• Journal of Plant Biotechnology
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• v.33 no.4
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• pp.297-302
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• 2006

In general, the proliferation of orchids via somatic embryos has been used for mass production of somatic clones because of high propagation efficiency. In spite of high propagation rate, this method often brings somaclonal variation, especially polyploid frequency. Therefor we here concentrated to investigate the relationship between endopolyploidization patterns of explants and the occurrence of tetraploid variant in clonally proliferated Doritaenopsis via somatic embryo regeneration system. In the fully developed somatic embryo, upper part contained 2C to 16C while middle and lower parts showed 2C to 32C DNA content. Two-week-old embryo contained 2C to 16C, whereas those regenerated after 4 to 10-week-old contained 2C to 64C nuclei. Results showed that endoreduplication was variable depending upon tissue types, ages, and parts in one species. lower part of somatic embryo having high endoreduplication degree increased the regeneration of tetraploid variants by about 3-fold comparing to upper part of somatic embryo culture. polyploid frequency occurrence might be closely related to the high levels of endoreduplication of somatic embryos used as explant. It suggested that the upper part of somatic embryo having comparatively low endoreduplication degree is suitable for the stable in vitro propagation system.

• Korean Journal of Medicinal Crop Science
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• v.11 no.2
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• pp.161-166
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• 2003

Korean ginseng(Panax gmseng C.A. Meyer) is very difficult to obtain stable production of qualified ginseng roots because of variable stresses in soil environments. In environment stresses, soil condition is the most important factor, among which nutrients, especially inorganic materials such as N, P, K, Ca, Mg, Fe, etc., influence greatly on the ginseng growth. However, present ginseng field soils in Korea contain so much amount of such inorganic materials that a variety of remarkable disorders were noted in many ginseng plantations, resulting in decrease of qualitative ginseng production. Therefore, it is required to search for genetic resources and genes tolerant to salt stress for the development of ginseng cultivars. Selection of stress-tolerant ginseng lines in fields is very difficult because it is almost impossible to control properly the environmental conditions of soil. On the contrary, it can be studied with ease to search for stress-tolerant ginseng lines through in vitro culture because of easy manipulation of stress conditions. Murashige & Skoog(MS) media with 2.5 folds of $KNO_3,\;NH_4NO_3,\;MgSO_4\;7H_2O,\;KH_2PO_4,\;and\;CaCl_2\;2H_2O$ was established for the selection of ginseng lines tolerant to salt stress under the embryo culture.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.131-138
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• 2001

This experiment was designed to evaluate effects of nonspecific immunostimulator(NIS) Barodon-FX(equation omitted), anionic alkali mineral complex and far-infrared radiation solution on in vivo-produced mouse and in vitro-produced bovine embryos to blastocyst development. Proportion of mouse embryos developing into blastocyst was not greater in BSA- and Barodon-added medium than in BSA-control, but there was signifcantly different(P ＜ 0.05) in hatching and hatched blastocyst development between 0.25% Barodon-and PVP-contained medium(54.7%) than PVP-control(32.5%). BOEC and GC resulted in higher proliferation rate(24∼40% and 17∼22%, respectively) in 0.25∼0.5% Barodon-added medium than in controls, but proliferation of GC and CC greatly decreased in 1∼2% Barodon-added medium. Effect of Barodon on cell proliferation greatly varied among somatic cells. Proportion of early bovine embryos developing into morula and blastocyst was significantly greater(P ＜ 0.05) in 0.5% Barodon-added medium(50% and 63.6%) than in control(31.6% and 27.4%) under co-culture with BOEC and GC, but developmental rate was not different between other Barodon treatments and control. These data indicate that effect of Barodon on cell proliferation significantly varied between somatic cells and that addition of 0.5% Barodon in BOEC-coculture system may further improve blastocyst development in early bovine embryos.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.289-292
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• 2008

Trisodium phosphate 12 hydrate and citric acid monohydrate mixture showed the strong anti-sticking effect on Streptococcus mutans, Streptococcus mitis, and Streptococcus salivarius, which are adhered to glass beads. Each Streptococcus species was shaking-cultured in brain heart infusion broth containing three glass beads. After 18 hr, glass beads were slightly washed into normal saline by three-pin-pointed pincette. Each three glass-beads set was put into reagent -containing tubes, which have 40 mg of bits of weighing paper for gaining brushing effect as similar as brushing one's teeth. The tubes were shaken by vortex mixer for 10 min except non-oral microbe, Streptococcus agalactiae (5 min). The samples were colony-counted by serial agar dilution method. Experiment was repeated three times for each Streptococcus species. The relative ratios of bacterial de-adherence by reagents were calculated in comparison with normal saline control. The de-adherence degree of citric acid-trisodium phosphate-saline mixture (CTS, pH 6.0) against Streptococcus mutans came to an average of 12.5 times compared with normal saline control. Trisodium-saline (TS, pH 8.4) showed the average of 7.5 times, and citric acid-saline (CS, pH 4.6) showed 6.0 times compared to the control group. The bacterial de-adherence degree against Streptococcus salivarius was each 7.2,2.6 and 2.8 times in above reagent sequence in comparison with saline control. CTS and TS showed 2.4 and 3.4 times of anti-sticking effect on Streptococcus mitis respectively, but CS had no anti-sticking effect on this bacterium. CTS, TS and CS showed 0.7, 0.6, and 0.6 times on non-oral microbe, Streptococcus agalactiae, separately compared with saline control. These results show that oral Streptococcus mutans, Streptococcus salivarius, and Streptococcus mitis, which are causative of dental caries or subacute endocarditis, may be easily removed from oral cavity by CTS mixture. It is conceivable that our experimental results will enable the development of a new conceptive toothpaste to prevent dental caries or subacute endocarditis after drawing teeth.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.25-33
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• 2005

This study is a part of research that development of effective genetic resources preservation system using the in vitro spermatogenesis, in vitro insemination and culture system. We aimed for establishment of in vitro culture system with in vitro activated porcine oocytes. The porcine oocytes were matured for 48 hours in $TCM199+10\%$ FCS and activated with $7\%$ ethanol. The activated oocytes were cultured for 7 days in $TCM199+10\%$ FCS or $NCSU23+0.4\%$ BSA medium. The activated oocytes were not developed to the blastocyst stage in $TCM199+10\%$ FCS medium. However in $NCSU23+0.4\%$ medium, those were developed to blastocyst with $3\%$ of treated oocytes. We extended maturation duration of porcine follicular oocytes fur 48, 52, 56, 60, 64, 68, and 72 hours and activated with $7\%$ ethanol and cultured using $NCSU23+0.4\%$ BSA medium. The six percents of activated oocytes were developed to blastocyst in 48 hours and $10\%$ in 52 hours with comparatively low rates suggested to be not fully activated by regenerated MPF. Maturation durations from 56 hours to 68 hours supported to develop upto $11.9\~18.3\%$ of blastocysts. However the developmental rate was declined to $7.2\%$ at 72 hours of maturation duration because of cytoplasmic deterioration. The assumed time window for activation will be $56\~68$ hours of maturation duration. When the matured oocytes were activated with electric pulse of 1, 1.2, 1.4, 1.6, 1.8 and 2.0kV/cm for $80{\mu}s$, although appling the electric current once was not enough for activation, appling twice with 1.6kV/cm for $80{\mu}s$ was shown the highest developmental rate with $11.3\%$. When those were compared with activating methods, $15.7%$ of blastocyst rate was obtained in the $7\%$ ethanol. That was higher than those in electric pulse with $9.5\%$ and calcium ionophore method with $5.8\%$. In this experimental condition, the $7\%$ ethanol treatment was the most effective method for activating porcine oocytes.

• Reproductive and Developmental Biology
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• v.39 no.3
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• pp.89-95
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• 2015

The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.171-178
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• 2003

This study was conducted to investigate the effect of electric fusion methods on cell fusion rate and embryo development by somatic cell nuclear transfer in Korean Native Cattle. The KNC ear cell was cultured in vitro for confluence in serum starvation condition(DMEM＋0.05％ FBS) for cell confluence. The zona pellucida of IVM oocytes were partially dissection using micro pipette. Ear cells were transferred into an enucleated oocyte. The reconstructed embryos were electrically fused with Zimmermann Cell Fusion Medium(ZCFM). Nuclear transfer embryos were activated with a combination of 10${\mu}{\textrm}{m}$ calcium ionophore(5 min) and 2.0mM 6-DMAP(3 hr). The activated embryos were cultured in CR1 -aa medium contains 0.3％ BSA or 10％ FBS at 37$^{\circ}C$, 90％ $N_2$, and 5％ $CO_2$in incubator for 6 days. The fusion rates were 51.6％(chamber) and 68.9％(needle), respectively and there were significantly difference between the fusion method(P<0.05). But, lysis rates were not significantly different(10.7％, 11.5％), respectively. The cleavage rates were significantly different between the chamber method(73.2％) and needle method(80.3％), respectively(P<0.05). The rates of early embryos(2∼4cells) and blastocysts of chamber and needle methods were 54.1％, 61.1％ and 18.4％, 26.3％ respectively, and needle method was significantly higher than chamber method(P<0.05). But, morulae formation rate were not significantly differences between the chamber(6.7％) and needle(6.2) method(P <0.05). These result suggest that electric fusion of needle method was to be profitable for nuclear transfer embryo fusion rate, blastocyst formation rate and reduce of oocyte lysis.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.311-317
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• 2000

This study was carried out to compare the temperature and time of heat shock, and the effect of heat shock on development of embryos after in vitro maturation and fertilization in bovine oocytes. The results obtained were as follows. 1. The optimum temperature and time of heat shock were 41$^{\circ}C$ and 30sec on in vitro development of embryos from 4~8 cell to blastocyst. 2. The rates of cleavage on zygotes produced on in vitro were significantly increased by heat shock after IVM than before IVM(P<0.05). 3. When the oocytes were treated heat shock after IVM and 5 days cultured, developmental rates to blastocyst were increased than other experimental treatments.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.124-124
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.124-124
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• 2005