• Archives of Pharmacal Research
• /
• v.15 no.1
• /
• pp.30-34
• /
• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were secreened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S rimosus ($pro^- \;his^-$) and S. aureofaciens ($ilv^-$) protoplasts with 40% (w/v) polythylene glycol 3350 (PEG) for 3 min gave $8.3\times10^{-7}$ of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisporus (leu-) protoplasts with 50% (w/v) PEG for 3 min at $30^\circ{C}$ gave $1.2\times10^{-6}$ of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. auroefaciens (2% frequency) lost their p-hydroxylation function.

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

• The Korean Journal of Mycology
• /
• v.17 no.3
• /
• pp.137-144
• /
• 1989

Effects of incompatibility existing between intra-and interspecies in Pleurotus spp. on protoplast fusion, clamp formation of their fusants and fruitbody production were investigated. Protoplast fusion between intra-and interspecies of the fungus was achieved by Poly ethylene glycol treatment. The fusion frequency between intraspecies was a little higher than that of interspecies. Fusion frequency between interspecies was not correlated with their similarities based on isozyme patterns. In case of protoplast fusion between intra-and interspecies, the fusants from the compatible isolates produced normal fruit bodies, while those from the incompatible isolates did not produce clamp connections and fruit bodies except those of a few isolates presumed mutants.

• Korean Journal of Microbiology
• /
• v.24 no.3
• /
• pp.197-203
• /
• 1986

Morphology and ultrastructure of protoplast fusion mode in Streptomyces lavendulae were studied by scanning and transmission electron microscopy. The isolated protoplasts were stable in some degree in hypertonic solution except that several protoplasts showed irregular morphology. Fusion events were occurred as follows; contact zone, fusion zone and separation zone were appeared sequentially. After formation of the separation zone, cytoplasm and DNA from both parents were mixed eventually. In the contact zone, two menbranes were still separated by electron transparent space. The contact zone changed to fusion zone by formation of fusion membrane that phospholipid molecules of two membranes were rearranged. Thereafter, nonmembraneous separation zone was formed by disappearance of fusion membrane. These changes were characterized by successive changes in typical membrane structure in fusion areas and by a progressive loss of bispherical shape.

• Korean Journal of Food Science and Technology
• /
• v.21 no.1
• /
• pp.37-44
• /
• 1989

This study aims at producing red pigment effectively by using protoplast fusion. Auxotrophic mutants of M. anka, $4478-27-37(Thi^-,\;Met^-),\;4478-27-62(Thi^-,\;Arg^-)$ were derived from M. anka, 4478-27. M. anka, $6540-185-24\;(Pan^-,\;Leu^-)$ and M. anka, $6540-185-72\;(Arg^-)$ were derived from M. anka, 6540-185. The optimal conditions of protoplast fromation were at time by using mixtures of chitinase (100mg/ml), cellulase (5mg/ml) and ${\beta}-glucuronidase\;(5mg/ml)$. The protoplast of those auxotrophic mutants were fused effectively, in the solution of 30% PEG $6,000,001M-CaCl_2$, 0.05M-glycine, pH 6.0. Fusion frequencies were 0.70%-0.85%. Fusants, No. 14 and 42 produced highest red pigment among them.

• The Korean Journal of Mycology
• /
• v.20 no.1
• /
• pp.44-50
• /
• 1992

The protoplast formation of Rhizoctonia solani in the fast growing anastomosis groups (AGs) 1 and 4, the intermediate AG-2 and AG-5, and the slow AG-3 yielded the most, moderate and the least in that order, respectively. Sclerotia formation varied with AGs. A high yield of protoplasts from AGs was obtained with a combined lytic enzyme system containing cellulase 'Onozuka' R-10, macerozyme R-10 and ${\beta}-glucuronidase$. When 3g (fresh weight) of 30 hr old mycelia was incubated for 3 hr at $32^{\circ}C$ with the enzyme mixture in 0.6 M mannitol, maximum protoplasts were obtained in the five AGs. A protoplast fusion between sclerotia forming AG-1 inactivated with heat and non-forming AG-5 was induced by polyethylene glycol and ${Ca}^{2+}$. Seven fusants obtained were based on characteristics of colony and sclerotium formation on culture plates. The fusants were confirmed by isozyme patterns of esterase and killing reaction between AG-1 and a fusant F1501.

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

• Current Research on Agriculture and Life Sciences
• /
• v.10
• /
• pp.147-155
• /
• 1992

This study was conducted to get basic information for factors affecting protoplast fusion between Petunia hybrida 'Titan red' and Nicotiana sanderae. The experiments such as fusogen, time of PEG treatment, temperature at fusion, $CaCl_2{\cdot}2H_2O$ concentration in fusion solution, and $CaCl_2{\cdot}2H_2O$ concentration and pH in eluting solution were carried out to increase the fusion efficiency. The results obtained were as follows; Fusion between P. hybrida and N. sanderae was accelerated when the mixture of the protoplasts was treated with 30% PEG 6,000 solution containing 5.5 mM $CaCl_2{\cdot}2H_2O$ for 10 minutes at $25^{\circ}C$, and subsequently eluted with a eluting solution containing 50 mM $CaCl_2{\cdot}2H_2O$ adjusted to pH 9.0.

• Journal of Environmental Science International
• /
• v.17 no.2
• /
• pp.163-170
• /
• 2008

Protoplasts of Panax ginseng C. A. Meyer and Aralia continentalis K. (Araliaceae) were isolated from callus cells and mesophyll cells, respectively. The maximum yield of protoplasts isolated from callus cells of P. ginseng were obtained by incubation for 3 hrs in the enzyme mixture of 0.5% macerozyme, 1.5% cellulase, and 0.5 M mannitol as an osmoticum. In the case of mesophyll cells of A. continentalis, the highest yield of protoplasts were obtained by incubation for 5 hrs in the enzyme mixture of 1% macerozyme, 2% cellulase, and 0.6 M mannitol. A polyethylene glycol (PEG) treatment induced an intergeneric fusion of the protoplasts. The fusion products, that is, heterokaryocytes were obtained by treatment of 50% PEG containing 0.05 M Ca salts.

• Microbiology and Biotechnology Letters
• /
• v.18 no.5
• /
• pp.460-465
• /
• 1990

Regeneration of protoplast was effective by preincubating spore suspension containing 30$\mu g$/ml of 2-DG for 4 hours, and CBE medium containing casamino acid, bovine serum albumin, ergosterol and myoinositol was found to be more efficient than any other regeneration medium tested in this experiment. The regeneration frequency was about 30%. Optimal conditions for conidial protoplast fusion were obtained by treatment of protoplasts with 10 mM $CaCl_2$ and 30% polyethylene glycol 4000 (pH 7.5) as fusogenic agent at $37^{\circ}C$ for 10 minutes. The fusion frequency was $8.2\times 10^{-4}$. The higher productivity of enzyme of fusant FWN-56 was achived: 2.3-fold for CMCase, 1.5-fold for avicelase, 1.8-fold for $\beta$-glucosidase and 2.5-fold for xylanase compared to that obtained in two parental strains. The genetic stability of fusant after maintenance on minimal medium for more than 4 weeks was high because segregant rate was below 1%. The conidial DNA content of fusant was 1.4-1.6 times higher than that of the parental strains, The nucleus size of fusants were also higher than that of each parental strains.