• Journal of Embryo Transfer
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• v.13 no.2
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• pp.139-145
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• 1998

In the present study, effects of interleukin-2 (IL-2), a differentiator and proliferator of T-cells, on nuclear maturation and sperm penetration of bovine oocytes was examined in a serum-free or serum-containing medium. Basic medium was used TCM-199 supplemented with 2.2g / ι sodium bicarbonate, 100 i.u. /rnl penicillin. 100$\mu$g /ml streptomycin, 0.25$\mu$g/ml Fungizone, this medium treated with FCS and IL-2. In experiment 1, we examined the effect of the addition of 0, 1, 5, 10 or 15nM /ml IL-2 to tissue culture medium (TCM-199) on nuclear maturation of oocytes Development of oocytes to the Metaphase II (M II) stage (%) was significantly (P<0.05) higher at 1, 5,10 and 15 nM /ml IL-2(54.2, 73.5, 80.0 and 69.6%, respectively) than at 0 nM /ml IL-2(35.7%). In experiment 2, we examined the effect of the addition of l0nM /ml IL-2 or 5% FCS in oocyte maturation. Nuclear maturation rates were significantly(P<0.05) higher l0nM /ml IL-2(80%) than non-treatment(35.7%) and 5% FCS(63.6%) treatment. On the other hand, there were no significant difference in the proportion of oocytes developed to the 2-cell stage after addition of IL-2 and/or FCS. These results suggest that IL-2 supports nuclear maturation of bovine immature oocytes in vitro. Serum-free maturation system using IL-2 might be useful for evaluation of various factors on oocyte maturation.

• Journal of Embryo Transfer
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• v.3 no.1
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• pp.13-23
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• 1988

These studies were undertaken to examine the interaction of tamoxifen with sex steroid hormones in rat uterine activity. The uterine wet weights of the immature Tat uterus were examined after the administration of estradiol-l7$\beta$(1$\mu$g), tamoxifen(50$\mu$g), progesterone(lmg). The uterotropic activity in immature ovariectomized rats was observed under various treatment conditions following pretreatment with above drugs. The results obtained were as follows:1) Tamoxifen produced significant increase (p <0.01) in uterine wet weight compared with control group, although the increase was not as great as that seen with estradiol-17$\beta$. Administration of estradiol-17$\beta$ together with tamoxifen inhibited significantly the increase of uterine wet weight by estradiol-17$\beta$ (p < 0.01). Coadministration of progresterone with tamoxifen partly blocked the increase of tamoxifen-induced uterine wet weights by progesterone. 2) Estradiol-17$\beta$after the estradiol-17$\beta$ pretreatment discontinued the declining uterine wet weights due to the absence of estrogen support, but uteri continued to increase in weight if daily estradiol-17 $\beta$ was maintained. Administration of tamoxifen on the fourth day of estradiol-17$\beta$ treatment reduced uterine wet weights within 24 hours, and the weights continued to decline with additional tamoxifen. 3) The modest growth of the uterus induced by three daily injections of 5Opg tamoxifen remained stable for five days, with or without additional tamoxifen treatment. Coadministration of tamoxifen with estradiol17$\beta$ increased slightly the increase of uterine wet weight by tamoxifen. Coadministration of tamoxifen with progesterone inhibited the increase of uterine wet weight by tamoxifen. 4) The modest growth of the uterus induced by three daily injections of lmg progesterone reduced uterine wet weight to the control level for five days. Commencement of tamoxifen or estadiol-17 $\beta$ injections on the fourth day of progesterone treatment rapidly elevated uterine wet weight.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.121-125
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• 2009

In the present study, effects of concentration of cryoprotectant solutions on the nuclear maturation of vitrifiedthawed bovine oocytes were examined. Also, the developmental capacity of vitrified-thawed immature oocytes following ICSI was investigated. Oocytes were cultured in TCM-199 medium supplemented with 5% FBS at $38^{\circ}$C in 5% $CO_2$ and air. The in vitro maturation rate of vitrified oocytes was 24.5 ${\pm}$ 4.2%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (72.0 ${\pm}$ 3.5%, p<0.05). The in vitro maturation rate of vitrified${\sim}$thawed oocytes incubated in TCM-199 medium supplemented with 1.0${\sim}$5.0 ug CB were 26.7 ${\pm}$ 3.2%, 35.7 ${\pm}$ 3.2%, 54.0 ${\pm}$ 3.0%, 42.5 ${\pm}$ 3.6%, respectively. The in vitro maturation rate (57.0 ${\pm}$ 3.0%) of the vitrified-thawed oocytes treated with 3.0 ${\mu}$g CB for 20 min was the highest of all vitrification groups, although the maturation rate were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation rates of the vitrified-thawed (with EDS and EDT) oocytes were 53.8 ${\pm}$ 3.4%, 51.1 ${\pm}$ 3.5%, respectively. This results were lower than the control group (72.0 ${\pm}$ 3.0%). The in vitro developmental rates of the vitrified-thawed oocytes following ICSI were 28.6 ${\pm}$ 4.5%, 25.6 ${\pm}$ 4.3%, respectively. This results were lower than the control group (40.0 ${\pm}$ 4.0%).

• Journal of Plant Biotechnology
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• v.32 no.2
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• pp.91-95
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• 2005

Agrobacterium tumefaciens-mediated immature embryo transformation was used to produce transgenic maize. Immature embryo of Hi II genotype were co-cultivated with strains Agrobacterium tumefaciens (C58C1) containing the binary vectors (pPTN290) carrying with Ubiquitin promoter-GUS gene as reporter gene and NOS promoter-nptll gene conferring resistance to paromomycin as selective agent. Seven embryogenic callus lines transformed showed the resistance in paromomycin antibiotics. Histochemical GUS assay showed that 7 individual lines transformed with the GUS gene were positive response among the transformants. Southern blot analysis revealed that the nptll gene segregated and expressed in their progeny.

• Korean Journal of Plant Tissue Culture
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• v.22 no.4
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• pp.189-194
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• 1995

Calli were successfully induced from immature embryos of Citrus junos SIEB. cultured on 1/2 MS medium supplemented with 40.4 BA. Plant were regenerated from immature embryo derived callus on MS medium with 5 $\mu$M BA. The calli were morphologically characterized by two types: one was whitish and the other was yellowish. After 16 weeks of culture, shoots and root were formed on calli. Plantlets were transplanted to soil and successfully grown to a whole plant Also, the arrangement of the cells showed many differences according to developmental stages of callus and organogenesis. The small cells were compact in callus cultured for 6 weeks and the extended cells which divided actively appeared in it after 8 weeks of culture. The globular protrusion of compacted cells occurred in callus after 10 weeks of culture, and the neighboring cells were liquefied. Oil sac surrounded by the liquefied cell was observed in the leaf and was formed by rupture of liquefied cells.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.37 no.3
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• pp.257-263
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• 1992

Immature embryos were tested for investigating the effect of cold treatment duration and GA$_3$ concentration on the vernalization response in barley. Immature embryos were cultured on B$_{5}$ medium with GA$_3$ for 5 days, and were transplanted under 20/15$^{\circ}C$ temperature after cold treatment. Germination rate and shoot length were increased more in 20-day-old embryos than those in 13-day-old ones but germination day was decreased. Addition of 1 or 10 ppm of GA$_3$ to B5 medium was effective on the growth of immature embryos. The higher GA$_3$ concentration was, the shorter time from embryo culture to flag leaf emergence and heading was. The earlist flag leaf emergence and heading were showed on treatment of 1 or 10 ppm of GA$_3$, but GA$_3$ did not completely replace vernalization. The days to flag leaf emergence of immature embryo-cultured barley with cold treatment for two and three weeks was shortened by 3 and 18 days at 1 ppm GA$_3$, and 16 and 20 days at 10 ppm GA$_3$, respectively, when compared to 0 ppm GA$_3$ culture with cold treatment for three weeks. It was at 10 ppm GA$_3$ with 3 weeks cold treatment that showed the lowest culm length, spike length and number of grain per spike. GA$_3$ concentration was not correlated significantly with each character in 1 week cold treatment, but was significant with each character in 2 weeks. In 3 weeks cold treatment, it was except for days to heading. Correlation between cold treatment duration and culm length was negative in 0 ppm GA$_3$. In 1 or 10 ppm of GA$_3$, all characters had highly negative correlation with cold treatment duration.n.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.283-291
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• 1997

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

• Journal of Plant Biotechnology
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• v.42 no.4
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• pp.388-395
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• 2015

Somatic embryogenesis is as an excellent technology for potential use in plant mass production, germplasm conservation, or genetic engineering. We examined the effect of cold storage using 3 embryogenic callus lines with different levels of embryogenesis competence derived from immature zygotic embryo cultures of Kalopanax setemlobus. Somatic embryo induction, germination and plant conversion were evaluated after 1, 3 and 6 months storage at $4^{\circ}C$ in the dark. Most cold-stored embryogenic calli formed somatic embryos normally even after 6 months; however, the induction rate was gradually decreased by increasing the storage period. The most competent line tended to show a slight decline in somatic embryo induction rate, as compared with other lines after cold storage. In general, cold storage resulted in reduced somatic embryo germination and plant regeneration, although 93% somatic embryo germination and 91% plant conversion were achieved regardless of the storage period. Cold storage led to cell browning and degradation. Additionally, the cell structures were confirmed by the aceto-carmine and evans blue dye evaluation. Collectively, our results showed that embryogenic callus of K. septemlobus could be preserved at $4^{\circ}C$ without subculture for 6 months, and suggested the need for storage of relatively more competent embryogenic calli lines to support somatic embryo induction.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.27-31
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• 1998

Immature hybrid embryos of H. hybridum, 'Picottee', 'White Christmas', 'Eldorado', 'Origin', 'Red Lion', 'elstar', 'Crypsy' were cultured on the MS medium supplemented with various concentrations of 2,4-D, NAA, BA and TDZ. Among the treatments, NAA were more effective for the shoot regeneration and bulblet formation than other treatment. Addition of 0.5 ㎎/L NAA was effective for bulblet induction from explant Shoot regeneration was most effective on the medium with 1.0㎎/L NAA and 2.0 ㎎/L TDZ. The addition of 1.0-2.0㎎/L TDZ induced numerous shoots per explant but strongly inhibited root development when compared to 1.0-2.0㎎/L BA. When bulb scale segments of 'Star Van Holland' was incubated, bulblet formation was the most effective on MS medium with 0.5㎎/L NAA.