• 한국해양바이오학회지
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• 제12권1호
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• pp.50-58
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• 2020

Sphacelaria is a filamentous brown algal genus that can be epibiotic on macroalgae, marine plants, and sea turtles. Its important role in benthic ecosystems, exposure to different stressors (e.g., grazing), and use as a model organism make Sphacelaria ideal for assessing physiological responses of organisms to environmental inputs. Single-cell RNA sequencing is a powerful new probe for understanding environmental responses of organisms at the molecular (transcriptome) level, capable of delineating gene regulation in different cell types. In the case of plants, this technique requires protoplasts ("naked" plant cells). The existing protoplast isolation protocols for Sphacelaria use non-commercial enzymes and are low-yielding. This study is the first to report the production of protoplasts from Sphacelaria fusca (Hudson) S.F. Gray, using a combination of commercial enzymes, chelation, and osmolarity treatment. A simple combination of commercial enzymes (cellulase Onozuka RS, alginate lyase, and driselase) with chelation pretreatment and an increased osmolarity (2512 mOsm/L H₂O) gave a protoplast yield of 15.08 ± 5.31 × 10⁴ protoplasts/g fresh weight, with all the Sphacelaria cell types represented. Driselase had no crucial effect on the protoplast isolation. However, the increased osmolarity had a highly significant and positive effect on the protoplast isolation, and chelation pretreatment was essential for optimal protoplast yield. The protocol represents a significant step forward for studies on Sphacelaria by efficiently generating protoplasts suitable for cellular studies, including single-cell RNA sequencing and expression profiling.

• 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.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2009년도 추계학술대회 및 정기총회
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• pp.14-16
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• 2009

Poria cocos is an importantant medical macrofungus,the sclerotium of Poria cocos has specific value as the drug material. There are few papers about its breeding and spawn rejuvenation. In this project, the protoplasts of cultivated strain T and wild strain L were prepared and treated separately by ultraviolet and heating, then fused with the PEG6000. The tural fusants were selected and identified by the affinity and ISSR analysis. 71 incompatibility strains between parents and reg regenerations were obtained from 118 regenerations by the affinity analysis. Five incompatibility strains were amplified with different primers, the results were showed that they had specific bands of both parents in the profile amplified with 3 primers, which proved these 5 strains were fusants by means of molecular biology marker. On the other hand, 25 strain were selected from 168 protoplast regenerations of cultivated strain T for cultivation experiment. The fresh sclerotium weight of these protoplast regenerations were better than the original strain.significantil 3 strains (T-1, T-4, T-7) increased respectively 118%, 73% and 73% than original strain. This method could be the effective in the rejuvenation Poria cocos.

• 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.

• 식물조직배양학회지
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• 제25권5호
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• pp.335-339
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• 1998

미숙배주조직 유래의 배발생캘러스를 이용하여 원형질체의 유리 및 배양에 미치는 요인을 조사하였다. 배발생캘러스로부터 원형질체를 유리시키는데 적당한 배양시간은 16시간이었고, 건전한 원형질체를 유리하는데 적당한 효소용액의 농도는 0.7M $\textrm{BH}_{3}$ 용액과 cellulase 1.0%, macerozyme 1.0%, pctolyase 0.2%가 혼합된 효소용액을 동량으로 조합하였을 때가 가장 효과적이었다. 유리된 원형질체는 MT기본배지에 0.1 mg/L 2,4-D를 첨가한 배지로 배양하면 캘러스 형성이 양호하였다. 유도된 캘러스는 고체배지에 계대배양하고 있으나 생육이 징약한 상태이다. 본 실험결과 배발생캘러스유래의 원형질체와 엽육세포유래의 원형질체 융합에 의한 배양도 가능할 것으로 생각되었다.

• Archives of Pharmacal Research
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• 제10권3호
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• pp.158-164
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• 1987

To obtain a new strain of Ganoderma lucidum by protoplast fusion technique, its protoplast formation and regeneration were studied. Several factors affecting the protoplast formation and regeneration were investigated to find their optimum conditions. The mycelium was grown for four days on the cellophane membrane placed on G. Incidum complete medium (GCM). When various commercial lytic enzymes were examined for protoplast isolation, the combination of Novozym 234 and $\beta$glucuronidase was found to be effective. An osmotic stabilizer, 0.6 M sucrose in 20 mM phosphate buffer pH 5.8, gave the highest yield of protoplasts. Three-hour incubation in shaking incubator was most suitable for releasing protoplasts. To increase the protoplast yield, pretreatment with 2-mercaptoethanol was carried out. The regeneration frequency in GCM containing 0.6M MgSO$_4$ 7$H_2O$ was shown to be 0.66%.

• 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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• 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.

• 한국미생물·생명공학회지
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• 제13권2호
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• pp.163-167
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• 1985

유전물질이 쉽게 교환될 수 없는 세균의 유전학적 연구와 새로운 균주개발에 유용한 protoplast 융합을 Streptococcus lactis에 적용하기 위하여 UV를 사용하여 streptomycin (200$\mu\textrm{g}$/$m\ell$)과 refampicin(200 $\mu\textrm{g}$/$m\ell$)에 각각 내성을 나타내는 변이주를 분리하였으며 이 변이주들을 사용하여 Protoplast 융합의 영향인자를 검토하였다. 두 약제내성 변이주의 Protoplast를 1:1의 비율로 혼합하고 CaCl$_2$와 polyethylene glycol(PEG)로 처리하여 융합세포를 얻을 수 있었으며, 이 때 CaCl$_2$의 최적 처리농도는 150nM (최종농도 : 25mM)이었 고 40% (w/v)의 PEG 4,000으로 2분간 처리하는 것이 가장 좋았다. 융합빈도는 최대 6.26$\times$$10^{-5}$이었다. Mating에 의한 약제내성 유전자의 재조합 현상은 일어나지 않았다.

• 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.