• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.359-363
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• 1986

S. diastaticus hydrolysised $\alpha$-1.4 linkage of the starch and was known fermenting yeast strain, but poorly hydrolized $\alpha$-1.6 linkage of the starch. To improve the starch fermentation ability of yeast, we tried that protoplast fusion between S. diastaticus and C. tropicalis and finally two starins of fusant (FPDC42, FPDC43) were obtained. C. tropicalis well hydrolysis both $\alpha$-1.4 and $\alpha$-1.6 linkanges in the starch. The protoplast of parental auxotrophic cells were fused in the presence of 10mM CaCl$_2$ and 35% of polyethylene glycol (M. W. 4,000). The fusion frequency was 10$^{-5}$ to 10$^{-6}$. Properties of the fusants(genetic stability, assimilation of carbon sources, random spore formation, copper resistance, NaCl tolerance, DNA content, cell size and growth rate) were investigated.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.575-584
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• 2019

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH₄Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 ㎍/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.339-343
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• 1995

This study was performed to investigate the effect of iron ion on the mesophyll protoplast culture of Arabidopsis thaliana. Mesophyll protoplasts were isolated and cultured in a modified IMH medium supplemented with various concentrations of Fe-EDTA. Relatively low concentration of Fe-EDTA (<0.02 mM) induced the low level (4.8%) of cell division. In addition the cell division and microcallus growth were dose-dependently stimulated by 0 to 1 mM of Fe-EDTA. In 0.5 to 1 mM concentration range of Fe-EDTA, microcolonies were readily formed and the plating deficiency (8.5%) also showed maximal rate. However more than 1 mM of Fe-EDTA inhibited the initial growth of protoplase. The overall results suggest that Fe2+ion concentration plays an important role at the early developmental stage of protoplast regeneration.

• The Korean Journal of Mycology
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• v.14 no.4
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• pp.273-280
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• 1986

The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{\sim}30^{\circ}C$ and $6.0{\sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{\circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.3
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• pp.306-316
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• 1997

Embryogenic calli were induced from mature seed scutella of anther culture-derived rice variety Zhonghua 8. Cell suspension cultures were initiated from friable embryogenic calli and utilized as source material for protoplast isolation. Generally, the older and finer cell suspensions gave higher protoplast yields than younger suspension cultures. Protoplasts exhibited sustained cell division and formed microcalli when cultured in KPR medium supplemented with 0.5 mg $l^{-1}$ 2,4-D, 1.0 mg $l^{-1}$ NAA and 0.5 mg $l^{-1}$ zeatin using the agarose embedding procedure without feeder cells. Protoplast plating efficiencies ranged from 0.20 to 0.54％. Microcalli were transferred to MS medium supplemented with 2.0 mg $l^{-1}$ kinetin and 0.5mg $l^{-1}$ NAA for plant regeneration. The regeneration frequencies were 2 to 12％, depending on the cell suspension lines of Zhonghua 8. The plants were transferred to the glasshouse and were fertile.

• Microbiology and Biotechnology Letters
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• v.17 no.3
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• pp.213-218
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• 1989

To investigate the possibility of using alkalophilic Bacillus sp. K-11 as a host for molecular cloning, plasmid pUB110 and pBD64 were introduced into alkalophilic Bacillus sp. K-17 by protoplast transformation system. Protoplasts of Bacillus sp. K-11 were prepared by treatment with 200 $\mu\textrm{g}$/$m\ell$ Iysozyme in SMM buffer containing 0.4M sucrose. Optimal temperature, pH and culture time for protoplast formation were 4$0^{\circ}C$, 7.0 and 4hrs, respectively. Cell wall was regenerated efficiently on DM3 medium containing 0.8% agar and 0.5M sodium succinate. Under these conditions for protoplast formation and regeneration, the highest transformation efficiency was obtained with cells incubated for 4hrs, and using 30%(V/V) of 40%(W/V) PEG6,000, In characteristics of transfer-mants, plasmid pUB110 was more stable than plasmid pBD64 in Bacillus sp. K-17. Maximum xylanase production of both transformants carrying pUB110 and pBD64, respectively was similar, but under the same conditions, enzyme secretion by transformant carrying pUB110 was earlier than that of transformant carrying pBD64.

• Korean Journal of Microbiology
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• v.28 no.4
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• pp.304-310
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• 1990

The conditions for protoplasts formation and regeneration of thermophilic anaerobic C. thermocellum and C. thermohydrosulfuricum were determined under the anaerobic growth conditions. The cells of C. thermocellum in initial exponential growth phase were identified to be the most suited for protoplast formation. The optimal conditions for protoplast formation were found to be at $37^{\circ}C$ for 2 hours with 0.5 mg/ml of lysozyme in TMG buffer (pH7.5). On the other hand, C. thermohydro-sulfuricum grown in the same medium but excluding glycine was optimally protoplasted at the same conditions but with 0.2 mg/ml of lysozyme. The protoplasts of both strains only subjected to lysozyme treatment of the short time were satisfactorily regenerated after 7-10 days incubation at $60^{\circ}C$ in regeneration medium containing 0.3-0.4 M sorbitol, 0.5% casamino acid, and high concentration of $CaCl_{2}$ and $MgCl_{2}$. The regeneration frequencies of the protoplasts of C. thermocellum and C. thermohydrosulfuricum were found to be very low level of $4.85{\times}10^{-3}$ and $4.23{\times}10^{-2}$, respectively. The nonregenerated L-form cells were also observed inregeneration medium together with regenerated cells.

• Korean Journal of Food Science and Technology
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• v.20 no.2
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• pp.164-169
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• 1988

Protoplast susion between Zygosaccharmoyces rouxii M-12 and Saccharimyces cerevusuare M-43 were investigated for breeding of a new brewing yeat strain for soy sauce. Auxotrophic mutants of Zygosaccharomyces rouxii ZRM-83 ($Met^-,\;Thr^-$) and Saccharomyces cerevisiae SCM-46 ($Lys^-,\;Arg^-$) were selected by treatment of 3.0% ethylmethane sulfonate and nutritional complementary method. Protoplast of both strains were more effective by treatment of 0.05mg/ml zymolase 20T for 60min. Fusion effeciency was much higher by treatment of 30% PEG 6,000 for 30min and fusion frequencies were $10^{-4}{\sim}10^{-5}$. These fusants originated from two protoplasts had properties of big cell size and much DNA content.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.27 no.2
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• pp.147-154
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• 1982

This study was conducted to investigate an effective method of protoplast isolation, the plating efficiency for cell division, and fusion of plant protoplasts by polyethylene glycol for somatic hybridzation. The effectiveness of protoplast isolation was different with the various enzyme concentrations, but, in the protoplast isolation from tobacco mesophyll, the enzyme solution with 0.5% macerozyme and 2.0% cellulase was very effective. The protoplast isolation from callus cultured in vitro for a long period was not obtained in any of the enzyme solution used. Protoplasts divided actively at cell densities above $10^44/ml and at $25^{\circ}C$ under 12hr illumination by inflorecient light (l50 Lux), regardless of presence of agar. The highest frequency of protoplast fusion was obtained after treatment with a solution of 0.33 M polyethylene glycol 1500.

• Applied Biological Chemistry
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• v.33 no.1
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• pp.93-100
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• 1990

The optimum conditions of electric stimulation for electrofusion of protoplasts of petunia, carrot and soybean, and the effects of calcium, magnesium, protease, trypsin, triton X-100, concanavalin A, dimethyl sulfoxide(DMSO), glycerol monooleate and spermine on fusion frequency and/or viability of petunia protoplast were investigated. The optimum frequencies(Hz)-amplitudes(V/cm) of AC Pulse for protoplast pearl-chain formation were 10 kHz-20 V/cm and 1 MHz-60 V/cm for petunia, 100 kHz-40 V/cm and $1\;MHz-40{\sim}60\;V/cm$ for carrot, and $1\;MHz-40{\sim}80\;V/cm$ for soybean, respectively. The optimum condition of DC pulse treatment at the 1 MHz-60 V/cm-15sec treatment of AC for electrofusion of petunia protoplasts was 2.5 kV/cm-40 sec, and under this condition the fusion frequency and viability of protoplasts were 45 % and 10 %, respectively, Both of the protoplasts of carrot and soybean were not fused under the AC and DC conditions tested in this experiment. The electrofusion of petunia protoplasts was stimulated by calcium, and the fusion frequency and the viability of the protoplasts were 43 % and 11 % , respectively at the calcium concentration of 140 mM. Although fusion frequency was not affected by magnesium only, magnesium stimulated fusion frequency in the presence of calcium, and the viability and fusion frequency of petunia protoplasts were 45 % and 13 %, respectively, at 140 mM of magnesium-140 mM of calcium. The relative fusion frequencies of petunia protoplasts to the controls were increased by 2.4, 2.1, 1.6, 1.4, 1.8, 1.5 and 2.2 folds, respectively, by the treatments of protease, trypsin, triton X-100, concanavalin A, DMSO, glycerol monooleate, and spermine. The viabilities of petunia protoplasts were decreased by these substances.