

• 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 Plant Tissue Culture
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• v.21 no.1
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• pp.23-28
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• 1994

This study was rimed out to examine the difference in the cell vitality between mesophyII protoplast (MP) and paraveinal mesophyII protoplast (PVMP) of Nicotiana tabaccum 'Xanti', Petunia hybrida 'Blue Star' and Chrysanthemum morifolium 'Baeckwang' by using urea permeability technique. The effects of various enzyme solutions and incubation time, NAA and thidiazron on plant regeneration from isolated protoplasts were also investigated. The vibratome technique was used for protoplast isolation and urea permeability test because the fresh living, thin tissue stripes (50 ${\mu}{\textrm}{m}$ of thickness) could be obtained with minimal damage with the vibratome. For the three plants examined, the urea permeability on the tested tissue stripes was relatively higher in PVMP than in MP by about Ks = 2.0 $\times$ 10$^{-5}$ cm/sec. The treatment of an enzyme mixture of 1.5% cellulase R-10, 1% Driselase, 0.5% Macerozyme R-10, and 0.5% Pectinase for 4 to 8 h was effective on the isolation of PVMP. The highest frequency of callus formation and plant regeneration from the isolated protoplasts was obtained with NAA 2 mg/L and thidiazuron 0.01 mg/L. Furthermore, the results demonstrated that cell devision and plantlet regeneration was more frequent in the PVMP than in the MP of the same leaf or plant We, therefore, conclude that UM is an excellent experimental material for the callus formation and regeneration from isolated protoplasts.

• Journal of Plant Biotechnology
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• v.7 no.3
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• pp.169-174
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• 2005

The optimal culture conditions were studied for plant regeneration from mesophyll protoplasts of Solanum sisymbriifolium. Axenic seedlings of S. sisymbriifolium were used as a explant for protoplast culture. Many viable protoplasts were isolated by incubating leaf slices in an enzyme solution containing 0.25% Meicerase and 0.05% Macerozyme for 16 hr at $25^{\circ}C$ without shaking. Protoplast density of $5.0{\times}10^4\;ml^{-1}$ in Kao medium containing 5.0 mg/L NAA, 1.0 mg/L 2,4-D and 1.0 mg/L BA was optimal for colony formation. Most colonies were formed when protoplasts were cultured at $25^{\circ}C$ after initial culture at $30^{\circ}C$ for one week. On the MS agar medium with 1.0 mg/L zeatin, 38.4% of protoplast-derived calli differentiated shoots. These shoots rooted on 1/2MS medium with 5.0 g/L sucrose and 2.5 g/L gellan gum, and developed into whole plants.

• Korean Journal of Plant Tissue Culture
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• v.22 no.1
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• pp.7-11
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• 1995

Protoplasts were isolated from hypocotyl tissues of 5-day-old Brassica oleracea var capitata Green Challenger seedlings. Several media were used for protoplast culture and shoot regeneration. The shoot-regeneration rapacity of protoplast derived callus depended on the initial culture medium. Protoplasts were cultured in liquid medium (B5 medium supplemented with CaCl2, 2H2O 600mg/L, g1ucose 20g/L, D-mannito1 70g/L, NAA lmg/L, BA lmg/L, 2.4-D 0.25 mg/L)at 27$^{\circ}C$ under the dark After 5 to 10 days, cultlues were diluted with medium with a reduced osmotic stabilizer and then transferred to illuminated conditions. The culture medium was changed with the fresh medium at 7- to 10-day-intervals until the formation of microcallus. Hypocotyl protoplast-derived callus proliferated when transferred to MS medium supplemented with NAA lmg/L, BA 1mg/L and GA$_3$ 0.02mg/L. Upon transfer to MS basal medium without growth regulators, roots were produced. In an attempt to increase the regeneration frequency, 10g/L polyvinylpyrrolidone was added to the regeneration medium, but the shoot regeneration was mot improved. The regenerated whole plants were acclimated in a sterized soilless mixture(vermiculite 2;perlite 2;peat moss1) in a culture room.

• 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}$.

• KSBB Journal
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• v.6 no.3
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• pp.289-297
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• 1991

The insecticidal protein gene in the pKL-20-1 clone derived from Bacillus thuringiensis serovar. kurstaki plasmid was subcloned in the plant shuttle vector, pGA643. The 7.3 kb fragment was cloned in the BglII and Hpal sites of pGA643 vector and expressed in E. coli S17-1, which produced insecticidal proteins killing Bombyx mori larvae. The clone was named pHL-20. The protoplast formation, calli induction and plantlet regeneration of Nicotiana tabacum was carried out. A tremendous number of mesophyll protoplasts of N. tabacum were formed, up to 7$\times$105 protoplast per ml, for 20 hours in darkness in the enzyme solution of 0.5% cellulase and 0.1% macerosin, pH 5.8. The viabilities of the protoplasts were maintained above 80% for 6 days in the media containing 2mg/1 of NAA and 1mg/1 of kinetin. Calli were induced from the protoplasts and leaves of the N. tabacum on MS medium containing 0.5mg/1 BAP. Under the culture conditions the protoplasts underwent repeated cell division into calli. Plantlets were regenerated from callus cultures derived from protoplast and leaves. Shoots were induced in a medium containing 1mg/1 of BAP.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.243-250
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• 1986

Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 72%, respectively. When bovine serum albumin(4mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol(0.5mg/ml), 96-99 % of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the addition of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and $CaCl_2$ are 20% and 100mM while optimal pH and exposure time are 6.0 and 30min. The fusion frequencies between complementary mutants ranged from $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.

• Korean Journal Plant Pathology
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• v.3 no.2
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• pp.124-130
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• 1987

The optimum conditions for protoplast formation and regeneration from Pyricularia oryzae Cav. were selected as follows. As a basic solution, 0.02M potassium phosphate buffer solution plus 0.6M KCl adjusted to pH 5.2 with 1N HCl was used. A mixture of enzyme combinations with 20mg Cellulase R-l0/ml, 5mg Macerozyme R-l0/ml and l0mg Driselase/ml used as a lytic enzyme showed better lytie effect than any single enzyme treatment for protoplast formation. Two-day-old mycelia of P. oryzae grown in the mixture of three lytie enzyme solution at $30^{\circ}C$ for 3 hr showed best condition for protoplasts formation. For regeneration from the protoplasts of P. oryzae, potato dextrose agar containing 0.02M potassium phosphate plus 0.6M KCl used as a stabilizer was best for regeneration medium.