• Korean Journal of Plant Tissue Culture
• /
• v.21 no.2
• /
• pp.85-90
• /
• 1994

Attempts were made to regenerate plants from petiole explane of Forest Pimpinella barchycarpa via repetitive somatic embryogenesis. Effective induction of somatic emb교ogenesis was achieved on both MS and modified $B_{5}\;(mB_{5})$ media containing BA + 2,4-D or BA + 2,4-D + NAA under light condition (16-h photoperiod/day) cultures. The explants exposed to the ligt produced numerous somatic embryos while those kept under the dark did not form any on the same medium. Somatic embryos at different developmental stages were observed to arise within a individual explants. Plantlets could be regenerated on $mB_{5}$ basal medium or $mB_{5}$ containing 0.1 mg/L NAA Secondary adventive embryos were formed on the surface of the somatic embryos. Therefore, repetitive somatic embryogenesis could be achieved by secondary embryogenesis. Although the treatment of 2,4-D or NAA alone was effective in callus formation and growth, but not in induction of somatic embryogenesis. Some explants, cultured on NAA-containing media in darkness, produced only adventive roots. The embryogenic potential was maintained for two years when subcultured to BA and 2,4-D containing media with 5 weeks inteval. Regenerated plantlets were maintained on $mB_{5}$ or MS basal media for 4 to 6 more weeks and transferred to soil of an artificial mixture for acclimation. Most plantlets (more than 97%) survived, and grew without any deformity.

• Clinical and Experimental Reproductive Medicine
• /
• v.28 no.1
• /
• pp.33-40
• /
• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

• Journal of Periodontal and Implant Science
• /
• v.46 no.6
• /
• pp.362-371
• /
• 2016

Purpose: The increasing demand for esthetically pleasing results has contributed to the use of ceramics for dental implant abutments. The aim of this study was to compare the biological response of epithelial tissue cultivated on lithium disilicate ($LS_2$) and zirconium oxide ($ZrO_2$) ceramics. Understanding the relevant physicochemical and mechanical properties of these ceramics will help identify the optimal material for facilitating gingival wound closure. Methods: Both biomaterials were prepared with 2 different surface treatments: raw and polished. Their physicochemical characteristics were analyzed by contact angle measurements, scanning white-light interferometry, and scanning electron microscopy. An organotypic culture was then performed using a chicken epithelium model to simulate peri-implant soft tissue. We measured the contact angle, hydrophobicity, and roughness of the materials as well as the tissue behavior at their surfaces (cell migration and cell adhesion). Results: The best cell migration was observed on $ZrO_2$ ceramic. Cell adhesion was also drastically lower on the polished $ZrO_2$ ceramic than on both the raw and polished $LS_2$. Evaluating various surface topographies of $LS_2$ showed that increasing surface roughness improved cell adhesion, leading to an increase of up to 13%. Conclusions: Our results demonstrate that a biomaterial, here $LS_2$, can be modified using simple surface changes in order to finely modulate soft tissue adhesion. Strong adhesion at the abutment associated with weak migration assists in gingival wound healing. On the same material, polishing can reduce cell adhesion without drastically modifying cell migration. A comparison of $LS_2$ and $ZrO_2$ ceramic showed that $LS_2$ was more conducive to creating varying tissue reactions. Our results can help dental surgeons to choose, especially for esthetic implant abutments, the most appropriate biomaterial as well as the most appropriate surface treatment to use in accordance with specific clinical dental applications.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.30 no.4
• /
• pp.283-290
• /
• 2010

Guineagrass (Panicum maximum Jacq.) is an important warm-season forage grass as well as biomass crop. It has both sexual and asexual mode of reproduction (apomictic) depending on cultivar. We developed efficient plant regeneration system for an apomictic (cv. Natsukaze) and a non-apomictic (Noh-PL1) guineagrass by optimizing the level of L-proline in the callus induction and that of $AgNO_3$ in plant regeneration medium. Among the L-proline concentrations tested, the best callus induction was achieved by using 2g/L L-proline in both the genotypes. Immature embryos proved to be the best explant source for tissue culture of guineagrass. The highest frequency of shoot regeneration was obtained on MS plant regeneration medium supplemented with 2 mg/L $AgNO_3$. These results provide a foundation for efficient tissue culture and genetic improvement of guineagrass.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.101-111
• /
• 2001

The present study was conducted to develop an in vitro culture system that would support bovine follicle growth from preantral to antral stage, oocyte maturation, fertilization, and embryonic development. Bovine preantral follicles (150$\pm$1.2 ${\mu}{\textrm}{m}$) surrounded by theca cell were isolated ezymatically and mechanically from ovarian cortical slides in Leibovitz L-l5 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium. The survival and growth rates of follicles cultured in the presence of FSH (10~150 ng/$m\ell$) were significantly higher than those of control group (P ＜ 0.001), but no significant differences were observed in survival and growth rates of follicles between the LH treatment groups (1~125 ng/$m\ell$) and the control. The survival (40%) and growth (244 $\pm$ 0.5 $\mu\textrm{g}$) of follicles cultured with FSH (90 ng/$m\ell$) and LH (25 ng/$m\ell$) were higher than those of control (25%, 160 $\pm$1.0 $\mu\textrm{g}$). Finally, 50% percent of healthy antral follicles were obtained, and almost 60% of them has complete meiotic division with 1st polar body (18.1%) and 10.0% have developed to the cleaved embryo and blastocyst stage. These results suggest that bovine preantral follicle with intact theca cell can grow to the antral stage using these culture conditions, and that oocytes from in vitro-matured bovine preantral follicle may acquire meiotic competence and can undergo fertilization and development.

• Development and Reproduction
• /
• v.5 no.1
• /
• pp.17-21
• /
• 2001

This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

• Korean Journal of Plant Tissue Culture
• /
• v.26 no.2
• /
• pp.121-126
• /
• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

• Journal of Korean Society of Forest Science
• /
• v.99 no.4
• /
• pp.633-637
• /
• 2010

This study was conducted to evaluate various effects of kinds of culture medium, concentrations of abscisic acid (ABA) or /kinds of osmotica on maturation of somatic embryos (SEs) with four (LL-L, LL-K, LL-P and LL-N) embryogenic tissue lines (ETLs) in Japanese larch (Larix leptolepis). In comparison of two culture medium, the LL-P produced the highest number of the cotyledon-staged SE (134.9/90 mg tissue) in 1/2LM medium. In contrast, no SEs were obtained except the LL-P (32.9) in medium of BLG. Effects of two concentrations of ABA in the medium with four ETL for SEs maturation were also compared. In the test of 60 or 100 ${\mu}M$ ABA, the highest result was obtained in 60 ${\mu}M$ ABA (142.9). However, the influence of ABA had little on SEs production except the LL-N regardless of concentrations of ABA. In comparison of different kinds/concentrations of osmotica, the best response was obtained from the treatment of 0.2 M maltose, the LL-K (540.5). In conclusion, the effects of production of SEs were greatly rely on the ETLs, rather than kinds of medium, concentrations of ABA or osmotica which were used in maturation of SEs.

• Molecules and Cells
• /
• v.23 no.3
• /
• pp.323-330
• /
• 2007

The mature wheat embryo is arguably one of the best explants for genetic transformation because of its unlimited availability and lack of growth season restriction. However, an efficient regeneration system using mature wheat embryos (Triticum aestivum L.) is still not available. To identify genes related to the tissue culture response (TCR) of wheat, QTLs for callus induction from mature embryos and callus regeneration were mapped using an RIL population derived from the cross of 'Wangshuibai' with 'Nanda2419', which has a good TCR. By whole genome scanning we identified five, four and four chromosome regions conditioning, respectively, percent embryos forming a callus (PEFC), percent calli regenerating plantlets (PCRP), and number of plantlets per regenerating callus (NPRC). The major QTLs QPefc.nau-2A and QPcrp.nau-2A were mapped to the long arm of chromosome 2A, explaining up to 22.8% and 17.6% of the respective phenotypic variance. Moreover, two major QTLs for NPRC were detected on chromosomes 2D and 5D; these together explained 51.6% of the phenotypic variance. We found that chromosomes 2A, 2D, 5A, 5B and 5D were associated via different intervals with at least two of the three TCR indexes used. Based on this study and other reports, the TCRs of different explant types of wheat may be under the control of shared or tightly linked genes, while different genes or gene combinations may govern the stages from callus induction to plantlet regeneration. The importance of group 2 and 5 chromosomes in controlling the TCRs of Triticeae crops and the likely conservation of the corresponding genes in cereals are discussed.

• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.17-23
• /
• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.