• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.3-7
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• 2002

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.281-286
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• 2018

Zona pellucida (ZP), a primarily representative coat of mammalian egg and embryo, has an extremely heterogeneous morphology during different developmental stages. The objective of the present study was to compare the morphological changes of the ZP surface of immature, in vitro and in vivo matured canine oocytes by using scanning electron microscopy (SEM). Canine ovaries were collected from local veterinary hospitals to recover immature oocytes. The ovaries were sliced and the released cumulus oocyte complexes (COCs) were washed with TL-HEPES. The selected COCs were randomly divided into two groups, first group was processed immediately at immature state and the second group was processed 72 h after in vitro maturation, and compared with in vivo derived oocytes. Oocytes were fixed, critical point dried and examined under SEM. The diameters of oocyte and outer holes of the ZP were measured on a total of 249 oocytes; the results were analyzed using One-way ANOVA. Our results showed that, the diameter of immature oocytes significantly differed (p < 0.05) from that of in vivo matured oocytes ($79.60{\pm}0.77{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Similarly, a significant difference (p < 0.05) in the diameters between those of in vitro and in vivo matured oocytes were found ($79.51{\pm}2.36{\mu}m$ vs. $101.46{\pm}1.07{\mu}m$, respectively). Moreover, the diameters of the outer holes of the ZP were significantly (p < 0.05) larger in in vivo matured ($1.48{\pm}0.42{\mu}m$) than in vitro matured for 72 and immature oocytes ($1.10{\pm}0.16$ and $0.43{\pm}0.12{\mu}m$, respectively). Taken together, these data indicates that the ZP surface is related to oocyte maturity in canine.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.258-258
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• 2004

Successful cryopreservation of mammalian oocytes would provide a source of materials for in vitro embryo production. This study was conducted to determine vitrification conditions for bovine immature oocytes using micro-drop method and, to examine maturation, fertilization and development of vitrified bovine immature oocytes. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1541-1546
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• 2011

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.299-302
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• 1994

Immature zygotic embryos (up to 4mm in length) were cultured on MS medium supplemented with 0.5 to 8mg/L 2,4-D. Up to 87% of them formed somatic embryos on the plumule without producing an intervening callus. The site of somatic embryo formation was confirmed by culturing plumule explants, which consisted of shoot apical meristem domes with 1 or 2 leaf primordia excised from 2-week-old seedlings. When the concentration of 2,4-D was increased over 4 mg/L, the plumule explants produced nonembryogenic calli only, whereas the distal end of the cotyledons directly formed numerous somatic embryos at frequencies of up to 60%. Upon transfer onto MS basal medium,2 out of 15 somatic embryos converted into plantlets. The plantlets were potted to a soil mixture and grown to maturity in a phytotron.

• Journal of Plant Biotechnology
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• v.48 no.1
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• pp.44-53
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• 2021

The present work aims to study the induction of somatic embryogenesis in cork oak (Quercus suber L.) from immature zygotic embryos and young apical leaves obtained from 2-month-old seedlings through acorn germination on sterilized peat. The immature zygotic embryos were grown for 1 month on the mineral solution of MS in the presence of 4.52 µM 2,4-D and 30 g/L sucrose. They were then transferred to the same mineral solution with no added growth regulators. In the third subculture, yellow somatic embryos, characterized by two voluminous cotyledons, were differentiated from the radicle of the immature zygotic embryos. The induction of somatic embryogenesis in young leaves required a series of transfers on different culture media containing 30 g/L sucrose and 100 mg/L myo-inositol. Secondary or recurrent somatic embryogenesis occurred within the immature somatic embryo radicles after 1 month of culture on growth regulator-free medium containing WPM macronutrients, MS micronutrients, and vitamins.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.58-58
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• 2006

• Journal of Ginseng Research
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• v.16 no.1
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• pp.37-43
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• 1992

Structural changes of the endosperm of Panax ginseng C.A. Meyer from fertilization to germination were investigated by light microscope. The endosperm of the ginseng seed is cellular type. Since endosperm cells adjacent embryo continuously breakdown and disappear with the elongation of embryo, the real of endosperm is gradually decreased. As the anatropous ovules of immature seed with green seed coat developes more and more, ovary cells adjacent ovary cavity become abundant by the periclinal division, their size is decreased, hypotrophy of cell wall discern, and they are gradually differentiated in seed coat. Though embryo responds strongly to basic dye at the stage of completion of endosperm formation, tissue of endosperm responds to acidic dye positively Cell wall of embryo and endosperm are composed of primary cell wall not lignified. Endosperm cells adjacent embryo begin to breakdown in the endosperm tissue of indehiscent seed before the beginning of the after-ripening. Dehiscent seed of which seed coat is opened through after-ripening represent the form as a seedling in the result of embryo developments with the formation of organs; radicle, cotyledon, plumule. Umbilifom layer represents strong positive response to the toluidine blue and the basic function. Umbiliform layer that endosperm cells breakdown and disappear is observed clearly at the periphery of the embryo cotylemon, while slightly at the periphery of the radicle.

• Journal of Plant Biology
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• v.39 no.2
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• pp.127-135
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• 1996

An efficient system of somatic embryogenesis was established for the red pepper plant (Capsicum annuum L. cv. Nokkwang) usign immature zygotic embryos. The size of the immature zygotic embryos and the concentrations of 2, 4-D and sucrose were found to be critical. Somatic embryos were induced via callus or directly from explants and regenerated into plantlets successfully. When zygotic embryos 1~2 mm long were cultured on the modified Murashige-Skoong (MS) medium supplemented with 2 mg/L 2, 4-D for 3 weeks in the dark, somatic embryos were induced directly from the apical region of zygotic embryos with the highest frequency being approximately 90%. To mature the somatic embryos, ABA and an ethylene inhibitor AgNO3 were used. The highest frequency of shoot regeneration (25% in each) resulted at 2$\mu$M ABA or 20$\mu$M AgNO3 treatment at rates 3.7 and 1.6 times control, respectively. Shoots developed mainly from the cotyledonary node on CoCl2-containing medium, and from the upper side of cotyledon on medium containing AgNO3 while the embryos on the control medium produced shoots from both the cotyledonary node and the upper region of cotyledons both at frequencies of 50%. Indirect somatic embryogenesis via callus was induced at an efficiency of approximately 10% with zygotic embryos 3~4 mm long cultured on MS medium containing 5~10 mg/L, 2, 4-D for 5~7 weeks under a continuous light condition. The plants regenerated from the somatic embryos were morphologically normal. Using scanning electron microscopy, the direct and indirect somatic embryogeneses were observed to follow the globular, heart and torpedo stages, similar to zygotic embryogenesis. Also, suspensors appeared in the early globular and ovoid-shaped late globular embryos during indirect somatic embryogenesis.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.43-48
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• 2013

Oreorchis coreana Finet is threatened globally by over-collection from its natural habitats for horticultural purposes. Its rarity in nature makes this plant one of the most endangered species in Korea. In this study, we investigated the effects of sodium hypochlorite (NaOCl) on orchid seed viability and seed germination. An in vitro bioassay swelling test using immature seeds was compared with a standard chemical procedure using triphenyl tetrazolium chloride (TTC) to test seed viability. In general, the bioassay was more appropriate for estimating embryo viability after a prolonged pre-treatment (more than 1 h) in 1% NaOCl, a surface sterilant often used to enhance germination of seeds of terrestrial plants. Therefore, an efficient method for investigating in vitro swelling of immature seeds is urgently needed. We established a method for determining the viability and swelling of O. coreana seeds via in vitro examination of immature seeds. Treatment of immature seeds with 1% NaOCl for 10 min greatly enhanced the extent of swelling of immature zygote embryos when compared to untreated seeds. These data obtained here appear to be comparable to viability and swelling that occurs in O. coreana seeds via asymbiotic germination.