• Microbiology and Biotechnology Letters
• /
• v.15 no.2
• /
• pp.104-111
• /
• 1987

As a trial method of breeding L-lysine producing strains, the intraspecific protoplast fusion bet-ween Brevibacterium flavum ATCC 21528R and Brevibacterium flavum ATCC 21529S and the intergeneric protoplast fusion between Brevibacterium flavum ATCC 21528R and Corynebacterium glutamicum ATCC 13058S were performed. The optimum conditions for protoplast formation of these strains were examined and the effect of plasma expander on regeneration and/or fusion was also observed. Both fusants No. CH23 and No. CH4l showed higher productivity of L-lysine than those of parental cells under the optimum cultural conditions at a rate of 21％ and 8.9％, respectively. And, activity of several enzymes in L-lysine biosynthetic pathway including aspartokinase, a rate-limiting enzyme, was determined. Besides, metabolic control mechanism of L-lysine biosynthesis in fusant No. CH23 and in No. CH41 was investigated to compare with that of parental strains.

• Korean Journal of Agricultural Science
• /
• v.19 no.1
• /
• pp.33-39
• /
• 1992

Effects of buprofezin, a chitin synthesis inhibitor of insects, on mycelial growth, protoplast formation and reversion of Coriolus versicolor, Ganoderma applanatum and G. lucidum were investigated. The mycelial growth of C. versicolor, G, applanatum and G, lucidum was severely inhibited by buprofezin treatment, and the inhibition rate severely as the concentration of the buprofezin increased. Aerial mycelia and oidia formation of the mushrooms were increased by buprofezin treatment, but mycelial morphology was not changed. The rate of protoplast formation and reversion of G, applanatum was greatly increased by the treatment of the buprofezin, while that of G. lucidium was decreased. The rate of protoplast formation of C. versicolor was also increased when buprofezin was added to the medium, but the rate of protoplast reversion was not affected by the treatment, From the results obtained in this experiment, we found that the rate of protoplast. formation and reversion was increased by the treatment of the buprofezin in the mushrooms such as C, versicolor and G, applanatum, whose mycelial growth was fast on the medium, while that of G. lucidum, whose mycelial growth was relatively slow, was decreased by the treatment.

• KSBB Journal
• /
• v.18 no.4
• /
• pp.289-293
• /
• 2003

Doxorubicin is an anthracycline-family polyketide compound with a very potent anti-cancer activity, typically produced by Streptomyces peucetius. In order to increase doxurubicin productivity, a semi-industrial doxorubicin-producing Streptomyces strain named BR-Dox was cultured in a R2YE liquid medium containing CaCO$_3$, and then converted to a cell wall-free protoplast using lysozyme treatment method, followed by PEG-mediated cell wall regeneration. Among several protoplast-regenerated Streptomyces BR-Dox strains, two independent isolates named BR-Dox4 and BR-Dox6 were visually selected using thin layer chromatography (TLC) based on the pigment overproducing phenotype. Comparing with Streptomyces BR-Dox parental strain, two protoplast-regenerated strains, BR-Dox4 and BR-Dox6 exhibited 25.2％ and 12.2％ higher doxorubicin productivity analyzed by high pressure liquid chromatography (HPLC), respectively. This result suggests that a protoplast-regeneration of an antibiotics-producing Streptomyces strain should be a promising strain development approach for antibiotics overproduction in Streptomyces species.

• The Plant Pathology Journal
• /
• v.17 no.1
• /
• pp.36-39
• /
• 2001

The secondary effects of pencycuron on cell membrane of Rhizoctonia solani AG4 were investigated by the observation of giant protoplast formation and lipid peroxidation. Compared to protoplasts of R. solani R-C (sensitive strain) and Rh-131 (non-sensitive strain) increased in their size by 2.0-3.5 times 12 h after incubation in potato-dextrose broth containing novozyme (7 mg/$m\ell$) and $\beta$-glucuronidase ($60\mu\textrm{g}/$\textrm{ml}) with 0.6 M mannitol (pH 5.2). The increase of protoplast size in R-C was slightly inhibited from $13.8\textrm{mg}/\textrm{ml}$ without pencycuron to 10.3 ${\mu}{\textrm}{m}$ with 1.0$\mu\textrm{g}$/$m\ell$ of pencycuron. However, the size of giant protoplast of Rh-131 was not affected by the pencycuron treatment. Both strains R-C and Rh-131 did not exhibit the lipid peroxidation 12 h after the application of 1.0 $\mu\textrm{g}$/$m\ell$ pencycuron. The remarkable peroxidation of membrane lipid was observed only in R-C 24 h after pencycuron application, but not in Rh-131. Althought the inhibition of giant protoplast formation and the membrane lipid peroxidation were observed only in the sensitive strain R-C by pencycuron, it is difficult to conclude that these are the primary mechanism of pencycuron. The mild activity of pencycuron on the inhibition of giant protoplast formation and late membrane lipid peroxidation in the fungicide-sensitive strain did not noincid with the dramatic activity of pencycuron in R. solani. Therefore, our results suggest that inhibition of giant protoplast formation and membrane lipid peroxidation is the secondary effect of pencycuron.

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

• Microbiology and Biotechnology Letters
• /
• v.16 no.2
• /
• pp.150-155
• /
• 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
• /
• v.18 no.3
• /
• pp.115-126
• /
• 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.

• Journal of Plant Biology
• /
• v.15 no.3
• /
• pp.14-18
• /
• 1972

This paper includes a review on recent development on protoplast culture, regeneraton of plant from protoplast, and fusion of isolated protoplasts, and also describes the possibility of obtaining interspecific hybrid plants through asexual fusion of protoplasts of cells from distantly related plants which are not crossed by the ordinary sexual method.

• The Korean Journal of Mycology
• /
• v.16 no.1
• /
• pp.21-25
• /
• 1988

This experiment was carried out to investigate proper conditions for protoplast isolation and reversion from Ganoderma lucidum and Gctnoderma sp.. In G. lucidum, 10 mg. $ml^{-1}$ Novozyme 234 with 0.6 M sucrose was proper for protoplast isolation. The optimal reaction time of mycelium with lytic enzyme was five hrs. Protoplast isolation from four-day-old mycelium was the most effective. Protoplast isolation from four-day-old mycelium in G. sp. was optimum in the combination of N ovozyme 234 and ${\beta}-glucuronidase$ with 0.6 M sucrose. MCM was suitable for reversion in G. lucidum while SCM was good for G. sp.. The most effective osmoticum stabilizer for protoplast reversion in G. lucidum and G. sp. was 0.6 M sucrose.

• The Korean Journal of Mycology
• /
• v.22 no.2
• /
• pp.130-137
• /
• 1994

This experiment was carried out to investigate proper conditions for protoplast isolation and regeneration from mycelia of Lyophyllum decastes. Novozym 234(10 mg/ml) with 0.6 M $MgSO_4$ in phosphate buffer(pH 4.0) was proper for protoplast isolation. The optimal reaction time of the mycelium with the lytic enzyme was four hours in shaking condition at 120 strokes per min. When the mycelium of L. decastes was cultured at $24^{\circ}C$ for 5 days, the formation of protoplasts was effective. The liquid medium was more effective for protoplast isolation than the solid medium. In the liquid medium, high yields of protoplasts were obtained from 0.6 M $MgSO_4$ osmotic stabilizer. Protoplasts of L. decastes were regenerated to normal hyphal growth and the regeneration frequency of the protoplasts in the complete agar medium containing Triton X-100(0.0025%) was $5.94{\sim}8.32%$. The regeneration medium stabilized with 0.6 M sucrose was the best for regeneration of the protoplasts. In contrast to protoplast formation, regeneration was inhibited by the inorganic salts used as osmotic stabilizer.