• Archives of Pharmacal Research
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• v.20 no.3
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• pp.206-211
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• 1997

The optimal conditions for the production and regeneration of the protoplasts from Lentinula edodes were studied. Protoplast formation from the mycelia of L. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at $30^{\circ}C$ and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or $MgSO_{4}$ More than 90% of the protoplasts contained nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was $3-5{\mu}m$ and it had a well defined cell structure.

• Korean Journal of Microbiology
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• v.27 no.4
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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.

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

• Applied Microscopy
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• v.14 no.2
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• pp.38-51
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• 1984

Fine structure of dormant and swollen conidiospore from Trichoderma koningii and the mechanism of protoplasting from the conidiospore were studied by scanning and transmission electron microscopy. The cell wall of dormant conidiospore was two-layered structure which consisted of electron dense outer layer and electron transparent inner layer. After 8.5 hrs incubation. the conidiospore was swollen and the outer layer of cell wall shown unequal thickness and partial breakage. Protoplast was released through the pore which has been formed by the breakage of outer layer and dissolution of newly synthesized cell wall for germ-tube formation. Swollen conidiospore and protoplast in releasing process contained various cell organelles and vacuoles with electron dense materials. The protoplast contained looser cytoplasm and had no cell wall materials outside of plasmamembrane.

• The Korean Journal of Mycology
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• v.15 no.1
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• pp.14-18
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• 1987

This experiment was undertaken to investigate proper conditions for protoplast formation from Lyophyllum ulmarium. Combination of Novozym 234, ${\beta}-Glucuronidase$ and ${\beta}-D-Glucanase$ with 0.6 M Sucrose was the most effective for isolation of protoplasts. The optimal reaction time of mycelium with the lytic mixture was 3 hrs in shaking condition at 120 strokes $min-^1$. When the mycelium of L. ulmarium was cultured for 6 days on yeast glucose agar medium at $25^{\circ}C$, the formation of protoplasts was effective. The yeast glucose agar medium stabilized with 0.6 M sucrose was the most effective for reversion of protoplasts.

• Journal of Plant Biotechnology
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• v.49 no.4
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• pp.331-338
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• 2022

Sunflower (Helianthus annuus L.) protoplasts were isolated from seven-day-old etiolated hypocotyls of 10 A line and four-week-old fully expanded young leaves of PI 441983 line in vitro seedlings using an enzymatic method. Purified protoplasts were collected by filtration and floatation in sucrose solution. Semi-solid protoplast culture was performed using the L4 regeneration protocol with various culture media and plating densities to achieve the highest efficiencies for protoplast culture of hypocotyl and mesophyll protoplasts of 10 A and PI 441983 lines, respectively. The concentrations in liquid L'4M medium and different plating densities were evaluated in two types of cytokinins, the adenine-type 6-benzyladenine (BA) and the phenylurea-type thidiazuron (TDZ). The highest colony formation was achieved in both sunflower lines when 0.5 mgL^-1 BA and 0.5 mgL^-1 TDZ were applied with a high plating density (3 × 10⁵ protoplasts mL^-1). These conditions led to 38.45% and 39.40% colony formation for hypocotyl protoplasts of the 10 A line and mesophyll protoplasts of the PI 441983 line, respectively. Moreover, many hypocotyl protoplast-derived colonies developed into micro-calli. In addition, superior development of both sunflower protoplasts was observed with all plating densities when BA was used in combination with TDZ. This finding will be applicable to future sunflower hybrid production via somatic hybridization.

• 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.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.150-155
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• 1988

In order to produce L-lysine from cellulosic substrates by the intergeneric protoplast fusion between cellulolytic bacteria, Cellulomonas flavigena KFCC31221 and amino acid producing bacteria, Brevibacterium flavum ATCC14067, Corynebacteriurn glutamicum ATCC13032, conditions for protoplast formation and regeneration of these strains were investigated. After the strains were mutated with 500$\mu\textrm{g}$/$m\ell$ N-methyl-N'-nitro N-nitrosoguanidine for 30 min and the mutants were enriched by treating 300$\mu\textrm{g}$/$m\ell$ penicillin-G for 2 hrs, B. flavum Hse- Str$^{r}$ , C. glutamicum Met$^{-}$Thr$^{-}$ Rif$^{r}$ and Cellulomonas flavigena Thr$^{-}$Val$^{-}$Kan$^{r}$ were isolated. The rate of protoplast formation ranged from 95 to 98% when strains were treated at the concentration of 500$\mu\textrm{g}$/$m\ell$ of lysozyme, pH 6.5, 33$^{\circ}C$, for 6 hrs. in Tris- malate buffer supplemented with 0.4M sucrose as osmotic stabilizer. Approximately 30-33% protoplast was regenerated on the regeneration complete medium(RCM) containing 1.5% agar and 0.5M sodium succinate overlaid with the same medium except 0.7% agar.

• The Korean Journal of Mycology
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• v.18 no.3
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• pp.115-126
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• 1990

To establish basic techniques for protoplast fusion of Coriolus versicolor several factors affecting protoplast formation and regeneration were investigated. Protoplast isolation was at maximum with 2.5-day cultured mycelia of C. versicolor treated with the combination of two enzymes, Novozym 234 (10 mg/ml) and cellulase Onozuka R-10 (15 mg/ml), for 3-4.5 hours at $30^{\circ}C.$ As an osmotic stabilizer for stabilizing the protoplast, 0.6 M sucrose was the best for formation and regeneration of the protoplast from the mycelia of the fungus and the regeneration frequency was 3.48%. Protoplast fusion was made by a modified method of Peberdy using PEG (M.W. 4,000). The fusion frequency between two mutants of C. versicolor was 1.86% and the fusion products showed differences in growth rate and colony morphology.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.377-382
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• 1985

As an essential previous step towards the development of cell fusion to breed a new brewing yearst strain, several factors predicted to affect the protoplast formation of S. cerevisiae, C. tropicalis and E. fibuligera were investigated in order to obtain the protoplasts in high yields. The optimum pH and temperature for the protoplas formation were 7.5 and 35$^{\circ}C$, respectively. Pretreatment of the yeast cells with 2-mercaptoethanol stimulated the protoplast formation and 50mM of the reagent was found as effective. Among several osmotic stabilizers tested for their effect on protoplas formation, 0.6M KCI was comparatively favorable.