• 미생물학회지
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• 제30권6호
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• pp.514-518
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• 1992

The present study has been perromed to investigate the optimal conditions for protoplast formation and regeneration of oleandomycin-producing Streptomyces antibioticus (S. antibioticus) KCTC 1081. Mycelia were grown in YME medium containing 0.2% (w/v) glycine and converted into the protoplast by incubating at 35.deg.C for 60 minutes in protoplast buffer (P buffer) containing 4 mg/ml lysozyme. The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 mM $Mg^{++}$, 5 mM $Ca^{++}$ and 0.3 M sucrose at 28.deg.C for 5 days. From these experiments, we established the improved regeneration medium and a protocol which supports higher and more consistent levels of regeneration of S. antibioticus protoplasts. The regenerant showed an increased antimicrobial activity compared with that of the initial strain.n.n.

• Journal of Plant Biology
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• 제32권4호
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• pp.313-322
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• 1989

A system was established for induction of multi-shoots and plant regeneration from mesophyll protoplasts of alfalfa, Medicago sativa L. cv. Vernal. Different hormonal effects were tested at each step of protoplast culture, i.e. cell division in modified Kao's liquid medium (K566-7). calli formation on SH semi solid medium, and multi-shoot regeneration from calli on SHa and SHb solid media. Frequency of multi-shoots and plant regeneration was affected by various combinations of phytohormones in final step. The evaluation of multi-shoots induction systems via protoplast culture was discused.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.527.3-527
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• 1986

Several factors predicted to affect the protoplast formation and regeneration were investigated. The optimum lysozyme, casamino acid and PVP concentration were 0.5 (mg/$m\ell$), 0.1 (%) and 1.5(%). In B. pumilus, Penicillin-G treatment concentration was 0.3 (U/$m\ell$) and optimum treatment period was transit log. phase. And in the case of Celm. fimi, 0.3 (U/$m\ell$) and initial log. phase. Osmotic stabilizer and di-cation for OSM medium of B.pumilus and Gelm .fimi were 25mM CaCl2, 0.5M sodium sucinate and 50mM MgCl$_2$, 100mM CaCl$_2$, 0.4M sodium succinate. The regeneration frequency of B.pumilus and Celm. fimi were 14.6(%) and 6.9(%).

• 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.

• 한국균학회지
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• 제22권2호
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• pp.130-137
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• 1994

담자균류인 잿빛 만가닥버섯 Lyophyllum decastes은 이미 항암작용이 있다고 보고된 바, 이 버섯의 유전연구와 균주개발을 위해 기초적 연구인 원형질체 분리와 재생에 관하여 실험하였다. 원형질체 분리의 최적 조건으로는 여러 효소의 혼용보다 Novozym 234(10 mg/ml)를 단용하였을 때 더 우수하였고, 삼투압 안정제로 0.6 M $MgSO_4$에서 효과적이었으며, $24^{\circ}C$ 에서 5일 배양한 균사와 효소액을 4시간 반응 시켰을 때 수득율이 높았다. 완충용액과 pH는 Na-phosphate buffer(pH 4)에서 원형질체 분리가 양호 하였으며, 고체배지보다 액체배지에서 자란 균사를 완충용액과 pH를 조절하지 않은 상태에서 효소와 반응시켰을 때 $12.5{\times}10^6\;cell/ml$로 높았다. 원형질체 재생시 균사가 배지 전면에 확산되어 성장하여 성장억제제로 Triton X-100(0.0025%)을 사용하였다. 원형질체 분리시와는 대조적으로 0.6M sucrose와 mannitol에서 재생이 효율적이었고, 각각 8.32%와 5.94%의 재생 빈도를 나타내었다.

• Food Science and Biotechnology
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• 제14권4호
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• pp.543-546
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• 2005

Optimized conditions for protoplast preparation and regeneration from young hyphae of Monascus ruber were established. Heat shock treatment of spores gave rapid and synchronized germination. Spores collected from cultures grown for 7-8 days at $30^{\circ}C$ were germinated until over 70% germ tubes reached to 3-5 spore length. Enzymatic digestion of young hyphae was optimal with 50 mg/mL Glucanex in 0.1 M sodium citrate buffer containing 0.8 M mannitol as an osmotic stabilizer. Regeneration rate was around 10% when 0.8 M sorbitol was used as an osmotic stabilizer in regeneration medium. These conditions will be applied in genetic study of M. ruber that produces citrinin at high level and thus is good model strain for molecular genetic dissection of citrinin biosynthesis.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.246-250
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• 1991

Conditions for the production and regeneration in Lactobacillus bulgaricus protoplasts were investigated. Protoplasts of L bulgaricus strains were obtained by treatment with mutanolysin and lysozyme together in a protoplast forming buffer containing 0.02 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.0) and 0.5 M sucrose. High protoplast yield was obtained from cells cultured in the de Man, Rogosa and Sharpe(MRS) medium at the middle to late logarithmic growth phase. Regeneration was efficiently accomplished with a complex medium containing 1% sucrose, 20 mM $MgCl_2$, 5% gelatin, and 0.5% bovine serum albumin. The frequency of regeneration of protoplasts was 10~20% after 5 days of incubation at $30^{\circ}C$.

• 미생물학회지
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• 제27권4호
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• pp.422-429
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• 1989

Auxotrophic mutant were isolated from wild types by the treatment with NTG as a mutagen, and the conditions of protoplast formation for them were established. The protoplasts of killer yeast Saccharomyces cerevisiae K52 were formed to the level of above 70% when cells grown for 20 hr in PM medium were treated with 200 unit/ml Lyticase 50,000 at $30^{\circ}C$ for 60 min after pretreatment of 50 mM 2-mercaptoethanol in 10mM potassium phosphate buffer (pH 7.5) containing EDTA and 0.6 M sorbitol for 15 min. Also, the protoplast of the recipient S. cerevisiae S 29 were formed to the level of above 85% as it was cultured to the log phase of 24 hr in PM medium under the same conditions. The fusion frequency between the protoplast of killer yeast S. cerevisiae K 52 and the protoplast of recipient S. cerevisiae S 29 was reached to $8.2\times 10^{-6}$ when the hypertonic regeneration medium embeded with the fused protoplasts after mixing the parental protoplasts to 10$^{8}$ cells/ml in SP buffer containing 20 mM $CaCl_{2}$ and 30% PEG 6,000 for 15 min at $30^{\circ}C$ were incubated.

• Plant Biotechnology Reports
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• 제2권3호
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• pp.171-177
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• 2008

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of microcolonies with plating efficiencies 3-10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.

• 한국미생물·생명공학회지
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• 제16권2호
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• pp.150-155
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• 1988

L-Lysine 생산균주인 Brevibacterium flavum ATCC 14067 과 Corynebacteriurn glutamicum ATCC13032에 섬유소 자화능이 우수한 Cellulomonas flavigena KFCC31221를 속간 융합시켜 섬유소로부터 L-lysine을 생산할 목적으로 이들 균주의 영양요구성 변이주 분리, 원형질체의 형성 및 재생의 조건을 조사하였다. NTG(500$\mu\textrm{g}$/$m\ell$)로 유기하여 penicillin-G(300$\mu\textrm{g}$/$m\ell$)로 농축한 변이주에서 B. flavum은 hse- str$^{r}$(100$\mu\textrm{g}$/$m\ell$), C. glutamicum는 Met$^{-}$T$hr^{-}$ Rif$^{r}$(5$\mu\textrm{g}$/$m\ell$), C. flavigena 는 T$hr^{-}$Val$^{-}$Kan$^{r}$(50$\mu\textrm{g}$/$m\ell$)의 gene marker를 가지는 영양요구성과 약제내성균주를 분리하였다. 공시균주의 원형질체 형성은 osmotic stabilizer로서는 0.5M sucrose, 배양기간은 대수증식기 중기의 균이 양호하였고, lysozyme 최적 처리 pH, 온도, 농도 및 반응시간은 각각 pH6.5, 33$^{\circ}C$, 500 $\mu\textrm{g}$/$m\ell$, 6시간으로 나타났으며, 이때의 원형질체형성율은 95-98%였다. 원형질체를 1.5% agar가 함유된 완전재생용 배지위에 0.7% agar가 함유된 완전재생용 배지로 중층했을 때 약 30~33%의 재생율을 보였다.