• Korean Journal of Microbiology
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• v.24 no.3
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• pp.228-235
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• 1986

The aims of the present studies were to develop conditions for the spheroplast formation of Zymomonas mobilis and regeneration of the spheroplasts to normal cells in synthetic media. Z. mobilis cells harvested from exponential growth phase were treated with lysozyme, mutanolysin, and glycine in various conditions. It was found that spheroplasts were formed only with the treatment of glycine but not with the enzymes treatments. It was therefore considered that the tetrapeptide strand of peptide strand of peptidoglycan might play more important roles than the glycan strand in maintaining the vital mechanical function of Z. mobilis. It was found also that removal of outher membrane was the major problem in protoplast formation of Z.mobilis. As results, It was observed that over 85% of cells were readly converted to spheroplasts with sole glycine treatment for 4 hr and 7-10% of the spheroplasts were regenerated to normal cells in synthetic media.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.157-167
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• 1987

The have been many reports that phenoloxidase are correlated with development in many fungi. C. congregatus, one of nushroom-forming basidiomycetes, which requires light for its development also has phenoloxidases. In C. congragatus, there are two sets of membrane-associated phenoloxidase (PHO I and PHO II) which are differentiated by their isozyme patterns, and each enzyme set consists of two different subtrate specific enzyme protein; o-tolidine reacting enzyme, and DOPA reacting enzyme. PHO I which is localized by a protoplast-concanavalin A technique by using a new solidifying agent, Pluronic Polyol F 127, instead of agar appears in the vegetative hyphae, and PHO II appears at the early primordial stage on agar and at the sclerotial stage of liquid shake cultures. Inhibition of PHO I with the enzyme inhibitors inhibits mushroom formation as well as melanization of the vegetative hyphae at concentrations which do not inhibit the vegetative growth. PHO I deficient mutants do not form mushrooms or melanins, and the mutants show abnormal nuclear migration patterns. PHO II has roles; possibly cementing the adjacent hyphae during the actual three dimensonal structure formation, and melanizing mushrooms and sclerotia. The possible roles of PHO I in the light reception complex and in melanin formation, the function of malanin, and possible roles of postulated post translational modifying enzymes which regulate the phenoloxidases, nuclear migration pattern, and self-nonself recognition mechanism are discussed.

• Korean Journal of Microbiology
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• v.23 no.4
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• pp.309-314
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• 1985

Autoxotrophic mutants of a cellulolytic baterium Cellulomonas sp. CS 1-1 were grown at $30^{\circ}C$ for 6hr using a complete medium containing 0.5M sucrose and for another 90 min after addition of 0.3 U/ml penicillin G, and were protoplasted by 0.2mg/ml lysozyme for 2hr. Prototrophic recombinants were obtained at the rates of $10^{-3}$ to $10^{-5}$by fusing the protoplasts in the presence of 40% polyethyleneglycol3350. Nystatin could be used to eliminate fungal contamination during the regeneration of the plotaplasts.

• Korean Journal of Microbiology
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• v.23 no.4
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• pp.265-270
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• 1985

Overall procedure of cell fusion between Brevibacterium flavum and Corynebacterium glutamicum was morphologically observed by transmission electron microscopy. Protoplasts formed by treatment of cells with penicillin G and lysozyme in order were released through the pores generated on a certain region of cell walls to be spherical form. When two different protoplasts were met, cell wall and membrane in the contact zone was disappeared and followed by the mutual exchange of cytoplasmic and/or chromosomal materials. Cell xall regeneration speed of the protoplasts fused was slower than that of the non-fused, whereas the size of the former was confirmed as bigger than that of the latter.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.274-276
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• 1997

Transformation of Coprinus congregatus with a linearized plasmid has been successfully carried out using phosphinothricin resistance gene as a dominant selectable marker. The transforming frequency was about 500 transformants per microgram of DNA using the protoplast-$CaCl_2$ method. The transforming vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from a transformant by Southern hybridization.

• Journal of Life Science
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• v.7 no.4
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• pp.282-288
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• 1997

To obtain a basic information on the development of Genus Viola, ultrastructure and electrofusion process between the two protoplasts from wild Viola callus cells and pansy mesophyll cells were observed with a scanning electron microscopy(SEM) and transmission electron microscopy(TEM). In the ultrastructural observation of wild viola callus protoplasts and pansy mesophyll protoplasts using SEM, their cell walls were removed completely. A knob-like formation was observed on the enlarge surface of viola callus protoplasts. On the surface of pansy mesophyll protoplasts net-like chloroplasts were observed. In SEM observation of pansy mesophyll protoplasts, chloroplasts devoid of membrane were observed on the surface the protoplasts. Pearl chain was formed by applying AC field of 200 V/cm at 1.0 MHz for 43 sec. The lysis of plasma membranes and fusion process occurred by applying a 1,600 V/cm DC pulse twice for 1 sec. After 1-2 hours of a DC pulse application, it was observed that the two protoplasts were fused completely into one cell. In TEM observation of the fused cell, many small vacuoles were located in the fusion area of the two protoplasts. Indeed, two distinct regions were observed during fusing process; in one region, a nucleus was found, while in the other region, both nucleus and nucleous were found.

• Molecules and Cells
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• v.27 no.4
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• pp.443-447
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• 2009

This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.137.1-137
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• 2003

A pepper strain of Cucumber mosaic virus (Pf-CMV) induces a mild chlorotic spot symptom in zucchini squash at 9 days post-inoculation (dpi), wile Fny strain of CMV causes severe mosaic and stunting symptom at 4 dpi in this host. Pseudorecombinants were constructed between the two strains, and assessments of symptom severity were indicated that both RNA2 and RNA3 were responsible for both mildness and the slow appearance of symptom elicited by Pf-CMV in zucchini squash. With various RNA2 and RNA3 chimeras between two strains of CMV, the genetic symptom determinants of phenotype of Pf-CMV were mapped to Tyr residue at positions amino acid 267 in 2a protein and at positions amino acid 168 in 3a movement protein (MP). Chimeras changed the sequences (both changed Tyr to lie) in the codons of both amino acid 168 of 3a MP and amino acid 267 of 2a protein were resulted in the high RNA accumulation, severity of symptom, and the rapid systemic spread, suggesting that 2a replicase as well as MP is involved in virus movement. The RNA accumulation pattern of all pseudorecombinants and chimeras are identical in protoplast of zucchini squash, indicating the virus movement is responsible for the phenotypes of two CMV strains rather than virus replication.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.68.2-69
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• 2003

Genetic transformation carried out to induce the pathogenicity mutants from the two isolates, Elsinoe fawcettii R-34 and MUD of citrus scab fungus to hygromycin resistant by transferring plasmides (pUCATPH) that contain hygB gene. We produced protoplast for transformation by using of combinations of available enzymes including ${\beta}$-D-glucanase, ${\beta}$ -glucuronidase, Iyticase and driselase. The protoplasts regenerated at 64 $\mu\textrm{g}$/ml of hygromycin B but not 128 $\mu\textrm{g}$ in sensitivity test to identify the concentration of useful marker for the selection of transformants. Approximately 1200 and 67 hygromycin resistant isolates from strain R-34 and strain MUD, respectively, were isolated on PDA added with 200 $\mu\textrm{g}$ /ml of hygromycun B. Fifty seven and 4 of hygromycin resistant isolates from strain R-34 and MUD, respectively, did not produce necrotic lesions on the leaf in detached-leaf assay. Finally, 9 isolates were isolated from strain R-34, and these Isolates produced non or very few symptoms on seedlings of citrus in greenhouse pathogenicity test. And it's very interesting that some isolates produced melanose-like symptom on very young leaves which it was not typical symptom and somtimes produced on only expanded leaf.

• Journal of The Korean Society of Grassland and Forage Science
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• v.15 no.1
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• pp.1-12
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• 1995

This study was to investigate an effective biological control of forage diseases and provide a basic data and a model in improving variety of antagonistic bacteria, with growth promoting effect on forage, through cell fusion. The results obtained were summarized as follows; 1. The antagonistic himbacterium against soil-borne phathogenic fungi Fusarium oxysporum and Rhizoctonia solani was isolated from continuous cropping himsphere soil of forage, and its biological and physiological characteristics were investigated. This bacterium was identified as Bacillus subrilis and named BS 101. Another strain for cell fusion was Bacillus thur ingiensis ssp. kurstaki HD-I(BT 37669) with insecticidal crystal. 2. The auxotropic mutants of BS 101 and BT 37669 were derived after mutagenesis using N-methyl-N'nitro- Nitrosoguanidine(NTG) to give amino acid requirement marker. n e s e auxotropic mutants of BS 101 and BT 37669 were named BS 1013(his-) and BT 69(asp-), respectively. 3. The best protoplast requirement was obtained using DM 3 medium, containing 5% casamino acid, 1 M $MgCI_2$ and 2% bovine semm albumin, to give Fusant 3, 7 and 8. BT toxin gene was not identified with fusants by Southern blotting. However, SDS-PAGE analysis of strains showed various protein patterns among fusants. 4. From the dark culture experiment, growth of forage in inoculated soil with antagonistic bacteria was delayed than that of non-inoculated soil with antagonistic bacteria in each continuous cropping soil and in each sterilized soil. On the other hand, growth duration of forage was different between continuous cropping soil and sterilized soil. 5. Seed germination of Alfalfa, Italian ryegrass and Orchardgrass were significantly improved by inoculation of antagonistic bacteria(p< 0.05).