• Journal of Plant Biology
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• v.32 no.4
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• pp.215-226
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• 1989

The effects of Ca2+ on polyamines on callose contents of carrot suspension cultured cells were studied. The regeneration process of the cell wall of carrot protoplast observed through the electron microscope. Treatment of the carrot suspension cultured cells with Ca2+ and polyamines resulted in considerable increase on callose contents at 0.1 mM of Ca2+ and polyamines, particulary spermidine. Poly-L-lysine and poly-L-ornithine increased about 30% and 100% of callose contents than that of the control respectively, whereas verapamil and flunarizine markedly decreased the callose contents. These effects of Ca2+ of free ion rather than as Ca2+-calmodulin complex. During the cultivation of the protoplast, the regeneration of the cell wall was somewhat observed on the 4th day, however, it was inhibited by verapamil. These results suggested that the promotive action of Ca2+ and polyamines were manifested in the callose contents and the regeneraton of the cell wall.

• Archives of Pharmacal Research
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• v.15 no.1
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• pp.35-40
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• 1992

S. fradiae showed the highest acetanilide p-hydroxylation activity in the tested strains. And S. fradiae was well characterized genetically, especially with respect to tylosin production. Two mutants, which lost hydroxylation, were isolated in 140 regenerated colonies from protoplasts. In restriction enzyme digesion of total DNAs, isolation of giant linear plasmid DNA and determination of antibiotic resistances to chloramphenicol, tylosin, hygromycin B and mitomycin C, any differences among mutants and a wild type strain were not detected. These facts suggest that lesion on 6, 000 Kb chromosomal DNA was responsible for the lack of p-hydroxylation activity induced by protoplast formation and regeneration.

• Archives of Pharmacal Research
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• v.20 no.4
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• pp.384-386
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• 1997

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.869-872
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• 2009

The cyclic undecapeptide cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. To increase CyA productivity by wild-type T. niveum (ATCC 34921), random mutagenesis was first performed using an antifungal agar-plug colony assay (APCA) selection approach. This generated a mutant strain producing more than 9-fold greater CyA than the wild-type strain. Additionally, a foreign bacterial gene, Vitreoscilla hemoglobin gene (VHb), was transformed via protoplast regeneration and its transcription was confirmed by RT-PCR in the UV-irradiated mutant cell. This led to an additional 33.5% increase of CyA production. Although most protoplast-regenerated T. niveum transformants tend to lose CyA productivity, the optimized combination of random mutagenesis and protoplast transformation described here should be an efficient strategy to generate a commercially valuable, yet metabolite low-producing, fungal species, such as CyA-producing T. niveum.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.20-28
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• 1999

To improve Bacillus strains producing biopolymer, conditions for protoplast formation and regeneration were investigated in biopolymer producing Bacillus subtilis K-1 and lactose utilizing Bacillus coagulans. Bacillus subtilis K-1 mutant (SM-2) and Bacillus coagulans mutants (CM-12) were marked auxotrophic and antibiotics-resistant (SM-2) and an antibiotics-resistant mutants, respectively. To formate protoplasts derived from the mutants, conditions were established as follows. For B. subtilis mutant SM-2, its culture in mid-logarithmic phase was added with penicillin G (1.0 unit/ml) and further reacted for 1.5 hr. Cells were collected and then treated in lysis fluid (pH 7.0) containing 0.4 M sucrose and lysozyme $25\;{\mu}g/ml$ for 40 min at $37^{\circ}$. Protoplast formation was very successful (99.6%) and the ratio of cell wall regeneration was 2.4%. For Bacillus coagulans mutant CM-12, its mid-logarithmic phase culture was treated with penicillin G (0.3 unit/ml) and glycine (0.5%) for 1hr. Cells were collected and then resuspended in lysis buffer (pH 7.0) containing 0.6 M lactose and lysozyme $(300\;{\mu}g/ml)$ for 30 min at $37^{\circ}$. Protoplast formation was also successful (90.8%) and cell wall regeneration ratio was similar to SM-2 (2.2%). To improve regeneration frequency, regeneration medium was obtained as followed condition,. Cell wall regeneration was improved 2-4 folds with 5.1% for B. subtilis SM-2 and 10.3% for B. coagulans CM-12 when protoplasts mixed with soft top agar(0.4%) was overlaid onto trypticase soy broth medium containing 0.4 M sucrose, 0.7% casamino acid, 1% PVP, 25 mM $MgCl_2,\;25\;mM\;CaCl_₂$ and 1.5% agar.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.218-223
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• 1993

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

• The Korean Journal of Mycology
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• v.13 no.3
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• pp.169-177
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• 1985

The studies were carried out to obtain the basic data for maximizing the protoplast yields from the mycelia of P. ostreatus and P. sajor-caju. Some factors affecting the regeneration of the protoplast of both species and the productivity of their reversion were also examined. The maximum yields of protoplasts were obtained from four days cultured mycelia of both species on cellophan membrane placed on the surface of PSA or MCM media in a petri dish. The optimal concentration of lytic enzyme Novozym 234 for protoplast releasing was 5 mg per ml of 0.5 M phosphate buffer solution with 0.6 M sucrose or 0.6 M $MgSO_4$ at pH 6.0. The greatest number of protoplasts was released 3 hours after incubation of the mycelia of P. ostreatus and after 4 hours for the P. sajor-caju in the lytic enzyme solution. Among the osmotic stabilizer solutions tested 0.6 M sucrose and 0.6 M KCl showed the best regeneration rates of the protoplasts of both species. When 0.75 % agar solution was over-layed on the regeneration media immediately after inoculation of the protoplast the regeneration rates were greatly enhanced. The ampicillin added to the agar solution prevented bacteria from infection. The reverted isolates produced the sporophores and basidial spores just like their parents without any mutations when they were cultivated in a broad mouth bottle with sawdust substrates.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.69-76
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• 2011

This study describes the effect of myo-inositol on sustained cell division and plant regeneration from cotyledon-derived protoplast of cabbage (Brassica oleracea var. capitata). Freshly isolated protoplasts were cultured in modified Murashige and Skoog (MS) medium removed ammonia ions and containing $0.4\;mg\;l^{-1}$ thiamine HCl, $100\;mg\;l^{-1}$ myo-inositol, $2\;mgl^{-1}$ 2,4-D, $0.5\;mgl^{-1}$ BA, $30\;gl^{-1}$ sucrose and several concentrations of myo-inositol (2, 4, 6, 8, 10% (w/v)) as an osmotic stabilizer. After 3 weeks of culture in the dark at $25^{\circ}C$, the plating efficiency of cabbage protoplasts reached to $22.5{\pm}2.9%$ when cultured in modified MS medium supplemented with $2\;mgl^{-1}$ 2,4-D, $0.5\;mgl^{-1}$ BA, $30\;gl^{-1}$ sucrose and 8% (w/v) of myo-inositol at a density of $2{\times}10^5$ protoplasts/ml. Rapidly growing cell colonies after 3 weeks of culture were transferred to the same culture medium removed osmoticum. To induce shoot regeneration from calluses, calluses with about 2 mm in diameter were transferred to the MS medium containing $2\;mgl^{-1}$ BA and $0.5\;mgl^{-1}$ NAA. After further three weeks of incubation onto the medium in the light, green shoots were formed on the surface of calluses at a frequency of 30%. Upon transfer to half-strength MS basal medium, roots were formed onto the bottom of regenerated shoots without auxin treatments. These regenerated plantlets were successfully acclimatized to soil transfer, grown to normal mature plants. The cabbage protoplast culture system established in this study could be applied for production of somatic hybrids or cybrids by asymmetric protoplast fusion and mass proliferation of elite somatic clones of cabbage.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.47-54
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• 1990

In order to investigate physiological characteristics of fusants by interspecific protoplast fusion of the genus Cellulomonas, protoplasts of Cellulomonas flavigena NCIB 12901 and Cellulomonas bibula NCIB 8142 were fused and cell wall regenerated. To give gene maker, C. bibula was treated with 500 ug/ml NTG for 1 hr and arginine requiring auxotrophic mutants were isolated. Protoplasts of the genus Cellulomonas were obtained by treatment with $600{\mu}{\textrm{g}}$/ml lysozyme, and 0.5M sorbitol was optimal for osmotic stabilizer on protoplast fromation. Protoplast fusion was enhanced by 40% PEG)M.W.6,000) containing 25 mM $CaCl_{2}$ at $30^{\circ}C$ for 30 min and fusion frequency between C. bibula and C. flavigena was $5\times 10^{-4}$. Processes of protoplast formation, cell wall regeneration and protoplast fusion were obsdrved by scanning electron microscope. By comparing enzyme activities of cellulase, exocellobiohydrolase, .betha.-glucosidase of the parent strains of Cellulomonas with those of thier mutants and fusants, fusants with increased enzyme activity were obtained. By the studies on nutritional requirement, antibiotic resistance, cellulolytic enzyme activities, type of peptidoglycan and motility of two mutants and fusants, fusants were proved to be recombinant of both mutant strains.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.295-303
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• 1997

To investigate the response on feeder cell cultures, protoplasts isolated from cell suspensions initiated from mature seed scutellum-derived callus of the Japonica rice variety Taipei 309, were cultured on filter membranes under various conditions. The effects of various factors, such as gelling agents, feeder cell and protoplast densities, species of feeder cells and heat shock treatment, have been investigated to improve protoplast plating efficiencies on filter membranes. Maximum protoplast plating efficiencies were obtained when protoplasts were cultured on KPR medium semi-solidified with Sea Plaque agarose at a density of $5\;\times\;10^{5}\;ml^{-1}$ protoplasts in the presence of Lolium multiflorum as feeder cells (0.5 ml pcv per 10 ml of protoplast culture medium). Pre-culture heat shock treatments for 1 min. and 5 min. to the protoplasts did not give any appreciable increase on the plating efficiency of protoplasts in the presence of feeder cells. Maltose-supplemented medium was superior for plant regeneration from protoplast-derived colonies compared with medium containing only sucrose. The plants were transferred to the glasshouse, flowered and were fertile.