• The Plant Pathology Journal
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• 제17권1호
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• pp.36-39
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• 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.

• 미생물학회지
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• 제29권2호
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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.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.251-257
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• 1986

Lactobacillus casei 균주의 protoplast 형성과 정상세포로의 재생에 관한 최적조건이 연구되었다. Lactobacilus casei 균주들은 sucrose 1 mole이 함유된 20mM potassium phosphate완충액 (pH6.8)에서 mutanolysin을 10 $\mu\textrm{g}$/$m{\ell}$ 농도로 처리하였을 때 용이하게 protoplast가 형성되었다. Protoplast의 최대형성은 균주가 0. 5%의 glycine이 함유된 TCM 배지에서 생장이 대수기 중기에서 말기에 도달된 세포를 수확하여 사용했을 때에 이루어졌다. 세포 재생은 6 mM CaCl$_2$, 6mM MgCl$_2$, 0.8 M sucrose 그리고 horse serum 10%가 함유한 재생 배지에서 효율적으로 이루어 졌다. Protoplast의 세포벽 재생 빈도는 3$0^{\circ}C$에서 3-4 일간 배양후 2-5% 범위로 나타났다.

• 한국균학회지
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• 제21권4호
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• pp.323-331
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• 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.

• 한국미생물·생명공학회지
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• 제13권4호
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• pp.391-395
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• 1985

내산성이 우수하고 citric acid와 SCP를 많이 생산할 수 있는 효모균주를 육종하기 위하여 구연산 생성효모인 Candide lipolytica의 종내 원형 질체융합 조건을 검토하였다. 배양기간과 효소처리조건을 최적화함에 의해 98%의 protoplast가 형성되었다. 3 % agar와 30mM $CaCl_2$를 함유한 재생용최소배지에 동일배지의 중층에 의해 약 20-30%의 protoplast가 재생되었다. 2개의 영양요구성이 상보적인 영양요구성원형질체, L-14($lys^-$)와 T-24($try^-$)에 100mM $CaCl_2$를 함유하는 30% PEG 6000을 $30^{\circ}C$ 에서 20분간 처리하여 4-5${\pm}$$10^{-4}$의 융합빈도가 얻어졌다.

• 한국미생물·생명공학회지
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• 제13권2호
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• pp.145-149
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• 1985

전분자원의 효율적 이용을 위한 방법의 일환으로 분리동정된 전분이용성 효모 H. anomala var. anomala FRI YO-32와 S. cerevisiae와의 세포융합가능성을 검토하기 위하여, 두 효모원형질체의 재생을 위한 최적조건들이 검토되었다. S. cerevisiae에 비하여 FRI YO-32균주의 원형질체가 삼투압안정성이 더 좋았으며 원형질체 재생에 영향을 주는 중요한 인자로서 agar와 삼투압안정제의 농도, 원형 질체 배양방법 등이 검토되었다. 또한 원형 질체 형성을 위한 효소처리 시간이 길어질수록 원형 질체 형성수율은 증가하나 재생효율은 감소하였다.

• 한국미생물·생명공학회지
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• 제15권2호
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• pp.89-94
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• 1987

Streptomyces mitakaensis 균주의 원형질체 형성과 정상세포로의 재생에 관한 최적조건을 연구했다. S. mitakaensis 균주를 GBYN 배지(glycerol 20g, beef extract 5g, yeast extract 5g과 NaCl 5g, 증류수 1,000$m\ell$)에 glycine 0.5% 함유된 배지에서 대수증식 기말까지 배양한 뒤에 lysozyme(1mg/$m\ell$)을 35$^{\circ}C$에서 60분간 처리를 했을 때에 원형질체 형성은 최고치를 나타냈다. 정상세포로의 재생은 R2 평판배지에 원형질체를 접종한 후 10일이 됐을 때에 재생이 되는 것을 관찰했고, H2액체 배지에서는 3일 후에 재생되는 것을 관찰했다. 세포재생 비율은 0. 1% 정도였다.

• 농업과학연구
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• 제17권2호
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• pp.77-81
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• 1990

Chitin 합성 저해제인 buprofezin이 P. ostreatus 및 P. sajor-caju 균사의 생장과 원형질체 나출 및 재생에 미치는 영향을 조사한 바를 요약하면 buprofezin은 P. ostreatus 및 P. sajor-caju.의 균사생장을 심히 저해하였고 buprofezin의 첨가농도가 높을수록 균사생장 억제가 심하였다. Buprofezin 처리에 의하여 기균사가 증가하였으나 균사의 형태에는 변화가 없었다. 원형질체의 나출량은 buprofezin 200-500 ppm 첨가구의 균사에서 무처리보다 현저히 높은 결과를 보였고, 원형질체의 재생율은 buprofezin의 처리에 의하여 증가하였다.

• 한국초지조사료학회지
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• 제17권1호
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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 $37^{\circ}C$.

• 한국미생물·생명공학회지
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• 제22권2호
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• pp.143-151
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• 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.