• Journal of Marine Bioscience and Biotechnology
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• v.12 no.1
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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.

• The Korean Journal of Mycology
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• v.21 no.4
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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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• 2009.10a
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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.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.391-395
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• 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.

• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.335-339
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• 1998

The factors affecting the isolation and culture of the protoplast of embryogenic callus, derived from immature ovule in Citrus junos, were examined. An incubation time in enzyme solution of 16 hrs was preferable for protoplast isolation. Efficient protoplast yields were obtained from the treatment of equal concentration of 0.7 M $\textrm{BH}_{3}$ to the enzyme solution containing 1.0% cellulase, 1.0% macerozyme and 0.2% pectolyase. Protoplast cultured in MT medium with 0.1 mg/L 2,4-D showed vigorous division and some of them formed callus. Induced callus was subcultured on solid MT medium but the callus showed very slow growth. The above results show the possibility to culture from protoplast fusion in Citrus genera.

• Archives of Pharmacal Research
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• v.10 no.3
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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%.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.145-149
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• 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
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• v.14 no.3
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• pp.251-257
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• 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$0^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.163-167
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• 1985

To investigate the condition for the protoplast fusion of Streptococcus lactis, streptomycin(200 $\mu\textrm{g}$/$m\ell$) resistant and rifampicin (200$\mu\textrm{g}$/$m\ell$) resistant mutants were isolated. By using these markers, protoplast fusion was carried out in the presence of CaCl$_2$ and polyethylene glycol. The optimal conditions for the protoplast fusion were obtained by treatment of protoplasts with 150 mM CaCl$_2$ (final concentration; 25 mM) and 40％ (w/v) PEG 4, 000 for 2 min. At the optimal conditions, the fusion frequency was 6.26$\times$10$^{-5}$ . On the other hand, genetic recombination between the antibiotic resistant mutants by mating was not observed.

• Korean Journal of Microbiology
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• v.29 no.2
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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.