• 한국균학회소식:학술대회논문집
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• 2009.10a
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• pp.14-16
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• 2009

Poria cocos is an importantant medical macrofungus,the sclerotium of Poria cocos has specific value as the drug material. There are few papers about its breeding and spawn rejuvenation. In this project, the protoplasts of cultivated strain T and wild strain L were prepared and treated separately by ultraviolet and heating, then fused with the PEG6000. The tural fusants were selected and identified by the affinity and ISSR analysis. 71 incompatibility strains between parents and reg regenerations were obtained from 118 regenerations by the affinity analysis. Five incompatibility strains were amplified with different primers, the results were showed that they had specific bands of both parents in the profile amplified with 3 primers, which proved these 5 strains were fusants by means of molecular biology marker. On the other hand, 25 strain were selected from 168 protoplast regenerations of cultivated strain T for cultivation experiment. The fresh sclerotium weight of these protoplast regenerations were better than the original strain.significantil 3 strains (T-1, T-4, T-7) increased respectively 118%, 73% and 73% than original strain. This method could be the effective in the rejuvenation Poria cocos.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.318-325
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• 1993

The optima] conditions for the formation, the regeneration. and the spheroplast fusion of Flavimonas aryz/habitans spheroplasts were investigated. Cells were transformed to spherop]asts effectively by treatment of 0.5% volume (v/v) of 0.] M EDTA and ]00 flg/ml lysozyme at $37^{\circ}C$ for 30 min without shaking. Magnesium chloride and calcium chloride were effective on the stabilization of spheroplasts. and 20 mM calcium chloride in the rich regeneration medium improve the yield of regenerants as much as 3.5-fo]d. Addition of 0.8% bovine serium albumine (BSA) in dilution buffer for spheroplast formation improved the stabilization of spheroplasts over extended periods (4-6 hr) at room temperature. and thus increased the yield of recombinants to 4.5-fold. The spheroplast formation frequency and regeneration frequency of F aryzihabitans strain was 90.10% and 3.800/." respectively. The first regenerated cell of F. aryzihabitans spheroplasts were appeared 6 hours after plating. By I I hours after plating, 80% of spheroplasts were regenerated on thc rich regeneration medium containing 0.5 M sucrose. The intraspeci11c spheroplast fusion of F urvz/habitans was carried out and the properties of obtained fusants were investigated. Formation of fusion products was effective when the Flav/munas spheroplast mixture was treated with 40%(w/v) PEG6000 and 20 mM CaCl, for 10 min at room temperature. and thc formation of frequency of recombinants were $2.0{\times}10^{-5}~3.6{\times}10^{-5}$. All tested recombinant clones were very stable on further propagation.

• Korean Journal of Food Science and Technology
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• v.23 no.5
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• pp.561-567
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• 1991

This study was designed to investigate the productivity of L-methionine by the method of protoplast fusion between Brevibacterium flavum ATCC 14067 and Corynebacterium glutamicm ATCC 13032, and then L-methionine production was performed to continuous fermentation using the immobilized fusant cells. Mutants B. flavum K 104($thr\;met\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}\;as\;genetic\;marker$) and C. glutamicum B 70($thr\;Hos\;Km^{r}\;Et^{r}\;Sm^{r}\;Tm^{r}as\;genetic\;marker$) were isolated by MNNG treatment. On the other hand, protoplast of mutants were formed to treat with lysis solution containing $500{\mu}g/ml$ of lysozyme. The ratios of protoplast formation and regeneration were 99% and $64{\sim}66%$ respectively. Fusion frequency between B. flavum K 104 and C. glutamicum B 70 showed the $3.5{\times}10^{5}$ in the 35% polyethylene glycol(PEG6000) containing 3% PVP solution. The productivity of L-methionine by fusant BFCG 37 immobilized with sodium alginate was 0.89 g/l the batch fermentation and was $18.75mg/^{1}hr\;^{1}$ on the continuous fermentation at $30^{\circ}C$ for 72 hr.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.12-19
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• 1999

To improve biopolymer productivity and properties of Bacillus strains, protoplast fusion was performed between biopolymer producing Bacillus subtilis K-1 and lactose utilizing Bacillus coagulans. The results were as follows; Protoplasts mixed in fusion fluid containing 33% PEG 6000, 1% PVP and 10 mM $CaCl_2$ were reacted for 5 min at $37^{\circ}C$ and then centrifused protoplasts were directly overlaid on the selective media containing $100\;{\mu}g/ml$ antibiotics and incubated for 3 days. At this conditions, the frequency of protoplast fusion was generally in the range of $4.6{\times}10^{-5}\;to\;1.8{\times}10^{-7}$ in ratio. Segregation ratio was observed between 1 to 6% indicating genetic stability of all the fusants. Fusants growth were also observed on the media contained amino acid and antibiotics as required marked materials. DNA contents of the selected fusants were 1.6 to 2.7 times more than that of parent strains. With observation by TEM microscopy, spherical protoplasts were first released from the swollen parental cells and then contracted to fuse in the process of fusion. And fused cells were observed representative vesicle. Originally, the parental cells were observed as in the morphology of thick-walled and double membrane-surrounded rod shape with TEM microscopy.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.45 no.1
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• pp.50-54
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• 2000

The elongation of mesocotyl and coleoptile plays important roles in the seedling emergence and stand establishment of dry direct-seeded rice. Experiments were carried out to elucidate the effects of seed-presoaking treatments of GA$_3$ and some inorganic salts on the mesocotyl, and coleoptile elongation of rice. Seed-soaked GA$_3$ promoted the elongation of mesocotyl, but little effect on the coleoptile elongation. The stimulation effects of GA$_3$ were found to be enhanced by addition of CaCl$_2$ However, the sole treatment of CaCl$_2$ showed no stimulating effect on the mesocotyl and coleoptile elongation. Mesocotyl elongation was most prominent in the combined treatments of 50ppm GA$_3$ with 100 mM CaCl$_2$. The synergistic effects of GA$_3$ and CaCl$_2$ on mesocotyl elongation varied with varietal groups. The stimulating effects of GA$_3$ were enhanced significantly by the addition of CaCl$_2$ in japonica varieties, Dongjinbyeo, Ilpumbyeo and Milyang 95, and tall indica variety, Labelle, but not in semidwarf Tongil type varieties, Tongilbyeo, Milyang 23, and Nampungbyeo, and semi-dwarf indica, Short Labelle. The promoting effects of GA$_3$ on the mesocotyl elongation were decreased in proportion to the lowered osmotic potential by PEG 6000 on the contrary to CaCl$_2$ This implies that the synergistic effects of CaCl$_2$ with GA$_3$ on mesocotyl elongation was not caused by osmotic potential lowered by CaCl$_2$ addition but by the salt itself. Salts such as Ca(NO$_3$)$_2$, MgCl$_2$ BaCl$_2$, NaCl, KCl and KNO$_3$ showed the synergistic effects with GA$_3$ on mesocotyl elongation as well. The degree of synergistic effects showed no differences among salts tested, implying that there is no specificity of ions constituting the salts.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.37 no.2
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• pp.134-140
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• 1992

Carrot(Daucus carota L.) seeds (CV Danver 126) were primed and then separated by density differences to improve both the percentage and time of radicle emergence. Seeds for priming were soaked in aerated distilled water for 2 days (water imbibed), 25% solution of polyethylene glycol(PEG) 6000 for 6 or 10 days, salt solution of 0.2M KNO$_3$＋0.1M $K_2$HPO$_4$ for 6 or 10 days, or mixed with Agro-Lig with 90% moisture content for 6 days and 70% moisture content for 6 or 10 days (SMP) at 2$0^{\circ}C$, respectively. The greatest embryo growth and the highest radicle emergence were observed from the seeds treated SMP with 90% moisture content for 6 days among the primed treatments. After the SMP treatment, the seeds were separated into density classes with a float-sink procedure using aqueous solution of Maltrin 600 with 0.02/cm$^3$ density increments. The lower density classes of the carrot seeds, the more embryo growth, the higher and the faster rates of radicle emergence were exhibited in order from 1.06 to 1.14 density classes of the carrot seeds treated SMP.

• The Korean Journal of Nuclear Medicine Technology
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• v.20 no.2
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• pp.62-68
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• 2016

Purpose In a cyclosporine experiment using a robotic liquid handing system has found a deviation of its standard curve and low reproducibility of patients's results. The difference of the test is that methanol is mixed with samples and the extractions are used for the test. Therefore, we assumed that the abnormal test results came from using methanol and conducted this test. In a manual of a robotic liquid handling system mentions that we can choose several setting parameters depending on the viscosity of the liquids being used, the size of the sampling tips and the motor speeds that you elect to use but there's no exact order. This study was undertaken to confirm pipetting ability depending on types of liquids and investigate proper setting parameters for the optimum dispensing ability. Materials and Methods 4types of liquids(water, serum, methanol, PEG 6000(25%)) and $TSH^{125}I$ tracer(515 kBq) are used to confirm pipetting ability. 29 specimens for Cyclosporine test are used to compare results. Prepare 8 plastic tubes for each of the liquids and with multi pipette $400{\mu}l$ of each liquid is dispensed to 8 tubes and $100{\mu}l$ of $TSH^{125}I$ tracer are dispensed to all of the tubes. From the prepared samples, $100{\mu}l$ of liquids are dispensed using a robotic liquid handing system, counted and calculated its CV(%) depending on types of liquids. And then by adjusting several setting parameters(air gap, dispense time, delay time) the change of the CV(%)are calcutated and finds optimum setting parameters. 29 specimens are tested with 3 methods. The first(A) is manual method and the second(B) is used robotic liquid handling system with existing parameters. The third(C) is used robotic liquid handling system with adjusted parameters. Pipetting ability depending on types of liquids is assessed with CV(%). On the basis of (A), patients's test results are compared (A)and(B), (A)and(C) and they are assessed with %RE(%Relative error) and %Diff(%Difference). Results The CV(%) of the CPM depending on liquid types were water 0.88, serum 0.95, methanol 10.22 and PEG 0.68. As expected dispensing of methanol using a liquid handling system was the problem and others were good. The methanol's dispensing were conducted by adjusting several setting parameters. When transport air gap 0 was adjusted to 2 and 5, CV(%) were 20.16, 12.54 and when system air gap 0 was adjusted to 2 and 5, CV(%) were 8.94, 1.36. When adjusted to system air gap 2, transport air gap 2 was 12.96 and adjusted to system air gap 5, Transport air gap 5 was 1.33. When dispense speed was adjusted 300 to 100, CV(%) was 13.32 and when dispense delay was adjusted 200 to 100 was 13.55. When compared (B) to (A), the result increased 99.44% and %RE was 93.59%. When compared (C-system air gap was adjusted 0 to 5) to (A), the result increased 6.75% and %RE was 5.10%. Conclusion Adjusting speed and delay time of aspiration and dispense was meaningless but changing system air gap was effective. By adjusting several parameters proper value was found and it affected the practical result of the experiment. To optimize the system active efforts are needed through the test and in case of dispensing new types of liquids proper test is required to check the liquid is suitable for using the equipment.

• Korean Journal of Medicinal Crop Science
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• v.21 no.1
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• pp.7-19
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• 2013

This study was carried out to select new pelleting binder and material for seeds from Fraxinus rhynchophylla Hance and Alnus sibirica Fisch. ex Turcz. The optimum treatments of the various concentrations and species of priming agents to improve seed germination of both woody medicinal plants were also estimated. Germinability was increased when the seeds of Fraxinus rhynchophylla Hance was soaked in -1.0 MPa of PEG6000 solution at $15^{\circ}C$ for 4 days significantly, the optimum treatment for improving germination of Alnus sibirica Fisch. ex Turcz was observed when the tested seeds was soaked in 100 mM of KCl at $15^{\circ}C$ for 4 days. The influence of physical and chemical properties of pelleting solid materials, the mixture of gypsum, diatomaceous earth, dalma ceramic and vermicuolite (6:1:1:1 ratio) were found as the best pelleting materials for Fraxinus rhynchophylla Hance and Alnus sibirica Fisch. ex Turcz. seeds. To satisfy the requirements of absorption and compatibility for multi-layer seed pelleting, SGPA (Starch-grafted cross-linked polyacrylates) hydrogel was prepared using starch, acrylonitrile, ceric ammonium nitrate, nitric acid, methyl alcohol and potassium hydroxide. The resulting SGPA hydrogel showed high water absorption but not plant compatibility. It suggested that seed pelleting using pelleting materials and SGPA hydrogel (multi-layer coating) after priming agent treatment is to increase germinability and seedling growth and it can reduced irrigation labours and can also save seed.

• Korean Journal of Weed Science
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• v.9 no.2
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• pp.123-129
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• 1989

1) Optimum temperature was $15^{\circ}C$ for C. bursa-pastoris, $20^{\circ}C$ for C. album, $30^{\circ}C$ for P. oleracea, A. lividus, E. crus-galli, D. sanguinalis, and 4 showed wide range of germination temperature. 2) Emergence of C. bursa-pastoris, and C. album was best at $14.8^{\circ}C$ of soil temperature, E. crus-galli at $23^{\circ}C$, E. indica, A. lividis and P. oleracea at $27.1^{\circ}C$ and A. retroflexus and D. sanguinalis at $31.1^{\circ}C$. 3) A. retroflexus and P. oleracea started to germinate at 30% water absorption stage and A. lividus, C. album, S. viridis, and D. sanguinalis at 40% and E. indica at 70%. 4) Germination of weed species was decreased as PEG 6000 induced osmotic potential lowered. C. album, P. oleracea, D. sanguinalis, A. lividus, and Solanum nigrum were germinated at -5.0 bar osmotic potential and C. album and P. oleracea were germinated at -7.0 bar.

• Korean Journal of Food Science and Technology
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• v.22 no.6
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• pp.611-617
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• 1990

This study was conducted to breed a high vitamin $B_{12}$ producer by the fusion of protoplasts between Bacillus natto and Bacillus megaterium. Auxotrophic mutants of Bacillus natto SH-34 ($thr^-try^-rif^r$) and Bacillus megaterium BK-13 ($arg^-ade^-lys^-str^r$) which showed high protease activity and production of vitamin $B_{12}$, respectively, were isolated for the fusion experiment. Protoplasts were induced by incubating the cells with lysis solution containing $500{\mu}/ml$ lysozyme, and the ratio of protoplast and regeneration formation were ranged from 99% and 67%, respectively. Fusion frequencies of fusants between Bacillus natto SH-34 and Bacillus megaterium BK-13 were appeared in the ranges of $1.0{\times}10^{-5}$ under the treatment of 30% PEG 6000 containing 3% PVP. The fusant, MNF-72 showed the highest product yield of $7.85{\mu}g/g-cell\;vitamin\;B_{12}$ in production medium. For the improvement of productivity, the immobilization of fusants with sodium alginate was carried out. In batch and continuous fermentation systems, the productivity were determined to be $0.58{\mu}g/ml.hr\;and\;0.80{\mu}g/ml.hr\;vitamin\;B_{12}$ under optimum condition, respectivity.