• Journal of Embryo Transfer
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• v.31 no.3
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• pp.207-213
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• 2016

The objective of this study was to investigate to influence of glutathione (GSH) on development and antioxidant enzyme activity in tetraploid porcine embryos. Tetraploid embryos were produced using parthenogenetic 2-cell embryo by electrofusion method. Tetraploid embryo development was observed every 24 hours and intracellular antioxidant enzyme activity was measured at 120 hours after electrofusion. The 4-cell to 16-cell stage tetraploid embryos was increased in 100 and $500{\mu}M$ GSH-treated groups compared control group at 48 hours (P < 0.05) but cleavage rates were not significantly different among the GSH treatment groups at 48, 72, 96, and 120 hours. Blastocyst formation was significantly increased by 300 and $500{\mu}M$ GSH at 120 hours in tetraploid embryos (P < 0.05). But blastocyst cell number were not significantly different among the GSH treatment groups ($16.4{\pm}0.8$, $16.8{\pm}2.6$, $18.5{\pm}2.8$ and $17.5{\pm}1.8$). The intracellular antioxidant enzyme level was increased in $500{\mu}M$ GSH compared to 0 and $100{\mu}M$ GSH (P < 0.05). We suggest that GSH may be improve development of tetraploid embryo in pigs.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.121-121
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• 2003

• Korean Journal of Poultry Science
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• v.17 no.1
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• pp.1-6
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• 1990

For the production of tetraploid embryos in chicken through induction of diploid gametes. The experiment was found results as follows; 1. Induction of diploid sperm was observed 11% from embryos of normal females and males at 12 days after injection of 0.37mg per 2kg body weight for three days. 2. 3 among 109 embryos from crosses of diploid gametes induced by TEM were found as tetraploid. 3. Genetic structures of tetraploid embryos were indentified as normal 4 pairs chromosomes without certain variation.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Horticultural Science & Technology
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• v.31 no.3
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• pp.352-358
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• 2013

For the germination and differentiation of immature embryos obtained by artificial crossing between tetraploid grape 'Fujiminori' (Vitis vinifera ${\times}$ V. labruscana) and triploid 'Summer Black' (V. labruscana ${\times}$ V. vinifera), were incubated in vitro using MS medium supplemented with $GA_3$ or coconut water (CW) at various concentrations. The percentage of embryo formation of 'Fujiminori' ${\times}$ 'Summer Black' was 64.3%. Embryo germination percentage was higher than 95% in all the $GA_3$ treatments at the concentrations of 0.01, 0.05, 0.25, and $1.25mg{\cdot}L^{-1}$. However, only 15.8-31.6% of the germinated embryos successfully developed into normal plantlets. At higher concentration of $GA_3$, the plantlets developed infirm hypocotyls with over elongated and less enlarged structure. Among the treatments of CW at the concentrations of 5, 10, 15, and 20% (v/v), 10% and 15% were more effective and plantlet achievement percentage were 68.4 and 66.7%, respectively. The addition of 10% CW was most effective to obtain plantlets with optimal shoot length, node and root numbers. 15% CW was suitable to obtain plantlets with longer roots. Accordingly, the embryo culture using the MS medium supplemented with 10-15% CW was observed to be more efficient for germinating and growing the immature embryos produced from artificial crossing between tetraploid grape 'Fujiminori' and triploid 'Summer Black'.

• 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.

• Journal of Animal Science and Technology
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• v.61 no.4
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• pp.225-233
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• 2019

The aim of this study was to produce porcine tetraploid (4N) parthenogenetic embryos using various methods and evaluate their developmental potential. In method 1 (M1), porcine 4N parthenogenetic embryos were obtained by inhibiting extrusion of both first (PB1) and second (PB2) polar bodies; in methods 2 (M2) and 3 (M3), 4N parthenogenetic embryos were obtained by electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 or PB1 extrusion, respectively. We found no differences in the rates of cleavage or blastocyst formation or the proportion of 4N embryos among M1, M2, and M3 groups. The different methods also did not influence apoptosis rates (number of TUNEL-positive cells/number of total cells) or expression levels of apoptosis-related BAX and BCL2L1 genes. However, total cell and EdU (5-ethynyl-2'-deoxyuridine)-positive cell numbers in 4N parthenogenetic blastocysts derived from M1 were higher (p < 0.05) than those for M2 and M3 groups. Our results suggest that, although porcine 4N parthenogenetic embryos could be produced by a variety of methods, inhibition of PB1 and PB2 extrusion (M1) is superior to electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 (M2) or PB1 (M3) extrusion.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.93-98
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• 2009

In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

• Korean Journal of Plant Tissue Culture
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• v.21 no.4
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• pp.215-220
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• 1994

Pollen-derived embryos cultured on the hormone-free medium showed a low germination frequency (12.5%) and poor growth response after germination. The greatest frequency of germination (81.3%) was obtained from the embryos cultured on medium with 0.3mg/L GA$_3$.The greatest frequency of generation (81.3) was obtained from embryos cultured on medium with 0.3mg/L GA$_3$. The embryos precultured for 20 days on medium with 0.3mg/L GA$_3$were transferred to the medium with various combination of hormones such as IAA, kinetin, zeatin, 6-benzylaminopurin (BA) and Gh$_3$. The germination frequency of cotyledonary stage embryos showed above 72% on media with all of the hormonal combinations, but the embryos germinated on medium with 2mg/L BA or 0.1mg/L kinetin and 0.3mg/L GA$_3$ developed more vigorously into plantlets than those of other hormonal combinations. Torpedo-stage embryos cultured on medium with 0.3 mg/L Gh$_3$ were pretreated for 8 weeks at 2-week intervals at 4$^{\circ}C$, The germination frequency of the cold-preheated embryos increased with the increment of pretreatment period from 2 to 8 weeks. The greatest frequency of germination (73.3%) was obtained from the embryos pretreated for 8 weeks at 4$^{\circ}C$. The chromosomes of the root-tip cells of W plane grown for 40 days after germination were observed. Most of the regenerated plants were haploid (55.8%) or diploid (315%), but triploid (1.3%), tetraploid (5.2%), or aneuploid (6.5%) were also detected among them.

• Journal of Plant Biotechnology
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• v.49 no.1
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• pp.74-81
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• 2022

Brassica napus, an oil crop that produces rapeseed oil, is an allotetraploid (AACC, 2n = 38) produced by natural hybridization between B. rapa and B. oleracea. In this study, microspore was cultured using the F₁ developed from a cross between 'EMS26' line with high oleic acid content and 'J8634-B-30' lines. The flower bud size showing the nuclear development at the late uninucleate and binucleate stage with high embryogenesis rate was 2.6 ~ 3.5 mm. Microspores were cultured using only this size and after then most microspore embryo developed into secondary embryos and then regeneration plants obtained from the developed multilobe. The analysis of the ploidy of the plants revealed that 66.7% and 27.8% of the total lines were tetraploids and octoploids, respectively. The sizes of stomatal cells in tetraploids, octoploids, and diploids were 25.5, 35.6, and 19.9 ㎛, respectively, indicating that ploidy level was positively correlated with cell size. Furthermore, 62 tetraploid doubled haploid (DH) lines were selected. The average oleic acid (C18:1) and linolenic acid (C18:3) concentrations of DH were 72.3% and 6.2%, respectively. Oleic acid and linolenic acid concentrations exceeded the two parental values in 5 and 14 DH lines, respectively, suggesting that these two fatty acids had transgressive segregation. Therefore, the DH population can be utilized for the biosynthesis of unsaturated fatty acids in rapeseed and related genes. It can also be used as a breeding material for varieties with high oleic acid concentrations.