• The Plant Pathology Journal
• /
• v.17 no.1
• /
• pp.36-39
• /
• 2001

The secondary effects of pencycuron on cell membrane of Rhizoctonia solani AG4 were investigated by the observation of giant protoplast formation and lipid peroxidation. Compared to protoplasts of R. solani R-C (sensitive strain) and Rh-131 (non-sensitive strain) increased in their size by 2.0-3.5 times 12 h after incubation in potato-dextrose broth containing novozyme (7 mg/$m\ell$) and $\beta$-glucuronidase ($60\mu\textrm{g}/$\textrm{ml}) with 0.6 M mannitol (pH 5.2). The increase of protoplast size in R-C was slightly inhibited from $13.8\textrm{mg}/\textrm{ml}$ without pencycuron to 10.3 ${\mu}{\textrm}{m}$ with 1.0$\mu\textrm{g}$/$m\ell$ of pencycuron. However, the size of giant protoplast of Rh-131 was not affected by the pencycuron treatment. Both strains R-C and Rh-131 did not exhibit the lipid peroxidation 12 h after the application of 1.0 $\mu\textrm{g}$/$m\ell$ pencycuron. The remarkable peroxidation of membrane lipid was observed only in R-C 24 h after pencycuron application, but not in Rh-131. Althought the inhibition of giant protoplast formation and the membrane lipid peroxidation were observed only in the sensitive strain R-C by pencycuron, it is difficult to conclude that these are the primary mechanism of pencycuron. The mild activity of pencycuron on the inhibition of giant protoplast formation and late membrane lipid peroxidation in the fungicide-sensitive strain did not noincid with the dramatic activity of pencycuron in R. solani. Therefore, our results suggest that inhibition of giant protoplast formation and membrane lipid peroxidation is the secondary effect of pencycuron.

• Korean Journal of Microbiology
• /
• v.29 no.2
• /
• pp.123-129
• /
• 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.

• Microbiology and Biotechnology Letters
• /
• v.14 no.3
• /
• pp.251-257
• /
• 1986

Optimum conditions for the protoplast formation and regeneration of Lactobacillus casei have been searched for. L. casei cells were converted to protoplast by treating with 10$\mu\textrm{g}$/$m{\ell}$ of mutanolysin in 20mM potassium phosphate buffer (pH6.8) containing 6mM CaCl$_2$, 6mM MgCl$_2$ and 1M sucrose. Maximum number of protoplasts was obtained when cells were taken from Tomochika's medium containing 0.5% glycine at the middle to late logarithmic growth phase. Regeneration was efficiently accomplished on the regeneration medium containing 6mM CaCl$_2$, 6mM MgCl$_2$, 0.8M sucrose and 10% of horse serum. The efficiently of the cell wall regeneration from protoplasts was 2-5% after 3-4 days of incubation at 3$0^{\circ}C$.

• The Korean Journal of Mycology
• /
• v.21 no.4
• /
• pp.323-331
• /
• 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.

• Microbiology and Biotechnology Letters
• /
• v.13 no.4
• /
• pp.391-395
• /
• 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
• /
• v.13 no.2
• /
• pp.145-149
• /
• 1985

Studies were conducted on the conditions for yeast protoplast regeneration in Hansenula anomala var.anomala FRI YO-32 and Saccharomyces cerevisiae. Protoplasts lysed when suspended in hypotonic solutions of KCI, and the least degree of osmolysis was shown in the hypertonic solution containing 1.4M KCI for the strain FRI YO-32 or 0.8M KCI for S. cerevisiae. It was considered that the concentration of agrar and KCI, and protoplast plating method were the main factors influencing regeneration of yeast protoplasts. Yeast protoplasts were regenerated very favorably when embedded in the complete protoplast regeneration media containing 3％ agar as well as 0.4M KCI for the strain FRI YO-32 or 1.0M KCI for S. cerevisiae. It was shown from the relationship between protoplast formation and regeneration that the higher extent of protoplast formation, the lower extent of protoplast regeneration.

• Microbiology and Biotechnology Letters
• /
• v.15 no.2
• /
• pp.89-94
• /
• 1987

The optimal conditions for the protoplast formation and regeneration of Streptomyces mitakaensis have been investigated. S. mitakaensis cells were converted to protoplast by treating with 0.1 mg/$m\ell$ of lysozyme in phosphate-tris buffer (pH 7.2) to the cells grown at the late logarithmic growth phase in the GBYN medium (gycerol 20g, beef extract 5g, yeast extract 5g, NaCl 5g in 1 liter of distilled water) contained 0.5% glycine. Cell regeneration from protoplast was accomplished in 10 days post inoculation on the R2 regeneration agar medium and at 3 days post inoculation on the H2 regeneration liquid medium. The efficiency of the regeneration was 0.l% in 3 days at 35$^{\circ}C$.

• Korean Journal of Agricultural Science
• /
• v.17 no.2
• /
• pp.77-81
• /
• 1990

Effects of buprofezin, an inhibitor of chitin synthesis, on mycelial growth, protoplast formation and reversion of Pleurotus ostreatus and P. sajor-caju were investigated. The mycelial growth of Pleurotus ostreatus and P. sajor-caju was the inhibited by buprofezin treatment, and the inhibition rate was severer as the concentration of the buprofezin increased. Aerial mycelium formation was increased by buprofezin treatment, but mycelial morphology was not changed. Protoplast formation of Pleurotus ostreatus and P. sajor-caju. was significantly increased when buprofezin was added to the culture medium at the concentration of 200~500 ppm and the protoplast reversion of the mushrooms was also increased by the treatment of the buprofezin.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.17 no.1
• /
• pp.11-18
• /
• 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 $37^{\circ}C$.

• Microbiology and Biotechnology Letters
• /
• v.22 no.2
• /
• pp.143-151
• /
• 1994

In the course of the study on strain inprovement by protoplast fusion, Lactobacillus acidophilus 88 protoplasts production and regeneration conditions were investigated. This strain produced a bacteriocin that revealed strong inhibitory activity against various indicator strains, especially L. helveticus CNRZ 1096. Protoplasts of L. acidophilus 88 strains were very efficiently obtained by treatment with 125 $\mu $g/ml lysozyme in a protoplast forming buffer containing 20 mM N-2 hydroxy-ethtl-piperazine-N'-2-ethane-sulfonic acid(HEPES, pH 7.0) and 1M sucrose at 37$\circ $C for 30 min. Hovever, treatment with mutanolysin was not effective for the production of L. acidophilus 88 protoplasts under the same conditions. High protoplast yield was obtained form the cells at the middle to late logarithmic growth phase in the de Man, Rogosa and Sharpe(MRS) medium. Regeneration was efficiently accomplished with the MRS medium containing 10% sucrose.